Presynaptic sites of isolated canine saphenous veins are more sensitive to protein kinase C than postsynaptic ones.

Effects of protein kinase C (PKC) activators and inhibitors on both tritium overflow and contraction evoked by 40 mM KCl were studied in canine saphenous veins preloaded with [3H]norepinephrine (NE). 12-O-Tetradecanoylphorbol 13-acetate (TPA) and phorbol 12,13-dibutyrate (PDBu) at 10(-11)-10(-7) M enhanced concentration-dependently the KCl-evoked tritium overflow, which was antagonized by polymyxin B (10(-5) M) and staurosporine (10(-7) or 10(-6) M). PDBu (10(-8) and 10(-7) M), but not TPA, potentiated the KCl-induced contraction. Only staurosporine reduced the KCl-induced contraction in the presence of PKC activators. Polymyxin B (3 X 10(-5) M) which failed to inhibit exogenous NE-induced contraction attenuated both responses to KCl. Staurosporine (10(-6) M) suppressed not only both the responses to KCl but also exogenous NE-induced contraction. Phentolamine (10(-6) M) inhibited almost completely the KCl-induced contraction and augmented remarkably the evoked tritium overflow. PDBu (10(-8) and 10(-7) M) still potentiated both responses to KCl in the phentolamine-treated veins. An additional treatment with nifedipine (10(-6) M) inhibited markedly the potentiation of the KCl-induced contraction by PDBu in the presence of phentolamine without affecting the evoked overflow. These results suggest that PKC may modulate KCl-evoked NE release from the adrenergic nerve endings of canine saphenous veins and that PKC is more sensitive to presynaptic than postsynaptic sites.

Although it is well-known that a rise in the intraneuronal concentration of Ca 2+ triggers exocytotic norepinephrine (NE) release from adrenergic nerve endings, the intraneuronal processes linking an increased Ca 2+ concen tration and NE release are not established. Protein kinase C (PKC), which is a Ca 2+ and phospholipid-enzyme, is distributed in pre synaptic nerve endings (1) and various other tissues (2), and it is activated by both tumor promoting phorbol esters (3) and the endo genous PKC activator 1,2-diacylglycerol (4).
Activation and inhibition of PKC have been reported to facilitate and to suppress depolarization-evoked release of neurotrans mitters, respectively (5)(6)(7)(8)(9)(10). These findings suggest that PKC may play a role in the regu lation of neurotransmitter release. Phorbol esters have been also utilized to evaluate the role of PKC in neurotransmitter-induced con traction of vascular smooth muscle (11 13). There are, however, few reports that have simultaneously investigated the involvement of PKC in vascular adrenergic neurotransmission and associated contraction of smooth muscle. Recently, we have reported that PKC activa tors enhance electrically evoked NE release from adrenergic nerve endings of the canine saphenous veins, but have no apparent effect on the corresponding contraction (14).
In the tissues with rich adrenergic innerva tion, KC1 causes exocytotic release of NE through depolarization of adrenergic nerve endings (15). Thus, KC1 as well as electrical nerve stimulation has been extensively em ployed for the studies on the effects of various agents on neurotransmitter release from cen tral (16,17) and peripheral (15,18) adrenergic nerve endings. In the present study, to further investigate the role of PKC in presynaptic and postsynaptic sites, we studied simultaneously the effects of PKC activators and inhibitors on both NE release and contraction in response to KC1 in the canine isolated saphenous veins preloaded with [3H]NE.

Isotope experiments
Mongrel dogs of either sex, weighing 8 17 kg, were anesthetized with sodium pentobar bital, 32 mg/kg, i.v. Helical strips (3 X 30 mm) of the canine saphenous veins were incu bated at 37°C for 2 hr in Krebs-bicarbonate (Krebs) solution containing 1.34 X 10-7 M 1 [7_3 H]NE (10,uCi in 5 ml of incubation medium) and 5.7 X 10-5 M ascorbic acid. This concentration of [3H]NE ensures ad equate labelling of the tissues (19). The Krebs solution had the following composition: 118.2 mM NaCI, 4.6 mM KC1, 1.2 mM MgSO4, 2.5 mM CaC12, 1.2 mM KH2PO4, 24.8 mM NaHCO3 and 10.0 mM dextrose. After the in cubation, the strips were mounted for superfu sion according to our previous report (20) and superfused at a rate of 3.6 ml/min with the Krebs solution maintained at 37C and bub bled with 95% 02 and 5% CO2. After a 60 min equilibration period, the veins were su perfused with 40 mM KCl solution for 6 min followed by a 16-min exposure to normal Krebs solution. The 6-min application of 40 mM KCl was repeated either 3 or 7 times as described below. The 40 mM KC1 solution was prepared by substituting NaCI with equimolar KC1. Developed tension was recorded isometrically on an ink-writing oscillograph (Nihon Kohden, WI-640G) through a force displacement transducer (Nihon Kohden, TB 611T). The superfusate samples were con tinuously collected every 2 min from 4 min be fore the second application of 40 mM KC1 throughout the experiment. One ml of each sample was mixed with 6 ml of scintillation mixture (5.5 g DPO, 0.1 g POPOP, 667 ml toluene and 333 ml Triton X-100) and then counted in a liquid scintillation counter (Alo ka, LSC-903). The tritium overflow evoked by 40 mM KC1 was calculated by subtracting the radioactivity present in the 2-min sample just before KC1 application from that in the 6-min of KCl application plus subsequent 2-min sam ple, and the sum of the differences was taken as the KCl-evoked tritium overflow. Thus, we have measured total evoked tritium overflow, and avoided a chromatographic separation of the metabolites, because it more closely re flects neurotransmitter release than does the overflow of [3H]NE (21).

Experimental protocol
The first series of experiments was carried out to study the effect of PKC activators, 12 0-tetrad ecanoylphorbol 13-acetate (TPA) and phorbol 12,13-dibutyrate (PDBu), on the 40 mM KCl-evoked tritium overflow and contrac tion in the presence or absence of PKC inhibi tors, polymyxin B and staurosporine. The stimulation with 40 mM KCl was repeated 7 times (S1-S7). Either PKC activators (10-11 10-7 M) or their vehicle, dimethyl sulfoxide (DMSO), at stepwise increasing concentra tions was superfused for 22 min each from 10 min before the onset of each stimulation of S3 to S7. The effect of PKC activators on the depolarization -evoked tritium overflow and contraction was expressed as the ratios of the responses to each stimulation (S3 S7) during the application of PKC activators to the re sponse to S2, that is S3/S2 S7/S2, and they were evaluated by comparing them with the corresponding ratios obtained from the vehicle-superfused veins. PKC inhibitors were superfused from 10 min before S3 throughout the experiment. The effect of PKC inhibitors in combination with PKC activators was also expressed as the ratios S3/S2-S7/S2. These ratios were compared with the corresponding ratios obtained in the veins superfused with PKC activators alone. Radioactivity obtained from the 2-min sample just before each stimu lation of S2 S7 was considered as the spon taneous tritium overflow and designated as Sp2 Sp7, respectively. The effect of drugs on the spontaneous tritium overflow was esti mated from the ratios, Spa/Sp2-Sp7/Sp2, and they were compared with the corresponding ratios in the absence of the drugs.
In the first series of experiments, higher concentrations of PDBu potentiated both trit ium overflow and contraction in response to 40 mM KCI. Therefore, the second series of experiments was performed to examine whether the PDBu-potentiated contraction is due to increased NE release or increased Ca 2+ influx as a result of depolarization of the venous smooth muscle cells, because these two mechanisms are responsible for the contrac tion elicited by KC1 in the canine saphenous vein (18) and because PDBu activates the voltage-dependent Ca 2+ channels in the smooth muscle of the canine saphenous vein (22). The stimulation with 40 mM KCl and the superfusion with PDBu (10-11-10-7 M) were identical to those described for the first series of experiments. Phentolamine and nifedipine at 10-6 M were superfused from 10 min before S3 throughout the experiment in order to block the components in response to released NE and depolarization of the smooth muscle cells, respectively, of the 40 mM KCl-induced contraction.
The third series of experiments was per formed to investigate the effect of PKC inhibi tors on the 40 mM KCl-evoked tritium over flow and contraction. The veins were stimu lated 3 times (S1 S3) with 40 mM KCI. PKC inhibitors were superfused 10 min before S3. The effects of the inhibitors on the responses to 40 mM KC1 and on the spontaneous tritium overflow were expressed as the ratios S3/S2 and Spa/Sp2, respectively, and they were com pared with the corresponding ratios in the vehicle-treated veins.
The fourth series of experiments was carried out to investigate whether inhibition of K+ channels leads to an enhancement of the KC1 evoked tritium overflow, because PDBu in hibits K+ channels following PKC activation (23). In these experiments, the 40 mM KCl treatment was repeated 3 times (S1 S3) and 10-3 M tetraethylammonium (TEA), a K+ channel blocker, was superfused 10 min before S3. The effect of TEA was expressed and evaluated, as in the third series of experiments.

Non-isotope experiments
To further examine whether the inhibition of the KCl-induced contraction by higher con centration of PKC inhibitors is only due to a decrease in the evoked tritium overflow, the effect of PKC inhibitors on exogenous NE was investigated. In these experiments, the 6-min application of 40 mM KC1 was repeated twice, and NE was then injected 3 times at 30 min in tervals in a volume of 0.1 ml into the superfu sion stream (NE1-NE3). The amount (6-20 nmol) of NE used was sufficient to cause a contraction similar to that elicited by the second application of 40 mM KC1. PKC inhibi tors were superfused 20 min before NE3. The effect of PKC inhibitors on the contraction in duced by exogenous NE was expressed as the ratio NE3/NE2, which was evaluated by com paring it with the corresponding ratio in the vehicle-treated veins.

Statistical analysis
All data are expressed as the mean ± S.E.M. Statistical analyses were performed us ing an unpaired Student's t-test for two-sample comparison and one-way analysis of variance followed by Dunnett test or the wholly signifi cant difference method for multiple compari sons. In each case, P values less than 0.05 were considered significant.
Nifedipine (3 X 10-3 M) was freshly dissolved in ethanol. Handling of nifedipine was carried out under light from a sodium vapor lamp. Polymyxin B and TEA were dissolved in dis tilled water. All drugs used were diluted with Krebs solution just before use.

RESULTS
Effects of PKC activators on 40 mM KC1 evoked tritium overflow and contraction Figure 1 shows the effects of stepwise in creasing concentrations of PKC activators on both tritium overflow and contraction induced by 40 mM KCI. TPA and PDBu (10-11-10-7 M), PKC activating phorbol esters, enhanced significantly the KCl-evoked tritium overflow in a concentration-dependent manner, com pared with the vehicle (DMSO)-superfused veins. The augmentation of the KCl-evoked tritium overflow by PDBu (10-8 and 10-7 M) was significantly (P < 0.05) greater than that by the same concentration of TPA. TPA at all concentrations used had no significant effects on the KCl-induced contraction, whereas PDBu at 10-8 and 10-7 M potentiated signifi cantly the contraction. Phorbol (10-11-10-7 M), which is inactive on PKC (24), affected neither the tritium overflow nor the contrac tion induced by KCI.
TPA, PDBu and phorbol at all concentra tions employed failed to alter the spontaneous tritium overflow (the ratio Sp7/Sp2 for DMSO was 0.77 ± 0.06; the ratios Sp7/Sp2 for 10-7 M of TPA, PDBu and phorbol were 0.72 ± 0.02, 0.73 ± 0.02 and 0.76 ± 0.02, respective ly. PDBu at 10-8 M or more elevated the resting tension. The elevation in the resting tension by PDBu corresponded to 15.5 ± 1.9% for 10-8 M and 62.0 ± 5.2% for 10-7 M of the contraction (3.56 ± 0.53 g, n = 6) induced by S2. TPA caused no marked rise in the resting tension. As shown in Fig. 2, the facilitatory effects of TPA and PDBu on the KCl-evoked tritium overflow were significantly reduced by polymyxin B (10-5 M) and staurosporine (10-7 or 10-6 M). There was no difference between the inhibitory actions of the two in hibitors. The KCl-induced contraction in the presence of TPA and PDBu was suppressed by staurosporine but not by polymyxin B (Fig.  2). The inhibitory action of staurosporine was concentration and superfusion time-depend ent under stepwise increasing concentrations of TPA or PDBu (the ratios S3/S2 and S7/S2 for 10-7 staurosporine were 0.96 ± 0.02 and 0.39 ± 0.05 in the TPA-treated veins, respec tively; the corresponding ratios for 10-6 M staurosporine were 0.82 ± 0.05 and 0.14 ± 0.08 in the PDBu-treated veins, respectively). Neither Polymyxin B nor staurosporine altered the spontaneous tritium overflow. The PDBu induced increase in the resting tension was completely abolished by concurrent superfu sion with 10-6 M staurosporine.

Effects of phentolamine or phentolamine plus nifedipine on the action of PDBu on 40 mM KC1-evoked tritium overflow and contraction
Phentolamine (10-6 M) alone facilitated sig nificantly the KCl-evoked tritium overflow, whereas it almost completely inhibited the contraction (Fig. 3). The inhibitory effect of phentolamine on the contraction progressively increased with time: the ratios S3/S2 and S7/S2 were 0.13 ± 0.03 and 0.02 ± 0.01, respective ly. On the other hand, the tritium overflow enhancing effect of phentolamine was almost constant regardless of the superfusion time. In the phentolamine-treated veins, PDBu at 10-11-10-7 M caused a significant and concentration-dependent enhancement of the KCI-evoked tritium overflow; and at 10-8 and 10-7 M, it potentiated significantly the KCI induced contraction (Fig. 3). Nifedipine (10-6 M) failed to affect the facilitatory effect of PDBu (10-11-10-7 M) on the KCI-evoked tritium overflow in the presence of phentol amine, but this Ca entry blocker inhibited markedly the KCI-induced contraction observed in a combined superfusion with PDBu (10-8 and 10-7 M) and phentolamine (Fig. 3). Neither phentolamine nor nifedipine had a significant effect on the spontaneous trit ium overflow from the PDBu-treated veins. Although phentolamine did not affect the rise in the resting tension by PDBu (10-8 and 10-7 M), nifedipine inhibited significantly (P < 0.01) the elevating action of PDBu on the resting tension in the presence of phentola mine: the elevation of the resting tension by 10-8 M and 10-7 M PDBu in combination with nifedipine and phentolamine corres ponded to 2.7 ± 2.0% and 16.8 ± 8.1%, re spectively, of the contraction (3.07 ± 0.29g, n = 5) induced by S2.

Effects of PKC inhibitors on 40 mM KCl evoked tritium overflow and contraction
Polymyxin B at 10-5 M had no effects on the tritium overflow and contraction in re sponse to 40 mM KCI, but at 3 X 10-5 M, it caused a significant inhibition of both re sponses to 40 mM KC1 (Table 1). Staurospor ine at 10-6 M but not at 10-7 M inhibited sig nificantly the KC1-evoked tritium overflow and contraction ( Table 1). The two PKC inhibitors did not alter the spontaneous tritium overflow and the resting tension.
Effects of TEA on 40 mM KG-evoked tritium overflow and contraction TEA (10-3 M) had no significant effects on the 40 mM KCI-evoked tritium overflow and contraction (the ratios S3/S2 in overflow were 0.99 ± 0.03 (n = 5) for the control and 1.04 ± 0.04 (n = 5) for TEA; the ratios S3/S2 in contraction were 0.99 ± 0.03 for the control and 1.01 ± 0.02 for TEA). This indicates that blockade of K+ channels is not responsible for the 40 mM KCI-evoked tritium overflow mechanisms.

Effects of PKC inhibitors on exogenous NE induced contraction
As shown in Table 2, there was no signifi cant difference between tensions developed by the 2nd 40 mM KCI and the 2nd NE before superfusion with polymyxin B or staurospor ine. Polymyxin B (3 X 10-s M) did not re duce the contractile response to exogenous NE, while staurosporine (10-7 and 10-6 M) caused a significant and concentration-depend ent inhibition of the contraction induced by exogenous NE (Table 2).

DISCUSSION
The involvement of PKC in stimulation secretion coupling processes has been demon strated by using tumor-promoting phorbol es ters that mimic the action of the endogenous PKC activator 1,2-diacylglycerol (see the In troduction). In the present study, PKC activat ing phorbol esters, TPA and PDBu, produced a significant and concentration-dependent fa cilitation of the 40 mM KC1-evoked tritium overflow in the canine saphenous veins pre loaded with [3H]NE. Phorbol, which lacks the ability to activate PKC (24), failed to enhance the KCl-evoked tritium overflow. PKC inhibi tors, polymyxin B (25) and staurosporine (26), by themselves, inhibited the evoked tritium overflow and counteracted the enhancing ac tion of PKC activators on the evoked tritium overflow. These results consistently suggest that PKC may be involved in exocytotic NE release from the canine saphenous veins.
Although TPA has been reported to be more potent or equivalent to PDBu in activat ing PKC in vitro (24,27), PDBu (108 and 10-7 M) potentiated the KC1-evoked tritium overflow more notably than did TPA, as pre viously reported in NE release from the rabbit hippocampus (6) and the mouse atria (28). Allgaier et al. (6) speculated that the differ ence in the ability of TPA and PDBu to po tentiate NE release may be due to the higher lipid solubility of TPA, because lipophilic drugs need more time to equilibrate in the ex tracellular space compared with more hydro philic analogues (29). On the other hand, Musgrave et al. (28) suggested that sympathe tic nerves have multiple forms of PKC which are differentially activated by TPA or PDBu, because the enhancing effect of TPA on NE release was identical when the pretreatment time was prolonged from 15 to 75 min and be cause PDBu facilitated significantly NE re lease even in the presence of the maximally effective concentration of TPA. These two views also might account for the results that PDBu (10-8 and 10-7 M), but not TPA, pro duced the rise in the resting tension and the enhancement of the KCl-induced contraction.
The accurate mechanism underlying the en hancing effect of PKC activators on NE re lease remains unclear. PDBu also enhanced significantly the KCl-evoked tritium overflow in the presence of phentolamine, indicating that the enhancement caused by PDBu is not due to blockade of presynaptic a 2-adreno ceptors. Furthermore, it has been reported that inhibition of neuronal NE reuptake is not responsible for the phorbol ester-potentiated NE release (30,31). PKC activation causes blockade of K+ currents (23) or facilitation of Na+ (32) and Ca 2+ (33) currents, each of which results in an enhancement of NE re lease. The present study with TEA, a K+ channel blocker, reveals that blockade of K+ channels is not responsible for the 40 mM KCI-evoked tritium overflow mechanisms. We have shown that the 40 mM KC1-evoked trit ium overflow from the canine saphenous veins is not modified by the drugs affecting Na+ channel function (20). Therefore, it seems that neither blockade of K+ channels nor facilitation of Na+ channels is the mechanism of the enhancing action of PKC activators on the 40 mM KCI-evoked tritium overflow. Ca 2+ and PKC act synergistically on stimulus-secretion coupling processes (34), which is due to an increase in the sensitivity to Ca 2+ of exocytotic NE release processes fol lowing the enzyme activation (35). PKC activation enhances Ca 2+ currents and 45Ca2+ accumulation in Aplysia neurons, cultured sympathetic neurons of the chick embryo and rat adrenal chromaffin cells (27,33,36). Since phorbol esters facilitate Ca 22+ dependent but not Ca 2+ independent NE release (6,30), it is most likely that PKC activation may result in either sensitization to Ca 2+ of exocytotic pro cesses or increased influx of Ca 2+ into the nerve endings to potentiate NE release.
PKC activators and inhibitors had no effect on the spontaneous tritium overflow in agree ment with the results in other tissues (10,30,31,35,37), suggesting that PKC may be re sponsible for modulating rather than for mediating NE release from the canine saphe nous veins.
TPA (10 11 10-7 M) and PDBu (10-" 10-9 M) failed to potentiate the 40 mM KC1 induced contraction in spite of a significant en hancement of the evoked tritium overflow. Recently, we reported that PDBu (10-7 M) did not increase the contractile response of canine saphenous vein to exogenous NE (14). These results suggest that presynaptic sites may be more sensitive to PKC activators than postsynaptic ones. When the KCI concentra tion in the superfusion medium was increased from 40 to 50 mM, the evoked tritium over flow and contraction increased by about 2.7 and 1.4-fold over the 40 mM KCl responses, respectively (Y. Takata et al., unpublished data). Therefore, the enhancement of the 40 mM KCI-evoked overflow by PKC activators seems to be insufficient for a significant poten tiation of the contraction. This view allowed us to speculate that the enhancing effects of PDBu (10-s and 10-7 M) on the contraction produced by 40 mM KCl are not due to in creased tritium overflow. Recent biological studies indicate that brain PKC can be sepa rated into three distinct fractions, designated types I, II and III, by chromatography on a hydroxyapatite column (38,39). Brandt et al. (40) suggested that PKC-I is located post synaptically, while PKC-II and PKC-III are lo cated presynaptically. The distinct distribu tions of the PKC family might be responsible for the higher sensitivity of presynaptic sites to PKC activators; however, whether multiple forms of PKC exist in pre and postsynaptic sites of the canine saphenous vein remains un known.
It has been reported that the KC1-induced contraction of the canine saphenous vein is caused not only by release of endogenous NE but also by depolarization of the smooth mus cle cells (18). The present results with phentol amine suggest that under the experimental conditions used, a large part of the contraction elicited by 40 mM KCl is attributable to NE released endogenously. Although the contrac tion which was still observed in the presence of phentolamine is thought to occur via de polarization of the smooth muscle cells, the possibility that the reversal of the inhibitory effect of phentolamine on postsynaptic a -ad renoceptors by increased release of NE may contribute to the residual contraction cannot be excluded, because nifedipine did not re duce significantly the KCl-induced contraction observed under a combined superfusion with PDBu (up to 10_9M) and phentolamine. Even in the phentolamine-treated veins, PDBu (10-8 and 10-7 M) potentiated signifi cantly the KCl-induced contraction and an additional superfusion with nifedipine suppres sed markedly the increased contraction with no significant inhibition of the evoked tritium overflow. Therefore, the potentiation of the KCl-induced contraction by PDBu in the pres ence of phentolamine, presumably as well as that in the absence of phentolamine, seems to be mainly due to an activation of the mecha nism of contraction through depolarization of the venous smooth muscle. This notion is sup ported by the findings (22) that PDBu acti vates the voltage-dependent Ca 2+ channels in these smooth muscle cells and that a combined treatment with PDBu and 20 mM KCl results in a marked contraction.
Polymyxin B at 10-5 M had no effects on the KCl-induced contraction regardless of the presence of PKC activators, while at 3 X 10-5 M, it inhibited significantly the KCl-evoked tritium overflow and contraction. The inhibi tory effect of polymyxin B on the contraction is exclusively due to a decrease in the evoked tritium overflow, because polymyxin B at 3 X 10-5 M did not reduce the contraction elicited by exogenous NE. The contraction induced by exogenous NE was significantly reduced by the 20-min pretreatment with staurosporine (10-7 M), but the contraction induced by 40 mM KC1 was not inhibited by the 10-min pre treatment, although the magnitude of the con tractile response to exogenous NE was similar to that induced by 40 mM KCI. The differ ences in the staurosporine effects are most likely due to staurosporine requiring a longer time to induce a significant inhibition. This view is supported by the present result that the inhibition of the KCl-induced contraction by staurosporine was time-dependent despite the superfusion with stepwise increasing con centrations of TPA or PDBu. The inhibitory effect of staurosporine (10-7 M) on the KCI induced contraction in the presence of TPA is not attributable to decreased release of NE, because polymyxin B (10-5 M), which inhib ited the evoked tritium overflow to a similar extent, failed to suppress the KCl-induced contraction. Staurosporine at 10-6 M reduced significantly the KCl-evoked tritium overflow and contraction regardless of the presence of PDBu. Together with the result that stauro sporine inhibited the exogenous NE-induced contraction, it appears that staurosporine (10-6 M) decreases the KCl-induced contrac tion through its action at pre and postsynaptic sites. Staurosporine has been reported to also possess a direct inhibitory action on myosin light chain kinase (41). PDBu at 10-8 and 10-7 M elevated the resting tension without any change in the spontaneous tritium over flow, which was not modified by phentol amine. The elevation of the resting tension produced by PDBu was inhibited by nifedipine and was abolished by staurosporine. These re sults suggest that PDBu may activate the voltage-dependent Ca2+ channels, presumably via PKC stimulation. A direct inhibitory effect on myosin light chain kinase may be also re sponsible for the abolition of increased resting tension.
In conclusion, the present study indicates that PKC modulates depolarization-evoked NE release from adrenergic nerve endings of the canine saphenous veins and presynaptic sites are more sensitive to PKC than post synaptic ones.