Site(s) and ionic basis of α‐autoinhibition and facilitation of [3H]noradrenaline secretion in guinea‐pig vas deferens

Abstract

1. Mechanisms controlling the secretion of [³H]noradrenaline from the noradrenergic nerves of guinea‐pig isolated vas deferens, prelabelled by incubation with [³H]noradrenaline, were studied using (a) different modes of (extramural or transmural) electrical nerve stimulation (a total of 300 shocks of varying strength, and a duration of 2 msec) at 1‐30 Hz, or (b) depolarizing concentrations of K⁺ (60‐110 m m). 2. The fractional rise in efflux of ³H‐labelled material (Δt) was used to measure the secretion of [³H]noradrenaline. 3. The dependence of [³H]noradrenaline secretion on the external Ca²⁺ concentration (1‐8 m m) was essentially hyperbolic. Double reciprocal plot analysis (1/Δt vs. 1/Ca²⁺) of the data yields that blockade of α‐autoinhibition (phentolamine 1 μ m) does not increase the maximal secretory velocity, but does enhance the apparent affinity of the secretory mechanism for external Ca²⁺. Exogenous noradrenaline has (qualitatively) opposite effects. The interaction between α‐autoinhibition and external Ca²⁺ thus shows a ‘competitive’ pattern, indicating that restriction of the utilization of external Ca²⁺ is a major mechanism in α‐autoinhibition of noradrenaline secretion, in this system. 4. Phenoxybenzamine (10 μ m) and phentolamine (1 μ m) increased the secretion of [³H]noradrenaline evoked by depolarization with K⁺ much less than that caused by electrical nerve stimulation (frequencies up to 10 Hz). Exogenous noradrenaline (1‐5 μ m) depressed the secretion evoked by both modes of stimulation. The results indicate that α‐autoinhibition of [³H]noradrenaline secretion is mainly operative when the secretory stimulus requires conduction of nerve impulses between varicosities. 5. The frequency dependence of [³H]noradrenaline secretion was hyperbolic, both in the presence and in the absence of α‐autoinhibition; at each frequency the secretion (Δt per shock) increased with the Ca²⁺ concentration in the medium (0·6‐8 m m). Double reciprocal plot analysis (1/Δt vs. 1/frequency) of the data yields that the pattern of interaction between external Ca²⁺ and facilitation depends on the presence or absence of α‐autoinhibition (phentolamine 1 μ m); in the former case it is ‘non‐competitive’, in the latter ‘competitive’. Similar analysis of the effect of facilitation by increasing the length of stimulus trains (from 5 to 300 pulses) at a constant frequency (5 Hz), on the Ca²⁺ dependence of Δt (1/Δt vs. 1/Ca²⁺) in the absence of α‐autoinhibition also yields that facilitation promotes utilization of external Ca²⁺. These results apparently imply that a rise in external Ca²⁺, in the presence of α‐autoinhibition, augments the secretory response to electrical nerve stimulation mainly by promoting recruitment of active units (varicosities?), without markedly altering their ‘affinity’ for facilitation. In the absence of autoinhibition (when all units are already recruited?), the results seem to imply that facilitation promotes depolarization‐secretion coupling in each, by more efficient utilization of external Ca²⁺. 6. The pattern of interaction between α‐autoinhibition and facilitation depends on the Ca²⁺ concentration in the medium. At or below the physiological level of Ca²⁺ in extracellular fluid (1·2 m m) it is ‘non‐competitive’, indicating that α‐autoinhibition and facilitation act, at least in part, at separate targets under these conditions. At high (5·4 m m) external Ca²⁺ the pattern becomes almost purely ‘competitive’, indicating that facilitation can, under suitable conditions, overcome all manifestations of α‐autoinhibition. 7. The secretion evoked by electrical nerve stimulation (Δt per shock, at 1 or 10 Hz) increased with the strength of applied shocks, both when applied extra‐ or transmurally, in the presence or absence of α‐autoinhibition. In the former case the rise in (Δt per shock) vs. (current strength) was hyperbolic, in the latter it followed a biphasic pattern. Double reciprocal plot analysis (1/Δt vs. 1/current) of the data yields a ‘non‐competitive’ pattern of interaction between facilitation or α‐autoinhibition, and exogenous current, when stimulation was extramural. When it was transmural the pattern is ‘competitive’. The results seem to imply that hyperpolarization, or depolarization, of nerve terminals are major mechanisms whereby α‐autoinhibition and facilitation, respectively, exert their effects on the secretory response to electrical nerve stimulation. 8. Neither activation of Na⁺, K⁺‐ATPase, nor promotion of G_Cl appear to be critically involved in α‐autoinhibition. Experiments with known blockers of G_K (tetraethylammonium, 4‐aminopyridine and Rb⁺) did not give support to the notion that promotion of K⁺ efflux is a mechanism whereby prejunctional α‐adrenoceptors cause (hyperpolarization of nerve terminals and) autoinhibition of secretion. If α‐autoinhibition does involve K⁺ channels in the nerve...