Effect of NO donors on protein phosphorylation in intact vascular and nonvascular smooth muscles

It is generally well accepted that nitrovasodilator-induced relaxation of vascular smooth muscle involves elevation of cGMP and activation of a specific cGMP-dependent protein kinase [protein kinase G (PKG)]. However, the protein targets of PKG and the underlying mechanisms by which this kinase leads to a relaxant response have not been elucidated. Several types of smooth muscle, including rat myometrium and vas deferens, are not relaxed by sodium nitroprusside, even at concentrations that produce marked elevation of cGMP and activation of PKG. The main objective of our studies was to compare PKG-mediated protein phosphorylation in intact rat aorta, rat myometrium, and rat vas deferens using two-dimensional gel electrophoresis. In intact rat aorta, seven PKG substrates were detected during relaxation of the tissue. None of the PKG substrates identified in the rat aorta appeared to be phosphorylated in the myometrium or vas deferens after administration of various cGMP-elevating agents. Thus the failure of the rat myometrium and rat vas deferens to relax in the face of cGMP elevation and PKG activation may be due to a lack of PKG substrate phosphorylation.     

Inhibition of smooth muscle 5'-nucleotidase by imidazole and its reversal by magnesium

Imidazole, commonly used as an effective pH-buffering reagent in aqueous media maintained at pH 7-8, was found to depress the 5'-nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) activity of microsomal membrane fraction isolated from rat vas deferens smooth muscle in a dose-dependent manner in the absence of added Mg2+. Such an inhibitory effect of imidazole on the smooth muscle 5'-nucleotidase was not dependent upon the purity or integrity of the membrane fractions used and could be fully reversed by the inclusion of 5-10 mM Mg2+ in the assay medium. Of the five different pH-buffering reagents tested, imidazole was specific in exerting inhibitory effect on the 5'-nucleotidase in the absence of Mg2+ and this inhibition could not be accounted for by the impurities present in the imidazole. Differential effects of chelating reagents and other divalent metal ions on the 5'-nucleotidase activity were also observed in imidazole and Tris buffer solutions. The 5'-nucleotidase activity was not affected if the membranes were preincubated and washed with a large volume of 50 mM imidazole and subsequently assayed in 50 mM Tris in the absence of Mg2+. Similar findings were obtained with EDTA treated membrane. These results suggest that imidazole does not act by removal of the activating metal ion but rather interacts directly with 5'-nucleotidase and alters the metal-enzyme interactions.     

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.     

Differential contractile response of normal vas deferens of rodents in correlation to their calcium and electrolyte levels

The contractile pattern of the vas deferens in three different rodents, rat, guinea pig and mouse was studied in response to adrenaline and noradrenaline. The left vas deferens of rat was more responsive to the graded doses of adrenaline and noradrenaline than the right. The same was also true for guinea pig and mouse vas deferens. This differential response has been correlated with the greater concentrations of calcium and sodium in the right vas deferens in rats and guinea pigs and it might also be related to the levels of membrane-bound and intracellular calmodulin-bound calcium. It is suggested that the left vas deferens might possess more calmodulin-bound calcium than the right, which might have instead, more membrane-bound calcium.     

The effect of hypertonic solution on the wet weight and contractions of rat uterus and vas deferens

The role of propagated activity in the responses to agonist drugs was studied for the rat uterus and vas deferens. Hypertonic solutions were used to inhibit propagation of activity by shrinking cells. Tissue weight was used to indicate cell volume. Hypertonic solutions after 10 min caused weight loss and reduced the size of contractions in response to submaximal doses of drugs, to KCl, and to external electrical stimulation. Contractions in response to KCl and drugs were diminished to a similar degree in the vas deferens, but in the uterus, drug contractions were depressed much more. Prolonged action of hypertonic solution also differed for the two tissues. In the uterus, weight changes correlated with changes in size of the drug-induced contractions. Uterine contractions reduced in hypertonic solution could be increased by using supramaximal doses of drug. When stimulation was applied to one end of the uterus in a three compartment bath, propagation of spontaneous drug- and KCl-induced contraction occurred, but it was prevented by placing hypertonic solution in the center compartment. An increase of the KCl to 44 mM in the hypertonic solution restored propagation. These experiments yielded no evidence of propagated responses in the rat vas deferens. It was concluded that propagated activity plays a role in drug-induced contractions in the rat uterus but not in the rat vas deferens. Hyperpolarization of shrunken cells might be involved in inhibition of propagation by hypertonic solutions.     

Do endogenous prostaglandins modulate noradrenergic transmission in the rat isolated perfused vas deferens?

The effects of the prostaglandin synthetase inhibitors aspirin, indomethacin and meclofenamic acid have been studied on the response of the rat isolated perfused vas deferens. None of these drugs, up to a concentration of 5 x 10(-5) M affected either phase of the biphasic constrictor response to 30 s periods of field stimulation. In the same preparations an inhibition of the response to field stimulation was seen in the presence of prostaglandin E1 at concentrations of 1 to 5 ng ml-1. All three prostaglandin synthetase inhibitors, at 5 x 10(-5) M, caused significant reduction of prostaglandin biosynthesis by homogenates of rat vas deferens. It is, therefore, suggested that stimuli which activate directly the noradrenergic nerves in the rat vas do not activate simultaneously a release of endogenous prostaglandins.     

Cyclic nucleotide levels during carbachol-induced smooth muscle contractions.

Cyclic nucleotide levels and tension were measured at various times during carbachol-induced smooth muscle contractions. Cyclic GMP levels were markedly increased during contractions of rat vas deferens, guinea pig myometrium and guinea pig taenia coli, but were unchanged during contractions of rat uterus or guinea pig ileum. No significant changes in cyclic GMP levels could be detected in estrogen-primed rat uteri at any of the times or drug concentrations studied. Even in tissues in which large increases in cyclic GMP levels could be detected during carbachol-induced contractions (i.e. guinea pig myometrium and taenia coli) the contractions appeared to precede the cyclic GMP increases by several seconds. No significant changes in cyclic AMP levels were observed during carbachol-induced contractions in any of the smooth muscles studied. Thus, changes in tissue levels of the cyclic nucleotides do not appear to be responsible for the initiation of carbachol-induced smooth muscle contractions.     

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.     

Isolation and properties of plasma membrane from smooth muscle

A generalized approach to obtain relatively pure fractions of plasma membrane from smooth muscle tissues for studying calcium transport is described. The use of various markers for cellular membranes to establish the purity of various fractions is critically considered. Plasma membranes from rat myometrium have been isolated in a purity estimated to be 95-99%. Plasma membrane purifications to 70-80% have been achieved from rat mesenteric arteries and veins, canine tracheal smooth muscle, rabbit intestinal muscle, rat vas deferens, rat fundus, and dog gastric corpus. The ATP-dependent transport of Ca is correlated with the distribution of plasma membrane markers. Ca gradient of greater than 1000-fold have been achieved. ATP-dependent active Ca transport by plasma membranes could sometimes be stimulated by oxalate or phosphate. Anion activation of Ca active transport is not a marker for endoplasmic reticulum. In some smooth muscles (e.g., rat vas deferens) ATP-dependent Ca uptake did not correlate exclusively with the distribution of plasma membrane markers. Instead, the correlation seemed to be with NADPH-cytochrome reductase EC 1.6.2.5 activity (putative endoplasmic reticulum marker) as well as with plasma membrane markers. In all smooth muscles, active Ca transport appears to be a property of the plasma membrane; in some it may also be a property of the endoplasmic reticulum. Mitochondria actively transport Ca, but in most systems studied to date, the Km for Ca2+ for this transport is higher than that for plasma membrane. Thus the plasma membrane may be the major physiological mechanism of active transport for Ca out of cytoplasm of smooth muscle cells. In two plasma membrane fractions (from rat myometrium and mesenteric arteries) it has been possible to demonstrate the existence of an Na-Ca exchange system. Its contribution to lowering cytoplasmic Ca is unknown.     

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.     

An endogenous brain substance, CDS (clonidine-displacing-substance), inhibits the twitch response of rat vas deferens

The effect of CDS, an endogenous brain substance that specifically displaces bound [3H]clonidine and [3H]rauwolscine in rat brain membranes and human platelets, has been tested in isolated, field-stimulated rat vas deferens. CDS, obtained after an extensive purification procedure as a single peak from an HPLC sizing column, inhibited the electrically stimulated rat vas deferens similarly to the inhibitory action of clonidine, an alpha 2-agonist. The effective dose of CDS as an inhibitor of the vas deferens is equivalent to its effective dose in displacing specifically bound [3H]-clonidine in rat brain membranes. Furthermore, the CDS inhibition of the twitch response is reversed by two alpha 2-adrenergic antagonists, yohimbine and phentolamine. From these results, it is suggested that CDS extracted from brain, with affinity for clonidine sites, may be involved in the nonadrenergic fast response of the sympathetic transmission of the vas deferens.     

3H-noradrenaline metabolism in the isolated epididymal and prostatic portions of the rat vas deferens

The spontaneous ³H-noradrenaline efflux from the isolated epididymal and prostatic portions of the rat vas deferens, was investigated. The spontaneous tritium efflux was higher in the epididymal portion than in the prostatic one from normal animals. Such differences were abolished after castration. On the other hand, acetylsalicylic acid enhanced the total tritium efflux only in the epididymal portion, whereas phentolamine and phenoxybenzamine increased the total tritium efflux from the two portions of the rat vas deferens in both experimental groups. The prostatic portion released a similar amount of ³H normetanephrine than the apididymal end, whereas other metabolic fractions (3,4-dihydroxyphenylglycol; 3,4-dihydroxymandelic acid and O-methylated deaminated metabolites) were smaller in the prostatic portion in comparison with the epididymal portion from control vas deferens. The results presented in the isolated rat vas deferens suggest the existence of a prostaglandin as well as an alpha adrenoceptive modulation of the spontaneous total tritium efflux.     

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.     

Effects of veratrine and paeoniflorin on isolated mouse vas deferens

In this study, we attempted to identify the interactions and mechanisms between veratrine and paeoniflorin on isolated mouse vas deferens. Paeoniflorin had no effect on isolated mouse vas deferens. Veratrine (1 x 10(-5) approximately 1 x 10(-3) g/ml) could directly induce contraction of isolated rat and mouse vas deferens. The concentration induced by veratrine (1 x 10(-5) g/ml) was completely inhibited by Ca2+-free solution and verapamil (1 x 10(-5) M), in both the epididymal and the prostatic portions of isolated mouse vas deferens. Naloxone (1 x 10(-5) M) did not alter the contraction induced by veratrine (1 x 10(-5) g/ml) in either the epididymal or the prostatic portions of isolated mouse vas deferens. Paeoniflorin (4.8 x 10(-5) g/ml) inhibited the contraction induced by veratrine (1 x 10(-5) g/ml) in both the epididymal and the prostatic portions of isolated mouse vas deferens. Paeoniflorin (4.8 x 10(-5) g/ml) potentiated norepinephrine (1 x 10(-5) M)-induced phasic contraction in the epididymal portion, but decreased contractions in the prostatic portion. Paeoniflorin (4.8 x 10(-5) g/ml) increased KCI (56 mM)-induced phasic contraction in the epididymal portion, but decreased the tonic contraction in either the epididymal or the prostatic portion. Veratrine (1 x 10(-5) g/ml)-induced contractions could be decreased by pretreatment with ryanodine (1 x 10(-5) M) in both the epididymal and the prostatic portions. Pretreatment with the combination of paeoniflorin (4.8 x 10(-5) g/ml) and ryanodine (1 x 10(-5) M) did not potentiate the inhibition of paeoniflorin in the veratrine-induced contraction in both the epididymal and the prostatic portions of isolated mouse vas deferens.     

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.     

The androgenic effect of norethisterone and 5alpha-norethisterone on the contractile response of the rat vas deferens to methoxamine and serotonin.

Norethisterone (NET) and its metabolite 5alpha-norethisterone (5alpha-NET) are competitors for the androgen receptor. The sensitivity of the rat vas deferens to the contractile action of methoxamine and serotonin is regulated by hormonal and anatomical factors. The aim of this study was to evaluate the ability of NET and 5alpha-NET to induce the androgen-regulated contractile response to methoxamine and serotonin in the epididymal and prostatic portions of rat vas deferens. Adult male rats either intact, castrated or steroid-treated castrated were used. The contractility was recorded isometrically, and non-cumulative concentration-response curves to either methoxamine or serotonin were obtained. NET and 5alpha-NET partially restored the sensitivity to methoxamine and serotonin in the epididymal portion of castrated rats. The maximal responses to both agonists were significantly higher than those observed in castrated rats, and significantly lower than the responses observed in either intact or androgen-treated castrated rats. The prostatic portion was less responsive to both agonists than the epididymal portion, in all groups but castrated rats, as castration induced sensitivity to both agonists. NET and 5alpha-NET displayed a partial though similar androgenic activity in the rat vas deferens. These results contrast with previous reports where a decrease of androgenic effect due to the 5alpha-reduction of NET has been found.     

The vas deferens of the spider crab,Libinia emarginata

Sperm enter the anterior vas deferens individually in the spider crab male. There they become surrounded by secretion products from the cells of the vas deferens, and are compartmentalized into spermatophores of varying size. The anterior vas deferens can be divided into three regions. The epithelium of the anterior vas deferens varies regionally from low to high columnar. The cytoplasm contains vast arrays of rough endoplasmic reticulum and Golgi complexes but few mitochondria. Intercellular spaces contain septate junctions, gap junctions and vesicles. Once the spermatophores have been formed in the anterior vas deferens, they are moved posteriorly to the middle vas deferens where they are stored and surrounded by seminal fluids. The epithelial cells of the middle vas deferens contain large amounts of rough endoplasmic reticulum and Golgi complexes. Numerous micropinocytotic vesicles appear, forming at the cell surface and within the apical cytoplasm. Their suggested function is the resorption of secretion products of the anterior vas deferens which initiated compartmentalization of the spermatozoa into spermatophores. The posterior vas deferens functions primarily as a storage center for spermatophores until they are released at the time of copulation. Seminal fluid surrounding the spermatophores is produced in this region as well as in the middle vas deferens. The cells of this region contain vast arrays of vesicular rough endoplasmic reticulum and Golgi complexes. The cells are multinucleate. Microtubules are numerous throughout the length of the cells and appear to insert on the plasma membrane.     

Beta-endorphin. Biological activity of analogs containing dermorphin and dynorphin sequences: ileum and vas deferens assays

Biological activity of synthetic beta-endorphin (beta-EP) analogs containing dermorphin or dynorphin-A-(1-13) structure has been investigated using the guinea pig ileum and the vas deferens of the mouse, rat and rabbit. Replacement of NH2-terminal 1-7 segment of camel beta-EP [beta c-EP-(1-7)] with dermorphin caused a great increase in opiate potency of the analog. [Dermorphin (1-7)]-beta c-EP was 120 times more potent than beta c-EP in the guinea pig ileum assay, 49 times more potent in the mouse vas deferens assay; and only 4 times more potent in the rat vas deferens assay. Replacement of NH2-terminal 1-13 segment of human beta-EP [beta h-EP-(1-13)] with dynorphin-A-(1-13) caused an increase in opiate potency in both the guinea pig ileum and rabbit vas deferens assays, a complete loss of potency in the rat vas deferens assay, and no change in the mouse vas deferens assay. In comparison with dynorphin-A-(1-13), the hybrid peptide was less potent in the guinea pig ileum assay as well as in mouse and rabbit vas deferens assay. It is suggested that beta c-EP-(8-31) facilitates the dermorphin moiety to act on opiate mu and delta receptors but not on the epsilon receptor, while beta h-(14-31) reduces the action of dynorphin on mu, delta and kappa receptors.     

Comparative study on the effect of morphine and the opioid-like peptides in the vas deferens of rodents: species and strain differences, evidence for multiple opiate receptors

The effect of an opiate alkaloid and an opioid-like peptide was studied on the electrically evoked twitching of the vas deferens of 3 common laboratory rodents. Normorphine and the synthetic opioid peptide D-Alanine₂ methionine enkephalinamide (D-Ala₂) produced dose dependent inhibitions of the twitching response in the mouse vas deferens. In the rat vas, while β-endorphin (β-EP) caused an inhibitory effect in three strains of rats to a similar degree, morphine produced a dose related enhancement of the twitching. In the guinea pig, both morphine and β-EP caused an increased in the muscular twitch. The results are interpreted in terms of an heterogenous mixture of opiate receptors present in the vas deferens from these rodents. The mouse appears to contain mainly δ receptors while the rat has mostly ε receptors characterized by their specificity and sensitivity to the action of β-EP. The guinea pig vas deferens has apparently lost the sensitivity to the inhibitory influence of the opioids, suggesting the absence of μ or δ opiate receptors in this tissue.