Opioid effects in developing rat vas deferens

The inhibitory effects of various opiates on developing rat vas deferens were studied by determining the degree of depression of mechanical responses elicited by electrical field stimulation. All agonists showed decreased effects with maturation, but the decrease occurred at different times. With normorphine the loss of agonist activity was greatest at days 12–16, while withd-Met²,Pro⁵-enkephalinamide it was greatest at days 16–30. -Endorphin also was less effective in adult than 30-day preparations, but methionine enkephalin was ineffective at all ages. Morphine and normorphine were weak antagonists of opiate agonists in the adult preparations. These results indicate that the nature and pharmacologic sensitivity of opiate actions change with development.     

The effects of the ionophore A-23187 on the rat vas deferens

The calcium ionophore A-23187 induced spontaneous, rhythmic contractions in the rat isolated vas deferens in a concentration-dependent manner. Contractions were blocked by phentolamine and were abolished following pretreatment with reserpine. In tissues preloaded with [3H]noradrenaline, A-23187 (10 microM) caused a time-dependent increase in the release of tritium. The findings suggest that A-23187-induced contractions in the rat vas deferens are secondary to the release of endogenous noradrenaline from the adrenergic nerves, as are contractions induced in this preparation by X-537A (another calcium ionophore) described earlier by other investigators.     

The effects of pancreatic polypeptides and neuropeptide Y on the rat vas deferens

In order to study the physiological significance of the coexistence of pancreatic polypeptide and norepinephrine (NE) in peripheral noradrenergic nerves, the effects of pancreatic polypeptides of several species were tested on the isolated rat vas deferens. Neuropeptide Y (NPY) was also studied because of its sequence homology to the pancreatic polypeptides. The contractile responses, which were mediated predominantly by activation of noradrenergic nerves following electrical stimulation, were inhibited by bovine pancreatic polypeptide (BPP), human pancreatic polypeptide (HPP), avian pancreatic polypeptide (APP) and NPY in a dose-dependent manner using a constant flow bath. The decreasing order of the inhibitory responses was as follows: BPP = HPP greater than NPY greater than APP. The inhibitory responses produced by BPP and HPP lasted more than 1 hr and displayed a marked tachyphylaxis. In contrast, the inhibitory effects induced by NPY and APP usually returned to the control level after 20-30 min and had minimal tachyphylaxis. The inhibitory action of NPY was still present during alpha-adrenergic blockade. Contractions produced by a single submaximal dose of exogenous NE or serotonin (5-HT) in unstimulated preparations were not affected by pretreatment with NPY. The amplitude of contractions was partially reduced 1 min after pretreatment with BPP or HPP; recovery occurred about 15 min after peptide pretreatment in a constant flow bath. These results suggest that an NPY receptor exists presynaptically in the rat vas deferens and that stimulation of the receptor by NPY inhibits the release of NE from noradrenergic nerves.(ABSTRACT TRUNCATED AT 250 WORDS)     

Action of pro-opiomelanocortin products on the rat vas deferens

Behavioral study of pro-opiomelanocortin products indicates that beta-endorphin and corticotrophin-like peptides have antagonistic effects. However, these peptides have similar actions on the rat vas deferens. beta-endorphin, alpha-MSH and ACTH each inhibit electrically evoked contraction of the duct, but the corticotrophin derived peptides are tenfold more potent on a molar basis (ED50 = 9 nM). Pharmacological analysis shows that the action of corticotrophin-derived peptides does not involve an opiate receptor mechanism. The results are discussed in terms of the central action of the peptides.     

Functional effects of alcohol withdrawal syndrome on peripheral sympathetic neurotransmission in vas deferens of adult rats

Aims

Alcohol withdrawal syndrome (AWS) is characterized by a set of physiological modifications triggered by abrupt withdrawal and/or decreasing consumption of ethanol (EtOH), which may manifest 16–48 h after ceasing consumption. The relationship between the effects of AWS and central and peripheral sympathetic neurotransmission is unknown. This study investigates the possible mechanisms on the sympathetic system during periods of AWS.

Main methods

Male Wistar rats were treated with EtOH (6–10 g/kg/day/v.o. 5 days). Subsequently, 1 h, 24 h, 48 h and 120 h after administration of the last dose of EtOH, the animals were sacrificed, and their vas deferens (VD) were removed to perform the following evaluations: (a) concentration–effect curves of sympathetic agonist; (b) activity of α₂-adrenoreceptor; (c) function of voltage-dependent calcium channels (Cav); and (d) release of endogenous catecholamines measured in real time coupled to HPLC.

Key findings

The results showed that the maximum effects of contraction were increased by agonists tested in at 24 h and 48 h EtOH withdrawal. The inhibitory affinity (pIC₅₀) of guanfacine was decreased 24 h EtOH withdrawal. The function of Cav was also decreased as pIC₅₀ values dropped 24 h and 48 h EtOH withdrawal. The release of catecholamines increased 48 h after EtOH withdrawal. It is suggested that AWS triggers hyperactivity in peripheral sympathetic neurotransmission.

Significance

The mechanisms underlying hyperactivity are possibly explained by a failure of autoregulation from catecholamines released by α₂-adrenoreceptors and/or an increase of Cav function, which may be potential targets to attenuate the symptoms of AWS at the peripheral level.     

In-vitro prostanoid production by the rat vas deferens

Prostaglandins (PGs), E-2, F-2 alpha, 6-keto-F-1 alpha and thromboxane B-2 (TXB-2), produced in vitro by the vas deferens of rats at different stages of sexual maturation (10, 35 and 100 days of age), were estimated by radioimmunoassay. Reversed-phase high-performance liquid chromatography was used to evaluate the RIA and to give a more complete profile, after incubation of the vas deferens with [14C]arachidonic acid. The amount of PGE-2 released into the medium after incubation at 37 degrees C for 5 min increased with age, and was the predominant prostanoid in the adult vas deferens. In prepubertal organs, 6-keto-PGF-1 alpha predominated. TXB-2 was always a minor product. The addition of exogenous arachidonic acid (10 micrograms/ml/20 mg tissue), provoked a higher production of the four compounds. PGE-2 and PGF-2 alpha levels were reduced after castration or hypophysectomy and were re-instated after treatment with testosterone propionate. PGE-2 was much more sensitive to hormonal deprivation than PGF-2 alpha. The production of 6-keto-PGF-1 alpha was not significantly affected by the above treatments.     

Selective receptors for beta-endorphin on the rat vas deferens.

The isolated rat vas deferens, being insensitive to morphine, contains selective binding sites for β-end-orphin. A half-maximal inhibition of twitch tension evoked by electrical stimulation is established with 100 nM β-endorphin, while fragments of β-endorphin, that is, methionine-enkephalin, α- and γ-endorphin, are almost ineffective. The opiate alkaloid etorphine, a powerful inhibitor of guinea-pig ileum and mouse vas deferens, is 100-fold less potent on the rat vas deferens. The unique β-endorphin activity suggests very specific binding sites for this peptide, which cannot be related to the μ- or δ-receptors so far described for opiods on isolated preparations.     

Phosphatidylinositol-cleaving activity in smooth muscle from rat vas deferens

• 1.1. Phosphatidylinositol-cleaving activity was studied in subcellular fractions from smooth muscle of rat vas deferens.
• 2.2. In the presence of calcium ions and deoxycholate most of the endogenous phosphatidylinositol was broken-down in 60 min, whilst the other phospholipids were stable.
• 3.3. The enzymatic activity responsible for this breakdown catalyses a phospholipase C-type cleavage of the glycerol-phosphate bond, the water soluble products from exogenous [³²P]-labelled phosphatidylinositol being d-myoinositol 1:2-cyclic phosphate (702-80%) and d-myoinositol 1-phosphate (202-30%).
• 4.4. Activity was abolished by 1 mM ethanedioxybis(ethylamine)tetra-acetate (EGTA) and in the presence of deoxycholate both the soluble and total particulate fractions showed maximum activity at pH 6.52-6.8. The soluble fraction showed a second peak of activity at pH 5.52-5.8 that was independent of deoxycholate; this was not observed in the particulate fraction.
• 5.5. About two-thirds of the activity was soluble. The remaining activity was particulate, with a preferential concentration in the microsomal fraction.

     

Comparative effects of somatostatin and enkephalins on the guinea pig ileum and the rat vas deferens

The action of somatostatin (SRIF (somatotrophin release inhibiting factor)) was compared with that of Met-enkephalin (Tyr-Gly-Gly-Phe-Met) in the electrically stimulated guinea pig ileum myenteric plexus longitudinal muscle and with that of an enkephalin analogue (FK 33-824 (Tyr-D-Ala-Gly-MePhe-Met-(O)-ol)) in the rat vas deferens. In both tissues SRIF produced a twitch inhibition which was not antagonized by naloxone and which showed a long-lasting tachyphylaxis. The enkephalins tested produced a naloxone-antagonizable inhibition of twitch in both tissues but no tachyphylaxis. Therefore we conclude that SRIF is not acting at opiate receptors in these tissues.     

Hormonal influence on rat vas deferens responses induced by field stimulation

The effect of castration and treatment with testosterone propionate (PT), estradiol benzoate (E2) and ciproterone acetate (CPA) on the responses of the vas deferens in rat has been studied. Castration produces a time-dependent decrease of the response amplitude. PT augments the response amplitude in normal rats and reverses the effect of castration. E2 augments the response amplitude in normal rats without modifying the castration effect. CPA does not change the response of the vas deferens. The results suggest that PT and E2 through possible different mechanisms, facilitate the transmission in rate vas deferens, whereas castration obstructs it.     

Testosterone stimulates prostanoid production by rat vas deferens.

The rat isolated vas deferens produces and releases prostanoids into an incubation medium. Production of these substances from the exogenous precursor 14C arachidonic acid was studied in prepubertal, pubertal and adult animals. Synthesis of prostaglandin F, prostaglandin E, prostaglandin D and thromboxane B2 is lower in prepubertals arid increases significantly in pubertals, with no further modifications in adults. Castration of pubertals and adults dramatically reduces the production of all measured arachidonic acid metabolites but does not modify it in prepubertals. Replacement therapy with testosterone propionate significantly enhances prostanoid production in pubertal and adult castrated rats. Similar treatment on normal prepubertals also increases synthesis, indicating that androgens could be modulators of prostanoid synthesis in vas deferens. The lower effects obtained treating castrated adults with progesterone and with 17-beta estradiol suggest an action, at least partially specific for androgenic steroids. It is concluded that prostanoid production by the rat vas deferens from an exogenous precursor is closely related to the presence of androgens.     

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.     

Interaction between 2-chloroadenosine and alpha-adrenoceptors in rat vas deferens

The effect of 2-chloroadenosine (2CA) on the binding of alpha 1- and alpha 2-adrenoceptor ligands in the rat vas deferens was investigated. In homogenates of vas deferens, 2CA (10(5)M) increased 3H-clonidine maximal binding sites from an undetectable level to 0.71 +/- 0.08 pmol/g. wet weight or 10.1 +/- 1.1 fmol/mg protein (N=12). This effect lasted for at least 5 hours after removal of 2CA. Concurrent addition of 1.25 mM theophylline completely abolished the effect of 2CA. A similar effect of 2CA on 3H-clonidine binding was observed following incubation of intact tissues with 2CA prior to homogenization. The effect of 2CA were similar in potency in the homogenate to that in the intact organ, suggesting that 2CA-sensitive sites are located on the outer surface of the plasma membrane. The binding of 3H-prazosin was not influenced by the presence of 10(-5)M 2CA. Contractions of isolated vasa deferentia induced by norepinephrine and phenylephrine were not changed by 10(-5)M 2CA, but the inhibition by clonidine of contractions induced by electric stimulation was enhanced by preincubation for 30 min with 10(-5)M 2CA. The results suggest that 2CA increases the number of available alpha 2-adrenoceptors and this interactions occurs, at least in part, presynaptically.     

Zinc and copper content in rat epididymis and vas deferens

The zinc and copper content in the different epididymal segments and vas deferens of castrated rats were investigated with the help of atomic absorption spectrophotometer. The vas deferens showed maximum zinc content as compared to that of different parts of epididymis in all groups whether castrated unilaterally, bilaterally or in the intact control. Zinc content was reduced in the epididymis and vas deferens ipsilateral to the castrated side as compared to that of contralateral control and intake animals. Lowest zinc content was observed in the epididymis and vas deferens of bilaterally castrated animals from that of other groups. Absence of sperms was observed in all segments of epididymis and vas in bilaterally castrated animals and from the unilaterally castrated side. Copper content was unaltered in all epididymal segments and vas deferens. There appears to be a correlation between the absence of sperms in the male genital tract and the decrease in zinc content.     

The effect of sympathomimetic drugs on contractility of the vas deferens in vitro and in vivo.

The sympathomimetic drugs noradrenaline, methoxamine, tyramine and norephedrine caused rhythmic contractions in isolated human vasa deferentia. Provided the drug was not washed out, these contractions lasted for the entire duration of the experiment (4-6 h). These contractions were mediated via alpha-adrenoreceptors. Intravenous administration of methoxamine or oxymetazolene to rats or guinea-pigs produced contractions of the vas deferens in vivo in some experiments but was accompanied by severe cardiovascular side effects. A local method of application was developed, using mixtures of tyramine with Silastic prepared as collars specially designed to fit round the vas deferens. Acute and chronic insertion of these slow-releasing devices around the vas deferens of rats produced rhythmic contractions of the vas deferens without any serious side effects.