Fluorescent viscoelastic enhancement.

Fluorescent viscoelastic enhancement. By inserting an Erreger 485 exciter filter into the operating microscope, translucent yellow Healon (sodium hyaluronate) transforms into a brilliant opaque green viscoelastic. We have developed this technique and termed it "fluorescent viscoelastic enhancement." Using the technique, we demonstrated that the time required to remove Healon from the anterior chamber after intraocular lens insertion varies. Healon is usually aspirated quickly, in less than 17 seconds. Otherwise it traps behind the intraocular lens and requires more time for irrigation/aspiration (I/A) and manipulation of the I/A tip. Fluorescent viscoelastic enhancement minimized I/A time, reducing excess turbulence and manipulation in the anterior chamber, and thus may reduce corneal endothelial cell loss. This study also demonstrated that fluorescent viscoelastic enhancement prevented postoperative intraocular pressure rise, compared to the conventional removal of clear Healon. Fluorescent viscoelastic enhancement assures the surgeon that a large amount of Healon is not left behind.


Visual outcome following complicated extracapsular cataract extraction. In a prospective study of 1,597 consecutive extracapsular cataract extractions (ECCE), we review 49 cases complicated by capsular or zonular rupture with or without vitreous loss. The ECCEs were divided into two groups depending on the type of intraocular lens (IOL) implanted. In Group A, whose patients received an anterior chamber lens, we found a significantly (P < .05, t-test) worse postoperative visual acuity than in Group B whose patients received a posterior chamber lens. We therefore suggest that in the presence of a capsular tear and vitreous loss, one should try to implant a posterior chamber lens rather than an anterior chamber lens to obtain as good a visual acuity as possible.


Combined trabeculectomy, cataract extraction, and foldable lens implantation. The efficacy of foldable silicone intraocular lenses in combined glaucoma/cataract surgery was retrospectively analyzed in 19 consecutive operations. Preoperatively all patients were receiving medical treatment for glaucoma; 21% had intraocular pressures above 21 mm Hg despite the medical therapy. Postoperatively, reduction of intraocular pressure to 21 mm Hg or less was achieved in 84% of eyes, with 58% requiring no antiglaucoma medications. Mean improvement of Snellen visual acuity was 4.9 lines; 84% of eyes achieved a final acuity of 20/40 or better. Median follow-up was 59 weeks (range 25 to 88 weeks). Thirteen eyes (68%) had a filtration bleb present when last examined. These preliminary results suggest that foldable silicone IOLs are an effective alternative to poly(methyl methacrylate) lenses in combined glaucoma/cataract surgery.


Incidence of postoperative posterior capsular opacification with types of senile cataracts. The most frequent postoperative complication in extracapsular cataract surgery is opacification of the posterior capsule. The purpose of this study was to correlate the frequency of capsular opacification with each kind of senile cataract. A total of 566 eyes with cataracts were studied. They had been operated on between 1980 and 1990 using the extracapsular, intercapsular, and phacoemulsification techniques. Our results confirm that senile complete cataracts (mature cataracts) had a significantly lower tendency to produce postoperative capsular opacification than other cataract types (nuclear, cortical, posterior subcapsular).


Panoramic specular microscopy of lens epithelial cells in the postoperative pseudophakic eye. Using the Konan-Keeler specular microscope, panoramic specular microscopy covering almost one quadrant of the anterior capsule was performed. Regional changes in the retained lens epithelial cells were recorded and compared simultaneously. The nearer the rim of the anterior capsular opening, the greater the decrease of cell density, increase of average cell size, and variety of cell size. The results indicate that the first postoperative changes in lens epithelial cells following phacoemulsification with subsequent capsular bag intraocular lens implantation occur in the area close to the anterior capsular opening, with subsequent changes in peripheral areas.


Refractive bifocal intraocular lens and pupillary diameter. To assess the relationship between pupillary diameter and focusing for distance and near vision with the refractive bifocal intraocular lens (IOL), we studied visual acuity for far and near distance, near point, and depth of focus with normal pupillary movements and after pharmacological miosis and mydriasis (pilocarpine 2% and phenylephrine 10% eyedrops, respectively) in 20 patients implanted with an IOLAB refractive bifocal IOL. Pharmacological miosis and mydriasis did not change far and near visual acuity values from the values with normal pupillary diameter. After pharmacological miosis, the near point position and the width of the depth of focus were unchanged. After pharmacological mydriasis, near point distance was unchanged but depth of focus dropped in a statistically significant manner (P < .01). We concluded that the pupillary diameter alone cannot produce distance or near vision focusing. The choice depends on the brain's preference. Pupillary diameter increases the quality of vision through the blur circles mechanism.


In vitro adhesion of Pseudomonas aeruginosa and Staphylococcus aureus to surface passivated poly(methyl methacrylate) intraocular lenses. Bacterial attachment to intraocular lenses (IOLs) can be a cause of infectious endophthalmitis following cataract surgery. In this study, Pseudomonas aeruginosa and Staphylococcus aureus adhered in vitro to untreated poly(methyl methacrylate) (PMMA) and to surface passivated PMMA lenses. The IOLs were placed in bacterial suspensions (1 x 10(7) cfu/ml) in vitro. Significantly fewer S. aureus attached (P < .05) to the normal PMMA IOLs (4,535 +/- [SD] 3,052 bacteria/mm2) and to passivated PMMA IOLs (8,720 +/- 10,040 bacteria/mm2) than did P. aeruginosa (normal PMMA: 67,808 +/- 45,070 bacteria/mm2, passivated PMMA: 85,795 +/- 70,647 bacteria/mm2). The differences in bacterial attachment to surface passivated IOLs and to untreated PMMA lenses were not significant.


Cellular reaction following cataract surgery with implantation of the heparin-surface-modified intraocular lens in rabbits with experimental uveitis. The inflammatory response after cataract surgery and intraocular lens (IOL) implantation was studied in rabbits with endotoxin-induced uveitis. On days 1, 3, 7, 14, and 30 postoperatively the rabbits were sacrificed and the number of white blood cells in the aqueous humor and cellular deposits on the IOLs were estimated. On days 14 and 30 the rabbits also had slitlamp examination to study the clinical outcome of the surgery. At day 1 after lens extraction and IOL implantation, the number of white blood cells in the aqueous humor was significantly lower (P < .05) in eyes with heparin-surface-modified (HSM) IOLs (795.2 +/- 262.9; mean +/- SEM) than in eyes with poly(methyl methacrylate) (PMMA) lenses (1386.5 +/- 247.9). No differences were seen at day 3, 7, 14, or 30 postoperatively. The choice of IOL material had no effect on the amount of cell deposits on the IOL surface or on clinical parameters such as anterior synechias, posterior synechias, fibrosis, and posterior capsular opacification. There was a trend toward a greater number of cellular deposits on the PMMA lenses, but this was not statistically significant. This study provides further evidence of improved biocompatibility of the HSM PMMA lens, as demonstrated by a decreased acute inflammatory response.


Optical testing and inspection methodology for modern intraocular lenses. A method of in vitro optical performance evaluation of intraocular lenses (IOLs) is discussed. The IOLs are tested in the eye cell, which is designed to be optically equivalent to the theoretical eye model. The eye cell is a combination of a wet cell and the optical element (the "conjugation lens") that converges a beam of light, similar to the action of the cornea in the theoretical eye model. The eye cell is designed so the spatial frequency scale (modulation transfer frequency [MTF] testing), expressed in terms of line pairs per millimeter (linear scale), equals the scale produced by the theoretical eye model. The most common way to express spatial frequencies in visual science is in line pairs per degree or cycles per degree (angular scale). The problem with in vitro testing is that the corresponding angular scale varies with the dioptric power of the conjugation lens. This paper provides a simple method of mapping the angular scale of the eye cell onto the theoretical eye model, allowing a comparison with clinical results as well as with MTFs measured in different eye cells. All corresponding formula for calculating the eye cell optical characteristics are provided.


Down slope sculpting.

Down slope sculpting. A method of sculpting and fracturing that greatly enhances the efficiency and safety of in-situ nuclear fracturing (nucleofractis) techniques is presented. By nudging the nucleus of the lens inferiorly, sculpting can be accomplished very deep in the upper part of the lens to facilitate early and efficient fracturing. The benefits, advantages, and safety of this "down slope" sculpting technique are described.


Simplified insertion technique for the SI-26NB intraocular lens. Inserting the 13 mm SI-26NB intraocular lens is a challenging task complicated by the need to tuck the modified C-configuration haptics within a folded optic. This step causes undue stress on the haptic and may result in asymmetrical distortion and subsequent decentration. Excluding this step simplifies the technique, as well as decreases the degree of acute angle stress on the haptic as it is passed through the scleral tunnel.


Mechanics of capsulorhexis.

Mechanics of capsulorhexis. Continuous (central, circular) curvilinear capsulorhexis (CCC), first introduced by Gimbel and Neuhann, has become increasingly popular, especially for endolenticular phacoemulsification with intracapsular intraocular lens implantation. The principles of simple mechanics are used to facilitate teaching the procedure and to reduce the incidence of complications. A safe and simple method of performing CCC based on these principles is described.


Effect of captopril and the nonpeptide angiotensin II antagonists, SK&F 108566 and EXP3174, on renal function in dogs with a renal artery stenosis. Treatment with an angiotensin converting enzyme (ACE) inhibitor can result in acute renal failure in patients with a renal artery stenosis. In the present study the effects of the selective nonpeptide angiotensin II antagonists, SK&F 108566 ((E)-alpha-[[2-Butyl-1-[(4-carboxyphenyl)methyl]-1H-imidazol-5-yl] methylene]-2-thiophenepropanoic acid) and EXP3174 (2-n-Butyl-4-chloro-1-[(2'-(1H-tetrazol-5-yl)-biphenyl-4-yl) methyl]imidazole-5-carboxylic acid hydrochloride) (the active metabolite of DuP 753, losartan) were compared with the ACE inhibitor, captopril, in the anesthetized dog which had been uninephrectomized and the remaining renal artery clamped to reduce renal blood flow (RBF) by approximately 50%. All three agents resulted in dose-dependent reductions in mean arterial pressure (MAP), glomerular filtration rate (GFR) and RBF. The maximum responses to captopril and SK&F 108566 were similar with MAP, RBF and GFR all decreasing approximately 30%. EXP3174 also resulted in decreases in GFR and RBF of approximately 30%; however, there was a smaller (approximately 17%) decrease in MAP. The data indicate that the possible bradykinin enhancing activity of ACE inhibitors may not provide any moderating activity of ACE inhibitor-induced reduction in GFR observed in dogs with a renal artery stenosis.


Effect of FR121196, a novel cognitive enhancer, on the memory impairment of rats in passive avoidance and radial arm maze tasks. Passive avoidance performance was impaired in young rats by scopolamine (1.0 mg/kg) or by nucleus basalis magnocellularis lesions and in aged rats (24-26 months old). FR121196[N-(4-acetyl-1-piperazinyl)-4-fluorobenzenesulfonamide+ ++], a newly introduced cognitive enhancer, ameliorated the failure in memory retention with bell-shaped dose-response curves in doses ranging from 0.1 to 10 mg/kg. Similar dose-response curves also were obtained with methamphetamine (0.1-10 mg/kg), whereas physostigmine (0.01-1.0 mg/kg) attenuated the amnesia in scopolamine-treated rats, but hardly affected that in nucleus basalis magnocellularis-lesioned or aged rats. In radial arm maze tasks, behavioral indices of spatial memory, such as first correct choices and number of errors, were impaired by an injection of scopolamine (0.5 mg/kg) or by lesions of the medial septum or fimbria-fornix. FR121196 ameliorated the scopolamine-induced memory deficit with a bell-shaped dose-response curve, whereas methamphetamine had the opposite effect. These two drugs had little effect on memory deficits brought about by medial septum or fimbria-fornix lesioning, whereas physostigmine ameliorated these deficits in scopolamine-treated and medial septum-lesioned rats, but not in fimbria-fornix-lesioned rats. Behavioral studies using Animex demonstrated that FR121196, in contrast with methamphetamine, does not increase locomotor activities in rats up to a dose of 10 mg/kg. These results are discussed in relation to the cholinergic and dopaminergic mechanisms of memory.


L-dopa induces opposing effects on pain in intact rats: (-)-sulpiride, SCH 23390 or alpha-methyl-DL-p-tyrosine methylester hydrochloride reveals profound hyperalgesia in large antinociceptive doses. The present study shows that during the time course of the action of single doses, L-dopa induces multiphasic opposing effects on pain, recorded as vocalization during the presentation of electrical stimulation applied to the tail of normal rats. This indicates that two or more functional systems contribute to produce the net response. A small dose (15 mg/kg) of L-dopa facilitates pain slightly, whereas larger doses (100-200 mg/kg) can produce an antinociceptive effect following an initial hyperalgesia. Moreover, profound hyperalgesia is revealed by either dopamine (DA) D1 and D2 receptor blockade by means of SCH 23390 [R-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetra-hydro-1H- 3-benzazepine hydrochloride] or (-)-sulpiride, respectively, as well as after a reduction of the presynaptic synthesis of catecholamines after pretreatment of the animals with the tyrosine hydroxylase inhibitor alpha-methyl-DL-p-tyrosine (alpha-MPT). The enhancement of L-dopa's hyperalgesic effect after SCH 23390 treatment is maximal already at the onset of the effects, whereas (-)-sulpiride or alpha-methyl-DL-p-tyrosine precipitates the hyperalgesia after a certain temporal delay during defined phases of the time course of the effects of large L-dopa doses. The D1 receptor agonist (+)-SKF 38393 potentiates both the hyperalgesic and antinociceptive effects of 100 mg/kg of L-dopa. It is suggested that L-dopa's net effect on pain is modulated from concentration-dependent, opposing effector systems involving both DA stimulatory and inhibitory receptor mechanisms. At high dosing, activation of D2 receptors enhancing DA functional activity produces an antinociceptive response that normally outweighs the hyperalgesia, but this effect becomes dissociable with inhibition of central DA activity.


Effects of chronic treatment with ethanol on the development of cross-tolerance to other alcohols and pentobarbital. The development of cross-tolerance to various alcohols and pentobarbital was examined in ethanol (EtOH)-treated mice. Chronic EtOH treatment (dosage rising in steps from 3.5-4.5 g/kg i.p. daily during a 23-day period) produced tolerance to its hypnotic effect. Such tolerance was seen as a reduction in the duration of loss of righting reflex (LRR), as well as higher blood EtOH levels at the offset of LRR, in EtOH-treated mice as compared to saline-treated controls. Cross-tolerance was shown by shifts in dose-response curves for the LRR induced by n-propanol and t-butanol. Such treatment, however, did not confer functional cross-tolerance to n-butanol and pentobarbital. Because n-butanol and pentobarbital are more lipid-soluble, whereas EtOH, n-propanol and t-butanol have low degrees of lipid solubility, the development of cross-tolerance among these sedative-hypnotic drugs might be related to their relative degrees of lipid solubility.


5-Hydroxytryptamine 1a receptor agonists block prepulse inhibition of acoustic startle reflex. Presentation of a nonstartling stimulus (prepulse) 100 msec before a startle-eliciting auditory stimulus (pulse) reduces startle reflex amplitude in mammals. Prepulse inhibition of acoustic startle reflex is smaller in schizophrenics than in nonschizophrenics, a phenomenon that has been hypothesized to reflect sensorimotor gating deficits underlying schizophrenic psychosis. Five 5-hydroxytryptamine1a (5-HT1a, serotonin) receptor agonists: 8-hydroxy-2-(di-n-propylamino) tetraline (8-OHDPAT), 5-methoxydimethyltryptamine, buspirone, gepirone and ipsapirone, were tested for effects on prepulse inhibition and startle reflex amplitude in rats. All five agents reduced prepulse inhibition at doses that had no effect on startle reflex amplitude or motor activity. Reduction of prepulse inhibition by 8-OHDPAT was antagonized by (-)propranolol, a 5-HT1a receptor antagonist, and partially by haloperidol, a dopamine D2 receptor antagonist, but not by ketanserin or methysergide, 5-HT2 receptor antagonists. 8-OHDPAT did not reduce prepulse inhibition in subjects pretreated with reserpine or tetrabenazine to deplete neuronal amines, but interpretation of this result is complicated because reserpine and tetrabenazine given alone reduced prepulse inhibition. The results indicate that 5-HT1a receptor agonists block prepulse inhibition of acoustic startle reflex, possibly via dopaminergic mechanisms.


Tolerance to the cataleptic effect of the N-methyl-D-aspartate (NMDA) receptor antagonists in pigeons: cross-tolerance between PCP-like compounds and competitive NMDA antagonists. The effects of chronic administration of phencyclidine (PCP) or CGS 19755 (cis-4-phosphonomethyl-2-piperidine-carboxylic acid) on the cataleptic effects of N-methyl-D-aspartate (NMDA) receptor antagonists were studied in pigeons. PCP, a channel blocker of the NMDA receptor complex, or CGS 19755, a competitive NMDA antagonist, was administered i.m. to separate groups of pigeons each day. Tolerance developed to the cataleptic effects in both PCP- and CGS 19755-treated pigeons. PCP tolerance was characterized initially by 5-fold rightward shift and, with an increased chronic PCP dose, a complete downward shift of the PCP dose-effect curve. CGS 19755 tolerance was indicated by a 10-fold rightward shift of its dose-cataleptic effect curve. Cross-tolerance was obtained from PCP to other PCP-like compounds including dizocilpine (MK 801), ketamine, dextrorphan, 1-(2-thienyl)-cyclohexyl-piperidine and [(+)-SKF 10047] [(+)-N-allyl-normetazocine] as well as to the competitive NMDA antagonist, CGS 19755. Cross-tolerance also developed from CGS 19755 to another competitive NMDA antagonist, CGP 40116 [D-(E)-2-amino-4-methyl-5-phosphono-3-pentenoic acid] as well as to PCP-like compounds. The pharmacological selectivity of tolerance was evident because there was equal sensitivity to etomidate or pentobarbital in tolerant and nontolerant pigeons. The symmetric cross-tolerance between PCP-like compounds and competitive NMDA antagonists suggests the cataleptic effects of the two classes of NMDA antagonists are probably mediated via a similar mechanism of inhibition of neurotransmission at the NMDA excitatory synapse.


Prolonged stimulation of bovine adrenal chromaffin cells with arachidonic acid and prostaglandin E2 increases expression of the proenkephalin gene and the secretion of [Met5]-enkephalin. The effects of long-term exposure of bovine adrenal medullary chromaffin (BAMC) cells to arachidonic acid (AA) and prostaglandin E2 (PGE2) on [Met5]-enkephalin (ME) secretion and expression of the proenkephalin A (proENK) gene were studied. Treatment with various concentrations of AA or PGE2 for 24 hr increased the secretion of ME in a concentration- and time-dependent manner. At high concentrations (10-100 microM), but not low (1-3 microM), AA significantly increased ME secretion by 1 hr. In contrast, the onset time for increase of ME secretion by PGE2 was 3 hr after exposure. The magnitude of increase in ME secretion in the presence of AA or PGE2 continued to increase with time. However, intracellular ME levels in AA- or PGE2-treated cells were not significantly different from that of controls, indicating that elevated levels of ME secretion into the media may be a result of increased biosynthesis of ME. In addition, AA or PGE2 increased proENK mRNA level in a concentration- and time-dependent manner. The onset time for the increase in proENK mRNA in response to PGE2 was 6 hr after exposure. The treatment of BAMC cells with 20 microM cycloheximide (a protein synthesis inhibitor) inhibited both the increased secretion of ME and proENK mRNA level induced by AA and PGE2 in a time-dependent manner, indicating that the delayed secretion of ME and the increase in proENK mRNA level induced by AA and PGE2 require protein synthesis. Indomethacin (a cyclooxygenase inhibitor, 10 microM) effectively inhibited AA-induced responses, whereas 10 microM nordihydroguaiaretic acid (a lipoxygenase inhibitor) was inactive.(ABSTRACT TRUNCATED AT 250 WORDS)


Effects of in vivo and in vitro administration of morphine sulfate upon rhesus macaque polymorphonuclear cell phagocytosis and chemotaxis. The effects of morphine administration were evaluated upon the polymorphonuclear (PMN) cell activities of rhesus macaques. Sixteen animals were used in the study. Seven macaques were treated with saline and nine animals were administered morphine in the form of morphine sulfate, following a control period of saline injections. The dosage regime was initiated at 1 mg of morphine sulfate per kg of b.wt., 3 times daily, with incremental steps to a stable dose of 5 mg/kg, 3 times daily. PMN cells prepared from morphine-treated animals showed a marked, transient reduction in their ability to kill ingested yeast blastospores, although the ingestive capacity of the PMN cells was not impaired. PMN cells prepared from three of the nine morphine-treated animals showed a transient reduction in chemotaxis toward a complement-derived chemotactic factor. In vitro studies on PMN cells prepared from morphine-naive animals suggested that the killing activity and the chemotaxis of the cells were reduced by treatment with 50 pM morphine sulfate, but not with 50 fmol of morphine sulfate.


Platelet-activating factor stimulates arachidonic acid release and enhances thromboxane B2 production in intact fetal rat brain ex vivo. The ability of brain preparations from 20-day-old rat fetuses to synthesize eicosanoids in the presence of platelet activating factor (PAF) was investigated. A rise (49%) in thromboxane B2 (TxB2; the stable thromboxane A2 metabolite) was observed after 30 min in the presence of 0.6 microM PAF. Repetitive administration of PAF did not rise TxB2 production above a certain level, suggesting desensitization. 1-O-alkyl, sn-glycero-3-phosphocholine (lyso-PAF) at 0.6 microM had no effect, whereas selective PAF antagonists, i.e., BN52021, BN50739 and BN50727, or indomethacin, a general cyclooxygenase inhibitor, blocked completely TxB2 synthesis. The calcium ionophore A23187 (10 microM) stimulated production of TxB2, prostaglandin E2 and 6-keto-prostaglandin F1 alpha eicosanoids, whereas extracellular calcium deprivation did not impair eicosanoid release. The effects of PAF and A23187 on TxB2 synthesis were not additive and were not dependent on extracellular calcium. Chelation of intracellular Ca++ by Fluo-3/AM reduced production of TxB2 and prostaglandin E2 eicosanoids. Fluo-3/AM also blocked effectively PAF-dependent TxB2 release, indicating that production of TxB2 was almost entirely dependent on free intracellular calcium levels. PAF-dependent changes in brain phospholipids, prelabeled with [3H]arachidonic acid, were examined. One hour after in vivo injection of the isotope, fetal brains were removed and incubated in vitro for 30 min with carbamyl-PAF. Radioactivity in arachidonic acid and diglyceride fractions increased (35% and 30%, respectively), whereas radioactivity in phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol decreased. BN50726 antagonist abolished the effect of PAF. The radioactivity in poly-phosphoinositides was diminished (30-40% decrease) after PAF addition.(ABSTRACT TRUNCATED AT 250 WORDS)


Effects of various serotonin receptor subtype-selective antagonists alone and on m-chlorophenylpiperazine-induced neuroendocrine changes in rats. Administration of m-chlorophenylpiperazine [m-CPP, a serotonin (5-HT) agonist] to rats increases plasma concentrations of prolactin and corticosterone. Pretreatment with various doses of ritanserin (5-HT1C/5-HT2 antagonist), ICS 205-930 and MDL-72222 (5-HT3 antagonists), iodocyanopindolol or CG361A (beta adrenoceptor antagonists) and spiperone (5-HT1A/5-HT2 antagonist) did not attenuate m-CPP-induced increases in plasma concentrations of prolactin. In contrast, pretreatment with various doses of metergoline (5-HT1/5-HT2 antagonist), propranolol (beta adrenoceptor antagonist that also has binding affinity for 5-HT1A, 5-HT1B and 5-HT1C sites), mesulergine and mianserin (5-HT1C/5-HT2 antagonists) attenuated m-CPP-induced increases in plasma prolactin. On the other hand, m-CPP-induced increases in corticosterone concentrations were attenuated only by pretreatment with a low dose of mianserin and a high dose of spiperone. When administered without m-CPP, metergoline, mesulergine, ritanserin, ICS 205-930 and high doses of mianserin, spiperone and propranolol increased plasma corticosterone secretion. On the other hand, none of the antagonists used in the present study, except spiperone, had any significant effect on plasma prolactin secretion. These findings suggest that m-CPP-induced prolactin secretion is mediated by stimulation of 5-HT1C receptors while corticosterone secretion may be mediated either by an antagonistic effect at 5-HT3 receptor subtype or by nonserotonergic mechanisms. Alternatively, enhancement of corticosterone secretion by the 5-HT antagonists when administered alone may be responsible for their failure to block m-CPP-induced corticosterone secretion.(ABSTRACT TRUNCATED AT 250 WORDS)


Functional analysis of brain dopamine systems in a genetic mouse model of Lesch-Nyhan syndrome. The Lesch-Nyhan syndrome is a neurogenetic disorder caused by congenital deficiency of the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT). The disorder is characterized by prominent neurobehavioral abnormalities which appear to result in part from dysfunction of striatal dopamine systems. HPRT-deficient (HPRT-) mutant strains of mice have been produced as animal models for this syndrome, but these animals exhibit none of the neurobehavioral abnormalities seen in Lesch-Nyhan patients. The present studies describe the behavioral responses of three strains of mice carrying one of two mutations in the HPRT gene to agents which interact with brain dopamine systems. HPRT- mice are more sensitive than age- and sex-matched littermates to the motor-activating properties of dopamine-releasing agents (amphetamine, amfonelic acid and methylphenidate), but not dopamine uptake inhibitors (GBR 12909 and nomifensine). The enhanced sensitivity of the HPRT- mice to the dopamine-releasing agents is not caused by dopamine receptor supersensitivity, because the HPRT- mice do not show enhanced motor responses to the direct D1/D2 dopamine receptor agonist apomorphine or to the selective D1 dopamine receptor agonist SKF 38393. The function of regulatory dopamine autoreceptors, as assessed by suppression of spontaneous motor activity by low doses of R(-)-propylnorapomorphine, also appears normal in the HPRT- mice. Biochemical analysis shows that the HPRT- mice have significantly lower levels of dopamine (-45%), but normal levels of tyrosine, 3,4-dihydroxyphenylacetic acid, homovanillic acid and 3-methoxytyramine in the caudoputamen. In contrast to the deficit in caudoputamen dopamine, no deficits were noted in the accumbens of the HPRT- mice. These results indicate the existence of an inherent abnormality in the dopamine systems in the brains of HPRT- mice, despite their apparently normal spontaneous behavior.


Motor impairments after methamphetamine-induced neurotoxicity in the rat. Methamphetamine is a psychomotor stimulant which, given in high doses, produces neurodegenerative changes in the dopamine and serotonin systems. This study was designed to assess motoric deficits in the rat following a high-dose regimen of methamphetamine. Long-Evans male rats received either four injections of saline or methamphetamine (12.5 mg/kg, every 2 hr), a dose sufficient to produce 45 and 36% reductions in striatal dopamine and serotonin, respectively. Before treatment, subjects were trained to perform one of the following motor tasks: one-way active avoidance, inhibitory avoidance, rotorod or the balance beam. After recovery, performance under base-line and drug challenge conditions revealed that methamphetamine treatment caused significant deficits in active avoidance performance (24% increase in response latency) and balance beam performance (2- to 3-fold increase in footfalls), but had no effect on inhibitory avoidance or rotorod performance. Administration of l-dopa (100 mg/kg) significantly improved the methamphetamine-treated subjects' performance on the balance beam, but had no effect on the control subjects' performance. The methamphetamine-treated subjects exhibited a significant decrease in sensitivity to the effects of fenfluramine in comparison to the controls on both the rotorod and active avoidance tasks. We conclude that high-dose methamphetamine treatment produces long-lasting motor deficits associated with chronic reductions of striatal dopamine and serotonin. These data and the utility of the motor tasks are discussed in reference to a laboratory model of Parkinson's disease.


Interactions between zinc and spermidine on the N-methyl-D-aspartate receptor complex: clues to the mechanism of action of 1,10-bis(guanidino)decane and pentamidine. This study investigated the mechanism of action of two novel, noncompetitive antagonists of the N-methyl-D-aspartate (NMDA) receptor using [3H]dizocilpine binding to rat brain membranes. Pentamidine and 1,10(bisguanidino)decane (BG10) inhibited [3H]dizocilpine binding in the concentration range of 1 to 100 microM, as did Zn++ and the competitive polyamine antagonist arcaine. The action of each of these agents was sensitive to the addition of spermidine to the assay. However, only arcaine interacted with spermidine in a competitive fashion. Spermidine decreased the apparent affinity of Zn++ and also increased the Hill slope of the Zn++ inhibition curves. BG10 and pentamidine inhibition of [3H]dizocilpine binding was less sensitive to spermidine. Calcium had similar effects to spermidine on the inhibition of [3H]dizocilpine binding by Zn++, arcaine, pentamidine and GB10, but was less potent than spermidine. Treating membranes with diethylpyrocarbonate, a histidine-modifying reagent, decreased the apparent affinity of Zn++ by more than 4-fold while having very modest effects on the actions of BG10, pentamidine and arcaine. In addition, Zn++ failed to slow the dissociation of [3H]dizocilpine in diethylpyrocarbonate-treated tissue, whereas the action of BG10 and pentamidine was qualitatively unaffected. These data show that the effects of BG10 and pentamidine on the NMDA receptor are complex and may involve more than one binding site for each drug. In addition, this study shows that the action of Zn++ on the NMDA receptor is modulated by polyamines. Finally, the mechanism of action of pentamidine and BG10 cannot be attributed to an action at the Zn++ recognition site.


Amiloride-sensitive lithium reabsorption in rats: a micropuncture study. It has been suggested that in sodium-restricted rats lithium is being reabsorbed in the collecting tubule, and that such reabsorption can be prevented by amiloride. We studied these options in rats by means of cortical micropuncture. Indeed, an important fraction of the filtered lithium (10.5%) was reabsorbed beyond the early distal tubule in sodium-restricted rats. Amiloride infusion abolished this reabsorption. The dosage used did not affect the reabsorption of water, sodium and lithium in the proximal tubule, and also did not change the reabsorption of water and sodium in the loop segment. However, the reabsorption of lithium in this segment tended to be higher during amiloride (P = .06), and the delivery of lithium to the early distal puncture site was significantly less (26.1 +/- 1.2%) than in the control rats (31.4 +/- 1.8%, P < .05). The reason for this apparent increase in lithium reabsorption in the loop segment is not clear. Interestingly, amiloride increased urine flow and decreased urine osmolality. The mechanism underlying this effect needs further exploration. We conclude that in sodium-restricted rats lithium reabsorption beyond the early distal tubule level occurs. Lithium clearance data therefore underestimate end-proximal delivery under these conditions. Lithium reabsorption beyond the early distal tubule can be prevented by amiloride treatment. However, because amiloride may stimulate lithium reabsorption further upstream, lithium clearance data obtained during amiloride infusion may still underestimate end-proximal delivery of water and sodium.


Potentiation of adriamycin toxicity by ethanol in perfused rat liver. Adriamycin, which has a quinone nucleus, damages periportal regions of the lobule in perfused rat liver in an oxygen-dependent manner, presumably by redox cycling. Because redox cycling requires reducing equivalents, we investigated whether ethanol, which generates NADH via alcohol dehydrogenase, would increase hepatotoxicity due to concentrations of adriamycin which by themselves were not toxic in perfused rat liver. Perfusion with adriamycin (100 microM) alone did not significantly alter oxygen uptake or cell death evaluated by release of lactate dehydrogenase or uptake of trypan blue. In contrast, oxygen uptake due to adriamycin was increased about 35 mumol/g/hr and lactate dehydrogenase release was elevated to values around 240 U/g/hr in the presence of ethanol (10 mM). As expected, ethanol increased NADH fluorescence detected from the liver surface due to reduction of NAD+ in a concentration-dependent manner (half-maximal effect = ca. 1 mM). The increase in NADH fluorescence due to ethanol and the stimulation of oxygen uptake due to adriamycin had similar dependencies on ethanol concentration. Upon infusion of adriamycin, oxygen uptake increased concomitantly with a decrease in NADH fluorescence, most likely due to utilization of NADH. The half-maximal change in both processes also occurred with concentrations of ethanol around 1 mM. Furthermore, methylpyrazole (4 mM), an alcohol dehydrogenase inhibitor, prevented the increase in NADH fluorescence due to ethanol as well as the stimulation of oxygen uptake due to adriamycin in the presence of ethanol. Ethylhexanol, another agent which increased NADH, also potentiated oxygen uptake due to adriamycin.(ABSTRACT TRUNCATED AT 250 WORDS)


An experimental paradigm for investigating the role of endogenous adenosine/A1 receptor interactions in vivo. The purpose of the present study was to develop a pharmacological method for determining in the rat in vivo whether endogenous adenosine participates in a given process via activation of A1 adenosine receptors. In anesthetized rats, A1 receptors were activated by infusing the highly selective A1 receptor agonist N6-cyclopentyladenosine, and A2 receptors were stimulated by infusing the highly selective A2 receptor agonist CGS21680C. The bradycardic response to N6-cyclopentyladenosine and the hypotensive response to CGS21680C were used to assess A1 receptor and A2 receptor activation, respectively. After control responses to these purinergic agonists were elicited, animals were given infusions for several hours of either vehicle or one of six dosage levels of FK453 (a potent, selective, nonxanthine A1 receptor antagonist), one of three dosage levels of FR113452 (the S-enantiomer of FK453) or one of seven dosage levels of DPCPX (a potent, selective, xanthine A1 receptor antagonist). Antagonists were infused for > 4 hr, and at various times during the infusions, bradycardic and hypotensive responses to N6-cyclopentyladenosine and CGS21680C, respectively, were reassessed. Both FK453 and DPCPX were highly potent A1 receptor antagonists in vivo, and complete inhibition of bradycardic responses to N6-cyclopentyladenosine were obtained with 3 and 1 micrograms/kg/min, respectively. FR113452 was a very weak antagonist and only slightly reduced bradycardic responses to N6-cyclopentyladenosine at 100 micrograms/kg/min. In vivo FK453 and DPCPX were > 300 and 1000 times selective for the A1 receptor, respectively, compared with the A2 receptor.(ABSTRACT TRUNCATED AT 250 WORDS)


Decreased plasma cholinesterase activity enhances cocaine toxicity in mice. The illicit use of cocaine continues in epidemic proportions. Despite the incidence of life-threatening complications from cocaine use, little is known of the individual determinants of cocaine toxicity. In vitro analysis demonstrating that cocaine is poorly metabolized by the serum of patients with low plasma cholinesterase (PCh) activity (succinylcholine sensitivity) led to the hypothesis that altered PCh activity might modulate cocaine toxicity. An in vivo mouse model was created to test this theory. Mice were pretreated s.c. with either parathion [a mixed plasma and red blood cell cholinesterase (RBCCh) inhibitor], tetraisopropyl pyrophosphoramide (a selective PCh inhibitor) or placebo, and cholinesterase activity was determined at 24 hr. Incremental doses of i.p. cocaine were administered in a controlled and blinded fashion, and lethality was observed. Ten mg/kg s.c. parathion produced a mean suppression of 68 +/- 9 and 61 +/- 8% of PCh and RBCCh activity, respectively. One mg/kg s.c. tetraisopropyl pyrophosphoramide produced a mean suppression of 78 +/- 3 and 9 +/- 8% of PCh and RBCCh activity, respectively. Each pretreatment produced a statistically significant increase in cocaine lethality throughout the dose-response curve. Our results suggest that PCh activity is an important determinant of cocaine toxicity. This effect appears to be independent of either RBCCh activity or manifestations of organophosphate intoxication.


Propylbenzilylcholine mustard has greater specificity for muscarinic m2 receptors than for m3 receptors present in cerebellar granule cell culture from rat. Both muscarinic m2 receptors that inhibit adenylyl cyclase and m3 receptors that stimulate the hydrolysis of inositol phospholipids are expressed in cerebellar granule cells. In order to determine whether a reserve population of either of these receptors is present in this cell culture, the irreversible muscarinic receptor antagonist, propylbenzilylcholine mustard (PBCM), was used at different concentrations to bind various proportions of available muscarinic receptors. After pretreating the cell cultures with low concentrations of PBCM (< 1 nM), the muscarinic m2 receptor-mediated response decreased. Higher concentrations of PBCM (1-3 nM) were needed to reduce the muscarinic m3 receptor-mediated response. These results suggested that either a reserve population of muscarinic m3 receptors is present or that PBCM shows greater specificity for muscarinic m2 receptors. Because the muscarinic m2 receptor is a 66 kDa protein, whereas the muscarinic m3 receptor is a 92 kDa protein, these receptors can be separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis after being labeled with [3H]PBCM. PBCM appears to specifically bind the 66 kDa m2 receptor at concentrations lower than those required to bind to the 92 kDa m3 receptor. A linear correlation was found between the increased binding of [3H]PBCM to each receptor and the proportional loss of that receptor-mediated response. Thus, a reserve population of either muscarinic m2 or m3 receptors does not appear to exist in cerebellar granule cells. These studies also show that PBCM has greater affinity for the muscarinic m2 receptor than the muscarinic m3 receptor.


Cardiovascular responses to cocaine are initially mediated by the central nervous system in rats. Cocaine produces a pressor response reportedly resulting from both potentiation of peripheral catecholamine activity and a centrally mediated sympathoexcitation. In the present study we sought to differentiate the central nervous system and peripheral contributions to the hemodynamic effects of cocaine. In conscious rats, cocaine (5 mg/kg i.v.) produced a pressor response with two distinct components consisting of a brief, substantial increase in mean arterial pressure (MAP) associated with hindquarters and mesenteric vasoconstriction followed by a sustained, modest response associated with mesenteric vasoconstriction and bradycardia. Pentolinium (7.5 mg/kg i.v.) or adrenal demedullation attenuated the peak increase in MAP by attenuating increases in mesenteric and hindquarters vascular resistance, but did not affect the sustained increase in MAP. Methyl atropine (0.5 or 1 mg/kg i.v.) pretreatment reduced the cocaine-induced increase in systemic vascular resistance and enhanced the hindquarters vasodilation during the sustained MAP response. In contrast, adrenal demedullation abolished the hindquarters vasodilation. The bradycardic response was prevented by pentolinium and reduced by methyl atropine. Sympathetic nerve activity was reduced dramatically after cocaine or procaine administration for several minutes in conscious and in chloralose-anesthetized rats. In several anesthetized rats, the sympathoinhibition was preceded by a brief (3-8 sec) increase in renal sympathetic nerve activity. Procaine or cocaine produced little change in cortical cerebral blood flow as estimated by using a laser Doppler flowmeter. These data suggest that cocaine produces an initial, brief centrally mediated sympathoexcitation, but the sustained, modest pressor response is dependent upon peripheral actions that are diminished by baroreflex activation.


Polymorphism of dextromethorphan metabolism: relationships between phenotype, genotype and response to the administration of encainide in humans. The polymorphism of dextromethorphan and encainide metabolism is genetically determined and is related to the activity of hepatic CYP2D6. In order to examine the relations between CYP2D6 phenotype, genotype and the electrocardiographic response to the oral administration of encainide, 110 healthy subjects were studied. Metabolic ratios were calculated in urine after oral administration of 40 mg of dextromethorphan and in plasma obtained 2.5 h after oral administration of 50 mg of encainide. Encainide-induced electrocardiographic changes were measured 2.5 h after oral administration of the drug. Genotype was determined in 52 subjects. Results showed that phenotype, either extensive or poor metabolizer, for CYP2D6-dependent metabolism could be identified from the dextromethorphan metabolic ratio calculated in urine, from the encainide metabolic ratio calculated in plasma and from the genotype. However, despite the fact that the changes in atrioventricular (PR) and intraventricular (QRS) conduction times produced by encainide were different in extensive and poor metabolizer subjects and correlated with CYP2D6 activity, the electrocardiographic response was never 100% specific and sensitive for the identification of either phenotype. Moreover, genotypic identification of heterozygous and homozygous extensive metabolizer subjects did not predict CYP2D6 activity, as determined by dextromethorphan and encainide metabolic ratios, or encainide response, as determined by intraventricular and atrioventricular changes. Thus, CYP2D6 activity does not fully predict the electrocardiographic effects of encainide, and genotype, as determined in our study, cannot replace the determination of metabolic ratio in predicting CYP2D6 activity and encainide response in extensive metabolizer subjects.


Effect of pinacidil on the membrane electrical activity of guinea pig detrusor muscle. The effects of pinacidil on the guinea pig detrusor smooth muscle membrane were studied to investigate the electrophysiological mechanisms by which this drug relaxes smooth muscle tissue and, thus, might be of value in the treatment of detrusor instability. Pinacidil (> or = 3 x 10(-7) M) hyperpolarized the membrane in a concentration-dependent manner, with a reduction in spontaneous spike discharges. The membrane hyperpolarization induced by pinacidil was consistently associated with an increase in membrane ionic conductance. Glybenclamide (10(-6) M) completely inhibited the membrane hyperpolarization induced by pinacidil (up to 10(-5) M). Membrane hyperpolarization with pinacidil was consistently greater in a low-K+ solution, and it decreased in the presence of a high-K+ solution, compared with that measured in normal Krebs solution. Pinacidil consistently suppressed carbachol-induced depolarization of the membrane, with a reduction in the frequency of spontaneous action potentials. Glybenclamide (10(-6) M) did not inhibit the effect of pinacidil on increased action potential frequency induced by carbachol but blocked the membrane hyperpolarization induced by pinacidil (10(-5) M). In addition, the amplitude and maximum velocity of depolarization of carbachol- and current-induced action potentials were significantly decreased by pinacidil in the presence of glybenclamide. These results suggest that pinacidil blocks action potential generation in detrusor smooth muscle by inducing membrane hyperpolarization secondary to an increase in K+ permeability. Pinacidil, at high concentrations (> or = 10(-5) M), might block action potentials by inhibiting the voltage-sensitive Ca++ influx independently of hyperpolarization of the membrane. These mechanisms might be of benefit therapeutically in relaxation of unstable detrusor contractions.


Ontogeny of N-methyl-D-aspartate-induced current in cultured hippocampal neurons. The ontogeny of the N-methyl-D-aspartate (NMDA) subtype of glutamatergic receptor/ion channel was studied by examining whole cell currents evoked by NMDA in cultured hippocampal neurons 1 to 30 days after plating of cells from 18- to 20-day-gestation rat fetuses. We observed a maturation-dependent increase in conductance, compatible with an increased density of NMDA receptors, which is in agreement with previous binding data. The whole cell currents evoked by NMDA (10-100 microM) in the presence of glycine (1-100 microM) had two components that contributed to the peak amplitude. The first was a rapidly decaying current (fast component) and the second a slowly decaying current (slow component), their ratio depending upon glycine concentration. The EC50 values for glycine were 1.8 and 0.3 microM for the fast and slow components of the current, respectively. The quantitative analysis of these components indicated the existence of two distinct glycine sites, which differ in their affinity for glycine. The fast component originates from the action of glycine at the site with lower affinity. Moreover, the ratio of the fast to the slow component was also dependent on the time lapsed after plating of the fetal hippocampal neurons. The slow component became more predominant and the fast component less predominant along with cell maturation in culture, a phenomenon which reflects a change in the ratio of high- to low-affinity glycine binding sites. In addition, studies on Zn2+ gave further evidence of a change in the NMDA receptor/channel properties related to maturation of the cultured neurons.(ABSTRACT TRUNCATED AT 250 WORDS)


Developmental change of the inhibition by lead of NMDA-activated currents in cultured hippocampal neurons. The inhibition of N-methyl-D-aspartate (NMDA)-activated current in cultured fetal rat hippocampal neurons by Pb2+ was investigated at various stages of cell development. Pb2+ selectively inhibited NMDA currents recorded from young cultured neurons. In the first week of culture, Pb2+ showed the most prominent inhibition, which was gradually attenuated in the following weeks. Pb2+'s action was selective for NMDA- as opposed to either kainate- or quisqualate-induced currents. The current-voltage relationship for NMDA-induced currents in the presence of Pb2+ revealed that the effect of this cation was voltage-independent, which suggested that the site of interaction of Pb2+ with the NMDA receptor/channel is located outside the membrane electric field. Single channel studies showed that Pb2+ reduced the frequency but not the lifetime of the NMDA-activated single channel currents. Further evaluation of the mechanism of action of Pb2+ on the NMDA receptor demonstrated that this cation is a noncompetitive antagonist of both NMDA and glycine. We have demonstrated that the NMDA-induced whole cell currents change along with cell development, and the effects of Pb2+ are also dependent upon age of culture. The NMDA-induced currents in cultured rat hippocampal neurons had two components, one that decayed rapidly and another that decayed slowly. The fast component was clearly observed at concentrations of glycine higher than 1 microM, whereas the slow component reached its maximum amplitude at the glycine concentration of 1 microM. Moreover, the rapidly decaying component of NMDA-evoked whole cell currents was predominant in young cultured neurons, and its contribution to the total current was reduced in old cultured neurons.(ABSTRACT TRUNCATED AT 250 WORDS)


Biotin delivery to brain with a covalent conjugate of avidin and a monoclonal antibody to the transferrin receptor. The OX26 mouse monoclonal antibody to the rat transferrin receptor undergoes transcytosis through the brain capillary endothelial wall, which makes up the blood-brain barrier (BBB) in vivo, owing to high concentrations of transferrin receptor on the BBB. This property allows the OX26 antibody to serve as a brain drug transport vector. To simplify coupling of therapeutics to the OX26 antibody, the present studies examine the use of the avidin/biotin system to promote coupling of biotin and biotinylated drugs to brain transport vectors. The OX26 antibody was affinity purified from ascites fluid and was covalently coupled via a thioether linkage to avidin, and the conjugate was purified to homogeneity by gel filtration fast protein liquid chromatography. The biotin binding capacity of the avidin-OX26 conjugate was measured, and 2.3 biotin binding sites per avidin-OX26 (1:1) conjugate were detected. Transcytosis through the BBB of [3H]biotin bound to either unconjugated avidin or to the avidin-OX26 conjugate was measured with an internal carotid artery perfusion/capillary depletion technique. [3H]Biotin bound to the avidin-OX26 conjugate was transported through the BBB at rates that equaled the rate of transcytosis of the unconjugated OX26 antibody. The clearance from plasma of [3H]biotin bound to the avidin-OX26 conjugate approximated the rate of clearance of the unconjugated OX26 antibody, and not the rate of clearance of [3H]biotin bound to avidin, which was cleared from plasma at much faster rates.(ABSTRACT TRUNCATED AT 250 WORDS)


GTP-binding protein involvement in membrane currents evoked by carbachol and histamine in guinea-pig ileal muscle. 1. Single smooth muscle cells obtained by enzymic dispersion of the longitudinal muscle layer of guinea-pig ileum were used for recording membrane currents under whole-cell voltage clamp in response to carbachol (100 microM, unless otherwise stated) or histamine (100 microM) applied extracellularly. 2. At a holding potential of 0 mV, a transient outward current was evoked by carbachol and histamine. Responses to the two agonists were very similar in size and time course to the current response to caffeine (10 mM). The response to carbachol was virtually absent in the presence of histamine, and vice versa. Caffeine was without effect in the presence of either of these agonists. Inclusion of EGTA (10 or 20 mM) in the pipette abolished the responses to carbachol, histamine and caffeine. Thus, the outward current responses were considered to represent opening of Ca(2+)-activated K+ channels in response to a massive release of Ca2+ from the same stores by these three agents. 3. An inward current was evoked by carbachol and histamine, but not by caffeine at a holding potential of -40 mV, which was considered to represent opening of cationic channels. The carbachol-induced inward current was much longer in duration and larger in size than the histamine-induced inward current. 4. Inclusion of GDP beta S (2 mM) in the pipette abolished the inward and outward current responses to histamine, but inhibited only part of those to carbachol. 5. When the holding potential was held at 0 mV with inclusion of GTP gamma S (0.1-1 mM) in the pipette, spontaneous transient outward currents appeared immediately after break-through but disappeared a few minutes later. Under these conditions, caffeine (10 mM) was almost without effect, suggesting that GTP gamma S had released Ca2+ stores. When the holding potential was held at -40 mV and GTP gamma S (0.1 or 0.2 mM) was present in the pipette, an inward current developed a few minutes after break-through. During the GTP gamma S-induced inward current, application of carbachol or histamine produced no further inward current. However, when 0.01 mM-GTP gamma S was included in the pipette solution, carbachol- and histamine-induced inward currents were potentiated. 6. Pretreated with 2-5 micrograms/ml pertussis toxin (PTX) did not change noticeably the outward current responses to carbachol and histamine, but abolished or markedly reduced the inward current responses.(ABSTRACT TRUNCATED AT 400 WORDS)


Mitochondrial function in type I cells isolated from rabbit arterial chemoreceptors. 1. In this, and the accompanying paper (Duchen & Biscoe, 1992), we test the hypothesis that the oxygen sensitivity of mitochondrial electron transport forms a basis for transduction in the carotid body, the primary peripheral arterial oxygen sensor. We here describe for isolated type I cells the changes in autofluorescence of mitochondrial NAD(P)H that accompany changes in PO2. 2. NAD(P)H autofluorescence (excitation, 340-360 nm; emission peak, 450 nm) increased with anoxia, reflecting a rise in the NAD(P)H/NAD(P) ratio. Graded increases in autofluorescence were seen in response to graded decreases in PO2, suggesting that mitochondrial function is progressively altered below a PO2 of about 60 mmHg. 3. A mitochondrial origin for the NAD(P)H autofluorescence was suggested by the mutual exclusion of the responses to anoxia and cyanide. 4. Oxidized flavoproteins fluoresce when excited at 450 nm with an emission peak at 550 nm. The small signals obtained under these conditions increased with uncoupler and showed a graded decrease with falling PO2 reflecting a rise in the FADH/FAD ratio. 5. Hypoxia raises [Ca2+]i. The hypoxia-induced changes in mitochondrial function were not secondary to this rise. A brief K(+)-induced depolarization leads to a transient increase in [Ca2+]i. At the same time there is a rapid decrease in NAD(P)H autofluorescence followed by an increase that far outlasts the rise in [Ca2+]i. This delayed increase in autofluorescence was smaller than was the increase with anoxia, even though K(+)-induced depolarization raised [Ca2+]i more than does anoxia. In Ca(2+)-free solutions the depolarization-induced changes were abolished, while those associated with hypoxia were maintained. 6. The changes of autofluorescence with K(+)-induced depolarization appear to reflect (i) oxidation of NAD(P)H by stimulation of respiration following mitochondrial Ca2+ uptake and (ii) reduction of NAD(P) by the Ca(2+)-dependent activation of mitochondrial dehydrogenases. This activation could last several minutes following only 100 ms depolarization, while the changes accompanying hypoxia closely followed the time course of the change in PO2. 7. In similarly isolated rat or mouse chromaffin cells and mouse dorsal root ganglion neurons under identical conditions, no measurable change in autofluorescence or in [Ca2+]i was seen until the PO2 fell below about 5 mmHg. 8. Carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP) increases O2 consumption, oxidizing mitochondrial NADH and hence decreasing autofluorescence, (delta FFCCP). Blockade of electron transport by anoxia or CN- decreases O2 consumption, increasing mitochondrial NADH/NAD and autofluorescence (delta FCN). The fractional change in autofluorescence with FCCP, delta FFCCP/delta FFCCP+FCN), is thus a measure of resting O2 consumption.(ABSTRACT TRUNCATED AT 400 WORDS)


All-or-nothing Ca2+ mobilization from the intracellular stores of single histamine-stimulated HeLa cells. 1. Histamine-stimulated mobilization of intracellular Ca2+ stores was monitored in intact and permeabilized populations of HeLa cells using both the fluorescent Ca2+ indicator Fura-2 and 45Ca2+ measurements. Digital video imaging of Fura-2-loaded cells was used to measure the intracellular calcium concentration ([Ca2+]i) of single cells. 2. In populations of HeLa cells, histamine caused a concentration-dependent increase in cytoplasmic [Ca2+]. The initial transient increase was independent of extracellular Ca2+ (Ca2+o) and was followed by a sustained increase that was abolished by removal of Ca2+o. 3. In Ca(2+)-free medium ([Ca2+]o < 1 microM), a maximal histamine concentration (25 microM) caused a transient increase in [Ca2+]i, and a subsequent challenge with histamine failed to evoke a further response indicating that the inositol 1,4,5-trisphosphate (InsP3)-sensitive Ca2+ stores had been completely emptied. Lower concentrations of histamine (0.5-10 microM) caused smaller, concentration-dependent increases in [Ca2+]i that were also transient. After exposure to these low histamine concentrations, where [Ca2+]i returned to baseline within 2 min, addition of a higher histamine concentration evoked a further increase in [Ca2+]i. The second increase in [Ca2+]i was inversely proportional to the increase caused by the first exposure to histamine, indicating that Ca2+ released in the initial response was not substantially resequestered into histamine-sensitive stores. 4. Single HeLa cells challenged with low concentrations of histamine in Ca(2+)-free medium responded with transient increases in [Ca2+]i, but individual cells differed in their sensitivity with 51% of cells responding to 1 microM, and 98% responding to 25 microM-histamine. 5. When single cells in Ca(2+)-free medium were challenged with stepwise increases in histamine concentration, they responded to each step with a transient [Ca2+]i increase after which [Ca2+]i returned to baseline within 1 min. Prolonging the interval between histamine additions by up to 25 min did not affect the [Ca2+]i increase evoked by a subsequent histamine addition. 6. Unidirectional 45Ca2+ efflux from saponin-permeabilized HeLa cells showed that, under conditions that prevented Ca2+ resequestration, submaximal concentrations of InsP3 rapidly emptied only a fraction of the InsP3-sensitive Ca2+ stores. The failure of low InsP3 concentrations to fully mobilize the InsP3-sensitive Ca2+ stores was not a consequence of InsP3 degradation. 7. We conclude that within single HeLa cells, intracellular Ca2+ stores are heterogeneous in their sensitivity to InsP3, and the fraction of Ca2+ stores mobilized by InsP3 increases as the InsP3 concentration increases.


A caffeine- and ryanodine-sensitive Ca2+ store in bullfrog sympathetic neurones modulates effects of Ca2+ entry on [Ca2+]i. 1. We studied how in changes in cytosolic free Ca2+ concentration ([Ca2+]i) produced by voltage-dependent Ca2+ entry are influenced by a caffeine-sensitive Ca2+ store in bullfrog sympathetic neurones. Ca2+ influx was elicited by K+ depolarization and the store was manipulated with either caffeine or ryanodine. 2. For a time after discharging the store with caffeine and switching to a caffeine-free medium: (a) [Ca2+]i was depressed by up to 40-50 nM below the resting level, (b) caffeine responsiveness was diminished, and (c) brief K+ applications elicited [Ca2+]i responses with slower onset and faster recovery than controls. These effects were more pronounced as the conditioning caffeine concentration was increased over the range 1-30 mM. 3. [Ca2+]i, caffeine and K+ responsiveness recovered in parallel with a half-time of approximately 2 min. Recovery required external Ca2+ and was speeded by increasing the availability of cytosolic Ca2+, suggesting that it reflected replenishment of the store at the expense of cytosolic Ca2+. 4. During recovery, Ca2+ entry stimulated by depolarization had the least effect on [Ca2+]i when the store was filling most rapidly. This suggests that the effect of Ca2+ entry on [Ca2+]i is modified, at least in part, because some of the Ca2+ which enters the cytosol during stimulation is taken up by the store as it refills. 5. Further experiments were carried out to investigate whether the store can also release Ca2+ in response to stimulated Ca2+ entry. In the continued presence of caffeine at a low concentration (1 mM), high K+ elicited a faster and larger [Ca2+]i response compared to controls; at higher concentrations of caffeine (10 and 30 mM) responses were depressed. 6. Ryanodine (1 microM) reduced the rate at which [Ca2+]i increased with Ca2+ entry, but not to the degree observed after discharging the store. At this concentration, ryanodine completely blocked responses to caffeine but had no detectable effect on Ca2+ channel current or the steady [Ca2+]i level achieved during depolarization. 7. We propose that, depending on its Ca2+ content, the caffeine-sensitive store can either attenuate or potentiate responses to depolarization. When depleted and in the process of refilling, the store reduces the impact of Ca2+ entry as some of the Ca2+ entering the cytosol during stimulation is captured by the store.(ABSTRACT TRUNCATED AT 400 WORDS)


Calcium requirements for secretion in bovine chromaffin cells. 1. Measurements of membrane capacitance and intracellular Ca2+ concentration, [Ca2+]i, were used to examine the Ca2+ dependence of secretion in single adrenal chromaffin cells. 2. Intracellular dialysis of Ca2+, through a patch pipette, promoted secretion; the rate of secretion increased monotonically as [Ca2+]i was elevated, while the total amount of secretion reached a maximum at 1.5 microM-Ca2+ and declined at high [Ca2+]i. 3. Release of Ca2+ from internal stores, using bradykinin or ionomycin, transiently elevated [Ca2+]i and the rate of secretion. 4. Considering responses to both Ca2+ dialysis and release from internal stores, it appears that the rate of secretion increases over a range of [Ca2+]i levels above 0.2 microM and saturates at concentrations greater than 10 microM, if at all. Secretion appears to have a Hill coefficient for Ca2+ of about 2. At [Ca2+]i greater than 1-2 microM, prolonged elevation of [Ca2+]i, via dialysis, produced lower rates of secretion than transient elevation of [Ca2+]i caused by release from internal stores. This may have been caused by a depletion of readily releasable chromaffin granules during prolonged elevation of [Ca2+]i. 5. Brief depolarizing pulses produced transient rises in both [Ca2+]i and the rate of secretion. The ability of these pulses to evoke secretion 'washed out' during prolonged intracellular dialysis, due to both reduced Ca2+ influx and a diminished ability of the cell to secrete in response to a given Ca2+ load. 6. The kinetics of the secretory response depended upon the size of the depolarization-induced Ca2+ load; small rises in [Ca2+]i increased membrane capacitance only during the depolarization, while larger rises in [Ca2+]i produced increases both during and following the depolarization. The secretory responses that outlasted the depolarization appeared to be due to persistent elevation of [Ca2+]i. Secretory responses were sometimes followed by a slower decline in membrane capacitance, probably due to endocytosis of membrane. 7. Comparison of the rates of secretion measured during depolarization to those produced by Ca2+ dialysis or release from internal stores suggests that [Ca2+]i at secretory sites can exceed 10 microM during depolarization. The spatially averaged measurements of [Ca2+]i indicate much smaller levels of [Ca2+]i; thus, there must be pronounced spatial gradients of [Ca2+]i during depolarization.


Synaptic interaction between medullary respiratory neurones during apneusis induced by NMDA-receptor blockade in cat. 1. Termination of inspiration is an essential component of respiratory rhythm generation and its perturbation can result in apneusis, i.e. significant prolongation of mechanisms, we studied the postsynaptic events in respiratory neurones during apneustic respiratory periods, and compared them to normal respiratory cycles. 2. Experiments were performed in pentobarbitone-anaesthetized, paralysed, thoracotomized cats ventilated with a constant volume or a cycle-triggered constant pressure pump. Apneusis, separated by normal cycles, was induced as follows: the animal was ventilated by a cycle-triggered pump that normally inflated the lungs during the inspiratory burst of phrenic nerve discharge. The NMDA-receptor blocker MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5, 10-iminemaleate] (0.3-0.7 mg/kg) was administered intravenously, and, for designated breaths, inflation of the lungs was withheld during neural inspiration. 3. Membrane potential trajectories of forty-one late expiratory (E-2) and eight postinspiratory (PI) neurones of the caudal ventral respiratory group were analysed before and/or after MK-801 administration, during normal and apneustic periods. 4. Before MK-801 administration, withholding lung inflation caused modest (10-20%) lengthening of the inspiratory period; after MK-801 administration, withholding inflation caused apneusis. Provided that the lungs were inflated during the inspiratory phase, the temporal pattern of phrenic nerve, recurrent laryngeal nerve and membrane potential trajectories of E-2 and PI neurones were not significantly altered by MK-801. Apneusis following NMDA-receptor blockade produced consistent changes in the synaptic activation patterns of E-2 neurones. In particular, the slow late inspiratory-related depolarization pattern of E-2 neurones was consistently retarded during apneustic inspiratory phases when compared to normal inspiratory phases. This was due to continuation of Cl(-)-mediated synaptic inhibition of E-2 neurones. Superior laryngeal nerve stimulation stopped apneusis and sustained membrane hyperpolarization of E-2 neurones similar to lung inflation. 5. During the plateau phase of apneusis, correlated 10-20 Hz oscillations could be observed in the integrated phrenic and recurrent laryngeal nerve activities as well as in the membrane potential of E-2 neurones. 6. We conclude that: (i) the prolonged inhibition of E-2 neurones during apneusis is indicative of the process responsible for the prolongation of the inspiratory phase.(ABSTRACT TRUNCATED AT 400 WORDS)


Pulsatile release of acetylcholine by nerve terminals (synaptosomes) isolated from Torpedo electric organ. 1. Electrophysiological detection of acetylcholine (ACh) release by synaptosomes from the electric organ of Torpedo was searched for by laying the isolated nerve terminals on a culture of Xenopus embryonic muscle cells (myocytes), and by recording the ACh-induced inward currents in the myocytes. 2. Whole-cell recording in one of the myocytes revealed rapid inward currents that where generated soon after synaptosome application. These pulsatile events strongly resembled those occurring normally during the early phase of synaptogenesis after nerve-muscle contact in Xenopus cell cultures. They were called spontaneous synaptic currents (SSCs). 3. The SSCs produced by the synaptosomes had a rapid time course, with mean time-to-peak and half-decay times of 2.6 +/- 0.4 ms and 6.0 +/- 1.1 ms, respectively. Most events had a falling phase that could be fitted with a single exponential. The mean time constant of decay was 6.2 +/- 1.1 ms. More than half of the SSCs (approximately 60%) constituted a rather homogenous population in which the time-to-peak versus amplitude showed a positive relationship, the smallest events displaying a shorter time course. The rest of the SSCs had a more variable and slower time course. Such events are also observed in young and mature junctions in situ. 4. The amplitudes of SSCs had a wide distribution which was skewed towards the smallest values. The mean amplitude was 65.2 +/- 16.1 pA. 5. During the minutes following an application of synaptosomes, the frequency of the SSCs tended to decrease, but their mean amplitude remained constant. Such behaviour could be reproduced during several successive additions of synaptosomes while recording in the same myocyte. 6. Just after synaptosome application, the SSCs were superposed to a noisy inward current that lasted for 20-60 s. Noise analysis of this current gave the values of 0.7 +/- 0.1 pA for the mean amplitude of the elementary event, and 4.7 +/- 0.2 ms for its mean duration, values that compare well with those reported for the activation of frog embryonic nicotinic receptor. This suggests that the noisy current was due to ACh molecules set free by synaptosomes which were either damaged or which released ACh at some distance. This view was strengthened by biochemical analysis of ACh release by synaptosomes in vitro. 7. Tubocurarine reversibly abolished the appearance of both the noise and the synaptosome-generated SSCs, showing that these currents were due to the action of ACh.(ABSTRACT TRUNCATED AT 400 WORDS)


Relative mitochondrial membrane potential and [Ca2+]i in type I cells isolated from the rabbit carotid body. 1. In the accompanying paper (Duchen & Biscoe, 1992) we have described graded changes in autofluorescence derived from mitochondrial NAD(P)H in type I cells of the carotid body in response to changes of PO2 over a physiologically significant range. These observations suggest that mitochondrial function in these cells is unusually sensitive to oxygen and could play a role in oxygen sensing. We have now explored further the relationships between hypoxia, mitochondrial membrane potential (delta psi m) and [Ca2+]i. 2. The fluorescence of Rhodamine 123 (Rh 123) accumulated within mitochondria is quenched by delta psi m. Mitochondrial depolarization thus increases the fluorescence signal. Blockade of electron transport (CN-, anoxia, rotenone) and uncoupling agents (e.g. carbonyl cyanide p-trifluoromethoxy-phenylhydrazone; FCCP) increased fluorescence by up to 80-120%, while fluorescence was reduced by blockade of the F0 proton channel of the mitochondrial ATP synthase complex (oligomycin). 3. delta psi m depolarized rapidly with anoxia, and was usually completely dissipated within 1-2 min. The depolarization of delta psi m with anoxia (or CN-) and repolarization on reoxygenation both followed a time course well characterized as the sum of two exponential processes. Oligomycin (0.2-2 micrograms/ml) hyperpolarized delta psi m and abolished the slower components of both the depolarization with anoxia and of the subsequent repolarization. These data (i) illustrate the role of the F1-F0 ATP synthetase in slowing the rate of dissipation of delta psi m on cessation of electron transport, (ii) confirm blockade of the ATP synthetase by oligomycin at these concentrations, and (iii) indicate significant accumulation of intramitochondrial ADP during 1-2 min of anoxia. 4. Depolarization of delta psi m was graded with graded changes in PO2 below about 60 mmHg. The stimulus-response curves thus constructed strongly resemble those for [Ca2+]i and NAD(P)H with PO2. The change in delta psi m closely followed changes in PO2 with time. 5. The rate of rise of [Ca2+]i in response to anoxia is strongly temperature sensitive. The rate of depolarization of delta psi m with anoxia similarly increased at least two- to fivefold on warming from 22 to 36 degrees C. The change with FCCP was not significantly altered by temperature. 6. These data show that the mitochondrial membrane potential changes over a physiological range of PO2 values in type I cells. This contrasts with the behaviour in dissociated chromaffin cells and sensory neurons, in which no change was measurable until the PO2 fell close to zero.(ABSTRACT TRUNCATED AT 400 WORDS)


Alveolar epithelial permeability in baboons: histamine and capsaicin. 1. We investigated if changes in alveolar epithelial permeability could be initiated by various doses of the irritant capsaicin through stimulation of pulmonary afferent nerves either directly, or indirectly through the release of an intermediate cell mediator, and whether mediation of permeability involved histamine H1 and/or H2 receptors. 2. Alveolar epithelial permeability was indicated by the clearance of peripherally deposited 99mTc-DTPA (technetium-99m-diethylenetriamine pentaacetate) aerosol in baboons. Aerosol challenge experiments were performed twice with histamine (32 mg/ml), once each with capsaicin (10(-6), 10(-5), 10(-4) and 10(-3) M), and once each with histamine (32 mg/ml) preceded by H1 (terfenadine) and H2 (ranitidine) antagonists alone and combined. 3. Mean half-time for 99mTc-DTPA clearance was 66 +/- 4 min. After histamine, it decreased to 41 +/- 5 min (P < 0.05), and after capsaicin it was 70 +/- 5 min (combined doses). Pretreatment with the H1 and H2 antagonists, either separately or in combination, did not consistently inhibit increases in 99mTc-DTPA permeability. 4. Capsaicin caused tachypnoea only at 10(-3) M (P < 0.05). Inhibition of histamine-induced tachypnoea required both antagonists (P < 0.05) suggesting the existence of a population of peripheral sensory neurons possessing H1 and H2 receptors. Changes in permeability were unrelated to the changes in respiratory frequency. Thus, neither the mechanisms that stimulate respiratory frequency, nor the respiratory frequency per se, were responsible for increased alveolar epithelial permeability. 5. Capsaicin did not change epithelial permeability at doses that cause capsaicin-sensitive C fibre stimulation as delineated by increases in respiratory frequency. Nor does C fibre stimulation, induced by mediators released from pulmonary cells activated by low doses of capsaicin, increase epithelial permeability. These data suggest that in the primate, histamine increases epithelial permeability to small solutes by a mechanism independent of the activation of capsaicin-sensitive C fibres and capsaicin-initiated release of cell mediators. 6. These findings do not support a role for C fibre activation in regulating epithelial permeability of small solutes.


Regulation by second messengers of the slowly activating, voltage-dependent potassium current expressed in Xenopus oocytes. 1. Voltage-clamp recordings of membrane current were made from Xenopus oocytes that had been injected with RNA which had been transcribed in vitro from a cloned complementary DNA. 2. Depolarization from -80 mV evoked outward potassium currents that developed very slowly. At -20 mV the time constant for activation was about 50 s, and at +40 mV about 6 s. 3. The potassium current was increased by the calcium ionophore A23187 or by intracellular injection of inositol 1,4,5-trisphosphate (IP3), each of which should increase the intracellular calcium concentration ([Ca2+]i). The current was decreased by injection of BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid). The current was also reduced by phorbol esters; this effect was blocked by staurosporine. 4. In oocytes that had also been injected with RNA encoding the 5-hydroxytryptamine (5-HT2) receptor, 5-HT increased the potassium current. After caffeine pretreatment, to block the release of intracellular calcium, 5-HT decreased the current; this decrease was prevented by staurosporine. 5. It is concluded that the slowly activating, voltage-dependent potassium current expressed in Xenopus oocytes is increased by increases in [Ca2+]i and is decreased by activation of protein kinase C. Stimulation of 5-HT2 receptors can have both these effects, but the former normally predominates.


Co-ordination of pumping in isolated bovine lymphatic vessels. 1. Segments of bovine mesenteric lymphatic of varying diameter taken from different parts of the lymphatic tree were cut to 20 mm in length and set up so that measurements could be made of spontaneous isometric contractions. 2. There was considerable variability in frequency of spontaneous contractions but this was independent of resting tension. There was no significant correlation between lymphatic diameter and inherent frequency of contraction. 3. Isolated segments of bovine mesenteric lymphatic 70-80 mm in length were cannulated and set up in a three-compartment organ bath which allowed independent temperature control in each compartment. Pressure was recorded at inflow and outflow ends and experiments were video recorded. 4. Contractile activity was normally initiated at the end of the lymphatic maintained at the higher temperature and the contractile wave was propagated along the length of the vessel. 5. Propagation could occur either in the direction of valve orientation (orthograde) or retrogradely. The volume of fluid pumped was not significantly affected by the direction of propagation. 6. Perfusion of the central compartment with Krebs solution at 0-2 degrees C disrupted normal propagation and allowed the two parts of the lymphatic to contract at different frequencies although the two parts maintained an approximately 2:1 ratio. 7. Perfusion of the central compartment with 10 mM-heptanol also disrupted normal propagation but the rates on either side of the partition bore no harmonic relationship to one another. 8. These results suggest that relatively short segments of lymph duct have the ability to contract spontaneously and that their inherent frequencies are not determined by their position in the lymphatic tree. The results are consistent with the existence of electrical coupling along the lymphatic's length and they suggest that over distances of at least 80 mm independent pacemakers are capable of mutual entrainment at a frequency representing a compromise between the fastest and slowest components.


Basal Ca2+ and the oscillation of Ca2+ in caffeine-treated bullfrog sympathetic neurones. 1. Effects of caffeine on the intracellular free Ca2+ concentration ([Ca2+]i) in single bullfrog sympathetic neurones in excised tissue were studied by recording Fura-2 fluorescence excited at 340, 361 or 380 nm and taking their ratios (R340/380 or R361/380). 2. Caffeine (3-10 mM) produced oscillation of [Ca2+]i and an 'apparent' decrease in the basal level of [Ca2+]i during a period between phasic rises. The mechanism of the latter effect was analysed in relation to the mechanism of the former. 3. Caffeine (3-10 mM) increased Fura-2 fluorescence in a range of excitation wavelength from 330 to 390 nm. The ratios of fluorescences, R340/380 and R361/380, however, were not significantly affected by caffeine. These results suggest that the 'apparent' reduction in the basal [Ca2+]i seen as a decrease in R340/380 or R361/380 results from a true decrease in [Ca2+]i. 4. Caffeine-induced decrease in [Ca2+]i persisted for every period between phasic rises of [Ca2+]i during [Ca2+]i oscillation, and after the blockade of [Ca2+]i oscillation by ryanodine. The decrease in the latter condition lasted for more than 20 min. 5. The decrease in the basal [Ca2+]i depended on the external Ca2+ concentration and was not mimicked by the action of cyclic nucleotides. 6. Possible mechanisms underlying the decrease in the basal [Ca2+]i produced by caffeine (effects on Ca2+ transport at the cell or Ca(2+)-storing organelle membrane) and their significance in relation to the [Ca2+]i oscillation were discussed.


Independent control of human finger-tip forces at individual digits during precision lifting. 1. Subjects lifted an object with two parallel vertical grip surfaces and a low centre of gravity using the precision grip between the tips of the thumb and index finger. The friction between the object and the digits was varied independently at each digit by changing the contact surfaces between lifts. 2. With equal frictional conditions at the two grip surfaces, the finger-tip forces were about equal at the two digits, i.e. similar vertical lifting forces and grip forces were used. With different frictions, the digit touching the most slippery surface exerted less vertical lifting force than the digit in contact with the rougher surface. Thus, the safety margins against slips were similar at the two digits whether they made contact with surfaces of similar or different friction. 3. During digital nerve block, large and variable safety margins were employed, i.e. the finger-tip forces did not reflect the surface conditions. Slips occurred more frequently than under normal conditions (14% of all trials with nerve block, <5% during normal conditions), and they only occasionally elicited compensatory adjustments of the finger-tip forces and then at prolonged latencies. 4. The partitioning of the vertical lifting force between the digits was thus dependent on digital afferent inputs and resulted from active automatic regulation and not just from the mechanics of the task. 5. The safety margin employed at a particular digit was mainly determined by the frictional conditions encountered by the digit, and to a lesser degree by the surface condition at the same digit in the previous lift (anticipatory control), but was barely influenced by the surface condition at the other digit. 6. It was concluded that the finger-tip forces were independently controlled for each digit according to a 'non-slip strategy'. The findings suggest that the force distribution among the digits represents a digit-specific lower-level neural control establishing a stable grasp. This control relies on digit-specific afferent inputs and somatosensory memory information. It is apparently subordinated to a higher-level control that is related to the total vertical lifting and normal forces required by the lifting task and the relevant physical properties of the manipulated object.


A study of glycogen depletion and the fibre-type composition of cat skeleto-fusimotor units. 1. We have used the glycogen-depletion technique, combined with myofibrillar ATPase (mATPase) staining for muscle fibre type, to study the fibre-type composition of four skeleto-fusimotor (beta) units in cat peroneus tertius, namely, one beta dynamic (beta d) unit and three beta static (beta s) units. 2. Depletion of glycogen was observed in serial cross-sections of thirty-four beta-unit extrafusal muscle fibres of various types traced from origin to insertion. No fibre was depleted of glycogen throughout its length; depletion was restricted to a number of zones, usually about five. Oxidative (type I) and oxidative-glycolytic (type IIA) fibres were depleted for a significantly greater proportion of their total length than glycolytic IIB fibres. 3. The fibre-type composition of the beta d unit was determined by tracing its fibres from end to end. The muscle unit consisted of one intrafusal bag1 fibre and ninety-three extrafusal muscle fibres comprising seventy-six type I fibres, eleven IIC fibres, and six fibres that changed from IIC to I during the course of their length (IIC/I fibres). The extrafusal fibre-type composition was thus 81.7% I plus 18.3% IIC and IIC/I. 4. The three beta s units (beta s1, beta s2, beta s3) were all fast-contracting and fatigued rapidly. Identification of their extrafusal fibre types, made in 1 mm2 areas sampled from different parts of each unit, gave mixed compositions as follows: beta s1, IIB + 6.7% IIA; beta s2, IIB + 5.8% IIA; beta s3, IIB + 29.9% IIA. The intrafusal component of each unit included either one or two long chain fibres. 5. In a discussion of the results, the fact that the continuous stimulation of extrafusal muscle fibres does not deplete them of glycogen throughout their length is examined in relation to the work of others who have assumed that it did. With regard to the finding of mixed extrafusal fibre types in the beta units, a distinction is drawn between minimal (around 5%) and moderate mixing. It is suggested that minimal mixing may occur in any motor unit as the outcome of endplate degeneration with foreign replacement, but that moderate mixing indicates an on-going process of conversion from one fibre type to another which in the adult may prove to occur only among beta units.


Single C nociceptor responses and psychophysical parameters of evoked pain: effect of rate of rise of heat stimuli in humans. 1. Effects of rate of rise of temperature stimuli applied to skin on (i) unitary receptor threshold and frequency response often single C nociceptors, and (ii) on magnitude and reaction times of evoked pain were studied in fifteen healthy human volunteers. 2. Temperature ramps of 32 to 45 or 47 degrees C were applied at three consistent rates of rise to receptive fields of C nociceptors in dorsum of foot (n = 9) or hand (n = 1). For rates of rise of 0.3, 2.0 and 6.0 degrees C/s, mean receptor threshold for heat was remarkably uniform: 41.5 +/- 0.57, 41.5 +/- 0.61 and 41.9 +/- 0.71 degrees C respectively. 3. The mean discharge rate of the ten cutaneous C nociceptors increased with rate of rise of temperature stimuli: 1.22 +/- 0.13, 4.57 +/- 0.49 and 13.45 +/- 0.71 impulses/s, respectively, for stimulus temperature rates of 0.3, 2.0 and 6.0 degrees C/s. 4. Magnitude estimates of pain for thirteen subjects also increased with rate of rise of temperature stimuli. Mean normalized magnitude estimates of heat pain were: 11.8 +/- 1.55, 15.1 +/- 0.84 and 28.0 +/- 1.87 for stimulus rates of rise of 0.3, 2.0 and 6.0 degrees C/s, respectively. 5. Results of simultaneous recordings of reaction time for pain and of C nociceptor responses to heat ramps given at 2.0 degrees C/s, in three subjects, indicate that under those circumstances heat pain messages are exclusively mediated by C nociceptors.


The minimal inhibitory synaptic currents evoked in neonatal rat motoneurones. 1. Tight-seal whole-cell recordings were made from lumbar motoneurones visually identified in thin slices of neonatal rat spinal cord. The inhibitory postsynaptic currents (IPSCs) were evoked by extracellular stimulation of a neighbouring internuncial neurone in the presence of glutamate receptor antagonists. 2. Glycinergic IPSCs were recorded in the presence of bicuculline. The IPSCs appeared in an all-or-none manner as the graded stimulus intensity exceeded a certain threshold. Their latencies showed a unimodal distribution with a mean of 0.81 ms at 37 degrees C. Thus, the observed IPSCs are suggested to be monosynaptically evoked unitary IPSCs. The mean conductance of unitary IPSCs was 2.9 +/- 1.2 nS (+/- S.D.). 3. When the external Ca2+ concentration ([Ca2+]o) was reduced, the number of failures in response to stimulation increased, thereby reducing the mean amplitude of IPSCs. The mean amplitude of IPSCs was linearly related to the [Ca2+]o (0.35-1.4 mM) with a mean slope of 3.1 +/- 0.67 on double logarithmic co-ordinates. 4. The amplitude of individual IPSCs decreased with decrease in [Ca2+]o. However, below 0.7 mM [Ca2+]o, the mean amplitude of IPSCs (excluding failures) reached a steady minimum level. The mean conductance of these IPSCs measured in 0.5 mM [Ca2+]o was 657 +/- 281 pS. 5. The minimal IPSCs had a coefficient of variation of 0.50 +/- 0.13. No clear correlation was observed between the rise time and the amplitude of minimal IPSCs evoked in individual motoneurones, indicating that the amplitude variability is not due to the different synaptic locations. 6. Spontaneous miniature IPSCs were recorded from motoneurones in the presence of tetrodotoxin. The miniature IPSCs had a mean conductance of 739 +/- 278 pS, being comparable to the minimal evoked IPSCs. 7. Under various internal and external Cl- concentration, the reversal potential of the IPSCs (EIPSC) approximately coincided with the Cl- equilibrium potential. A 730-fold change in the potassium concentration gradient across the membrane did not affect the EIPSC. The permeability ratio of K+ to Cl- (Pk/PCl) was less than 0.05. 8. It is concluded that the IPSCs are carried almost exclusively by Cl- and that the minimal evoked IPSCs represent the quantal response of the transmitter.


Serotonin regulates the phase of the rat suprachiasmatic circadian pacemaker in vitro only during the subjective day. 1. The suprachiasmatic nucleus (SCN) of the hypothalamus is the primary pacemaker for circadian rhythms in mammals. The 24 h pacemaker is endogenous to the SCN and persists for multiple cycles in the suprachiasmatic brain slice. 2. While serotonin is not endogenous to the SCN, a major midbrain hypothalamic afferent pathway is serotonergic. Within this tract the dorsal raphe nucleus sends direct projections to the ventrolateral portions of the SCN. We investigated a possible regulatory role for serotonin in the mammalian circadian system by examining its effect, when applied at projection sites, on the circadian rhythm of neuronal activity in rat SCN in vitro. 3. Eight-week-old male rats from our inbred colony, housed on a 12 h light: 12 h dark schedule, were used. Hypothalamic brain slices containing the paired SCN were prepared in the day and maintained in glucose and bicarbonate-supplemented balanced salt solution for up to 53 h. 4. A 10(-11) ml drop of 10(-6) M-serotonin (5-hydroxytryptamine (5-HT) creatinine sulphate complex) in medium was applied to the ventrolateral portion of one of the SCN for 5 min on the first day in vitro. The effect of the treatment at each of seven time points across the circadian cycle was examined. The rhythm of spontaneous neuronal activity was recorded extracellularly on the second and third days in vitro. Phase shifts were determined by comparing the time-of-peak of neuronal activity in serotonin- vs. media-treated slices. 5. Application of serotonin during the subjective day induced significant advances in the phase of the electrical activity rhythm (n = 11). The most sensitive time of treatment was CT 7 (circadian time 7 is 7 h after 'lights on' in the animal colony), when a 7.0 +/- 0.1 h phase advance was observed (n = 3). This phase advance was perpetuated on day 3 in vitro without decrement. Serotonin treatment during the subjective night had no effect on the timing of the electrical activity rhythm (n = 9). 6. The specificity of the serotonin-induced phase change was assessed by treating slices in the same manner with a microdrop of serotonergic agonists, 5-carboxamidotryptamine, that targets the 5-HT1 class of receptors, or 8-hydroxy-dipropylaminotetralin (8-OH DPAT), that acts on the 5-HT1A receptor subtype.(ABSTRACT TRUNCATED AT 400 WORDS)


Transient chloride binding as a contributory factor to corneal stromal swelling in the ox. 1. Investigations were made of the cation exchange capacity of fresh isolated ox corneal stroma (Q, units: mequiv fixed stromal charge/kg stromal fluid) at pH 7.4 over a variety of stomal hydrations (H, units: kg stromal fluid/kg dry tissue) both above and below the physiological hydration of 3.2, whilst the stromas were immersed in a variety of sodium chloride solutions (range 5-1000 mM). 2. At any particular salt concentration, the product QH (dry tissue exchange capacity, units: mequiv/kg dry tissue) appeared constant, over all the hydrations investigated. 3. Dry tissue exchange capacity (QH) varied, however, when the bathing salt concentration was altered. It varied between 55 mequiv/kg dry tissue (e.g. Q = 17 mequiv at H = 3.2) in 5 mM-NaCl to 240 mequiv/kg dry tissue (e.g. Q = 75 mequiv/l at H = 3.2) in 1000 mM-NaCl. 4. The variation of stromal exchange capacity in NaCl solutions of different concentrations was similar when detected by three independent procedures: stromal gel pressure measurements, intrastromal sodium ion distributions, and intrastromal electrical potentials. 5. Intrastromal chloride ion distributions were anomalous. Total chloride (measured by radio-isotopes) was consistently higher than that predicted by Donnan theory. 6. The data were consistent with Elliott's hypothesis that a fraction of intrastromal chloride ions bind to the corneal stromal matrix and in so doing contribute to the fixed negative charge of the stroma. 7. Our observations may be explained by a model of the cation exchange capacity of ox cornea which has two types of components. On is (at constant pH) invariant, and has a dry tissue exchange capacity of about 50 mequiv/kg dry tissue, and is probably generated by the sulphonic and carboxylic acid groups of the glycosaminoglycans. The other is explained by supposing it to consist of a chloride binding ligand which exhibits first order binding, is half occupied at ambient chloride concentrations of 300 mM, and has a total capacity of 240 mequiv/kg dry tissue. 8. Partial stromal extraction with 4 M-guanidine HCl indicated that the chloride binding ligand is not associated with the collagen molecules in the corneal stromal fibrils. 9. It is suggested that such a stromal chloride ion binding ligand would help to stabilize the hydration and transparency of the living cornea when it is exposed to environments of varying tonicity (such as in river or sea bathing).


Interface shear stresses during ambulation with a below-knee prosthetic limb. Shear stresses on a residual limb in a prosthetic socket are considered clinically to contribute to tissue breakdown in below-knee amputees. When applied simultaneously with normal stresses, they can cause injury within the skin or can generate an abrasion on the surface. To gain insight into shear stresses and parameters that affect them, interface stresses were recorded on below-knee amputee subjects during walking trials. On the tibial flares, resultant shear ranged from 5.6 kPa to 39.0 kPa, while on the posterior surface it ranged from 5.0 kPa to 40.7 kPa. During stance phase, anterior resultant shears on a socket were directed toward the apex while posterior resultant shears were directed downward approximately perpendicular to the ground. Waveform shapes were usually double-peaked, with the first peak at 25% to 40% into stance phase and the second peak at 65% to 85% into stance. Application of these results to residual limb tissue mechanics and prosthetic design is discussed.


Comparative effects of posture on pressure and shear at the body-seat interface. This study considers the effects of seated posture and body orientation on the pressure-distribution and surface shear (tangential) forces acting at the body-seat interface. Nine postures typically assumed by wheel-chair users were studied. Comparisons were made within and between two study groups, made up of 12 subjects with spinal cord injuries (SCI) and 10 nondisabled subjects. Both interface pressure and the surface shear were measured simultaneously in each of nine reproducible, seated postures. The same seat cushion was used for all trials. The Oxford Pressure Monitor, a pneumatic cell device, was used to measure and record the interface pressures. Instrumentation for measuring and recording the surface shear force was constructed specifically for the study. Analysis consisted of statistically comparing changes in pressure values and shear forces derived from eight sitting postures with reference to values recorded in a defined neutral sitting posture. The pressure-distribution findings suggest that in the postures studied SCI subjects have maximum pressures that are higher than nondisabled subjects in all postures, ranging from 6% to 46% depending on the posture. Maximum pressures can be reduced by postural changes: forward flexion to 50 degrees, -9%; backrest recline to 120 degrees, -12%; and, full body tilt, -11%. On average, the SCI group members have peak pressure gradients (PPG) that are 1.5 to 2.5 greater than the nondisabled group. The maximum reduction in PPG occurred at backrest recline of 120 degrees, -18%. Tangential shear force acts at the body-seat interface in all nine postures studied. Extrapolation of results suggests that full-body tilt to approximately 25 degrees reduces the surface shear force to near zero. In contrast, a backrest-only recline of 20 degrees causes a 25% increase in the surface shear force. These results suggest that caution must be taken when using nondisabled subjects as surrogates for people with SCI because of the inherent differences between the groups. Also, researchers and clinicians should recognize that posture and body orientation in space are additional variables that can have a profound effect on the interaction between a seated person and his or her supporting surface.


A manufacturing system for contoured foam cushions. The design, application and evaluation of a specialized, personal computer-based manufacturing system for contouring foam cushions is presented. The topics discussed include both the hardware configuration and the software design. The target applications of this device are local or centralized fabrication of custom-contoured seat cushions. Although the technologies used for the development and implementation of this system are not new, using a personal-computer-based (PC) controller in place of a stand-alone numerically controlled (NC) motion controller significantly reduced the cost associated with this component. Further reductions in cost resulted from an optimization of the mechanical configuration for the dedicated task of carving foam cushions.