Treatment of cultured bovine adrenal chromaffin cells with 12‐O‐tetradecanoylphorbol 13‐acetate (TPA), an activator of protein kinase C (PKC), decreased [3H]saxitoxin ([3H]STX) binding in a concentration (IC50 = 19 nM)‐ and time (t1/2 = 4.5 h)‐dependent manner. TPA (100 nM for 15 h) lowered the Bmax of [3H]STX binding by 53% without altering the KD value. Phorbol 12,13‐dibutyrate (PDBu) also reduced [3H]STX binding, whereas 4α‐TPA, an inactive analogue, had no effect. The inhibitory effect of TPA was abolished when H‐7 (an inhibitor of PKC), but not H‐89 (an inhibitor of cyclic AMP‐dependent protein kinase), was included in the culture medium for 1 h before and during TPA treatment. Simultaneous treatment with TPA in combination with either actinomycin D or cycloheximide, an inhibitor of protein synthesis, nullified the effect of TPA. TPA treatment also attenuated veratridine‐induced 22Na+ influx but did not alter the affinity of veratridine for Na channels as well as an allosteric potentiation of veratridine‐induced 22Na+ influx by brevetoxin. These results suggest that an activation of PKC down‐regulates the density of Na channels without altering their pharmacological features; this down‐regulation is mediated via the de novo synthesis of an as yet unidentified protein(s), rather than an immediate effect of Na channel phosphorylation.

We have previously reported that in bovine adrenal chromaffin cells Ptychodiscus brevis toxin-3 (PbTx-3) does not alter the veratridine-induced 22Na influx when given alone, but increases the influx of 22Na when co-applied with either alpha- or beta-scorpion venom (Wada et al. 1992). In the present study, we characterized [3H]PbTx-3 binding in bovine adrenal chromaffin cells. [3H]PbTx-3 binding was saturable, reversible and of high-affinity with an equilibrium dissociation constant (Kd) of 32.0 +/- 4.9 nmol/l and a maximum binding capacity (Bmax) of 6.2 +/- 1.2 pmol/4 x 10(6) cells (4.5 +/- 0.9 pmol/mg cell protein). A Hill plot revealed the lack of cooperative interaction among the binding sites. Unlabelled PbTx-3 inhibited [3H]PbTx-3 binding with an IC50 of 31 nmol/l. However, tetrodotoxin, veratridine, alpha- and beta-scorpion venom, or veratridine in combination with either alpha- or beta-scorpion venom did not alter [3H]PbTx-3 binding. All these results suggest that PbTx-3 binds to a site (site 5) distinct from the previously known four toxin binding sites, which does not gate voltage-dependent Na channels by itself, but is specifically involved in the allosteric modulation of Na channels in adrenal medullary cells.

1 The role of the vascular endothelium in the vasoconstrictor response to transmural nerve stimulation (TNS) was studied in isolated ring segments of rat mesenteric and femoral arteries. 2 In both types of artery, TNS (1 to 16Hz) produced frequency-dependent vasoconstriction, which was abolished by l00nM tetrodotoxin, 1OpM guanethidine or lOnM prazosin, indicating that the response was mediated by endogenous noradrenaline (NA) released from noradrenergic nerves. NA-mediated vasoconstriction in response to TNS was significantly potentiated by removal of the endothelium. 3 In the presence of 10 nm prazosin, the reduced vasoconstriction in response to TNS was restored by incubation with 10pM 5-hydroxytryptamine (5-HT) for 20min. Restoration of the response to TNS was markedly attenuated by treatment with 10nM ketanserin, 100nM tetrodotoxin, or 10puM guanethidine, indicating that the restored response was mediated by 5-HT released from noradrenergic nerves. Vasoconstriction mediated by 5-HT in response to TNS was not modified by removal of the endothelium. 4 In both types of artery with intact endothelium, treatment with 3 pM methylene blue potentiated the NA-mediated contractile response to TNS, but did not potentiate the 5-HT-mediated response to TNS. 5 In both types of artery, the contractile responses to exogenous NA and 5-HT were potentiated by removal of the endothelium. 6 These results suggest that endothelial cells regulate neurogenic vasoconstriction by releasing endothelium-derived relaxing factor. Furthermore, it appears likely that the response to neuronally released 5-HT is not affected by the endothelium.

SUMMARY The release of 5-hydroxytryptamine from the vascular adrenergic nerve by periarterial nerve stimulation in spontaneously hypertensive rats (SHR) was compared with that in normotensive Wistar-Kyoto rats (WKY). The isolated mesenteric vascular bed was perfused at a constant flow rate of 5 ml/min. Vasoconstrictor responses to periarterial nerve stimulation (4, 8, 12, and 16 Hz for 30 seconds) and 5-hydroxytryptamine (1 fiM), but not norepinephrine (1 nmol), were significantly greater in SHR than in WKY. After treatment with 5-hydroxytryptamine (1 fj.M) for 15 minutes, vasoconstrictor responses to periarterial nerve stimulation previously reduced by prazosin (50 nM) were restored and a frequency-dependent pressor response reappeared. However, 5-HT treatment did not significantly affect the pressor response to exogenously administered norepinephrine (1 nmol), which was previously inhibited by prazosin. The degree of the restoration in SHR was significantly greater than that in WKY at all frequencies used. The restoration of the pressor response to periarterial nerve stimulation after 5-hydroxytryptamine treatment did not occur in the presence of the selective 5-hydroxytryptamine 2 receptor antagonists ketanserin (10 nM) or LY53857 (10 nM). In the perfused mesenteric vascular bed of both WKY and SHR prelabeled with [ 3 H]5-hydroxytryptamine, periarterial nerve stimulation (4-16 Hz) evoked a frequency-dependent increase in tritium efflux that was abolished by Ca 2+ -free Krebs-Ringer solution or tetrodotoxin (100 nM) and treatment with 6-hydroxydopamine. The tritium efflux evoked by periarterial nerve stimulation was significantly greater in SHR than in WKY at all frequencies used. These results suggest that the release of 5-hydroxytryptamine from adrenergic nerve endings by periarterial nerve stimulation is enhanced in the mesenteric vascular bed of the SHR. (Hypertension 10: 321-327, 1987) KEY WORDS • 5-hydroxytryptamine • vascular adrenergic nerve • spontaneously hypertensive rats S PONTANEOUSLY hypertensive rats (SHR) have been used as a model for studying human essential hypertension. The increased total vascular resistance resulting from enhanced sympathetic adrenergic tone is postulated to be the cause of this hypertension.1 However, actual mechanisms underlying the development and maintenance of the hypertension remain unresolved. Several lines of evidence suggest that 5-hydroxytryptamine (5-HT) may be involved in the hypertension of SHR.2 In fact, the vasoconstrictor response to 5-HT has been shown to be

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