Mouse Postganglionic Sympathetic Neurons

Basic properties of noradrenaline release were studied in primary cultures of thoracolumbar postganglionic sympathetic neurons taken from 1-3-day-old NMRI mice. After 7 days in vitro, the cultures were preincubated with [3H]noradrenaline and then superfused and stimulated electrically. Conventional trains of pulses (for example, 36 pulses at 3 Hz) as well as single pulses and brief high-frequency trains (for example, four pulses at 100 Hz) elicited a well-measurable overflow of tritium, which was abolished by 0.3 microM tetrodotoxin or omission of Ca2+, but not changed by 1 microM rauwolscine. In trains of one, two, four, six, eight, or 10 pulses at 3 Hz, the evoked overflow of tritium remained constant from pulse to pulse at 1.3 mM Ca2+, but declined slightly at 2.5 mM Ca2+. Tetraethylammonium at 10 mM selectively increased the overflow elicited by small pulse numbers and especially by a single pulse. In trains of 10 pulses delivered at 0.3, 1, 3, 10, 30, or 100 Hz, the evoked overflow of tritium increased from 0.3 to 30 Hz and then declined at 100 Hz. This relationship was particularly pronounced at low Ca2+ concentrations (for example, 0.3 mM). Tetraethylammonium at 10 mM selectively increased the overflow elicited by low frequencies of stimulation. It is concluded that primary cultures of mouse postganglionic sympathetic neurons can be used to investigate release of [3H]noradrenaline. The release is well measurable, even upon a single electrical pulse. It agrees with release in intact sympathetically innervated tissues in a number of fundamental properties, including the pulse number and frequency dependence. The preparation may be of special interest in conjunction with genetic manipulations in the donor animals.     

Electrically evoked release of [(3)H]noradrenaline from mouse cultured sympathetic neurons: release-modulating heteroreceptors

Cultured neurons from the thoracolumbar sympathetic chain of newborn mice are known to possess release-inhibiting alpha(2)-autoreceptors. The present study was carried out in a search for release-modulating heteroreceptors on these neurons. Primary cultures were preincubated with [(3)H]noradrenaline and then superfused and stimulated by single pulses, trains of 8 pulses at 100 Hz, or trains of 36 pulses at 3 Hz. The cholinergic agonist carbachol reduced the evoked overflow of tritium. Experiments with antagonists indicated that the inhibition was mediated by M(2) muscarinic receptors. The cannabinoid agonist WIN 55,212-2 reduced the evoked overflow of tritium through CB(1) receptors. Prostaglandin E(2), sulprostone, and somatostatin also caused presynaptic inhibition. The inhibitory effects of carbachol, WIN 55,212-2, prostaglandin E(2), and somatostatin were abolished (at the highest concentration of WIN 55, 212-2 almost abolished) by pretreatment of the cultures with pertussis toxin (250 ng/ml). Several drugs, including the beta(2)-adrenoceptor agonist salbutamol, opioid receptor agonists, neuropeptide Y, angiotensin II, and bradykinin, failed to change the evoked overflow of tritium. These results demonstrate a distinct pattern of presynaptic inhibitory heteroreceptors, all coupled to pertussis toxin-sensitive G proteins. The lack of operation of several presynaptic receptors known to exist in adult mice in situ may be due to the age of the (newborn) donor animals or to the culture conditions.     

Protein phosphorylation in respiring slices of guinea-pig cerebral cortex. Evidence for a role for noradrenaline and adenosine 3'':5''-cyclic monophosphate in the increased phosphorylation observed on application of electrical pulses.

1. Exposure of slices of cerebral cortex from guinea pigs to electrical pulses for 10s or to noradrenaline, 5-hydroxytryptamine or histamine increases the rate of phosphorylation of unidentified proteins in the tissue; the increases in protein phosphorylation due to electrical pulses and noradrenaline were non-additive, whereas the increases due to pulses and 5-hydroxytryptamine or histamine were additive. 2. The stimulating effects of electrical pulses and noradrenaline on protein phosphorylation were antagonized by the beta-adrenergic blocking agents L-propranolol, dichloroisoprenaline, practolol and ICI 66082, but not by the alpha-adrenergic blocking agents, phentolamine and phenoxybenzamine. 3. The increase in protein phosphorylation associated with electrical pulses was antagonized by 10 mum-trifluoperazine and 0.5 mum-prostaglandin E1. 4. It is postulated that under the experimental conditions used the action of electrical pulses on protein phosphorylation is mediated by noradrenaline acting through a beta-adrenergic receptor mechanism probably involving adenylate cyclase.     

Rate and Duration of Stimulation Determine Presynaptic Effects of Haloperidol on Dopaminergic Neurons

Abstract: Superfused rabbit neostriatal slices prelabeled with [³H]dopamine ([³H]DA) were depolarized with electrical pulses (12 V, 1 ms). Although transmitter release showed a proportional increase with a greater number of pulses (30-360 pulses), flat frequency-release curves were obtained. Haloperidol (0.03–0.3 μ m ) enhanced ³H overflow without affecting its metabolism or time course, and antagonized apomorphine-induced inhibition of transmitter release. Maximal enhancement of release by haloperidol was obtained with 30–60 pulses delivered at a rate of 3 Hz, whereas much less facilitation of release was seen at 0.3 and 1 Hz (30–90 pulses) or with 360 pulses at either of the three frequencies. Therefore, the slope of the frequency-release curve was markedly increased by haloperidol. These results indicate that activation of presynaptic DA receptors, and thus facilitation of release by haloperidol was highly dependent on the rate and duration of stimulation of striatal dopaminergic terminals. In these neurons the feedback loop seems to act physiologically to depress the slope of the frequency-release curve.     

Bradykinin modulates the release of noradrenaline from vas deferens nerve terminals

To asses whether bradykinin influences the release of noradrenaline from the adrenergic varicosities of the vas deferens, tissues were loaded with 3H-noradrenaline. Upon electrical depolarization bradykinin increased in a concentration-dependent fashion, the overflow of tritium from the mouse or rat vas deferens. The 3H-overflow is dependent on the external Ca2+concentration suggesting neuronal release of 3H-noradrenaline. The present results add evidence to the hypothesis that bradykinin modulates the release of noradrenaline from peripheral sympathetic nerve terminals via the activation of a presynaptic mechanism.     

Inhibition of iloprost of the contractile effect of noradrenaline in mesenteric artery rings: evidence for a possible calcium-dependent mechanism.

Iloprost caused a concentration-dependent decrease in the response to noradrenaline in the rabbit isolated endothelium denuded rings from superior mesenteric artery but not thoracic aorta. Similar inhibition was obtained by verapamil using identical concentrations. In Ca(2+)-free EGTA containing medium noradrenaline both at lower and higher concentrations elicited a reduced contractile response and further addition of Ca2+ (2.5 mM) to the medium produced a second contraction in both mesenteric artery and aortic rings which was significantly and equally inhibited by iloprost and verapamil using identical concentrations in mesenteric artery but not in aortic rings. Prior addition of iloprost to the medium did not protect the inhibitory effect of phenoxybenzamine against noradrenaline-induced contraction. These results were taken as an evidence for the possible Ca2+ entry reducing effect of iloprost in mesenteric artery but not thoracic aorta. These results were also taken as an indirect evidence supporting the hypothesis that increased synthesis of prostacyclin by noradrenaline in the vascular wall may inhibit the contractile effect of the agonist by a (-) feedback mechanism mediated by Ca2+ entry into the vascular smooth muscle.     

Noradrenaline Release from Cultured Mouse Postganglionic Sympathetic Neurons

The possible existence of alpha2-autoreceptors, P2-autoreceptors, and adenosine A1- or A2A-receptors was studied in cultured thoracolumbar postganglionic sympathetic neurons from mice. The cells were preincubated with [3H]noradrenaline and then superfused. The selective alpha2-adrenoceptor agonist UK 14,304 reduced the electrically evoked overflow of tritium. When the cultures were stimulated by trains of increasing pulse number, ranging from a single pulse to 72 pulses at 3 Hz, the concentration-inhibition curve of UK 14,304 was shifted progressively to the right and the maximal inhibition obtainable became progressively smaller. Six alpha-adrenoceptor antagonists shifted the concentration-inhibition curve of UK 14,304 in a parallel manner to the right. Neither ATP (3-300 microM), adenosine (0.01-100 microM), the selective A1-receptor agonist cyclopentyladenosine (1-1,000 nM), nor the selective A2A-receptor agonist CGS-21680 (1-10,000 nM) changed the basal or the electrically evoked overflow of tritium. It is concluded that the cultured neurons possess presynaptic, release-inhibiting alpha2-autoreceptors. As in intact tissues, the effectiveness of presynaptic alpha2-adrenergic inhibition depends on the "strength" of the releasing stimulus. The pK(D) values of the six antagonists against UK 14,304 indicate that the autoreceptors belong to the pharmacological alpha2D and hence the genetic alpha(2A/D) subtype of alpha2-adrenoceptor. Neither P2-autoreceptors nor receptors for adenosine, the degradation product of ATP, were detected.     

Presynaptic modulation of evoked NE release contributes to sympathetic activation after pressure overload

Activation of the sympathetic nervous system is well documented in heart failure. Our previous studies demonstrated an increase in evoked norepinephrine (NE) release from left ventricle (LV) slices at 10 days of pressure overload. The purpose of this study was to test the hypothesis that presynaptic modulation of NE release contributes to sympathetic activation after pressure overload. We examined the functional status of the presynaptic alpha(2)- and beta(2)-receptors and ANG II subtype 1 (AT(1)) receptors in LV slices from 10-day aortic constricted (AC) and sham-operated (SO) rats. Evoked (3)H overflow from LV slices preloaded with [(3)H]NE was increased in AC rats. The alpha(2)-agonist UK-14,304 decreased evoked (3)H overflow with no differences between groups. The beta(2)-agonist salbutamol increased evoked (3)H overflow with greater sensitivity in slices from AC rats. The beta-antagonist propranolol decreased evoked (3)H overflow from LV slices of AC rats but not controls. ANG II increased evoked (3)H overflow with greater sensitivity in slices from AC rats. These data support the hypothesis that aberrant presynaptic modulation of catecholamine release contributes to sympathetic activation after pressure overload.     

Release and functional role of neuropeptide Y as a sympathetic modulator in human saphenous vein biopsies

Transmural electrical stimulation of the sympathetic nerve endings of human saphenous vein biopsies released two forms of NPY identified chromatographically as native and oxidized peptide. The release process is dependent on extracellular calcium, the frequency, and the duration of the stimuli. While guanethidine reduced the overflow of ir-NPY, phenoxybenzamine did not augment NPY release, but increased that of noradrenaline. Oxidized NPY, like native NPY, potentiated the noradrenaline and adenosine 5'-triphospahate-induced vasoconstriction, an effect blocked by BIBP 3226 and consonant with the RT-PCR detection of the mRNA encoding the NPY Y1 receptor. These results highlight the functional role of NPY in human vascular sympathetic reflexes.     

Fast and Local Electrochemical Monitoring of Noradrenaline Release from Sympathetic Terminals in Isolated Rat Tail Artery

Abstract: Noradrenaline release from sympathetic nerve terminals was evoked by electrical nerve stimulation of an isolated segment of rat tail artery. This release was recorded by a carbon fiber electrode combined with differential pulse amperometry. The active part of the electrode (one carbon fiber 8 μm in diameter and 50 μm in length) was placed in close contact with the arterial surface. The oxidation current appearing at +120 mV and corresponding to the local noradrenaline concentration at the electrode surface was recorded every 0.5 s. No oxidation current was detected under resting conditions, but electrical stimulation evoked an immediate increase in this current. This response was suppressed when tetrodotoxin was added to the perfusion medium and was enhanced when noradrenaline reuptake was inhibited by cocaine. The amplitude of the response was increased with increasing stimulation frequencies (2–25 Hz) and train lengths (1–16 pulses). Finally, the time resolution of the method (0.5 s) was good enough to show that noradrenaline release precedes the postsynaptic response, i.e., the electrically evoked contraction of the artery.     

Refractoriness of sural nerve in humans

High-frequency stimulation of peripheral nerve bundles is frequently used in clinical tests and physiologic experiments to study presynaptic and postsynaptic effects. To understand the postsynaptic effects, it is important to ensure that each pulse in the train is equally effective in stimulating the presynaptic nerve bundle; however, the optimal interpulse interval (IPI) and the stimulus intensity at which each pulse is equally effective in stimulating the same number of axons are not known. The magnitude of the compound action potential produced by each pulse in a train was tested on the sural nerve of 4 healthy human subjects. The stimulus train (2-4 pulses) was applied to the sural nerve at the lateral malleolus, and neural responses were recorded from just below the knee. With 2-pulse trains, families of curves between IPIs (1-6 ms) and normalized amplitudes of the second response were plotted for different stimulus intensities. Visual inspection of the data showed that the curves fell into 2 groups: with stimulus intensities <2.5x perception threshold (Th), the test response appeared partially at longer IPIs, whereas with stimulus intensities >=3x Th, partial recovery of the test response was earlier. The interval for complete recovery was statistically the same for low- and high-intensity stimulation. With more than 2 pulses in a stimulus train (IPI = 5 ms), the amplitude of the compound action potential (CAP) was not affected significantly. These results are important in understanding both the presynaptic and postsynaptic responses when presynaptic axon bundles are stimulated at high frequencies.     

Adrenergic activity of the avian oviduct in vivo

The effect of noradrenaline or isoproterenol, alone or in the presence of an alpha (phenoxybenzamine) or beta (propranolol) adrenergic blocking drugs on the oviducts of anesthetized laying hens was investigated. The results show that both alpha and beta adrenergic activity is present in the avian oviduct with the exception of the uterus which does not appear to have alpha excitatory activity. Norepinephrine induced a strong contraction followed by a brief relaxation period in the infundibulum, magnum and is thmus; administration of phenoxybenzamine blocked this response in all the three segments, indicating the presence of alpha adrenergic receptors. The uterus, however, exhibited an inhibitory response in the majority of the hens and this response was not affected by the administration of phenoxybenzamine. Isoproterenol always induced relaxation in all the four segments of the oviduct. This response was blocked by propranolol, a beta adrenergic blocker, indicating the presence of beta adrenergic receptors. The role of autonomic nerves innervating the reproductive tract in the regulation of reproduction is discussed.     

Turnover of protein-bound serine phosphate in respiring slices of guinea-pig cerebral cortex. Effects of putative transmitters, tetrodotoxin and other agents

1. The effect of various agents on the turnover of protein-bound phosphorus in respiring slices of cerebral cortex was studied. 2. Confirming previous work turnover was increased by the application of electrical pulses for 10s to the tissue. 3. Turnover was also increased by exposure of the slices for 10min to noradrenaline (0.5mm), 5-hydroxytryptamine (1mum) and histamine (0.1mm). 4. When slices were stimulated by electrical pulses in the presence of histamine the increase in turnover was the sum of the responses given by each agent above, suggesting that different phosphorylating systems were involved. 5. Tetrodotoxin (0.5mum) blocked the increased turnover due to electrical pulses, but not that due to histamine. Tetrodotoxin also prevented the increase in tissue cyclic AMP content caused by the application of electrical pulses. 6. Phosphoprotein turnover was not affected by adenosine, despite the increase in tissue cyclic AMP content given by this agent. 7. Adenosine blocked the phosphoprotein response to histamine, but did not affect the response to electrical pulses. 8. The results are discussed in relation to the hypothesis that the stimulation of protein phosphorus turnover by electrical pulses is secondary to the release of cyclic AMP in the tissue.     

Dynamic responses of electrically coupled systems

An identified pair of electrically coupled neurons in the buccal ganglion of the freshwater snail Helisoma trivolvis is an experimentally accessible model of electrical synaptic transmission. In this investigation, electrical synaptic transmission is characterized using sinusoidal frequency (Bode) responses computed by Laplace transforms and responses to brief stimuli. The frequency response of the injected neuron shows a 20-dB/decade attenuation and a phase shift from 0 degree at low frequencies to -90 degrees at high frequencies. The response of a coupled cell shows a 40-dB/decade attenuation and a phase shift from 0 degrees at low frequencies to -180 degrees at high frequencies. A simple mathematical model of electrical synaptic transmission is described that displays each of these crucial features of the measured frequency responses. Methods are described to estimate the frequency responses of coupled systems based on presynaptic measurements. The responses of the coupled system to brief pulses of current were computed using the principle of superposition. The electrical properties of coupled systems impose a minimum delay in reaching a peak in all postsynaptic responses. The delays in the postsynaptic responses to brief stimuli are related to the electrical and anatomical parameters of coupled networks.     

Differential inhibition of noradrenaline release mediated by inhibitory A1-adenosine receptors in the mesenteric vein and artery from normotensive and hypertensive rats

Mesenteric arteries and veins are densely innervated by sympathetic nerves and are crucial in the regulation of peripheral resistance and capacitance, respectively, thus, in the control of blood pressure. Presynaptic adenosine receptors are involved in vascular tonus regulation, by modulating noradrenaline release from vascular postganglionic sympathetic nerve endings. Some studies also suggest that adenosine receptors (AR) may have a role in hypertension. We aim at investigating the role of presynaptic adenosine receptors in mesenteric vessels and establish a relationship between their effects (in mesenteric vessels) and hypertension, using the spontaneously hypertensive rats (SHR) as a model of hypertension. Adenosine receptor-mediated modulation of noradrenaline release was investigated through the effects of selective agonists and antagonists on electrically-evoked [³H]-noradrenaline overflow. CPA (A₁AR selective agonist: 1–100 nM) inhibited tritium overflow, but the inhibition was lower in SHR mesenteric vessels. IB-MECA (A₃AR selective agonist: 1–100 nM) also inhibited tritium overflow but only in WKY mesenteric veins. CGS 21680 (A_2AAR selective agonist: up to 100 nM) failed to facilitate noradrenaline release in mesenteric veins, from both strains, but induced a similar facilitation in the mesenteric arteries. NECA (non-selective AR agonist: 1, 3 and 10 μM), in the presence of A₁ (DPCPX, 20 nM) and A₃ (MRS 1523, 1 μM) AR selective antagonists, failed to change tritium overflow. In summary, the modulatory effects mediated by presynaptic adenosine receptors were characterized, for the first time, in mesenteric vessels: a major inhibition exerted by the A₁ subtype in both vessels; a slight inhibition mediated by A₃ receptors in mesenteric vein; a facilitation mediated by A_2A receptors only in mesenteric artery (from both strains). The less efficient prejunctional adenosine receptor mediated inhibitory effects can contribute to an increase of noradrenaline in the synaptic cleft (both in arteries and veins), which might conduce to increased vascular reactivity.     

Factors determining post-stimulation dilatation

Post-stimulation dilatation (PSD) of the femoral artery and vein after cessation of postganglionic sympathetic stimulation were related to the frequency and pulse number of the preceding stimulation. It was found that: 1) A minimum number of pulses (MNP) is needed to evoke PSD. MNP is inversely related to the stimulation frequency. A marked PSD develops after stimulation at 1 Hz when only 100 pulses were applied, whereas, if stimulated at 4 Hz or at higher frequencies, even 2,000 pulses fail to induce PSD. 2) The maximum value, the maximum rate and the overall diameter change of PSD (expressed either in absolute values or in relation to the preceding contraction) are a) directly related to the number of pulses at a constant stimulation frequency, b) for a constant number of pulses the above values are inversely related to the stimulation frequency. 3) The relation of PSD values to the stimulation parameters contradict the assumption that PSD is elicited either by a neurogenic transmitter released by the stimulation, or by an extraneuronal transmitter whose release is associated with the release of noradrenaline. PSD is suggested to be due to a decreased noradrenaline level within the synaptic cleft due to persistence of the reuptake after the release of noradrenaline had ceased.     

Neuropeptide Y (NPY) and the pig heart: release and coronary vasoconstrictor effects

The effects of electrical stimulation of the stellate ganglia on the arterio-venous concentration differences of neuropeptide Y (NPY)-like immunoreactivity (LI) over the pig heart were studied in vivo in relation to changes in heart rate and left ventricular pressure. Furthermore, the effects of NPY on coronary vascular tone were analysed in vivo and in vitro. Stellate ganglion stimulation at a high frequency (10 Hz) caused a clear-cut, long lasting increase in plasma levels of NPY-LI in the coronary sinus compared to the aorta, suggesting release of this peptide from sympathetic terminals within the heart. The stimulation-evoked overflow of NPY-LI from the heart was enhanced about 3-fold by alpha-adrenoceptor blockade using phenoxybenzamine, suggesting that NPY release is under prejunctional inhibitory control by noradrenaline (NA). Combined alpha- and beta-adrenoceptor blockade abolished most of the positive inotropic response of the heart upon stellate ganglion stimulation, while a considerable positive chronotropic effect remained. After guanethidine treatment, stellate ganglion stimulation still produced a small positive inotropic and chronotropic effect on the heart. The stimulation evoked NPY overflow was markedly reduced by guanethidine indicating an origin from sympathetic nerve terminals. Injection of NPY into the constantly perfused left anterior descending artery in vivo caused a long lasting, adrenoceptor antagonist resistant increase in perfusion pressure, suggesting coronary vasoconstriction. NPY contracted coronary arteries in vitro via a nifedipine-sensitive mechanism. NA dilated coronary vessels both in vivo and in vitro via beta-adrenoceptor activation. It is concluded that sympathetic nerve stimulation increases overflow of NPY-LI from the heart suggesting release from cardiac nerves in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential Effects of Bromocriptine on Dopamine and Acetylcholine Release Modulatory Receptors

Rabbit neostriatal slices were prelabeled with [3H]dopamine (DA) and [14C]choline and then superfused. The electrical stimulation-evoked release of DA and of acetylcholine (ACh) was abolished by 0.33 microM tetrodotoxin and by low calcium concentrations (0.13 mM). Bromocriptine, a selective D2-DA receptor agonist, inhibited in a concentration-dependent manner the evoked overflow of DA and ACh, without affecting the basal efflux of both transmitters. The effects of bromocriptine were antagonized by sulpiride, a specific antagonist of D2-DA receptors. With stimulation at 0.3 Hz and 120 pulses, bromocriptine was eight times more potent in inhibiting the evoked overflow of DA (IC50: 11 nM) than that of ACh (IC50: 83 nM). Stimulations at 3 Hz and 360 pulses markedly reduced the potency of bromocriptine in inhibiting DA and ACh release, and diminished its selectivity for presynaptic receptors. These results indicate that DA receptors that modulate the release of DA and ACh are of the D2 subtype. The greater potency of bromocriptine at pre- than at postsynaptic sites suggests that these receptors may be different in quantity and/or quality [D2-alpha (presynaptic) versus D2-beta (postsynaptic)]. Finally, marked differences in the potency and efficacy of DA agonist actions on DA and ACh release modulatory receptors are obtained, depending on the parameters of stimulation used.     

NPY and NPY Y1 receptor effects on noradrenaline overflow from the rat brain in vitro

Neurotransmitters and neuropeptides play important roles in the regulation of various neuroendocrine functions particularly feeding. The aim of this study was to investigate whether a functional interaction occurs among neuropeptide Y (NPY) at NPY Y₁ receptors and noradrenaline overflow, as this may contribute to the regulation of appetite. The release of endogenous noradrenaline and its metabolite 3,4-dihydroxyphenylglycol (DHPG) were examined from hypothalamic and medullary prisms using the technique of in vitro superfusion and high performance liquid chromatography (HPLC) with coulometric detection. Noradrenaline and DHPG overflow was investigated at rest, in response to NPY (0.1 μM) and in response to the NPY Y₁ receptor agonist, [Leu³¹,Pro³⁴]NPY (0.1 μM). Perfusion with NPY and [Leu³¹,Pro³⁴]NPY significantly reduced noradrenaline overflow from the hypothalamus and medulla. Perfusion with NPY and [Leu³¹,Pro³⁴]NPY was without significant effect on hypothalamic DHPG overflow, while medullary DHPG overflow was significantly reduced by NPY and [Leu³¹,Pro³⁴]NPY. Results from this study provide evidence of NPY Y₁ receptor-mediated inhibition of noradrenaline release in the hypothalamus and medulla, further illustrating a complex interaction between neurotransmitters and neuropeptides within the rat brain.     

Inhibition by VIP and atriopeptin II on the field stimulation evoked release of [3H]noradrenaline in the rat portal vein.

The modulatory effects of vasodilatory peptides on noradrenaline release from sympathetic nerve terminals have been studied in the rat portal vein model. Transmural field stimulation of the longitudinally mounted vein preparation evoked concomitant increases in the [3H]noradrenaline overflow and the integrated tension. Both responses were abolished by guanethidine or tetrodotoxin, whereas only the tension response was blocked by phentolamine. CGRP and VIP, both being present in intramural nerve fibers in the rat portal vein, were compared with atriopeptin II for modulatory effects. CGRP (100 nM) had no effect on the overflow of [3H]noradrenaline or the integrated tension response to transmural stimulation. VIP (30 nM) and atriopeptin II (30 nM) both caused significant reductions of both [3H]noradrenaline overflow and the integrated tension. These results indicate that the decreased tension response to transmural stimulation in the presence of VIP or AP II reflects the sum of both pre- and postsynaptic inhibitions.