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During recent years several examples have been reported of neurons in which a neuropeptide coexists with a 'traditional' neurotransmitter of lower molecular weight, and evidence has been obtained that nerve stimulation may cause co-secretion of both (see Lundberg & Hokfelt 1983). In the postganglionic sympathetic nerves of rat vas deferens, the 36 amino acid peptide, neuropeptide Y (NPY, Tatemoto 1982) co-exists with noradrenaline (NA). Exogenous NPY has been reported to depress the twitch phase of the contractile response of rat vas deferens evoked by single shock electrical field stimulation, but not the contraction evoked by exogenous NA (Allen et al. 1982; Lundberg et al. 1982). This finding suggests that liberated endogenous NPY may interact with NA prejunctionally, by depressing the secretion of NA. In the present work we have examined this hypothesis more directly, by studying the effect of this peptide both on the secretion of 'H-NA and on the contraction evoked by electrical nerve stimulation, in rat isolated vas deferens.Briefly (for details concerning the experimental methods see Alberts et al. 19811, male Sprague Dawley rats were stunned and bled to death, and the vasa deferentia dissected out and preincubated at 37°C for 30 min with 2 pM 'H-NA (New England Nuclear Co.), in Tyrode's solution of the following composition (mM): NaCl 136.9, KCI 2.7, CaClz 1.8, MgCIz 0.5, NaHCO, 11.9, NaHzPOs 0.4, glucose 5.6, ascorbate 0.114. The preparation was then washed, mounted in a perspex chamber and perifused at 1 ml/min with Tyrode's solution. Desipramine (0.6 pM) and normetanephrine (10 pM) were added to block reuptake of liberated NA, and atropine (2.6 pM) to block possible interaction with cholinergic functions. The effluent was divided into 4 ml fractions and used for analysis of 'H-NA secretion. The nerves were excited by field stimulation with trains of 300 shocks at 1 Hz, via platinum ring electrodes at the top and bottom of the preparation, using a Grass S44 stimulator (90 V, 0.3 msec). The rise in longitudinal tension (resting tension adjusted to 10 mN) was recorded isometrically, using a force displacement transducer and amplifier (Hottinger Baldwin Messtechnik GmbH, Q 11/5 and KWS 3072, respectively) and an Omniscribe recorder (Houston Instrument). After addition of 2.5 ml Instagel (Packard Instruments) the ' H activity in 1 ml aliquots of the effluents and of extracts of the tissue, at the end of the experiment, was counted in a Packard liquid scintillation spectrometer. In order to separate 'H-NA from metabo-Fig. 1. Effect of NPY on the secretion of tracer NA evoked by electrical field stimulation (90 V, 0.3 msec) with trains of 300 shocks at 1 Hz. Open bars show fractional stimulus-evoked rise in 'H, hatched bars fractional rise in 'H-NA. Mean values, and range from two parallel experiments. NPY (0.2 pM) was present I5 min before and during the second stimulation period; after this NPYfree medium was admitted. Stimulation periods 3 and 4 started at 20 and 52 min, respectively, after discontinuing ...

SUMMARY1. 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 or (b) depolarizing concentrations of K+ (60-110 mM).2. The fractional rise in efflux of 3H-labelled material (At) was used to measure the secretion of [3H]noradrenaline.3. Thedependence of[3H]noradrenalinesecretionontheexternalCa2+ concentration (1-8 mM) was essentially hyperbolic. Double reciprocal plot analysis (1/At vs. 1/Ca2+) of the data yields that blockade of a-autoinhibition (phentolamine 1 /SM) does not increase the maximal secretary velocity, but does enhance the apparent affinity of the secretary mechanism for external Ca2+. Exogenous noradrenaline has (qualitatively) opposite effects. The interaction between a-autoinhibition and external Caa2+ thus shows a 'competitive' pattern, indicating that restriction of the utilization of external. Ca2+ is a major mechanism in a-autoinhibition of noradrenaline secretion, in this system. 4. Phenoxybenzamine (10 ,uM) and phentolamine (1 /LM) 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)(2)(3)(4)(5) /SM) depressed the secretion evoked by both modes of stimulation. The results indicate that a-autoinhibition of [3H]noradrenaline secretion is mainly operative when the secretary 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 ofa-autoinhibition; at each frequency the secretion (At per shock) increased with the Ca2+ concentration in the medium (0'6-8 mM). Double reciprocal plot analysis (1/At vs. 1/frequency) of the data yields that the pattern of interaction between external Ca2+ and facilitation depends on the presence or absence of a-autoinhibition (phentolamine 1 /SM); in the former case it is 'noncompetitive', in the latter 'competitive'. Similar analysis of the effect of facilitation byThe experiments reported in this paper were carried out at the Department of Physiology, Karolinska Institutet. Authors' names are in alphabetical order.0022-3751/81/3710-0802 $07.50 ©D 1981 The Physiological Society 298 P. ALBERTS, T. BARTFAI AND L. STJARNE increasing the length of stimulus trains (from 5 to 300 pulses) at a constant frequency (5 Hz), on the Ca2+ dependence ofAt (1/At vs. 1/Ca2+) in the absence ofa-autoinhibition also yields that facilitation promotes utilization of external Ca2+. These results apparently imply that a rise in external Ca2 , in the presence of a-autoinhibition, augments the secretary response to electrical nerve stimulation mainly by promoting recruitment of active units (varicosities?), without markedly altering their 'a...

2. With these stimulation parameters, which yielded about the same fractional secretion of [3H]ACh, and with eserine (10 /LM) present in the medium, atropine (1 /M) enhanced the 'indirectly', electrically evoked secretion 3-65 + 034 (n = 6) fold, and that caused by 40 mm or 80 mM-potassium 1-82+0-06 (n = 6) or 1P55+0-09 (n = 10) fold, respectively. Atropine thus enhanced 'indirectly', electrically evoked secretion 4-fold more than that caused by 'direct' depolarization of varicosities with high potassium (P < 0 001).3. This difference is not likely to be caused by depression of the sensitivity of the presynaptic muscarinic receptors to ACh released by nerve stimulation, caused by the hypertonicity of the medium in the potassium stimulation experiments. The medium made hypertonic by addition of Tris-HEPES (80 mM) did lower the binding affinity of membrane preparations of (pre-and post-synaptic) muscarinic receptors, to carbamylcholine, and also the contractile responsiveness of the longitudinal muscle to this agent, in both cases to about one half. But it did not appear to alter the responsiveness of either pre-or post-synaptic muscarinic receptors to endogenous ACh, released by nerve stimulation.4. The results support a dual-mode model for the muscarinic negative feed-back control of ACh secretion from the nerve terminals of this preparation, mainly operating by restriction of the invasion of terminals, and only secondarily by depression of the efficiency of depolarization-secretion coupling in invaded varicosities.5. Since this model has earlier been proposed to apply for the control of secretion of [3H]noradrenaline from the micro-anatomically similar nerve terminals of P. ALBERTS, T. BARTFAI AND L. STJARNE noradrenergic nerves, the present findings suggest that the model may have a wider biological significance, and possibly apply to the control of the secretary activity of bouton8-en-pa88ant nerve terminals in general.