Ethanol-induced spontaneous norepinephrine release from the rat vas deferens.

Acute ethanol exposure increases norepinephrine (NE) turnover both centrally and peripherally. One of the mechanisms by which ethanol could increase NE turnover is by increasing spontaneous NE release. The effect of ethanol on spontaneous NE release was investigated using tritium release from l-[3H]NE-labeled vasa deferentia as an index of NE release. Ethanol, 65 mM, increased spontaneous tritium release by 8% and pargyline, 100 mg/kg b.wt., pretreatment increased the ethanol effect by 63%. Ethanol alone (22 mM) had no effect on tritium release; however, with pargyline pretreatment tritium release increased by 13%. Combined reserpine, 5 mg/kg, and pargyline, 100 mg/kg, abolished these ethanol effects. Preincubation with tyramine, 1.8 microM, and omitting calcium from Krebs-bicarbonate buffer also abolished the ethanol effect. Butanol and propanol were more potent and methanol less potent than ethanol in increasing spontaneous tritium release. The mechanism consistent with all the above observations is that ethanol increases spontaneous exocytosis due to a nonspecific effect on presynaptic and vesicular membranes.     

Adrenergic nerves mediate angiotensin-induced prostaglandin release in the rabbit vas deferens

The effect of sympathectomy and norepinephrine depletion on prostaglandin (PG) synthesis in response to angiotensins II and III was examined in isolated vasa deferentia of the rabbit. Either 6-hydroxydopamine or surgical denervation significantly depressed norepinephrine concentrations in the vas deferens relative to contralateral controls, thus confirming an effective sympathectomy. Guanethidine also resulted in a significant reduction in norepinephrine concentrations in the vas deferens. Base-line PGE production by the vasa deferentia was not altered by guanethidine but was attenuated by 6-hydroxydopamine and increased by surgical denervation. All the treatments reduced angiotensin-induced PGE synthesis. The effect of denervation on PGE synthesis was greater than that of norepinephrine depletion. We interpret these results to indicate that angiotensins stimulate PGE production by adrenergic nerves in the vas deferens and that released norepinephrine mediates a part of the PGE production in response to the angiotensins. The 6-hydroxydopamine experiments are consistent with the adrenergic nerves being the predominant source of PGE in this preparation.     

Dihydroxyphenylglycol and intraneuronal metabolism of endogenous and exogenous norepinephrine in the rat vas deferens

To elucidate the origin and significance of dihydroxyphenylglycol (DHPG) as a metabolite of norepinephrine (NE), the isolated rat vas deferens was preloaded with tracer amounts of tritiated NE and examined for the release of radioactive and endogenous NE and DHPG before and during electrical stimulation or stimulation with excess K+. Tissues were incubated with desipramine or reserpine to determine the effects of blockade of neuronal uptake and of interference with vesicular translocation of NE. Radioactive NE appeared to distribute differently from endogenous NE into at least two pools, but for the most part endogenous NE and DHPG behaved similarly in response to pharmacological manipulations. Desipramine blocked completely the increased appearance of both radioactive and endogenous DHPG in the medium during electrical stimulation or K+ stimulation; DHPG responses to stimulation are thus dependent on recapture of NE at the synapse. Basal release of DHPG was increased by reserpine, and this increase was not affected by desipramine; therefore, reserpine-induced release of DHPG is independent of neuronal uptake consistent with formation of DHPG from NE leaking into the cytosol from vesicular stores. Reserpine enhanced the release of DHPG during stimulation, and concomitant desipramine treatment blocked this effect; thus, interference with NE translocation into storage vesicles increases the availability of recaptured NE for intraneuronal metabolism. During stimulation of NE release between 70 to 80% of the recaptured NE was estimated to be sequestered into storage vesicles for rerelease. Combined measurement of endogenous and labeled NE and DHPG provides a useful tool for examining neuronal uptake and intraneuronal disposition of NE.     

Dual effect of ouabain on the palytoxin-induced contraction and norepinephrine release in the guinea-pig vas deferens

Palytoxin (PTX), C129H223N3O54, isolated from marine coelenterates of Palythoa tuberculosa, caused a first rapid contraction followed by the slow phasic contraction of guinea-pig vas deferens. In the presence of ouabain (10(-5) M), PTX (10(-8) M) failed to cause the first contraction; however, the second contraction was potentiated. In the presence of phentolamine (10(-6) M), the second contraction was inhibited selectively. When ouabain was applied to the muscle in the presence of phentolamine, both first and second contractile responses to PTX were abolished. When the muscle was exposed to the potassium-depleted solution, the first contractile response to PTX was rather potentiated. PTX caused the release of norepinephrine from the muscle. Exposure of the muscle to ouabain (10(-5) M) markedly increased the PTX-induced release. It is indicated that the first and second contractile responses to PTX have entirely different properties. The second response is due to a release of norepinephrine from nerves and was potentiated by ouabain through the increase in the norepinephrine release, whereas the first response was not due to the norepinephrine release but presumably to a direct action on smooth muscle cell and was inhibited by ouabain. The mechanism of the action of PTX was discussed in the relation with Na,K-ATPase.     

Correlation between the release of the sympathetic neurotransmitter ATP and soluble nucleotidases from the guinea pig vas deferens

Recently, we have shown that by releasing specific nucleotidases the sympathetic nerves of the guinea pig vas deferens may regulate the metabolism of extracellular adenine nucleotides and consequently, the inactivation of neurotransmitter ATP. Based on the evidence for tetrodotoxin sensitivity and calcium dependence of the nerve stimulation-evoked overflow of enzyme activity, we have suggested that soluble nucleotidases may be stored in synaptic vesicles within the sympathetic nerves and released upon arrival of nerve action potentials by a mechanism similar to that for release of neurotransmitters. To further test this hypothesis we studied the time course of nerve stimulation-evoked overflow of ATP, norepinephrine (NE), releasable ATPase (r-ATPase) activity, and releasable AMPase (r-AMPase) activity under control conditions and in the presence of drugs known to selectively modulate sympathetic neurotransmission. The results show that the time course of overflow of r-ATPase and r-AMPase activities resembles the transient pattern of overflow of ATP but not the tonic pattern of overflow of NE. Vasa deferentia dissected from animals treated with reserpine release ATP, r-ATPase, and r-AMPase, whereas the overflow of NE is completely abolished. Guanethidine, on the other hand, inhibits equally well the overflow of the two neurotransmitters and the releasable nucleotidase activities. Agonists of the alpha(2)-adrenergic receptors abolish the overflow of ATP, r- ATPase, and r-AMPase but not the overflow of NE. This evidence supports the idea that the sympathetic nerves of the guinea pig vas deferens store and release ATP together with specific nucleotidases responsible for the inactivation of this neurotransmitter.     

Serotonergic modulation of the release of endogenous norepinephrine from rat hypothalamic slices

Ca+(+)-dependent release of endogenous norepinephrine (NE) and dopamine from superfused rat hypothalamic slices was stimulated by 40 mM K+. 20 mM K+ released only NE. Two consecutive exposures to 20 mM K+ (S1 and S2, respectively) produced NE release of similar magnitude (S2/S1 = 1.03 +/- 0.08). Serotonin (5-HT), 3 to 10 microM, in the presence of methylsergide or ritanserin (antagonists at 5-HT1-like and 5-HT2 receptors), caused a concentration-dependent decrease of K(+)-evoked NE release. 5-HT alone did not alter K(+)-evoked NE release. 2-Methyl-serotonin, 2-methyl-5-hydroxytryptamine, 3 to 10 microM (a selective 5-HT3 agonist), mimicked the 5-HT response in the presence and in the absence of ritanserin. A highly selective 5-HT3 antagonist, (3 alpha-tropanyl)1H-indole-3-carboxylic acid ester (ICS 205-930), 1 nM, inhibited the effect of both agonists. The isomers of another highly selective 5-HT3 antagonist, zacopride, inhibited the effect of 2-methyl-serotonin, 2-methyl-5-hydroxytryptamine, at a concentration range, 0.03 to 20 nM, characteristic of their interaction with 5-HT3 receptors. alpha-Methyl-serotonin, alpha-methyl-5-hydroxytryptamine, a selective 5-HT1-like/5-HT2 agonist, failed to affect the K(+)-evoked NE release, but antagonized the effect of 2-methyl-serotonin, 2-methyl-5-hydroxytryptamine. These observations provide direct evidence that, in rat hypothalamus, 5-HT modulates release of endogenous NE through activation of 5-HT3 and, possibly, 5-HT1C receptors.     

A possible crossed histamine-containing pathway adjacent to the sympathetic system of the rat vas deferens

Studies using nerve degeneration techniques (ganglionectomy, interganglionic section, postganglionic axotomy, uni- or bilateral hypogastric nerve section and right pelvic ganglionectomy) and fluorometric determinations of histamine and norepinephrine have shown the presence of nervous pathways containing histamine adjacent to the sympathetic system of the rat vas deferens. The findings suggest that these pathways cross between the ganglionic clusters located at the angle formed by the seminal vesicle and the vas deferens. They are not structurally related to the central nervous system by way of the hypogastric or pelvic ganglion. The histamine-containing pathways are independent of the noradrenergic pathways as dissociation between norepinephrine depletion and histamine depletion can be shown under nerve degeneration. The time course of nerve degeneration over a long period after sympathectomy shows a biphasic effect on histamine levels of the vas deferens. The early histamine depletion would be indicative of degeneration of histamine-containing pathways, and the delayed histamine increasing phase has been considered as due to accumulation of mast cells in the degenerating nerve sheaths. A possible role for the histamine-containing pathways in the modulation of sympathetic activity is envisaged.     

Imidazoline desensitization of epinephrine responses in rat vas deferens.

Repeated exposure of the rat vas deferens to the imidazoline oxymetazoline (OXY) results in a progressive loss of response which can appear selective for imidazoline agonists. The present study tests the hypothesis that imidazolines produce desensitization through prolonged blockade or inactivation of alpha-1 adrenoreceptors. Repeated exposure to OXY, naphazoline (NPZ) or tetrahydrozoline (THZ) produces a concentration- and time-dependent rightward shift and depression of the (-)-epinephrine concentration-effect curve, suggesting a mechanism of prolonged receptor blockade or inactivation. (-)-Epinephrine Kd values were similar when estimated after either receptor inactivation with phenoxybenzamine or repeated exposure to imidazolines. The differences in the ability of individual imidazolines to produce desensitization (order of potency: OXY greater than NPZ greater than or equal to THZ) do not follow their intrinsic activity (NPZ approximately THZ approximately OXY) or affinity (OXY greater than or equal to NPZ greater than THZ). The ability of individual imidazoline and phenethylamine agonists to produce a response in imidazoline-desensitized rat vas deferens reflects agonist intrinsic efficacy. Desensitization by imidazoline exposure does not affect contraction produced by either KCl or neurokinin A. Imidazolines produce effects similar to receptor inactivation and their desensitization in vas deferens can be explained without invoking an imidazoline subtype of alpha-1 adrenoreceptor.     

Presynaptic nicotinic receptors involved in release of noradrenaline and ATP from the prostatic portion of the rat vas deferens

A differential response to cholinomimetic agonists in epididymal and prostatic portions of rat vas deferens was characterized. The prostatic portion was less sensitive to acetylcholine and carbachol than the epididymal portion. The contraction induced by cholinomimetic agonists was inhibited in the epididymal portion by atropine (1.0-3.0 nM) and in the prostatic portion by hexamethonium (0.1 mM). The contractile response of the prostatic portion to exogenous acetylcholine was not inhibited by textrodotoxin (1.0 microM) but was attenuated by reserpine treatment (10 mg.kg-1 i.p. 24 h) and by prazosin or alpha, beta-methylene ATP. A combination of an alpha-1-adrenoceptor antagonist (prazosin) and P2 purinoceptor desensitization with alpha, beta-methylene ATP abolished the contractile response of the prostatic portion. The contraction induced by repetitive field stimulation of the prostatic portion was attenuated by hexamethonium whereas the response to a single stimulus was not modified. The data suggest that cholinomimetic drugs activate both nicotinic receptors located in nerve terminals of the prostatic portion and muscarinic receptors located in the smooth muscle cells of the epididymal portion, and that stimulation of nicotinic receptors induces the release of noradrenaline and ATP.     

Pharmacological characterization of purinergic receptors in the rat vas deferens

Using the isolated rat vas deferens, we have confirmed the existence of P1 purinergic receptors whose activation results in an inhibition of the neurogenic twitch of the vas deferens. The observed order of potency for agonists (adenosine ethyl carboxamide greater than 2-chloroadenosine greater than adenosine greater than 5'-AMP greater than 5'-ADP greater than ATP) and antagonism of these effects by theophylline supports a P1-mediated response. Metabolically stable analogs of ATP elicited dose-dependent contractile responses which were quantitatively greater than, but qualitatively comparable to, ATP-induced responses. The order of potency for the eliciting contraction was the following: adenylyl-5-imidodiphosphate = beta-gamma-methylene ATP greater than adenosine tetraphosphate much greater than ATP greater than ADP. Interestingly, these compounds also produced an inhibition of the neurogenic twitch with a similar rank order of potency. This response was not due to the activation of P1 receptors insofar as high concentrations of theophylline failed to attenuate either the inhibition of the neurogenic twitch or the contractile response induced by these agonists. Thus, these data demonstrate the presence of both P1 and P2 purinergic receptors in the rat vas deferens. In addition, the data are consistent with the idea that two distinct classes of P2 receptors exist in this tissue. Furthermore, these data suggest that the rat vas deferens provides a useful tissue for studying compounds which interact with both major subtypes of purinergic receptors.     

Presynaptic imidazoline receptors mediate inhibition of noradrenaline release from sympathetic nerves in rat blood vessels

Summary— In rat vena cava and aorta preincubated with [³H]noradrenaline the involvement of imidazoline receptors in modulation of [³H]noradrenaline release from sympathetic nerves was investigated. In the vena cava, the guanidine 1,3-di(2-tolyl)guanidine (DTG) inhibited the electrically evoked [³H]noradrenaline release; the inhibitory effect was more pronounced in the presence than in the absence of the α₂-adrenoceptor antagonist rauwolscine. The concentration-response curves of BDF 6143 [4-chloro-2-(2-imidazolin-2-ylamino)-isoindoline], and idazoxan for their facilitatory effect on electrically evoked [³H]noradrenaline release was bell-shaped; in the presence of rauwolscine, BDF 6143 inhibited the evoked [³H)noradrenaline release, whereas idazoxan did not. After blockade of α₂-autoreceptors by rauwolscine, the electrically evoked [³H]noradrenaline release from vena cava was inhibited not only by DTG and BDF 6143 but also by aganodine, clonidine and cirazoline; the rank order of potency of most of the drugs was similar to that found at the presynaptic imidazoline receptors in the rabbit aorta and pulmonary artery as well as in human atrial appendages. In the presence of rauwolscine, clonidine-induced inhibition of electrically evoked [³H]noradrenaline release was counteracted by 1 μM of the selective CB₁ receptor antagonist SR141716A (N-[piperidin-1-yl]-5-[4-chlorophenyl]-1-[2,4-dichlorophenyl]-4-methyl-1H-pyrazole-3-carboxamide). In the aorta, BDF 6143 and cirazoline did not modify [³H]noradrenaline release in the absence of α₂-adrenoceptor blockade; in the presence of rauwolscine, the electrically evoked [³H]noradrenaline release from aorta was inhibited by BDF 6143, cirazoline, aganodine and Clonidine with a rank order of potency similar to that in the vena cava. SR141716A 1 μM antagonized the inhibitory effect of BDF 6143 and Clonidine (in the presence of rauwolscine). In conclusion, noradrenaline release in rat vena cava and aorta is inhibited via presynaptic imidazoline receptors which appear to be related to those previously characterized in rabbit and human cardiovascular tissue.     

Release, metabolism and intraneuronal disposition of exogenous, endogenous and newly synthesized norepinephrine in the rat vas deferens

In the isolated rat vas deferens the release and intraneuronal disposition of endogenous norepinephrine (NE) were compared with those of newly synthesized or exogenous radioactive NE by preloading tissues with trace amounts of tritiated dopamine ([3H]DA) or tritiated NE ([3H]NE) and measuring release of radioactive and endogenous NE and dihydroxyphenylglycol (DHPG). Tissues were examined before and during electrical simulation, exposure to tyramine or exposure to depolarizing concentrations of K+. In [3H]DA-preloaded tissues the [3H]DA was converted readily to [3H]NE. Newly synthesized radioactive NE formed from exogenous [3H]DA was distributed differently from endogenous NE within at least two intraneuronal pools. One pool contained a high concentration (high specific activity) of newly synthesized [3H]NE and less than 3% of the total NE content of the tissues, and it released NE more readily than the larger low specific activity pool which contained over 95% of the total tissue NE content. Exogenous [3H]NE in [3H]NE-preloaded vasa deferentia was distributed among at least three different tissue pools, one consisting of extraneuronally bound NE containing [3H]NE of high specific activity, and two intraneuronal pools containing [3H]NE of intermediate and low specific activity in which NE was released from the intermediate specific activity pool more readily than from the low specific activity pool.(ABSTRACT TRUNCATED AT 250 WORDS)     

Platelet-generated thromboxane A2 enhances norepinephrine release from adrenergic nerves

Platelet-generated thromboxane A2 enhances norepinephrine release from adrenergic nerves. Microsomes were prepared from outdated human platelets and incubated with arachidonic acid to produce thromboxane A2. The incubate was added to isolated, electrically stimulated rabbit portal veins to determine its effect on adrenergic neurotransmission. Immunoreactive thromboxane B2 concentrations were measured to assess the generation of the precursor, thromboxane A2. Addition of microsomes in the presence of arachidonic acid (1 microM) caused a concentration-dependent increase in thromboxane B2 concentrations, electrically induced force and norepinephrine release. Inclusion of acetylsalicylic acid (10 microM) or the thromboxane synthase inhibitor, U63557A (100 micrograms/ml), in the arachidonic acid-microsome incubate eliminated the potentiation of force, norepinephrine release and thromboxane B2 generation. The thromboxane receptor antagonist, SQ30741 (1 microM), also eliminated the microsome effects on neurogenic force and norepinephrine release but not on thromboxane generation. The microsome-arachidonic acid incubate did not influence norepinephrine concentration-contractile response curves. These data are consistent with a potentiative action of thromboxane A2 on adrenergic neurotransmission, primarily mediated by enhanced release of norepinephrine. The physiological or pathological significance of this observation depends on the thromboxane concentrations in the vicinity of adrenergic nerves.