Development and characterization of rabbit proximal tubular epithelial cell lines. We have isolated rabbit kidney proximal tubular epithelial cell lines. The selection was based on their ability to form confluent monolayers on porous supports and to maintain receptor-mediated signal transduction and ion transport, characteristic of the proximal tubule. The isolation method consisted of several steps: (1) superficial cortical proximal tubule segments were microdissected and cultured on a matrix-coated porous support until cells formed a confluent monolayer; (2) primary cultures showing hormone-regulated ion transport typical for the proximal tubule were selected and co-cultured with irradiated fibroblasts; and (3) the epithelial cells surviving after several passages were expanded and passaged on porous substrates. Most of the cell lines developed in this manner were obtained by co-culture with irradiated fibroblasts producing a recombinant retrovirus encoding SV40 large T antigen and G418 resistance. However, SV40 T antigen expression was not essential for immortalization, since neither T antigen nor G418 resistance was detected in the isolated cell lines and co-culture with non-producing 3T3 cells gave similar results. One cell line (vEPT) has been characterized in some detail with respect to morphological, biochemical, and ion transport properties. This line forms confluent monolayers with apical microvilli, tight junctions, and convolutions of the basolateral plasma membrane. Once confluent, monolayers maintain conductances of 25 to 32 mS/cm2 for several weeks in culture and possess phlorizin-sensitive short-circuit current (Isc) in glucose containing media, indicative of apical Na(+)-glucose co-transport. vEPT cells also retain receptor and signaling mechanisms for angiotensin II (Ang II). Apical and basal Ang II and 5,6-epoxy-eicosatrienoic acid (5,6-EET) modulate the Isc in a manner similar to primary cultures. The cell lines share with primary cultures expression of the cytokeratins K8, K10/K11, and K19 ("nomenclature" [21]). They also retain several receptor and signal transduction mechanisms. For example, Ang II, arachidonate, bradykinin, 5,6-EET, parathyroid hormone (residues 1 through 34), and purine nucleotides increase cytosolic Ca2+, PTH elevates cAMP levels, and Ang II enhances proximal tubule-specific arachidonic acid metabolism.


Tetrandrine inhibits both T and L calcium channel currents in ventricular cells. Tetrandrine, a putative Ca2+ channel blocker, is extracted from the Chinese medicinal herb, Radix stephania tetrandrae. In the present study, the whole-cell version of the patch clamp technique was used to investigate the effects of tetrandrine on both T and L calcium channel currents in primary cultured neonatal rat ventricular cells. We show that tetrandrine inhibits both T and L calcium channel currents in ventricular cells. This inhibition of inward Ca2+ currents is concentration dependent and reversible. Tetrandrine does not shift the I-V relationship of the calcium currents. These results clearly demonstrate that tetrandrine acts as a calcium channel antagonist in ventricular cells. Previous data show that tetrandrine may be regarded as a wide-spectrum calcium channel antagonist.


Renal effects of alacepril in essential hypertension. Short-term effects of alacepril, an angiotensin-converting enzyme inhibitor (ACEI), on renal function and hemodynamics were investigated in 10 hypertensive subjects (aged 55.7 +/- 9.5 years, mean +/- SD). Renal plasma flow (RPF) and glomerular filtration rate (GFR) were examined before and after 12-week administration of alacepril, by [131I]hippuran and [99mTc]DTPA, respectively. Alacepril (50 mg/day) caused a significant decrease in both systolic and diastolic blood pressure (SBP and DBP, from 161 +/- 8 to 140 +/- 10 mm Hg and from 100 +/- 3 to 90 +/- 5 mm Hg, respectively). Alacepril increased GFR (from 63.4 +/- 22.2 to 69.1 +/- 22.1 ml/min/1.73 m2, p less than 0.05) without changing RPF (from 438 +/- 194 to 432 +/- 148 ml/min/1.73 m2, p greater than 0.05). Serum creatinine and electrolytes were not changed by alacepril administration. These data show that short-term alacepril administration improves renal function, probably owing to relaxation of renal vasoconstriction.


Hemodynamic and renal effects of the protein kinase inhibitor staurosporine in conscious rats. To evaluate the pattern of hemodynamic responses produced by an inhibitor of protein kinase C (PKC), staurosporine 0.03-0.55 mg/kg was administered intravenously (i.v.) to conscious, normotensive rats chronically instrumented with vascular catheters for direct measurement of blood pressure (BP) and i.v. administration of drugs and either an aortic flow probe for measurement of cardiac output (CO) or miniaturized pulsed Doppler flow probes for measurement of hindquarter, renal, and mesenteric vascular resistances. Staurosporine decreased mean arterial pressure (MAP) and total peripheral resistance (TPR) and increased heart rate (HR) in a dose-dependent manner. Because staurosporine decreased resistance in all three vascular beds monitored (hindquarter, renal, and mesenteric), staurosporine is probably a nonselective vasodilator that decreases MAP by decreasing resistance in a number of peripheral vascular beds. Staurosporine produced biphasic effects on CO, dF/dtmax and peak aortic blood flow; these parameters were significantly increased at doses less than 0.3 mg/kg and decreased to levels equal to or significantly less than control values at doses greater than 0.3 mg/kg. In comparison, the calcium channel blocker nitrendipine decreased MAP and TPR and increased HR, CO, dF/dtmax, and peak aortic flow in a dose-dependent manner over the entire dose range (0.01-1 mg/kg i.v.). Staurosporine (0.3 mg/kg) and nitrendipine (1 mg/kg) produced similar changes in MAP (-44 +/- 3 and -33 +/- 2 mm Hg, respectively), yet staurosporine affected dF/dtmax to a lesser extent than nitrendipine (-5 +/- 36 and 390 +/- 46 ml/s/s, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)


Progesterone potentiation of bupivacaine arrhythmogenicity in pentobarbital-anesthetized rats and beating rat heart cell cultures. The effects of progesterone treatment on bupivacaine arrhythmogenicity in beating rat heart myocyte cultures and on anesthetized rats were determined. After determining the bupivacaine AD50 (the concentration of bupivacaine that caused 50% of all beating rat heart myocyte cultures to become arrhythmic), we determined the effect of 1-hour progesterone HCl exposure on myocyte contractile rhythm. Each concentration of progesterone (6.25, 12.5, 25, and 50 micrograms/ml) caused a significant and concentration-dependent reduction in the AD50 for bupivacaine. Estradiol treatment also increased the arrhythmogenicity of bupivacaine in myocyte cultures, but was only one fourth as potent as progesterone. Neither progesterone nor estradiol effects on bupivacaine arrhythmogenicity were potentiated by epinephrine. Chronic progesterone pretreatment (5 mg/kg/day for 21 days) caused a significant increase in bupivacaine arrhythmogenicity in intact pentobarbital-anesthetized rats. There was a significant decrease in the time to onset of arrhythmia as compared with control nonprogesterone-treated rats (6.2 +/- 1.3 vs. 30.8 +/- 2.5 min, mean +/- SE). The results of this study indicate that progesterone can potentiate bupivacaine arrhythmogenicity both in vivo and in vitro. Potentiation of bupivacaine arrhythmia in myocyte cultures suggests that this effect is at least partly mediated at the myocyte level.


Effects of a combination of acidosis, lactate, and lysophosphatidylcholine on action potentials and ionic currents in guinea pig ventricular myocytes. The aim of this study was to determine the electrophysiological effects of a combination of factors that are of importance during myocardial ischaemia, i.e., acidosis, lactate, and lysophosphatidylcholine, in ventricular myocytes. Intracellular microelectrode techniques were used to record action potential and ionic currents in ventricular myocytes before, during, and after a 30 min exposure to a salt solution that was acidotic (pH 6.8), and contained lactate (10 mM) and lysophosphatidylcholine (5 microM). Single ventricular myocytes were dissociated enzymatically from guinea pig hearts, and perfused with either normal or modified physiological salt solution. Combined acidosis, lactate, and lysophosphatidylcholine resulted in a reduction in the resting membrane potential and maximum rate of depolarisation of phase 0, and flattening of the plateau but prolongation of the action potential duration at 90% repolarisation. Automatic activity was also induced in about one-third of the cells studied. Under voltage-clamp conditions, this combination of factors reduced the peak inward calcium current, on repolarisation after a depolarising step, reduced the steady-state outward current, and reduced the delayed rectifier current, measured as the tail current at the end of a depolarising clamp step. In some cells, a transient inward current was induced by the modified salt solution. It is concluded that the characteristic alterations in action potential characteristics induced by a combination of acidosis, lactate, and lysophosphatidylcholine are likely to result from reductions in the inward Ca current and the background and delayed rectifier K current.


Prostaglandin E2 in dogs with heart failure: hemodynamic, hormonal, and renal effects. To test the efficacy of exogenous prostaglandins for vasodilator therapy in heart failure, we studied the hemodynamic, hormonal, and renal effects of prostaglandin E2 (1.5-150 ng/kg/min) in six conscious dogs before and after induction of heart failure by right ventricular pacing (250 beats/min, 10 days). In healthy dogs, PGE2 decreased the mean arterial pressure (MAP) by a reduction in total peripheral resistance (TPR), increased cardiac output (CO), stroke volume (SV), and heart rate with no effect on right atrial pressure (RAP). Plasma levels of renin (PRC) and norepinephrine (NE) were increased at the highest dosage. Renal plasma flow (RPF) and sodium excretion (UNaV) were augmented without a change in the glomerular filtration rate (GFR) and urine flow (UF). In dogs with heart failure, PGE2 lowered the MAP and TPR and elevated the CO and SV without an effect on the RAP, PRC, and NE. The RPF and GFR were not changed, but the increase in UNaV was preserved and UF significantly augmented. In experimental heart failure, PGE2 increases the CO due to arteriolar dilation and afterload reduction without inducing further neurohumoral activation and exerts potent natriuretic and diuretic action. Therefore, PGE2 may have beneficial effects in heart failure therapy.


Differential effects of alpha 1- and alpha 2-adrenoceptor agonists on peripheral vasoconstriction in pithed diabetic rats. The pressor responses to selective alpha 1-adrenoceptor agonists (cirazoline and methoxamine) and to alpha 2-adrenoceptor agonists (UK-14,304 and B-HT 933) were investigated in pithed, streptozotocin-induced diabetic rats and age-matched control animals. Three months after induction of diabetes, the basal values of diastolic blood pressure (DBP) were the same in pithed diabetic and control rats (34.3 +/- 4.4 vs. 32.8 +/- 4.4 mm Hg, p greater than 0.05, n = 30). In pithed diabetic rats, dose-response curves for the vasoconstrictor effects of cirazoline and methoxamine were shifted to the right with a slight decrease in the maximum response as compared with those in control animals. In contrast, no shift in dose-response curves for the alpha 2-adrenoceptor-mediated vasoconstrictor effects was observed, as reflected by similar pD2 values. A pronounced decrease in the maximum pressor response to the alpha 2-adrenoceptor agonists could be demonstrated. This reduction was significantly greater than that of the alpha 1-adrenoceptor agonists. In conclusion, the functional role of alpha 1- and alpha 2-adrenoceptors in the peripheral resistance vessels appears to be differentially influenced by the diabetic state.


Enhanced inotropic responsiveness to alpha 1-adrenoceptor stimulation in isolated working hearts from diabetic rats. We compared the inotropic responsiveness to the alpha 1-adrenoceptor agonist cirazoline and the calcium entry promoter Bay K 8644 in isolated working hearts from streptozotocin (STZ) diabetic rats and age-matched controls. The maximal rate of contraction and cardiac output (CO) were unaffected by diabetes. The maximal rate of relaxation was significantly decreased in diabetic hearts as compared with controls. Cirazoline (10(-9)-10(-6) M) induced an increase in both maximal rate of contraction and relaxation in diabetic and control hearts. Notably, the maximal rate of contraction was significantly greater in diabetic hearts. Changes in maximal rate of relaxation were similar in both preparations. A significant increase in CO was observed in diabetic hearts, but not in controls. With Bay K 8644 (10(-9)-10(-6) M) added, the increase in maximal rate of contraction and relaxation as well as CO were not significantly different in the two preparations although the changes in maximal rate of relaxation tended to be decreased in diabetic hearts. Our results indicate that cardiac alpha 1-adrenoceptors appear to gain greater functional importance in regulation of myocardial inotropism in diabetes mellitus.


Bay K 8644 is a negative inotrope in the presence of arachidonic acid but not eicosapentaenoic acid. The inotropic and radioligand binding characteristics of arachidonic acid (AA) and the fish oil derivative, eicosapentaenoic acid (EPA), were studied. AA alone was a concentration-dependent negative inotrope in isolated hamster papillary muscles. The negative inotropic effect of AA was enhanced 500-fold by the coadministration of indomethacin (cyclo-oxygenase inhibitor) and caffeic acid (lipoxygenase inhibitor). The dihydropyridine calcium channel agonist Bay K 8644 (BK) was a concentration-dependent positive inotrope. BK in the presence of AA was a concentration-dependent negative inotrope. The negative inotropic effect of BK in the presence of AA was blocked by caffeic acid but not indomethacin. EPA alone was a concentration-dependent negative inotrope. EPA had no effect on the positive inotropic effect of BK. Scatchard analyses of [3H]nitrendipine (dihydropyridine) binding to cardiac membranes in the presence of AA or EPA resulted in a decrease in the KD (-1/slope) with no effect on Bmax (x intercept). Kinetic studies further revealed that both AA and EPA enhanced the off rate of [3H]nitrendipine binding. These data suggest that both AA and EPA mediate similar direct negative inotropic effects through cardiac sarcolemmal calcium channels. A unique effect on E-C coupling by AA, not shared by EPA, is mediated through the lipoxygenase pathway (leukotrienes). AA and its eicosanoid products may play critically important roles in regulating myocardial contractility during acute and chronic myocardial ischemia. Differences between AA and EPA may contribute to the beneficial effects of dietary fish oils enriched in EPA.


Hemodynamic and neurohumoral responses to intravenous nicorandil in congestive heart failure in humans. Nicorandil is a vasodilator drug that combines potassium channel opening properties with nitrate effects. The resulting potent and unique vasodilating properties suggest a potential therapeutic role in congestive heart failure. We therefore studied the acute hemodynamic and neurohumoral responses to nicorandil, given as single intravenous bolus doses of 158, 251, 398, or 630 micrograms/kg, to 22 patients with chronic congestive heart failure (ejection fraction less than 40%). Hemodynamic responses occurred within 5 min of dosing and terminated within 240 min. The heart rate was significantly increased only at 5 min after the 158 micrograms/kg dose, and was unchanged after all other doses. The mean arterial pressure was reduced only by the 398 and 630 micrograms/kg doses. The pulmonary capillary wedge pressure and right atrial pressure were significantly reduced by all doses within the initial 30 min; this reduction in pulmonary capillary wedge pressure was better sustained over time by the two larger doses, whereas the reduction in right atrial pressure was sustained only by the 158 micrograms/kg dose. The cardiac index was reduced by the 158 micrograms/kg dose, but increased after 251, 398, and 630 micrograms/kg of nicorandil. Plasma nicorandil concentrations were positively correlated with changes in cardiac index, systemic arterial pressure, pulmonary capillary wedge pressure, heart rate, and systemic vascular resistance. When measured 1 h after dosing, plasma immunoreactive ANF decreased, norepinephrine concentrations did not change, and plasma renin activity increased, but only at the 630 micrograms/kg dose level.(ABSTRACT TRUNCATED AT 250 WORDS)


Cardiovascular pharmacology of the vasodilator-cardiotonic agent, 349U85. 349U85 is a chemically novel, nonglycoside, noncatecholamine cardiotonic-vasodilator agent with a unique cardiovascular profile in vitro and in vivo. 349U85 and milrinone, 10(-6) to 3 x 10(-5) M each, produce concentration-dependent increases in tension development of 33-60% and 37-60%, respectively, with corresponding 5-18% and 17-55% increases in contractile rate, respectively, in guinea pig spontaneously beating isolated paired atria. In anesthetized dogs, 349U85 at 0.03-1.0 mg/kg i.v. produces dose-dependent increases in left ventricular contractility (dP/dt) of 12-159%, decreases in total peripheral resistance of 11-38%, and increases in heart rate of 3-26%. Milrinone, Cl-914, and enoximone produce comparable increases in dP/dt and decreases in peripheral resistance yet increase the heart rate a maximum of 71, 49, and 41%, respectively. Intra-arterial injection of 349U85 into the vascularly isolated hindlimb of anesthetized dogs produces dose-dependent direct vasodilation. The inotropic effect of 349U85, following a single intravenous dose, is sustained in excess of 4 h while comparable initial inotropic effects of milrinone and enoximone are sustained less than 1 and 2.5 h, respectively. 349U85 effectively reverses acute cardiac depression in anesthetized dogs with a duration exceeding that of milrinone. In conscious dogs, 349U85, at 0.1-1.0 mg/kg p.o., produces a dose-dependent positive inotropic effect (15-73%) with no significant effect on heart rate. Following a single oral dose of 349U85, the inotropic effect is sustained in excess of 6 h. Results of these studies indicate that 349U85 is a potent, long-lasting positive inotropic and vasodilator agent with minimal heart rate effect in vitro and in vivo and is different from a number of reference inodilators.


Comparison of negative inotropic and vasodilator effects of isradipine and nifedipine after complete autonomic blockade in ischemic heart disease. The cardiodepressant and vasodilator effects of an intravenous (i.v.) infusion of 0.5 mg isradipine or 2 mg nifedipine were interindividually (10 vs. 10 patients) compared in a double-blind study in patients with stable coronary heart disease. To minimize vasodilation-induced autonomic reflex mechanisms, which may counterbalance negative inotropic effects after acute administration of calcium antagonists, i.v. autonomic blockade was produced by 0.2 mg/kg propranolol and 0.04 mg/kg atropine. Systemic hemodynamics were measured before and 15 min after the end of the 15-min calcium antagonist infusion. After administration of both drugs, heart rate (HR) decreased similarly during the observation period (isradipine from 93 +/- 10 to 88 +/- 9 beats/min and nifedipine from 84 +/- 9 to 79 +/- 6 beats/min, both p less than or equal to 0.01). The reduction in total peripheral resistance (TPR) was significantly (p less than or equal to 0.01) greater after isradipine (from 1,376 +/- 285 to 1,002 +/- 224 dynes s cm-5) than after nifedipine (1,258 +/- 262 to 1,112 +/- 225 dynes s cm-5). Between the two drugs, the difference in the reduction of afterload independent dP/dt40, determined by tip-manometry, reached borderline significance (p = 0.08) (isradipine from 1,197 +/- 258 to 1,157 +/- 225 mm Hg/s, NS and nifedipine 1,228 +/- 226 to 1,109 +/- 227 mm Hg/s, p less than or equal to 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)


Contrasting effects of cyclic AMP increase caused by beta-adrenergic stimulation or by adenylate cyclase activation on ventricular fibrillation threshold of isolated rat heart. Increased myocardial tissue cyclic AMP has been associated with both a positive inotropic and a proarrhythmic effect. We wished to determine whether two agents that increase myocardial cyclic AMP levels by different mechanisms would induce comparable changes in vulnerability of the heart to ventricular fibrillation (VF) and in the inotropic status. Using an isolated perfused rat heart model, we studied the effects of beta-adrenoceptor stimulation by isoproterenol (ISO) and direct activation of adenylate cyclase by forskolin. The ventricular fibrillation threshold (VFT) was taken as an index of the vulnerability to VF and peak left ventricular systolic pressure (LVSP) as a measure of the force of LV contraction. ISO resulted in a dose-related increase in tissue cyclic AMP with a corresponding decrease in VFT and a marked increase in LVSP. Forskolin produced a delayed but exponential increase in cyclic AMP at concentrations greater than 3 x 10(-7) M with relatively small increases in LVSP. With forskolin, the VFT decreased only at extremely high cyclic AMP levels, suggesting that the drug had increased cyclic AMP in a compartmentalized manner. The discrepant effects of ISO and forskolin on VFT could not be explained by changes in heart rate (HR). These results show that an increase in tissue cyclic AMP can have markedly different arrhythmogenic effects depending on the mechanism by which cyclic AMP is increased.


Protective effect of cicletanine on hypertension-induced decreases in the renal kallikrein-kinin and prostaglandin systems in stroke-prone spontaneously hypertensive rats. We investigated the effect of two oral (p.o.) doses of cicletanine (5 and 30 mg/kg/day) for 4 weeks on urinary excretion (UKE), renal concentration (RKC) of kallikrein, and prostaglandin E2 (PGE2) and 6-keto-PGF1 alpha urinary excretion of stroke-prone (SP) spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) rats submitted to a high sodium intake (1%). Both doses of cicletanine induced a significant antihypertensive effect in treated SHR as compared with hypertensive untreated controls (HC). After 4-week treatment, a significant difference in mortality was observed between normotensive controls (NC) (0%) and HC (84%). Both doses of cicletanine reduced the mortality of hypertensive animals (8% SHR with 5 mg and 24% SHR with 30 mg vs. 84% in HC). Whereas UKE and RKC were decreased in HC during the progression of untreated hypertension from week 1 to week 4, both doses of cicletanine administration significantly prevented this decrease. Consistently with maintenance of UKE during the course of hypertension, the level of tissue kallikrein was higher in hypertensive cicletanine-treated than in untreated SHR. This increased RKC was associated with a significantly higher rate of kallikrein biosynthesis. The increased level of the urinary excretion and tissue concentration of PGE2 and 6-keto-PGF1 alpha in cicletanine-treated SHR as compared with untreated animals was also of interest. This protective effect on PG excretion correlated with that on kallikrein excretion. The results confirm the efficiency of cicletatine as an antihypertensive treatment. The antihypertensive action includes protective effects on potential vasodepressor kallikrein-kinin and prostaglandin systems.(ABSTRACT TRUNCATED AT 250 WORDS)


Effects of yohimbine on heart rate variability in panic disorder patients and normal controls: a study of power spectral analysis of heart rate. We studied the effects of yohimbine on heart rate (HR) variability in 13 normal controls and 13 patients with panic disorder. Yohimbine produced a significant increase in SD of HR in standing posture in both patients (p = 0.01) and normal controls (p = 0.025). Panic disorder patients had a significant increase in standing absolute midfrequency (MF) power (0.07-0.15 Hz) after administration of yohimbine (p = 0.002). The ratio of post- to preyohimbine standing MF power (0.07-0.15 Hz) during standing was significantly higher in patients as compared with controls (2.3 +/- 1.08 vs. 1.33 +/- 0.38; p = 0.01), which suggests an increased responsivity of panic disorder patients to the adrenergic effects of yohimbine.


Reduction of canine myocardial infarct size by CI-959, an inhibitor of inflammatory cell activation. CI-959, a cell-activation inhibitor that prevents the formation of oxygen-derived free radicals by inflammatory cells, was studied to determine its effects on myocardial infarct size and subsequent scar formation in dogs. The left circumflex coronary artery was occluded for 90 min, followed by 6 h of reperfusion. Drug infusion was started 15 min before reperfusion at a loading dose of 8 mg/kg i.v., followed immediately by 2 mg/kg i.v. infused over 80 min. The infarct size, assessed by TTC staining techniques, was significantly reduced in 12 dogs treated with CI-959 (23.3 +/- 3.6% of the area at risk) when compared to 11 vehicle-treated animals (35.5 +/- 4% of the area at risk, p less than 0.05). This reduction in infarct size was not attributed to changes in regional myocardial blood flow, as measured by radioactive microspheres, or to a reduction in myocardial oxygen demand, as estimated by changes in the rate-pressure product. The scar thickness, measured after a 6-week recovery period in 9 animals treated with CI-959, was not significantly reduced in comparison with 11 controls. In vitro, CI-959 effectively inhibited oxygen free radical formation by canine neutrophils. The results of this study show that CI-959 significantly reduces the myocardial infarct size without causing scar thinning, which might lead to ventricular aneurysm, and suggests the most likely mechanism for its beneficial action is the prevention of formation of toxic oxygen radicals.


Beneficial effects of a new prostacyclin analogue, KP-10614, on acute myocardial infarction in rats. The potential therapeutic value of a new prostacyclin analogue, (4z, 16s)-4,5,18,18,19,19-hexadehydro-16,20-dimethyl-delta 6(9a)- 9-(O)-methano-PGI1 (KP-10614), was studied in acute myocardial infarction in rats. Myocardial infarction was induced by ligation of the left coronary artery and ischemic injury was followed up to 4 h. The infarct size, evaluated by the area unstained by 2,3,5-triphenyltetrazolium chloride, reached 41.1 +/- 1.4% of the left ventricle at 4 h. KP-10614 (3 ng/kg/min x 4 h) reduced the infarct size at 4 h significantly (26.5 +/- 2.9%). At the same dose, KP-10614 inhibited ADP-induced ex vivo platelet aggregation significantly (21.5 +/- 4.0% of the control aggregation), but did not alter the arterial blood pressure or heart rate. To assess the role of platelets in myocardial infarction, circulating platelets were reduced by about 95% with rabbit antiserum to rat platelets. In platelet-depleted rats, the infarct size decreased significantly to 24.1 +/- 4.6% of the left ventricle at 4 h. These results suggest that platelets play an important role in expression of myocardial ischemic injury resulting from coronary artery occlusion in rats, and the ability of KP-10614 to decrease the infarct size appeared to be attributable, at least in part, to the inhibition of platelet aggregation or cellular metabolic effects produced by platelets at the site of tissue injury.


Diltiazem potentiates angiotensin II-mediated renal prostacyclin synthesis. Diltiazem (DIL), a calcium antagonist, has variable effects on renal hemodynamics, and has been considered to act independently of renal prostaglandins (PGs). Angiotensin II (AII) constricts renal vasculature but also increases renal vasodilator PG synthesis. We examined interactions between AII and DIL on [14C]p-aminohippurate ([14C]PAH) clearance, mean arterial pressure (MAP), and urine 6-ketoprostaglandin F1 alpha excretion (U6k) in groups of seven to nine conscious Sprague-Dawley rats. We calculated the renal vascular resistance (RVR) as the ratio of MAP/[14C]PAH clearance. AII infusion (10 ng/kg/min i.v.) increased the RVR by 50-80% for at least 120 min. DIL (1 mg/kg plus 2 micrograms/kg/min) reversed this vasoconstriction but adding indomethacin to DIL prevented the reversal. DIL alone did not change the RVR at 30-60 min after starting an infusion but increased it by 22% at 90-120 min. Adding AII to the DIL infusion actually decreased the RVR and this decrease was abolished by indomethacin. DIL alone had no effect on U6k while AII increased it slightly (1.36 +/- 0.12 to 1.86 +/- 0.22 ng/30 min, n = 6, p less than 0.05), and adding DIL to AII increased it further (3.19 ng/30 min, n = 6, p less than 0.05). We conclude that DIL enhances AII-induced renal prostacyclin synthesis and that this is functionally relevant. This mechanism may explain the reported variability of the renal hemodynamic effects of DIL. Furthermore, DIL-enhanced renal vasodilator PG synthesis may help protect the kidney during vasoconstrictor stress.


The role of the renin-angiotensin system in the control of cell communication in the heart: effects of enalapril and angiotensin II. The influence of the renin-angiotensin system on the control of cell communication was investigated in isolated ventricular cell pairs of adult rats. It was found that angiotensin II (1 microgram/ml) reduced the junctional conductance (gj) by about 55% within 20 s. This effect of angiotensin II was suppressed by DuP 753--an angiotensin receptor blocking agent. Enalapril (1 microgram/ml)--an angiotensin converting enzyme inhibitor--caused an increase in junctional conductance (106%) within 2 min. The effect of enalapril on gj was not related to activation of beta-adrenergic receptors or cAMP-dependent protein kinase. The effect of angiotensin II on gj was suppressed by staurosporine--a potent inhibitor of protein kinase C. This finding indicates that the peptide is changing gj through activation of protein kinase C. The increase in cell coupling caused by enalapril raises the possibility that the antiarrhythmic action of enalapril as well its effect in congestive heart failure are related to an increase in electrical synchronization of cardiac myocytes.


Prostacyclin and nitric oxide contribute to the vasodilator action of acetylcholine and bradykinin in the intact rabbit coronary bed. The relative contribution of nitric oxide (NO) and cyclo-oxygenase products in the dilator response to equieffective doses of acetylcholine (ACh) and bradykinin (Bk) was studied in the isolated, saline-perfused rabbit heart under constant flow conditions. ACh (1 microM) and Bk (10 nM) induced a similar vasodilation, with a maximum reduction in coronary perfusion pressure (CPP) of 27 +/- 2%. The vasodilation induced by both agonists was associated with an enhanced release of 6-keto-PGF1 alpha from the coronary bed, with the Bk-induced increase in 6-keto-PGF1 alpha being threefold greater than that induced by ACh. The angiotensin converting enzyme (ACE) inhibitor ramiprilat (0.3 microM) selectively enhanced both the 6-keto-PGF1 alpha outflow and the dilator response to Bk. The B2-receptor antagonist Hoe 140 (0.1 microM) blocked both Bk effects. The cyclo-oxygenase inhibitor diclofenac (1 microM) halved the dilator response to Bk, but did not affect the vasodilation to ACh. Both agonists induced the release of NO, as assessed by the increase in cyclic GMP content of platelets passing through the vascular bed. However, ACh induced a 2.5-fold greater increase in platelet cyclic GMP content, compared to Bk. Treatment of hearts with NG-nitro-L-arginine (L-NNA, 30 microM) halved the ACh- and Bk-induced maximum reduction in CPP. Combined infusion of L-NNA and diclofenac completely blocked the dilator response to Bk, and inhibited the vasodilation to ACh more efficiently than L-NNA alone. We conclude that both NO and PGI2 contribute to the coronary dilator response to Bk and ACh in the rabbit Langendorff heart.(ABSTRACT TRUNCATED AT 250 WORDS)


Endothelin-1 increases the pulmonary microvascular pressure and causes pulmonary edema in salt solution but not blood-perfused rat lungs. Endothelin-1 (ET-1) is a potent vasoactive peptide that has been reported to cause lung edema. This study tested if the edemagenic effect of ET-1 is due to preferential venoconstriction and, if so, whether the site of resistance is similar with salt solution (PSS) and more physiologic blood perfusate. ET-1 caused concentration-dependent contraction of pulmonary arterial and venous rings, with an EC50 of 1.3 nM in artery and 0.6 nM in vein (p less than 0.05). In PSS-perfused lungs, 5 nM ET-1 caused a 7.0 +/- 0.8 torr pressor response that was associated with a 5.0 +/- 0.3 torr increase in microvascular pressure and a 530 +/- 20 mg increase in lung weight within 10 min. In contrast, KCl-treated lungs had an equivalent pressor response (7.4 +/- 1.1 torr), yet the microvascular pressure increased by only 2.5 +/- 0.4 torr (p less than 0.05 from ET-1) and the lung weight was unchanged. Meclofenamate did not prevent the effect of ET-1 on microvascular pressure or lung weight. In blood-perfused lungs, ET-1 caused a 7.3 +/- 0.1 torr pressor response but only a 2.0 +/- 0.5 torr increase in microvascular pressure and no increase in lung weight. ET-1 had no effect on permeability either of cultured endothelial cell monolayers or in the pulmonary microvasculature in vivo. We conclude that the edemagenic effect of ET-1 in PSS-perfused lungs is mediated through venoconstriction and an increase in microvascular pressure.(ABSTRACT TRUNCATED AT 250 WORDS)


Endothelin plasma levels in old and young patients during open heart surgery: correlations to cardiopulmonary and endocrinology parameters. We compared endothelin (ET) plasma levels during and after aortocoronary bypass (CPB) grafting in old and young patients. Correlations to cardiopulmonary parameters and catecholamines were tested. The study included 22 patients (11 aged greater than 70 years and 11 aged less than 55 years). Measurements were performed after induction of anesthesia (baseline), before bypass, during bypass, after patients were weaned off bypass, at the end of operation, and after 4-h intensive care (IC) treatment. ET [radioimmunoassay (RIA) technique] and catecholamines [high pressure liquid chromatography (HPLC) technique] plasma levels were determined from arterial blood samples; hemodynamics (pulmonary artery catheter), oxygen data, and laser Doppler flow were also monitored. Baseline ET plasma levels were within normal range (young, 3.1 +/- 0.9 pg/ml, old, 4.5 +/- 1.4 pg/ml). Old patients had higher values during the entire investigation period. During CPB ET plasma levels increased to a maximum immediately after patients were weaned off bypass. A significant increase (twofold to baseline) was noted during IC therapy. ET plasma levels did not correlate to catecholamine plasma levels or to hemodynamic or laser Doppler flow parameters. A significant correlation existed between ET plasma levels and oxygen consumption. Monitoring ET plasma levels appears to be of minor value in predicting circulatory changes and assessing surgical stress. Further investigations must elucidate the increase in oxygen consumption and its correlation to ET plasma level.


Evaluation of inotropic effect of endothelin-1 in vivo. The vasoconstrictive peptide endothelin-1 (ET-1) has been reported to exert a very important positive inotropic effect in vitro. To assess the effect of ET-1 on myocardial contractility in vivo, we compared the effect of intracoronary infusion of 10(-8) M ET-1 (constant coronary blood flow) to that of 10(-8) M dobutamine in 8 swine. ET infusion did not produce changes in segmental shortening (control vs. drug, mean +/- SD): 33.8 +/- 14.3 vs. 30.8 +/- 12.1%, shortening velocity: 10.3 +/- 4.3 vs. 10.7 +/- 4.5 mm/s, or maximum +dP/dt: 1,691 +/- 701 vs. 1,772 +/- 773 mm Hg/s, whereas dobutamine infusion induced an important increase in these measurements; segmental shortening: 36.9 +/- 14 vs. 48.4 +/- 18.8%, shortening velocity: 10.1 +/- 2.6 vs. 14.7 +/- 4.5 mm/s, and maximum +dP/dt: 2,041 +/- 567 vs. 2,389 +/- 765 mm Hg/s (all p less than 0.05). Mean myocardial blood flow assessed by microspheres was unchanged by ET-1 despite a marked increase in coronary artery pressure (88.6 +/- 12.9 vs. 157 +/- 8.8 mm Hg, p less than 0.001). Regional infusion of ET-1 at a dose provoking extensive coronary vasoconstriction does not induce any change in regional or global myocardial function in swine.


Comparative cardiovascular actions of clentiazem, diltiazem, verapamil, nifedipine, and nimodipine in isolated rabbit tissues. Clentiazem is an 8-chloro-substituted derivative of diltiazem. We compared the relative potency of clentiazem with that of diltiazem, verapamil, nifedipine, and nimodipine in isolated rabbit right atria and vascular smooth muscle removed from various arterial beds. In experiments with isolated right atria, calcium channel blockers were added cumulatively to study relative cardiodepressive potencies (as compared with vascular effects) with the following results: verapamil greater than or equal to diltiazem greater than clentiazem greater than or equal to nimodipine much greater than nifedipine. The aorta, pulmonary, renal, mesenteric, coronary, and basilar arteries were removed, cut helically in strips, and mounted in isolated tissue baths to measure isometric force. Vessels were contracted with either 40 mM KCl (opening voltage-operated calcium channels) or 1 x 10(-5) M norepinephrine (NE, opening receptor-operated calcium channels). Cumulative dose-response curves were generated for relaxation with each calcium channel blocker. All compounds were more potent at relaxing potassium-induced contractions than NE-induced contractions. In vessels precontracted with KCl, neither diltiazem, verapamil, or nifedipine showed selectivity for basilar artery as compared with the mesenteric artery. Both clentiazem and nimodipine were selective (6- and 30-fold, respectively) for basilar artery in blocking potassium-induced contractions. When NE was used to contract the arteries, clentiazem (12-fold), diltiazem (8-fold), verapamil (8-fold), and nifedipine (153-fold) were all more potent in relaxing the contraction in basilar artery than in mesenteric artery. Nimodipine failed to demonstrate selectivity for basilar artery as compared with mesenteric artery contracted with NE.(ABSTRACT TRUNCATED AT 250 WORDS)


Short-term effects of celiprolol on blood pressure and left ventricular performance in hypertensive cardiomyopathy. Celiprolol (C) is a new selective beta 1-blocker with partial beta 2-agonistic activity. The purpose of this study was to explore its antihypertensive efficacy and its short-term effect on systemic vascular resistances (SVR), cardiac output (CO), left ventricular ejection fraction (LVEF), and left ventricular diastolic performance (LVDP). The Doppler technique was used. In an open-label study, 20 hypertensive patients (15 males, 5 females, age range of 29-68 years) with left ventricular hypertrophy detected by echography were daily treated with 400 mg of C in a single dose, for a period of 4 weeks. C reduced significantly the systolic blood pressure (SBP) and the diastolic blood pressure (DBP) (158 +/- 12 vs. 142 +/- 11 mm Hg, p < 0.05 and 101 +/- 6 vs. 87 +/- 4 mm Hg, p < 0.001, respectively) with a decrease in heart rate (74 +/- 12 vs. 67 +/- 8 beats/min, p = NS). The SVR decreased significantly (2,050 +/- 22 vs. 1,495 +/- 23 dyn/s/cm5, p < 0.001) with a slight but not significant increase in the CO (4.5 +/- 0.69 vs. 5.11 +/- 0.82 L/min). The LVEF did not decrease significantly (58 +/- 5 vs. 56.9 +/- 6%, p = NS) while the LVDP was modified favorably, significantly reducing the early diastolic deceleration time (EDDT) (199 +/- 61 vs. 132 +/- 80 ms, p < 0.01) and reducing the isovolumetric relaxation time (IVRT) (167 +/- 16 vs. 117 +/- 23 ms, p < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)


Contrasting influences of central and peripheral opioids on cardiac baroreflex sensitivity in rabbits. We studied the effects of naloxone [0.1 mg/kg intravenously (i.v.) or 7.5 micrograms/kg intracisternally (i.c.)], naloxone methyl iodide (NMI, 0.2 mg/kg, i.v. or 15 micrograms/kg i.c.) and morphine (2 mg/kg i.v.) on the cardiac baroreflex elicited in conscious rabbits especially bred for high and low baroreflex sensitivity (BRS) (group I, BRS > 5.5 beats/min/mm Hg and group II BRS < 4 beats/min/mm Hg, respectively). Full sigmoid barocurves were produced in 37 rabbits by i.v. injection of phenylephrine (1-15 micrograms/kg) and nitroglycerin (1-20 micrograms/kg) after pretreatment with saline or one of the above drugs. In group I, both naloxone i.v. and i.c. and NMI i.c. significantly reduced BRS and decreased the degree of bradycardia in response to a pressor stimulus; neither morphine nor NMI i.v. had any effect. In group II, naloxone i.v. and i.c. and NMI i.c. had no effect on BRS, but both morphine and NMI i.v. significantly increased BRS. An even greater increase was achieved by a combination of these drugs, which also increased specifically the degree of tachycardia in response to a decrease in blood pressure (BP). The results suggest that baroreceptor activation in group I released in the brainstem an opioid peptide that acts to increase BRS. In contrast, group II rabbits responded by peripheral opioid activation, which results in a decrease in BRS, possibly by inhibiting norepinephrine (NE) release from cardiac neurons. The predominance of peripheral or central opioid involvement in BRS modulation appears to be due to genetic factors.(ABSTRACT TRUNCATED AT 250 WORDS)


Protection of the ischemic myocardium by the converting-enzyme inhibitor zofenopril: insight into its mechanism of action. We assessed whether local inhibition of myocardial converting enzyme by captopril and zofenopril reduces the functional and metabolic damage caused by ischemia and reperfusion. First we investigated the effects of zofenopril and captopril on the mechanical function, cellular redox state, and norepinephrine (NE) content of isolated and aerobically perfused rabbit hearts. Both drugs failed to modify the myocardial redox state. At concentrations > 10(-6) M, zofenopril, but not captopril, caused a reduction in myocardial NE content. At 10(-4) M, both drugs caused a reduction in developed pressure and an increase in diastolic pressure and release of creatine phosphokinase (CPK). Second we investigated their effects on ischemic and reperfused myocardium. Both drugs exerted a cardioprotection; zofenopril was always more potent than captopril. Recovery of developed pressure on reperfusion improved, and peak release of NE was reduced, as was release of CPK. Calcium homeostasis and mitochondrial function were maintained. Captopril had no effect on occurrence of oxidative stress during reperfusion, whereas zofenopril reduced it. In hearts treated with the converting enzyme inhibitors, peak release of NE was correlated to mitochondrial calcium content, production of ATP, and recovery of mechanical function on reperfusion. These data suggest that the cardioprotective effect of zofenopril and captopril is independent of hemodynamic changes or reduction of the toxicity of oxygen free radicals and that it could be related to a reduction in release of NE.


In vitro pharmacology of R 80122, a novel phosphodiesterase inhibitor. The cardiac in vitro effects of R 80122, a novel phosphodiesterase (PDE) inhibitor, were investigated and compared with those of the reference compound milrinone and of the calcium-sensitizer adibendan. In guinea pig left atria, both milrinone and R 80122 increased contractile force; 10 microM milrinone was equieffective to 1 microM R 80122. The rate of spontaneously beating atria was not altered by R 80122 in the concentration range of 0.01-0.3 microM. Higher concentrations (1-10 microM) led to a statistically insignificant increase of 20%. Milrinone's effect on frequency was more pronounced and amounted to 21% at 10 microM and to 40% at 100 microM. Adibendan increased heart rate (HR) by 10% at a concentration of only 0.03 microM. This effect was not enhanced any further by increasing the concentration. In papillary muscle, the positive inotropic effects of both milrinone and R 80122 were inhibited by carbachol, indicating involvement of cyclic AMP. Further indications for a cyclic AMP-dependent action were obtained by induction of slow action potentials and synergism with isoprenaline. In electrophysiologic measurements, milrinone reduced action potential duration (APD) in a high concentration whereas R 80122 had no effect. Action potential changes elicited by a toxic concentration of ouabain were reduced by R 80122. Relaxation of rat aortic rings contracted by KCl and relaxation of guinea pig aortic rings contracted by norepinephrine (NE) was comparable for both milrinone and R 80122. R 80122 also caused relaxation of canine coronary arteries constricted with prostaglandin F2 alpha (PGF2 alpha) both with and without endothelium. NE-induced contractions in canine gastrosplenic arteries were not affected by R 80122. Cardiac contractility that had been impaired to various degrees by pentobarbital or by aging was restored to control values by both milrinone and R 80122. R 80122 enhanced cardiac contractility at lower concentrations than milrinone with no concomitant increase in frequency or shortening of the action potential, which may be advantageous for treatment of heart failure.


Effects of a cardiotonic quinolinone derivative Y-20487 on the isoproterenol-induced positive inotropic action and cyclic AMP accumulation in rat ventricular myocardium: comparison with rolipram, Ro 20-1724, milrinone, and isobutylmethylxanthine. The effect of a new cardiotonic agent Y-20487 [6-(3,6-dihydro-2-oxo-2H-1,3,4-thiadiazin-5-yl)-3,4-dihydro-2(1H)- quinolinone] on cyclic AMP levels of rat ventricular cardiomyocytes and the contractile force of papillary muscles was investigated in comparison with selective cyclic AMP phosphodiesterase (PDE) inhibitors, milrinone (PDE-III selective), rolipram and Ro 20-1724 (PDE-IV selective), and a nonselective inhibitor 3-isobutyl-1-methylxanthine (IBMX). Rolipram and Ro 20-1724 did not elicit cyclic AMP accumulation and positive inotropy, but they potentiated the isoproterenol (ISO)-induced cyclic AMP accumulation more effectively than IBMX. Rolipram was more effective than Ro 20-1724 in enhancing ISO-induced cyclic AMP accumulation but was less effective in enhancing the ISO-induced positive inotropic effect, indicating that these agents produce a differential action on cyclic AMP metabolism and inotropy. Milrinone and Y-20487 elicited cyclic AMP accumulation and positive inotropy by themselves. Whereas milrinone scarcely affected the ISO-induced effects, Y-20487 shifted the concentration-response curve for the positive inotropic effect of ISO to the left to the same extent that IBMX did. Y-20487, however, was much less effective than IBMX in enhancing the ISO-induced cyclic AMP accumulation. The present results indicate that in rat ventricular myocardium, PDE-IV may play a crucial role in breakdown of cyclic AMP generated by beta-adrenoceptor stimulation, whereas other types of PDE isoenzymes, including PDE-III, may be responsible for the cyclic AMP accumulation and direct positive inotropic effect induced by PDE inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)


Antihypertensive effects of MPC-1304, a novel calcium antagonist, in experimental hypertensive rats and dogs. Antihypertensive effects of a novel calcium antagonist, MPC-1304, (+-)-methyl 2-oxopropyl 1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-3,5- pyridine-dicarboxylate and its active metabolites were investigated in experimental hypertensive rats and dogs and compared with those of other dihydropyridine derivatives (nifedipine, nisoldipine, nicardipine, and nitrendipine). MPC-1304 had a dose-related antihypertensive effect with a slight increase in heart rate (HR) in rats. The antihypertensive effects of MPC-1304 were more potent than those of other dihydropyridines, and its active metabolites had antihypertensive effects comparable to those of other dihydropyridines. The hypotensive effects of MPC-1304 were stronger in hypertensive rats than in normotensive rats. During repeated oral administration of MPC-1304 to spontaneously hypertensive rats (SHR, once daily for 4 weeks, 0.3-3 mg/kg), dose-response curves of the antihypertensive effect did not change and body weight gain was equal to that of the vehicle-treated group. When given orally to conscious renal hypertensive dogs, MPC-1304 0.1-0.3 mg/kg had a potency and duration of antihypertensive action comparable to that of nitrendipine (1-3 mg/kg). MPC-1304 increased coronary blood flow (CBF) and aortic blood flow (ABF) in conscious normotensive dogs. In conclusion, MPC-1304 and its active metabolites have potent antihypertensive effects and cause slight tachycardia, and they may be useful in treating hypertension.


Effect of nilvadipine on balloon catheterization-induced intimal thickening of the coronary artery in miniature pigs. The effect of nilvadipine on balloon catheterization-induced intimal thickening of the coronary artery was examined in miniature pigs. A diffuse intimal thickening was observed in vehicle-treated controls 6 weeks after balloon catheterization. The histologic features of the intimal thickening resembled those of early atherosclerotic lesions and restenosis after percutaneous transluminal coronary angioplasty (PTCA). Nilvadipine given in a daily dose of 10 mg/kg (subcutaneously, s.c.) for 6 weeks significantly inhibited intimal thickening. The ratio of the cross-sectional area of the intima to the cross-sectional area of the media was significantly reduced by 62% in nilvadipine-treated animals. These results suggest that nilvadipine may prevent or exert beneficial effects on coronary arterial injuries such as atherosclerosis and restenosis after PTCA.


Acute inhibition of endopeptidase 24.11 in essential hypertension: SCH 34826 enhances atrial natriuretic peptide and natriuresis without lowering blood pressure. The acute renal, endocrine, and hemodynamic effects of the orally active endopeptidase inhibitor SCH 34826 (400 mg every 6 hours for five doses) were investigated in a group of 6 male patients [with established mild to moderate essential hypertension and left ventricular (LV) hypertrophy] in a balanced random-order double-blind, placebo-controlled cross-over study. Plasma atrial natriuretic factor (ANF) concentrations increased (p < 0.05) to fourfold control values after the first dose of inhibitor, but later postdose increments of ANF were less pronounced. Plasma cyclic GMP also increased significantly (p < 0.05). These effects were associated with a transient modest but significant (p < 0.05) increase in sodium excretion (50 mmol sodium in excess of placebo values) that was complete in 24 h. Mean 24-h urinary excretions of cyclic GMP and immunoreactive ANF were also significantly increased by 55 and 86%, respectively. Other urine indexes (including other electrolytes, volume, creatinine, aldosterone, and cortisol) and renal hemodynamics [including glomerular filtration rate (GFR) and effective renal plasma flow (RPF)] were unchanged. Renin-angiotensin-aldosterone system (RAAS) activity was not significantly altered. Plasma epinephrine increased after the initial three doses of SCH 34826. Systolic blood pressure (SBP) and heart rate (HR) were not altered by SCH 34826. Diastolic BP (DBP) increased slightly (p = 0.044). Acute inhibition of endopeptidase 24.11 by SCH 34826 in essential hypertension caused significant increments in plasma ANF and cyclic GMP together with modest natriuresis. No antihypertensive effect was observed in the first 30 h of treatment.(ABSTRACT TRUNCATED AT 250 WORDS)


Effects of S-dobutamine on venous blood return and organ nutrient blood flow. The selective contractile effects of s-dobutamine were studied in vitro in selected canine arteries and vein preparations; propranolol was included to block potential beta-mediated vasodilation. These in vitro data were expanded by quantifying the in vivo effects of s-dobutamine on venous blood return and redistribution of regional nutrient blood flow (NBF) and non-nutrient blood flow (non-NBF) in anesthetized dogs. In in vitro studies with isolated canine arteries and veins, s-dobutamine exhibited vein-selective constriction. At maximally efficacious concentrations of agonist, contractions of carotid, coronary, and femoral arteries in response to s-dobutamine were only 7, 25 and 45% as great as those elicited by norepinephrine (NE). Similarly, in jugular vein, s-dobutamine-mediated contractions were 55% as great as those obtained in response to NE. Coronary and femoral arteries precontracted with NE were relaxed in a dose-related manner by increasing concentrations of s-dobutamine. Effects of NE and s-dobutamine on venous blood return (VR) were compared in dogs. s-Dobutamine increased VR by 49 +/- 10 ml, whereas NE increased VR by 14 +/- 6 ml during 5-min infusion. s-Dobutamine significantly increased coronary NBF in left ventricular (LV) endocardium from 115 +/- 10 to 194 +/- 13 and 263 +/- 9 ml/min/100 g at doses of 10 and 20 micrograms/kg/min, respectively. In addition, LV epicardium flow was increased from 87 +/- 8 to 189 +/- 15 and 262 +/- 11 ml/min/100 g at 10 and 20 micrograms/kg/min, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)


Quality of life perception during antihypertensive treatment: a comparative study of bisoprolol and enalapril. The well-being of hypertensive patients may be adversely affected by the disease itself, its complications, and other concomitant processes such as anxiety, sedation, and side effects of prescribed drugs. Some recently developed antihypertensive agents have been suggested to be devoid of these deleterious effects on well-being expressed as quality of life. We compared the effect on quality of life of the angiotensin-converting enzyme (ACE) inhibitor enalapril to the effect of bisoprolol as a representative of a new class of selective beta 1-adrenoreceptor blocking agents. Fifty-seven patients with mild to moderate hypertension were eligible to enter an 18-week cross-over study, consisting of a single-blind 2-week run-in period and two 8-week double-blind cross-over periods. At the end of the run-in period and the two cross-over periods, both systolic and diastolic blood pressure (SBP, DBP) were assessed, as was quality of life perception by the Inventory of Subjective Health. During bisoprolol treatment, supine BP decreased from 163 +/- 2/102 +/- 1 to 144 +/- 3/86 +/- 1 mm Hg. The antihypertensive effect was at least as good with bisoprolol as with enalapril. Quality of life perception as measured with the Inventory of Subjective Health was comparable for the two drugs. Spontaneously mentioned adverse effects were more frequent (74%) during enalapril than during bisoprolol treatment. At the end of the study, 69% of patients chose to continue antihypertensive treatment with bisoprolol. Our results show that the highly selective beta 1-adrenoreceptor blocking drug bisoprolol is at least as effective as enalapril and has no deleterious effects on well-being.


Cardiovascular effects of omega-conopeptides in conscious rats: mechanisms of action. We examined the effects of omega-conopeptides, a novel class of neuronal voltage-gated calcium channel antagonists, on hemodynamic responses in rats. Intravenous (i.v.) injections of SNX-111 (omega-conopeptide MVIIA) dose-dependently decreased arterial blood pressure (BP) in conscious rats. Intracerebroventricular (i.c.v.) SNX-111 injections (580 pmol) tended to increase BP and, after an initial decrease, to increase heart rate (HR). The dose-response curve for SNX-111 administered i.v. in conscious rats was biphasic. Beginning at subdepressor doses, SNX-111 caused a long-lasting blockade of pressor responses elicited by sympathetic nerve stimulation in pithed animals but did not prevent increases in BP evoked by exogenously administered norepinephrine (NE). Pretreatment of rats with histamine antagonists partially blocked the hypotensive effects of the higher (870 and 2,900 nmol/kg) doses of SNX-111. Substitution of alanine for arginine at position 10 ([Ala10]-MVIIA) markedly attenuated the histamine-mediated component of the vasodepressor response. Together, these findings indicate that SNX-111 administered i.v. decreases systemic BP by a combination of blockade of sympathetic neurotransmission and mast cell degranulation; the latter function appears to be dependent on the arginine residue in position 10 of the amino acid sequence of SNX-111.


Nicorandil attenuates myocardial dysfunction associated with transient ischemia by opening ATP-dependent potassium channels. The objective of this study was to determine whether ATP-dependent potassium channel activation is involved in the mechanism by which nicorandil reduces postischemic contractile dysfunction produced by a brief period of ischemia (myocardial stunning). Barbital-anesthetized dogs were subjected to 15-min left anterior descending (LAD) coronary artery occlusion followed by 3-h reperfusion. Saline or nicorandil (100 micrograms/kg + 25 micrograms/kg/min) were infused 15 min before and throughout occlusion with or without addition of the KATP channel antagonist, glibenclamide 0.3 mg/kg as an intravenous (i.v.) bolus. Regional myocardial blood flow was measured by radioactive microspheres, and left ventricular (LV) segment function was measured by sonomicrometry. There were no significant differences between the groups in area-at-risk size or collateral blood flow. In contrast, nicorandil significantly reduced mean aortic blood pressure (BP) and the rate-pressure product (RPP) which persisted throughout the occlusion period. In addition, nicorandil markedly accelerated recovery of segment shortening in the ischemic/reperfused region as compared with control dogs. Pretreatment of dogs with glibenclamide blocked none of the hemodynamic effects of nicorandil, but it did prevent improvement in reperfusion segment function. The small dose of glibenclamide used had no effect on hemodynamics or the degree of stunning. Thus, these results suggest that nicorandil attenuates stunning in anesthetized dogs by a direct cardioprotective effect as a result of KATP channel activation in ischemic myocardium.


Angiotensin II receptor blockade after myocardial infarction in rats: effects on hemodynamics, myocardial DNA synthesis, and interstitial collagen content. Angiotensin-converting enzyme (ACE) inhibitors are widely used for treatment of heart failure after myocardial infarction (MI). The beneficial effects consist of a combination of hemodynamic effects and interference with cardiac structural alterations. These effects are believed to depend on inhibition of angiotensin II (AII) formation and thus diminished angiotensin receptor stimulation. We administered the angiotensin II-1 (AT-1) receptor antagonist losartan during and after completion of the repair phase of an MI to investigate involvement of the AT-1 receptor in the above described effects of captopril. MI reduced cardiac output (CO) (sham 94 +/- 4 ml/min, MI 78 +/- 5 ml/min) and maximal CO (sham 154 +/- 4, MI 107 +/- 5 ml/min, respectively). Losartan (15 mg/kg/day) resulted in a rightward shift of the AII pressor dose-response curve by a factor of 32-40. Neither CO nor COVL,max was affected by losartan treatment in either phase (late treatment CO = 78 +/- 5, COVL,max = 118 +/- 9 ml/min). Although early treatment with losartan reduced cardiac hypertrophy measured as heart weight, DNA synthesis was reduced only slightly. In contrast, collagen deposition was inhibited completely. The results suggest that the effects of captopril in rats after MI are not dependent on AT-1 receptor-mediated mechanisms.


Serotonin-induced vasoconstriction in dog kidney. Serotonin (5-HT 0.1 and 0.3 micrograms/kg) was injected as a bolus into the renal artery of anesthetized dogs. Renal blood flow (RBF) decreased initially after 5-HT injection and then increased. The 5-HT-induced decrease in RBF was potentiated during intrarenal infusion of a 5-HT1 and 5-HT2 antagonist methysergide at 3 micrograms/kg/min or of a 5-HT2 antagonist ketanserin (3-30 micrograms/kg/min), but methysergide at 30 micrograms/kg/min attenuated the decrease in RBF. The delayed increase in RBF was suppressed during ketanserin or methysergide infusion. In ketanserin-pretreated dogs, methysergide (3-30 micrograms/kg/min) dose-dependently suppressed the 5-HT-induced decrease in RBF. A 5-HT3 antagonist, ICS 205-930 (3-30 micrograms/kg/min), did not affect the 5-HT-induced RBF responses. A Ca2+ entry blocker CD-349 (30 and 100 ng/kg/min) and a Ca2+ release inhibitor TMB-8 (30 and 100 micrograms/kg/min) suppressed the 5-HT-induced decrease in RBF. These results suggest that 5-HT activates 5-HT1 receptors to induce vasoconstriction by mobilization of extracellular and intracellular Ca2+, but simultaneous activation of 5-HT2 receptors attenuates the vasoconstriction, probably by causing vasodilation in dog kidney.


Terbutaline-induced downregulation of beta 2-adrenoceptors without Gi-protein alterations in human lymphocytes. Recent evidence suggests that agonist-induced desensitization of Gs protein-coupled beta-adrenoceptors is accompanied by sensitization of Gi protein-coupled receptors and/or an increase in Gi protein. To find out whether such "cross-regulation" between Gs protein- and Gi protein-coupled receptors can be also demonstrated in vivo in humans, we studied the effects of a 2 week treatment of eight male volunteers with the beta 2-adrenoceptor agonist terbutaline (3 x 5 mg/day) on beta 2-adrenoceptor density and Gi-protein content in lymphocytes and on alpha 2-adrenoceptor density (Gi-coupled receptors) in platelets. Terbutaline decreased the lymphocyte beta 2-adrenoceptor density by about 30%, but had no significant influence on lymphocyte Gi-protein levels (assessed by pertussis toxin-catalyzed [32P]ADP ribosylation) or on platelet alpha 2-adrenoceptor density. We conclude that circulating blood cells are not suitable to demonstrate in humans in vivo a "cross-regulation" between Gs- and Gi-coupled beta- and alpha-adrenoceptors.


Modulation of antigen- and ischemia-induced effects by the platelet-activating factor antagonist WEB-2086 in isolated sensitized rat hearts. The modulatory role of the platelet-activating factor (PAF) antagonist WEB-2086 (30 microM) on the response to antigen-induced (trinitrophenyl-haptenized ovalbumin) and global ischemia (30 and 60 min)-induced changes in the response to antigen was studied in isolated hearts from actively sensitized rats. In sensitized normoxic hearts, both antigen (0.8 mg) and PAF (100 pmol) induced a short-term increase followed by a long-term decrease in coronary flow (CF). The antigen- but not the PAF-evoked increase in CF was accompanied by a substantial release of histamine. WEB-2086 enhanced the vasodilator effect and abolished the vasoconstrictor effect of 100 pmol of PAF but neither modified the coronary vascular effects of antigen nor the antigen-induced histamine release. Ischemia for 60 min followed by 30 min of reperfusion increased the diastolic left ventricular pressure but a 30-min period of ischemia and reperfusion had no effect on baseline cardiac function. WEB-2086 had no effect on ischemia-induced changes in cardiac function. A 30-min period of global ischemia enhanced the antigen-induced decrease in CF and systolic left ventricular pressure (SLVP). A 60 min period, however, suppressed the antigen-induced effects on CF and SLVP as well as antigen-induced histamine release. WEB-2086 partly protected the heart against the enhanced antigen-induced decrease in CF and SLVP after a 30-min period of global ischemia but no modulatory role of WEB-2086 was observed after 60 min of global ischemia. Our conclusions are that (a) PAF is not involved in rat cardiac anaphylaxis since WEB-2086 was proven to be inactive; (b) cardiac ischemia and cardiac anaphylaxis have interrelated mechanism of action since ischemia changed the anaphylactic response, which indicates that coincidence of these two pathological events could influence the clinical outcome; and (c) PAF is possibly involved in rat cardiac ischemia since WEB-2086 partly protected the heart against the enhanced antigen-induced decrease in CF and SLVP after 30 min of ischemia and reperfusion.


Effect of therapeutic dopamine administration on myocardial catecholamine and neuropeptide Y concentrations in the failing ventricles of patients with idiopathic dilated cardiomyopathy. The purpose of this study was to investigate the relationship between dopamine (DA) exposure and myocardial catecholamine and neuropeptide Y (NPY) concentrations in patients with severe congestive heart failure due to idiopathic dilated cardiomyopathy (IDC). Both nonfailing (NF) and failing (F) hearts were obtained in collaboration with the Utah Cardiac Transplantation Program and the Intermountain Organ Recovery System. The patients were stratified into five groups according to their preoperative exposure to dobutamine (DBT) and/or DA. Compared to 12 untreated, NF control hearts, norepinephrine (NE) concentrations were significantly decreased in 30 untreated F hearts obtained from patients with IDC. Norepinephrine concentrations were also significantly decreased in DA-treated NF hearts and in DA-treated F hearts compared to untreated NF and untreated or DBT-treated failing hearts, respectively. NPY concentrations were significantly decreased in untreated F hearts and were further decreased in dopamine-treated NF and DA-treated F hearts compared to untreated NF and untreated or DBT-treated F hearts. Thus, NE and NPY depletion related to DA administration was evident in both NF and F myocardium and was specific for DA in that it was not evident in patients who received the direct-acting beta-agonist inotrope DBT. These data suggest that the major inotropic mechanism of action of DA is through cardiac adrenergic neurotransmitter release. The data also provide further support for the concept that indirect acting inotropes such as DA may have limited inotropic potential in F hearts where neuronal NE has been depleted.


C14-aminosteroid LNF 209, an agent with positive inotropic and antimuscarinic activity. LNF 209 is a cardioactive steroid containing at position C14 a protonized amino group. It was tested whether LNF 209 has antimuscarinic properties besides its cardiotonic effect, which was also quantified. In guinea pig left atria (3 Hz, 0.9 mM Ca2+, 5.4 mM K+), LNF 209 increased the force of contraction at concentrations > or = 1 microM, thus having a tenfold lower potency than ouabain. Correspondingly, cardiac glycoside binding sites in guinea pig cardiac membranes labeled with [3H]ouabain were occupied by LNF 209 (Ki = 1.7 microM) at concentrations tenfold higher compared with ouabain (Kd = 0.14 microM). The negative inotropic effect of the muscarinic agonist oxotremorine was antagonized by LNF 209, but not by ouabain; in the Schild plot, the data for LNF 209 could be connected by a line with a slope of unity and a pA2 = 7.5. The binding of [3H]N-methylscopolamine ([3H]NMS) to the M2-cholinoceptors in guinea pig cardiac membranes was inhibited by LNF 209 with a Ki = 0.5 microM; LNF reduced the affinity of [3H]NMS binding without affecting the number of receptor sites. In isolated segments of guinea pig ileum, LNF 209 antagonized the effect of oxotremorine with a pA2 = 7.4. It is concluded that LNF 209 is a competitive antagonist at M-cholinoceptors without preference for the cardiac M2-cholinoceptors. Whether the antimuscarinic property is of significance in species highly sensitive to digitalis remains to be established.


Tumour necrosis factor alpha augments the release of an endothelium-dependent vasoconstrictor from human polymorphonuclear leukocytes. We have studied the effect of cytokine priming by recombinant human tumour necrosis factor alpha (rhTNF alpha) on the release of vasoactive mediators from human polymorphonuclear leukocytes (PMNs). Supernatants of human PMNs (100 microliters, 5 x 10(7) ml, from 12 donors) relaxed precontracted rabbit aortic rings. The relaxations were preceded by a transient contraction in four experiments. In contrast, supernatants of PMNs incubated with rhTNF alpha (0.3 nM, 30 min, 37 degrees C) elicited variable vascular responses that consisted of either a sustained contraction (n = 4), a transient contraction followed by a sustained relaxation (n = 2), or a sustained relaxation only (n = 6). The vascular tone produced by the rhTNF alpha-treated PMN supernatant was significantly greater over 30 min than that produced by the control supernatant (p < 0.01). Endothelium removal prevented the PMN-induced contractions and significantly decreased the vascular tone produced by all rhTNF alpha-treated PMN supernatants (p < 0.05). The PMN supernatants had no vasoactive effect on aortic rings at resting tension. Molecular size analysis indicated that the PMN-derived contractile mediator is > 30,000 Da. These results suggest that human PMNs, primed by rhTNF alpha, release a stable endothelium-dependent vasoconstrictor that opposes the action of a stable, spontaneously released direct vasodilator.


Enalapril increases cardiac refractoriness. The influence of enalapril--an angiotensin converting enzyme inhibitor--on cardiac refractoriness was investigated. Strength-interval curves were initially obtained under control conditions and after exposing the muscles to Tyrode solution containing 50 micrograms/ml of enalapril. The results indicate that enalapril displaced the strength-curves to the right. The minimal current intensity required to elicit a propagated response was clearly increased by enalapril at all the intervals used. No change in action potential duration was found with enalapril but the action potential amplitude and the resting potential were both increased. The rise in cardiac refractoriness caused by enalapril might indicate that the drug has anti-arrhythmic properties.


Central noradrenergic activity and the cardiovascular effects of nitroglycerin and amyl nitrate. Intracerebroventricular and i.v. administration of nitroglycerin in anesthetized Sprague-Dawley rats produced dose-dependent decreases in mean arterial pressure and increases in heart rate, but intracisternal injection of the drug induced hypotension and bradycardia. Hypotensive responses to intracisternal injection of nitroglycerin showed that the compound was two to three times more potent than by i.v. administration (six times when comparing the area under the curve). Intravenous sodium nitroprusside produced dose-dependent hypotensive and tachycardiac effects, but minimal responses were induced by i.c.v. and intracisternal injection. Central pretreatment with either yohimbine or rauwolscine antagonized hypotensive responses to i.c.v. and i.v. nitroglycerin, but did not alter the depressor responses induced by i.v. sodium nitroprusside. Tachycardiac responses to i.c.v. nitroglycerin are greater than the responses induced by i.v. administration. Inhalation of amyl nitrite produced marked hypotensive responses, with the dose-response curves being shifted to the right by i.c.v. pretreatment with rauwolscine, but not by i.v. pretreatment. The concentrations of 3,4-dihydroxyphenylglycol and 3-methoxy-4-hydroxyphenylglycol in cerebrospinal fluid were increased by i.v., i.c.v., and intracisternal administration of nitroglycerin, but were not altered by i.v. sodium nitroprusside. The concentrations of dihydroxyphenylacetic acid were elevated by i.v. and i.c.v. injection of nitroglycerin. Results suggest that nitroglycerin stimulates central noradrenergic activity, which may be involved in the component of hypotensive effects of the drug. Reflex tachycardiac responses to nitroglycerin may be further complicated by forebrain stimulation and medulla-mediated bradycardia. Sodium nitroprusside did not demonstrate central activity.


Absence of hemodynamic deterioration in the presence of amlodipine following experimental myocardial infarction. In general, calcium channel blockers have not been used in patients during acute myocardial infarction as they may exacerbate heart failure, possibly by neuro-humoral stimulation. Amlodipine, a new dihydropyridine calcium channel blocker without neurohumoral stimulation, was tested in an experimental model of acute myocardial infarction with assessment of hemodynamics, left ventricular (LV) function, and infarct size. Anesthetized dogs were subjected to 3 h of coronary artery occlusion followed by 3 h of reperfusion. At 2 h of occlusion, they were randomized to receive either amlodipine (250 micrograms/kg, n = 11) or saline (n = 11). Before treatment, all variables were similar in both groups. The diastolic pressure was unchanged following saline, but was reduced following amlodipine by 1 h after therapy (from 94 +/- 5 to 71 +/- 3 mm Hg, p < 0.0001 vs. saline) and for the duration of the protocol. Indices of left ventricular (LV) function did not deteriorate with amlodipine treatment compared with saline. After 3 h of reperfusion, the LV dP/dt was 1,720 +/- 114 mm Hg/s in the saline group and 1,958 +/- 167 mm Hg/s with amlodipine (p = ns). The area ejection fraction, assessed by echocardiography, was similar in both groups (43 +/- 5%, saline; 45 +/- 3%, amlodipine; p = ns), as was the LV end-diastolic pressure (8 +/- 1 mm Hg, saline; 7 +/- 1 mm Hg, amlodipine; p = ns). Subendocardial regional myocardial blood flow, measured by radioactive microspheres, was 0.75 +/- 0.08 ml/min/g with saline and 1.34 +/- 0.33 ml/min/g with amlodipine in the previously ischemic reperfused subendocardium (p = 0.1).(ABSTRACT TRUNCATED AT 250 WORDS)


The effect of transforming growth factor-alpha on airway angiogenesis. A problem in lung transplantation is tracheal or bronchial dehiscence from ischemia. To determine if an angiogenic factor applied to the airway would improve capillary regrowth, a three-ring segment of trachea was completely severed and sutured in rats. In one group of animals the ischemic segment was wrapped with Gelfoam soaked in an angiogenic factor, transforming growth factor-alpha. In a second group the ischemic area was wrapped with Gelfoam soaked with only the vehicle. In a third group the devascularized area received no additional treatment. One animal from each group was killed daily for 7 days after operation. The tracheal vasculature was cast and viewed by light and scanning electron microscopy. None of the four animals that died early were in the transforming growth factor-alpha group. All animals lost weight between the day of operation and death, but this was least in the transforming growth factor-alpha group (p = 0.05). The light microscopy showed ischemic changes and the development of granulation tissue. The scanning electron microscopy of the vascular casts showed extensive loss of the vessels in the cut area. On day 1 the vessels of all animals dilated and their walls became rough. By day 3 a few corkscrew vessels penetrated the ischemic zone. By day 4 the animal that received transforming growth factor-alpha had more capillaries than the others. By day 6 revascularization in the transforming growth factor-alpha animal was abundant. Besides budding, new capillaries appeared to develop by lateral growth. After the fifth day vessels about 30 to 50 microns in diameter bulged focally. On the bulges, ridges the size, shape, and pattern of capillaries formed. Capillary formation in this manner has not been reported previously. Revascularization emerged sooner and more extensively with transforming growth factor-alpha. No adverse effect of transforming growth factor-alpha was found.


Measurement of serum cytokine levels in patients with myelodysplastic syndromes. Sera of 25 healthy controls and 75 patients suffering from myelodysplastic syndromes (MDS) were investigated for serum concentration of interleukin-1 alpha (IL-1 alpha), IL-3, IL-6, granulocyte-colony-stimulating factor (G-CSF), granulocyte-macrophage-CSF (GM-CSF), erythropoietin (Epo), and tumor necrosis factor-alpha (TNF-alpha). According to French-American-British (FAB) classification, 21 refractory anemia (RA), seven refractory anemia with ring sideroblasts (RARS), 15 chronic myelomonocytic leukemia (CMML), 12 refractory anemia with excess of blasts (RAEB), and 20 RAEB in transformation (RAEBt) were examined. TNF-alpha levels were inversely correlated with lower levels of hemoglobin concentration (r = -0.31, p = 0.005), irrespective of the requirements for transfusion in anemic MDS patients. Significant differences in TNF-alpha levels between CMML (26.2 +/- 5.9 pg/ml) and the FAB subgroups (16.1 +/- 1.6 pg/ml) were detected. There was an overall inverse relationship between the level of erythropoietin and the degree of anemia, but a wide range of Epo response between patients with similar hemoglobin concentrations. Serum levels of IL-1 alpha and GM-CSF were undetected in most of the patients. In 57% of the samples there were detectable levels of G-CSF, without a correlation of the serum levels with blood cell counts, nor with any of the FAB subcategories. Overall, 29% and 25% of the patient sera exhibited elevated IL-3 and IL-6 levels, respectively. There was no correlation of the serum levels with any of the blood counts, other cytokines, nor FAB subcategories. In conclusion, simple negative feedback mechanism between a specific cytokine and the production of blood cells seems not to be the case in MDS, except for red cell production and erythropoietin concentration. Our data may suggest the involvement of TNF-alpha in the pathogenesis of anemia in MDS.


Utility of immediate on-site cytopathological procurement and evaluation in fine needle aspiration biopsy of head and neck masses. The utility of on-site microscopic evaluation of fine needle aspirates (FNAs) of the head and neck was assessed by comparing the diagnostic yield in 336 specimens obtained with immediate on-site cytopathological procurement and evaluation to that achieved in 548 cases performed without immediate on-site evaluation. Three hundred six (91%) of 336 immediate evaluation specimens were adequate for cytopathologic diagnosis, compared to 391 (71%) of 548 specimens not evaluated immediately (P < .001, chi-squared test). The higher satisfactory rate in immediate evaluation cases was related primarily to 1. immediate reaspiration of the masses until sufficient cytopathologic material was obtained for diagnosis; and 2. optimal specimen preparation. It is concluded that immediate on-site cytopathological procurement and evaluation of fine needle aspirates of head and neck masses is a valuable practice which assures a higher yield of adequate specimens compared to biopsies taken without immediate evaluation. The technique of immediate on-site evaluation of FNAs is discussed and a cost-benefit analysis of immediate on-site evaluation of FNAs is presented.


Laser-induced hyperthermia: comparison of two different methods. The effectiveness of two different techniques of laser-induced hyperthermia was analyzed in healthy piglet livers. Hyperthermia was produced using a continuous-wave Nd:YAG laser with both a contact sapphire probe and a bare quartz fibre. In both methods the tip was inserted into the liver, and the temperature 1 cm from the tip was raised and held between 42 degrees C and 44 degrees C for 600 seconds. Specimens were taken 0, 7 and 14 days after the treatment. The tissue effects of 36 treatment sites were analysed using standard van Gieson's method. In addition, a histochemical method for demonstrating lactate dehydrogenase activity was employed to show damage not observable by routine methods. No statistically significant difference in the extent of the treatment sites at d 0 and after 7 days was seen when comparing the two laser methods. After 2 weeks, the diameter of the lesion with the bare fibre was significantly larger (3,7 mm with both analysing methods) than by using the contact probe (2,6 with LDH demonstration; P < 0.01 and 2.1 mm with van Gieson's staining; P < 0.001). It can be concluded that the simple fibre tip seems more effective in causing tissue necrosis than the sapphire tip.


Possible involvement of nitric oxide in chlordiazepoxide-induced anxiolysis in mice. Mice challenged with the anxiolytic benzodiazepine chlordiazepoxide exhibited significant increases in the percent of total entries into and percent of total time spent on open arms of an elevated plus maze. Systemic pretreatment with the nitric oxide synthase-inhibitor L-NG-nitro arginine (L-NOARG) antagonized these effects of chlordiazepoxide. This inhibitory effect of L-NOARG was stereospecifically and completely reversed by intracerebroventricular administration of L-arginine but not D-arginine. These findings suggest a possible role of nitric oxide in the anxiolytic effect of chlordiazepoxide in the elevated plus maze.


Stimulus-specific inhibition of insulin release from rat pancreas by both rat and porcine galanin. The effect of the neuropeptide galanin on insulin and somatostatin secretion in the rat was studied under various conditions. In the perfused rat pancreas, insulin secretion stimulated by arginine, but not cholecystokinin-8 (CCK-8) or acetylcholine (ACh) was inhibited by both rat and porcine galanin, whereas ACh-stimulated somatostatin release was inhibited by rat but not porcine galanin. Neither arginine nor CCK-8 significantly altered somatostatin secretion and galanin was without effect under those conditions. Gastric inhibitory polypeptide-stimulated insulin release from cultured mixtures of purified rat beta- and non-beta-cells was inhibited by rat and porcine galanin in a concentration-dependent and equipotent manner. The results suggest that the inhibitory effect of galanin on insulin and somatostatin secretion may be stimulus-specific and species-specific.


Porcine endometrial epithelial cells immortalized by transfection with origin-defective, temperature-sensitive simian virus 40 DNA. The purpose of this study was to immortalize porcine endometrial cells and to characterize the transformed cells. Primary porcine endometrial cells were transfected with the plasmid vector (pmk16) containing SV40 DNA using a liposome-mediated method. The viral DNA was from a replication-defective, origin-minus, temperature-sensitive mutant strain (A58). One clone, designated PE-1, has been propagated for over 120 passages. PE-1 cells grown at 33C (33C cells) exhibit spindle-shaped morphology; when cultured at 40C (40C cells), they took on a polygonal or spherical shape. Morphology of 40C cells returned to the spindle shape after culture flasks were shifted back to 33C. During a 2-week period, 33C cells propagated approximately 30-fold faster than 40C cells, whereas protein concentration was higher in 40C cells. Southern blot analysis of PE-1 cells demonstrated successful integration of the ts-SV40 DNA sequence into the porcine endometrial cells, possibly at multiple sites. The presence of cytokeratin on PE-1 cell membranes was shown by immunocytochemical studies, suggesting that the PE-1 cell clone was of epithelial origin. Reverse phase (RP)-HPLC analysis of PE-1 cell extract indicated that the majority of immunoreactive beta-endorphin (ir-BEND) eluted with a hydrophobicity similar to that of synthetic BEND and alpha-N-acetylated BEND (Nac-BEND). These results demonstrate that a porcine endometrial cell line has been established, and that this cell line possesses characteristics of temperature sensitivity in cell morphology, growth rate, and protein synthesis.


Non-linear epitopes of the large subunit of Ku autoantigen recognized by monoclonal and autoantibodies. Sera from certain patients with SLE, scleroderma and other autoimmune diseases react with the two subunits of the Ku protein: 86 and 70 kDa. Previous experiments indicated that a region of 40 amino acids near the C-terminus of the 86 kDa subunit between amino acids 667 and 708 was critical for binding of monoclonal and some autoimmune antibodies. In the present study, a series of additional 5' deletions and site-specific mutations in the critical region were produced and the immunoreactivities of the recombinant proteins were examined. ELISA and immunoblot analyses showed that three non-competing monoclonal antibodies specific for the 86 kDa subunit require stretches of amino acids significantly longer than 40 amino acids for reactivity, suggesting that the antigen is recognized in a folded state with perhaps more than one contact point. The reactivities of 12 of 24 anti-Ku positive autoimmune sera screened depended on the same amino acid sequences required for binding of the monoclonal antibodies, site-specific mutations reduced the reactivities of monoclonal and autoantibodies in a similar way. Preincubation of native Ku protein with the monoclonal antibodies shifted the electrophoretic mobility of Ku protein-DNA complex, suggesting that these monoclonal antibodies bind to epitopes on the surface of the native Ku protein. Taken together, the results from the deletion and site-directed mutagenesis demonstrate that both monoclonal and autoantibodies recognize non-linear epitopes of the 86 kDa polypeptide. These findings indicate that in a large portion of patients the anti-Ku autoimmune response is similar to the normal immune response to the Ku antigen in mice.


Sequence diversity of T cell receptor alpha chain transcripts from BALB/c thymus. Most of the diversity in T cell receptor subunits resides in the region that is the equivalent of the CDR3 of immunoglobulins. In order to learn more about the relative contributions of the various mechanisms that generate this diversity we have analyzed the sequences of alpha chain transcripts from BALB/c thymus. The J alpha repertoire of BALB/c mice was examined by comparison of new J alpha sequences and previously published sequences. Among the 41 J alpha genes examined, most of the diversity is located at the 5' end, consistent with the notion that this region contacts the antigen. VJ junctional diversity was examined by sequencing various V alpha J alpha combinations derived from different stages of development. Deletion of bases from the ends of V and J genes does not occur with equal frequency. A greater number of bases were deleted on average from the ends of J genes. Bases were added at junctions frequently in isolates from adult animals, consistent with the presence of terminal deoxynucleotidyl transferase. However, there were short stretches of sequences at junctions which were also present at the 5' end of J genes. These findings extend recent observations that alpha chain genes use multiple mechanisms for generating diversity.


Intrinsic immunogenicity of an internal VP1 T-B epitope pair of type 1 poliovirus. We describe the intrinsic immunogenicity of a poliovirus T-B epitope pair that is located in the N-terminus of the capsid protein VP1. This peptide is unusual in that it is located on the interior of the native virion at the VP1-VP3 interface in a region that becomes exposed after cell binding, proteolysis, or heating of the virus. Immunization of mice with either the virion or free peptide leads to anti-peptide antibody production. Anti-peptide immunity is under genetic control and 1-Ak restricted T cell proliferative responses have been identified. SJL/J (H-2s) mice that are low responders to this T-B epitope pair are also low responders to PSV-1 itself, suggesting that this site may be important in the production of neutralizing anti-PSV-1 antibodies. Interestingly, seropositive humans also have significant anti-peptide titers suggesting that immunization with poliovirus in a species permissive for infection also leads to anti-peptide antibody production. Collectively, these data suggest that a T-B epitope pair located on the interior of a protein or virion can be immunogenic. Several mechanisms whereby internal T-B epitope pairs might become immunogenic are discussed.


Unique biochemical properties of a mutant MHC class I molecule, H-2Ksm1. This study describes serological and biochemical properties of a novel MHC class I molecule. The mutant H-2Ksm1 molecule was discovered in a mouse because of loss of reactivity of its peripheral blood lymphocytes to monoclonal antibodies. This mutation in the H-2Ks molecule is the first in vivo mutation described that has altered an amino acid residue (amino acid 107) distant from the regions generally considered to be peptide or TCR contacts. Cell surface expression of the mutant molecules remains high but the Arg107 to Trp substitution appears to alter the native protein conformation, markedly decreasing cell surface association with beta 2-microglobulin light chains and conferring a loss of recognition by Ks specific antibodies.


Human anti-acetylcholine receptor antibodies use variable gene segments analogous to those used in autoantibodies of various specificities. The production of autoantibodies to the nicotinic acetylcholine receptor are responsible for many of the neurological symptoms observed in myasthenia gravis. An understanding of the structural organization of the anti-receptor antibodies may help to define the role of these antibodies in the pathogenesis of this disease. The nucleotide sequences of the heavy and light chains of three human monoclonal anti-receptor antibodies isolated from peripheral blood lymphocytes from two patients with myasthenia gravis were analyzed. In addition, the structure of an anti-idiotypic antibody was studied. The VH and VL gene segments used in the anti-receptor antibodies appear to be derived from the same repertoire as gene segments that have been found in other autoantibodies isolated from patients with various autoimmune diseases. The IgM anti-receptor antibodies are direct copies of germline gene segments, while the structures of the IgG anti-receptor antibody and the anti-idiotypic antibody appear to be mutated suggesting that they have undergone antigenic selection.


Profile of the regions on the alpha-chain of human acetylcholine receptor recognized by autoantibodies in myasthenia gravis. Eighteen synthetic overlapping peptides encompassing the entire extracellular part (residues alpha 1-210) of the alpha-chain of human acetylcholine receptor (AChR) and a 19th peptide (residues alpha 262-276) corresponding to an extracellular connection between two transmembrane regions were prepared and used for the measurement, by solid-phase radioimmunoassay, of the binding of autoantibodies in plasma from myasthenia gravis (MG) patients. Autoantibodies were found to recognize only a limited number of the synthetic peptides. The regions recognized resided predominantly within the areas alpha 10-30, alpha 111-145 and alpha 175-198 and, less frequently, region alpha 45-77. Differences in the recognition profile of the peptides from patient to patient indicated that the autoantibody responses were under genetic control. However, by using a mixture of the appropriate peptides, it was possible to determine autoantibodies in all 15 myasthenia sera and to distinguish between these, normal human sera and other neurological or autoimmune diseases. The mapping of the continuous antigenic regions recognized by autoantibodies on the alpha-chain of human AChR has permitted a comparison of the regions recognized by autoantibodies and autoimmune T-cells from the same donor. It also provided a peptide-based direct antibody binding method for diagnosis of MG.


The natural history of community-acquired hepatitis C in the United States. The Sentinel Counties Chronic non-A, non-B Hepatitis Study Team. Chronic liver disease develops in more than half of patients with post-transfusion hepatitis C, but little is known about the natural history of community-acquired hepatitis C. In 1985 and 1986 we identified adults with acute non-A, non-B hepatitis in four counties in the United States and followed them prospectively. We used three markers to detect hepatitis C virus (HCV) infection in stored samples of serum: antibody to HCV (anti-HCV) detected by second-generation serologic assays; HCV RNA detected by polymerase-chain-reaction assay; and antibody to HCV antigen (anti-HCVAg) detected by fluorescent-antibody-blocking assay. Of 130 patients with non-A, non-B hepatitis, 106 (82 percent) had HCV infection, 93 were positive for anti-HCV, and 13 were positive only for HCV RNA or anti-HCVAg. Chronic hepatitis developed in 60 (62 percent) of 97 HCV-infected patients followed for 9 to 48 months, with no relation to the risk factors for infection. Ten of the 30 patients who had liver biopsies had chronic active hepatitis. In samples collected 42 to 48 months after the onset of hepatitis, HCV RNA was detected in 12 of 13 tested patients with chronic hepatitis and in all 15 tested patients with hepatitis that had resolved. Anti-HCV persisted in all but two of the initially positive patients, for a rate of antibody loss of 0.6 per 100 person-years. Patients with community-acquired hepatitis C have a high rate of chronic hepatitis. HCV may be a major cause of chronic liver disease in the United States, and in most patients HCV infection seems to persist for at least several years, even in the absence of active liver disease.


[Mobilization using incompatibility group P1 plasmids in strains of Pseudomonas pseudomallei and Pseudomonas mallei as potential vectors for DNA cloning]. The cells of Pseudomonas pseudomallei and Pseudomonas mallei have been shown to serve as recipients for the plasmid RSF1010 and its recombinant derivatives pVA1 and pVA4. The conjugative plasmids RP1 and pTH10 of the incompatibility group P1 are able to mobilize the nontransmissive vector plasmids for conjugation transfer into Pseudomonas pseudomallei and Pseudomonas mallei strains. The SmR determinant of the plasmid RSF1010 is expressed in the latter strains. These data makes the mentioned vector plasmids the candidates for DNA cloning in these strains.


Altered structure and expression of the p53 gene in human neuroepithelial tumors. The p53 gene, located on chromosome 17p13.1, may be important in the pathogenesis of human neuroepithelial tumors, because it is a tumor suppressor gene and genetic alteration is essential for certain human cells to acquire the neoplastic phenotype. The structure and expression of the p53 gene were investigated in cultured human glioma cells and biopsied specimens of neuroepithelial tumors. Immunocytochemical examination of p53 gene expression revealed positive nuclear staining in six of seven glioma cell lines tested. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis demonstrated unequivocal heterogeneity of migration rate in p53 bands. Pulse-chase analysis clearly showed an increased half-life of p53 in cultured human glioma cells. These abnormalities are presumably due to genetic alterations in the p53 gene. Nucleotide substitutions in exon 5, 7, or 8 of the p53 gene could be detected by polymerase chain reaction-single strand conformational polymorphic analysis in four of seven (57%) human glioma cell lines, and nine of 29 (31%) biopsied specimens of neuroepithelial tumors examined. The present results indicate that genetic alterations in the p53 gene are responsible for the tumorigenesis of at least some human neuroepithelial tumors.


Cerebral vein disorders and postoperative brain damage associated with the pterional approach in aneurysm surgery. The possible causes of postoperative brain damage were examined in 100 cases of cerebral aneurysms operated on by the pterional approach. Postoperative brain damage occurred in 15% of cases, located mostly in the inferior frontal lobe. Its incidence was higher in early than in delayed operation and increased with severity of preoperative clinical conditions but not correlated with patient age and aneurysm location. The venous perfusion patterns in the inferior frontal lobe were classified into three types based on preoperative venograms: Sylvian type drained mainly into the superficial Sylvian veins (SSVs), Frontal type drained mainly into the frontal bridging veins, and Intermediate type. Postoperative brain damage was most frequent in the Sylvian type with statistical significance (p < 0.01). The brain retraction procedure impairs regional cerebral blood flow (rCBF). Venous congestion in the retracted inferior frontal lobe, caused by stretching and narrowing of SSVs due to both brain retraction and dissection of the Sylvian fissure, also reduces rCBF. Thus, a marked reduction in rCBF in the retracted area causes postoperative brain damage. Postoperative venograms showed the SSVs to be obscured in 24% of patients, indicating that the pterional approach possibly influences the SSV perfusion. A venous perfusion disorder during the pterional approach is the most important factor in postoperative brain damage, and careful preoperative assessment of cerebral veins is indispensable.


Preliminary results of interstitial 192Ir brachytherapy for malignant gliomas. Twenty-six patients with recurrent or unremovable malignant gliomas were treated by interstitial brachytherapy with iridium-192 seeds. Stereotactic implantation of the afterloading catheters using the Brown-Roberts-Wells computed tomography (CT)-guided stereotactic system was performed in 24 patients and surgical implantation in two patients with pontine glioma. The response to therapy was measured by serial CT, magnetic resonance imaging, and clinical examination. Tumor regression was seen in 17 patients 1-3 months after implantation. Tumor progression was seen in only three patients. After interstitial brachytherapy, the most commonly observed CT finding was central low density. Median survival time was 18 months after implantation. Autopsies in five patients revealed the delayed effects of radiation injury such as typical vascular changes, microcalcification, and coagulative necrosis in the implant area and tumor recurrence at the periphery. The results suggest that brachytherapy is not curative but prolonged the median survival time by 6 months.