Purification and characterization of the inositol 1,4,5-trisphosphate receptor protein from rat vas deferens

Among rat peripheral tissues examined, Ins(1,4,5)P(3) receptor binding is highest in the vas deferens, with levels about 25% of those of the cerebellum. We have purified the InsP(3) receptor binding protein from rat vas deferens membranes 600-fold. The purified protein displays a single 260 kDa band on SDS/PAGE, and the native protein has an apparent molecular mass of 1000 kDa, the same as in cerebellum. The inositol phosphate specificity, pH-dependence and influence of various reagents are the same for purified vas deferens and cerebellar receptors. Whereas particulate InsP(3) binding in cerebellum is potently inhibited by Ca(2+), particulate and purified vas deferens receptor binding of InsP(3) is not influenced by Ca(2+). Vas deferens appears to lack calmedin activity, but the InsP(3) receptor is sensitive to Ca(2+) inhibition conferred by brain calmedin. The vas deferens may prove to be a valuable tissue for characterizing functional aspects of InsP(3) receptors.     

Effects of streptozotocin-induced diabetes and insulin on calcium responsiveness of the rat vas deferens

In the present study, effects of streptozotocin-induced diabetes and insulin treatment on the reactivity of rat vas deferens to KCl and calmidazolium, a calmodulin antagonist, were evaluated and calmodulin levels in vas deferens tissue from diabetic and insulin-treated rats were determined. Diabetes was induced in rats by a single injection of streptozotocin. Five weeks after the induction of diabetes, one group of diabetic rats was injected with insulin for 3 weeks. After 8 weeks, vas deferens tissues on one side of diabetic and insulin-treated diabetic rats and their controls were mounted in organ bath to measure isometric tension, while the tissues on the other side of rats were homogenized to determine calmodulin levels by radioimmunoassay. Concentration-response curves to KCl were obtained in vas deferens tissues in the absence and presence of calmidazolium. The effects of KCl and calmidazolium on vas deferens isolated from 8-weeks diabetic rats were decreased. Calmodulin levels were also found to be decreased in vas deferens from diabetic rats. Decreased calmodulin levels in diabetic rat vas deferens were not corrected by insulin treatment. Only a partial correction following insulin treatment was observed in contractile effect of KCl on diabetic rat vas deferens, whereas insulin treatment increases the affinity of calmodulin in this muscle. Experimental diabetes causes an impairment in calcium/calmodulin-dependent contractile process of vas deferens, which is correctable partially following insulin therapy. The changes in the function of rat vas deferens due to streptozotocin diabetes seem to be related to impaired sexual functions in human diabetes.     

NORADRENALINE METABOLIZING ENZYMES IN NORMAL AND SYMPATHETICALLY DENERVATED VAS DEFERENS

—A surgical technique for sympathetically denervating the vas deferens has been evaluated biochemically. A slight fall in soluble muscle protein content and no significant change in DNA content of the operated vas deferens were found. This indicates that the surgical procedure causes only a slight degree of tissue damage and may be useful for investigating the cellular localization and properties of noradrenaline metabolizing enzymes. In three species examined (rat, guinea pig and rabbit), monoamine oxidase activity of the vas deferens fell by approximately 50 per cent after denervation. The time course of the fall in monoamine oxidase activity of rat vas deferens was parallel to that of the disappearance of noradrenaline suggesting that this proportion of the total enzyme activity had a neuronal localization. The remaining enzyme activity is presumably located extraneuronally. Significant falls in catechol-O-methyl transferase activity were found in rat and rabbit vas deferens after denervation but not in guinea pig. The rabbit and rat vas deferens had respectively approximately 60 and 30 per cent of the catechol-O-methyl transferase activity associated with the sympathetic nerves. A complete loss of DOPA decarboxylase and tyrosine hydroxylase activities occurred in rat vas deferens after denervation, suggesting that these noradrenaline synthesizing enzymes have an entirely neuronal localization.     

Influence of nonakhlazin on adrenergic neurotransmission in the vas deferens of rats]

The effect of a new antianginal drug--nonachlazine on the adrenergic neurotransmission in the isolated rat vas deferens was studied by examining the vas deferens contractions in response to the transmural electric stimulation of the postganglionic sympathetic nerves and addition of noradrenaline (NA) or BaCl2. After the nonachlazine treatment the NA content in the vas deferens was also studied by the spectrofluorometric method. Besides, the effect of the drug on the uptake of the exogenous NA was investigated. Nonachlazine was found to possess some sympatholytic and spasmolytic effect and could block the uptake of the exogenous NA greatly.     

Immunolocalization of AE2 anion exchanger in rat and mouse epididymis.

A low-bicarbonate concentration and an acidic pH in the luminal fluid of the epididymis and vas deferens are important for sperm maturation. These factors help maintain mature sperm in an immotile but viable state during storage in the cauda epididymidis and vas deferens. Two proton extrusion mechanisms, an Na(+)/H(+) exchanger and an H(+)ATPase, have been proposed to be involved in this luminal acidification process. The Na(+)/H(+) exchanger has not yet been localized in situ, but we have reported that H(+)ATPase is expressed on the apical membrane of apical (or narrow) and clear cells of the epididymis. These cells are enriched in carbonic anhydrase II, indicating the involvement of bicarbonate in the acidification process and suggesting that the epididymis is a site of bicarbonate reabsorption. Previous unsuccessful attempts to localize the Cl/HCO(3) anion exchanger AE1 in rat epididymis did not investigate other anion exchanger (AE) isoforms. In this report, we used a recently described SDS antigen unmasking treatment to localize the Cl/HCO(3) exchanger AE2 in rat and mouse epididymis. AE2 is highly expressed in the initial segment, intermediate zone, and caput epididymidis, where it is located on the basolateral membrane of epithelial cells. The cauda epididymidis and vas deferens also contain basolateral AE2, but in lower amounts. The identity of the AE2 protein was further confirmed by the observation that basolateral AE2 expression was unaltered in the epididymis of AE1-knockout mice. Basolateral AE2 may participate in bicarbonate reabsorption and luminal acidification, and/or may be involved in intracellular pH homeostasis of epithelial cells of the male reproductive tract.     

Action of morphine on guinea-pig myenteric plexus and mouse vas deferens studied by intracellular recording.

Morphine reduces the output of transmitter from the myenteric plexus-longitudinal muscle preparation of the guinea-pig ileum and from the mouse vas deferens. Intracellular recordings were made from ganglion cells of the myenteric plexus and smooth muscle cells of the vas deferens. Synaptic transmission within the myenteric plexus was blocked by hexamethonium. Morphine did not change the properties of the ganglion cells, nor did it affect synaptic potentials. 5-Hydroxytryptamine inhibited acetylcholine release at intraganglionic synapses by an action which was unaffected by morphine. In the vas deferens, excitatory junction potentials were elicited by stimulation of postganglionic adrenergic nerve fibres. The junction potentials were depressed by morphine and levorphanol but not by dextrorphan. This depression was reversed by naloxone. The results indicate that morphine acts directly to reduce transmitter release at the neuro-effector junctions in the myenteric plexus-longitudinal muscle preparation and in the vas deferens in these species.     

A pharmacological investigation of the biphasic nature of the contractile response of rabbit and rat vas deferens to field stimulation

It has been demonstrated previously with the vas deferens of the guinea-pig that the first and second phases of the contractile response to motor nerve stimulation are preferentially antagonized by the P₂-purinoceptor antagonist arylazido aminopropionyl ATP (ANAPP₃), and the α₁-adrenoceptor antagonist prazosin, respectively. We have now investigated the effect of the two antagonists on the biphasic contraction in the vas deferens of two other species; rabbit and rat. ANAPP₃, in a concentration which antagonized responses to exogenously applied ATP but not those to exogenous norepinephrine, preferentially reduced the initial phasic response of the rabbit vas deferens to motor nerve stimulation without significantly reducing the secondary, tonic phase of the response. Prazosin had the opposite effect; antagonizing the response to norepinephrine but not to ATP and reducing the tonic response to motor nerve stimulation without significantly reducing the initial phasic response. Results obtained with the rat vas deferens were similar. The present results combined with previous findings suggest that ATP and norepinephrine act as cotransmitters in the vas deferens of several species.     

Morphological and biochemical changes in supersensitive smooth muscle.

The effect of postganglionic denervation on the incidence of nexal contacts in the smooth muscle of the rat vas deferens was investigated. The chronically denervated tissue exhibited twice as many nexuses as control. This increase in the incidence of cell contacts may contribute to the supersensitivity and/or the increase in maximum response of the denervated vas deferens. The effects of denervation, decentralization, and pretreatment with reserpine on the concentration of adenosine triphosphate (ATP) in vasa deferentia of rats and guinea pigs were also investigated. One day after denervation there was a substantial decrease in the endogenous norepinephrine and ATP concentrations. The norepinephrine concentration remained low (less than 10% of control) throughout subsequent days (up to 14 days) whereas the ATP concentration, after the first postoperative day, rose significantly. The rise in ATP concentration was temporally correlated with the development of postjunctional supersensitivity. Decentralization and pretreatment with reserpine both resulted in a significant increase in ATP concentration which preceded by 2 to 3 days a significant increase in sensitivity of the vas deferens. It appears that a change in the tissue concentration of ATP may be one of the initial events that occurs following interruption of the neural contact to the smooth muscle of the vas deferens.     

The effect of adenosine and beta-endorphin on the contractions of the vas deferens in rats with various durations of ethanol narcosis

Action of ethanol on animals with different predisposition to alcohol consumption depended on the level of ethanol metabolism or sensitivity of nervous system to alcohol. There was studied the ethanol effects on contraction of rat vas deferens. Such an approach enables to distinguish the metabolic effects from action on nervous system. It was shown, that dose of ethanol, which inhibits the contraction of rat vas deferens of by 50% is the same in both groups of rats, but the ED50 of adenosin and beta-endorphin were 1.7 and 4.7 times lower for vas deferens of predisposed rats. It's possible to suggest the differences between two groups of rats to be connected with the effects of ethanol on purinergic and endorphinergic system, but not with ethanol itself.     

Changes of norepinephrine levels, tyrosine hydroxylase and dopamine-beta-hydroxylase activities after castration and testosterone treatment in vas deferens of adult rats

Norepinephrine levels and tyrosine hydroxylase and dopamine-beta-hydroxylase activities have been used to evaluate the effect of castration and testosterone treatment on the sympathetic innervation of the adult vas deferens. Castration was followed by a decrease in both norepinephrine content and tyrosine hydroxylase activity, even though the changes were not concomitant. Treatment of castrated animals with testosterone reversed the effect of castration on organ weight and norepinephrine content, but only a short-lasting increase in tyrosine hydroxylase activity occurred at the beginning of testosterone treatment. In contrast, the testosterone-induced recovery of norepinephrine content observed at this time was accompanied by a marked increase in dopamine-beta-hydroxylase activity. The results suggest that in rat vas deferens, norepinephrine levels are under androgenic control and that this regulation mainly involves changes in dopamine-beta-hydroxylase activity rather than a modulation of tyrosine hydroxylase.     

Himbacine discriminates between putative muscarinic M1 receptor-mediated responses

This study describes the antagonistic properties of himbacine, in comparison with those of pirenzepine, at muscarinic receptors mediating the depolarization of rat superior cervical ganglion, the inhibition of electrically-induced twitch contractions of rabbit vas deferens and the contraction of dog saphenous vein, currently classified as putative muscarinic M₁ sites. The affinity of himbacine for the vas deferens site (pA₂ 8.08) was nearly ten times higher than those for the M₁ receptors of rat ganglion and dog saphenous vein (pA₂ 7.14 and 7.16, respectively); affinity estimates for pirenzepine were similar throughout the different preparations. The present data are consistent with the allocation of ganglion and saphenous vein receptors into the M₁ subclass; the profile of the vas deferens site, conversely, appears to be different, and possibly more closely related to that reported for the M₄/m4 receptor.     

Contractile responses of the rat vas deferens after epithelium removal

The purpose of the present investigation was to verify the role of the epithelium in the functional response of the rat vas deferens. Our results showed that the contractile effect of cumulative doses of clonidine (3.10(-5)-3.10(-3)) was increased after the removal of the epithelium. The effect of clonidine in epithelium-free vas deferens returned to normal values when an isolated epithelium from another vas deferens was added to the organ bath, showing that the epithelium is responsible for this increase of maximum effect for clonidine. Drugs functionally or structurally related to clonidine, such as oxymetazoline, alpha-methylnorepinephrine and moxonidine, did not have their dose-response curves altered. The curves for other contractile agents, such as noradrenaline, acetylcholine, ATP, 5HT, bradykinin and histamine, or the relaxation induced by isoprenaline and forskolin were also not modified. Electrically-induced contractions at frequencies from 0.1 to 20 Hz and the mechanism of negative feed-back, brought about by clonidine (10(-10)-10(-8) M) through pre-synaptic alpha2-adrenoceptors, were not changed after the removal of epithelium. In conclusion, a significant function of the epithelium in the contractility of the rat vas deferens was demonstrated for clonidine, but not for other agonists.     

Beta-endorphin: peripheral opioid activity of homologues from six species

The peripheral opioid activity of six homologous beta-endorphins (beta-EPs) were assayed on the guinea pig ileum and the vas deferens of the mouse, the rat and the rabbit. In the guinea pig ileum assay, human beta-EP (beta h-EP) was less potent than camel, turkey, and ostrich beta-EPs, of the same potency as equine beta-EP and more active than des-acetyl salmon beta-EP. In the rat vas deferens, mammalian beta-EPs showed higher activity than those from the bird and the fish, whereas in the mouse vas deferens assay, beta h-EP is more active than those from other species. In the rabbit vas deferens, however, all homologous beta-EPs show very weak activity. The relative potency of beta-EP homologues obtained from rat vas deferens assay is in good correlation with the analgesic potency, while the receptor binding activity does not correlate with any of the four bioassays, but appears to be related to the charge properties of the peptides.     

Long-term treatment with fluoxetine associates with peripheral effects on rat vas deferens contractility

The aim of this work was to study whether long-term treatment with fluoxetine could induce peripheral effects by modifying vas deferens contractile activity. For this purpose the contractile response to NE, and 5-HT of vas deferens isolated from male Wistar rats that received fluoxetine 10 mg/kg/day i.p., during 21 days, was studied using the isolated organ bath technique. Results show that vas deferens of treated rats presented spontaneous activity, an effect that was abolished by prazosin and isoproterenol and that was not affected by nitroprusside or indomethacin. In addition, fluoxetine did not modify the response to calcium suggesting that spontaneous activity was not a consequence of an abnormal calcium movement. Fluoxetine induced a significant increase in the response of vas deferens to 5-HT and to low NE concentrations while NE maximal effect was unaffected. Fluoxetine treatment did not modify the binding parameters of [3H]-prazosin to vas deferens. It is concluded that long-term treatment with fluoxetine modifies vas deferens contractile activity. This effect could be the result of an alteration of adrenergic neurotransmission and could account for some of the untoward effects observed during clinical course with fluoxetine.     

Interaction of xylamine with peripheral sympathetic neurons

Xylamine (XYL) administered to intact rats caused a 70-80% reduction in norepinephrine (NE) uptake by the vas deferens but had little or no effect on NE content in that tissue. The vas deferens accumulates 3H-XYL in vitro by a desmethylimipramine (DMI)-sensitive mechanism. Vasa deferentia from 6-hydroxydopamine (6OHDA) pretreated animals exhibited a 80% reduction in both NE content and XYL uptake activity. These results indicate that XYL is taken up by sympathetic nerve terminals and can reduce NE uptake activity without depleting terminals of neurotransmitter.     

Presynaptic effects of neuropeptide Y on [3H]noradrenaline and [3H]acetylcholine release

The electrically evoked release of radioactivity from mouse vas deferens and rat hypothalamic slices preloaded with [3H]noradrenaline was measured. In addition the release of [3H]acetylcholine from longitudinal muscle strip of guinea-pig ileum was also measured. Neurochemical evidence has been obtained that neuropeptide Y (NPY), although it co-exists and is released with (-)-noradrenaline (NA), it behaves differently as far as its effect on presynaptic modulation of chemical neurotransmission is concerned. It exerts a frequency-dependent presynaptic inhibitory effect on noradrenaline release from mouse vas deferens but has no effect on the electrically evoked release of NA from rat hypothalamus. Unlike NA, NPY does not influence the release of [3H]acetylcholine from the longitudinal muscle strip of guinea-pig ileum and does not potentiate the presynaptic effect of NA. It seems very likely, that the inhibitory effect of NPY is mediated via receptors. Its action is concentration dependent. While exogenous noradrenaline inhibited the release of noradrenaline by 91%, the maximum inhibition reached with NPY was not higher than 60%, indicating that either the intrinsic activity of NPY is lower or much less axon terminals are equipped with NPY receptors. Peptide YY (PYY) also reduced the release of NA from mouse vas deferens.     

Subacute reserpine treatment reveals preferential coupling between the M3 muscarinic receptor subtype and phosphatidylinositol turnover.

Muscarinic receptors in the rat cerebral cortex, cardiac atria and vas deferens were identified, quantitated, and characterized relative to phosphatidylinositol (PI) turnover as the functional response to stimulation of specific receptor subtypes. Receptor densities as determined by 3H-QNB binding were ranked: cerebral cortex greater than vas deferens greater than heart. Using displacement of 3H-QNB binding by the selective M1 and M2 muscarinic receptor antagonists pirenzepine and 11[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,11-dihydro- 6H-pyrido [2,3-b] [1,4] benzodiazepine-6-one (AF-DX 116) respectively, heterogeneous populations were found in the cerebral cortex and vas deferens. The M1 receptor subtype predominated in the former and the M2 predominated in the latter. An homogeneous M2 receptor population was present in the heart. Methacholine-stimulated accumulation of 3H inositol-1-phosphate was greater in the vas deferens than in the cerebral cortex, whereas PI turnover was not enhanced in cardiac atria. Reserpine treatment of rats (0.5 mg kg-1 day-1 for 7 days) increased muscarinic receptor density in the vas deferens coincident with a shift in the low affinity pKi for AF-DX 116 to a value comparable to high affinity binding, and abolished the enhanced PI hydrolysis. In the cerebral cortex, reserpine treatment shifted only the early portion of the methacholine dose-response curve to the right. These results are judged to be supportive of preferential coupling between the M3 muscarinic receptor subtype and PI turnover.     

Release of contractile agents from rat vas deferens by clonidine.

Clonidine induces contractile effects on the isolated rat vas deferens, but not on rat uterus or guinea-pig ileum. However, we have observed that if clonidine is incubated for about 10 min with a nutrient solution containing an isolated rat vas deferens, the resulting solution can contract an isolated rat uterus, or guinea-pig ileum indicating the involvement of a substance released from the vas. This contractile effect was partially reduced by naloxone and by serotonin antagonists, and by using a denervated vas, indicating that opioids, serotonin and eventually other substances released from nerve tissue of the vas can be involved.     

Regional distribution of dopamine, 5-hydroxytryptamine,and noradrenaline in the rat vas deferens

The concentrations of dopamine (DA), 5-hydroxytryptamine (5-HT) and noradrenaline (NA) in the rat vas deferens divided in eight or four sections were determined by high performance liquid chromatography with electrochemical detection. Dopamine and NA had the same regional distribution; their concentrations were maximal near the prostatic end and decreased towards the epididymis. The concentration of 5-HT also decreased from the prostatic to the epididimal end, but 5-HT did not follow the same regional distribution as DA and NA. Reserpine (0.02 or 0.2 mg/kg, i.p., 24 hr) and 6-hydroxydopamine (2×80 mg/kg, i.v., 6 days) decreased the contents of DA and NA; the concentrations of both amines were modified to a similar extent. Reserpine also diminished the content of 5-HT. Pargyline (200 mg/kg, i.p., 2 hr) increased the concentration of 5-HT whilep-chlorophenylalanine (300 mg/kg, oral, 3 days) decreased the contents of the amine in some sections of the vas deferens. This study suggests that DA and NA co-exist in the same sympathetic neurons. Some of the 5-HT could be stored in mast cells as previously proposed, but the finding that tissue content of 5-HT changes after inhibiting the deamination or synthesis of the amine suggests that other source(s) of 5-HT distinct from mast cells exist in the rat vas deferens.