Role of alpha-adrenergic receptors in denervation supersensitivity of rat vas deferens

Contractile responses of rat vas deferens were studied with particular attention directed to the role of receptors and neuronal control. Marked contraction of the vas deferens was observed with alpha-adrenergic agonists, depending on their concentrations. This tissue had a low sensitivity to ACh. Four days after denervation, this tissue showed a supersensitivity to alpha-adrenergic agonists and a high K+ concentration, but not to ACh. The increase in sensitivity to alpha-agonists resulted in an enhancement of the maximal response and a shift of the concentration response curve to lower concentrations of these reagents. Alterations were seen in the alpha-adrenergic receptors in the rat vas deferens, assayed by measuring the binding of [3H]WB4101. The maximal binding sites decreased significantly to 86 from 142 fmoles per mg protein. The affinity of the receptors for alpha-agonist, determined by measuring the ability of agonists to displace bound [3H] WB4101, increased significantly, while the affinity to alpha-antagonists remained unchanged. Studies on [3H]QNB binding indicated no significant change in muscarinic ACh receptors after denervation. Thus, supersensitivity of the alpha-adrenergic mechanism mediated by a specific change in affinity of alpha-receptors occurs after denervation of rat vas deferens. These changes in sensitivity and in receptors are discussed in relation to the characteristics and roles of alpha-receptors in the rat vas deferens.     

Absence of denervation supersensitivity to neurokinin A in the rat vas deferens.

1. Unilateral denervation of the rat vas deferens (RVD) was performed under anesthesia. The animals were allowed to recover 4 or 10 days and then concentration-effect (C-E) curves to noradrenaline (NA) and neurokinin A (NKA) were constructed in denervated and control RVD. 2. Tissues denervated 4 or 10 days produced NA responses shifted 20-fold to the left with maxima 130% of control. 3. NKA C-E curves in denervated RVD were not significantly different from control. 4. Phenylephrine exhibited a 6-fold increase in tissue sensitivity after denervation. 5. Chronic denervation of the vas deferens resulted in significant postsynaptic supersensitivity to alpha 1-adrenoceptor agonists but not to NKA.     

A role for protein kinase C in the supersensitivity of the rat vas deferens following chronic surgical denervation

Chronic surgical denervation of the rat vas deferens leads to an enhanced contractile response of the tissue to norepinephrine in vitro. Norepinephrine produces a higher rate of protein kinase C translocation to the particulate fraction of denervated tissues as compared with the paired, control vas deferens. Diacylglycerol generation in response to norepinephrine and contractile responses to phorbol diacetate were not altered by chronic denervation of the vas deferens. However, the contractile response to norepinephrine in these tissues was less susceptible to the inhibitory effects of the calcium channel blocker nifedipine. A potential role of protein kinase C in sensitizing the contractile apparatus to mobilized calcium in denervation supersensitivity is discussed.     

In vitro denervation of the rat vas deferens through hypothermic storage.

1. The rat vas deferens was excised, stored at 4-6 degrees C and tested after 24, 48, 72 or 96 h for its contractile activity and for the presence of innervation. 2. The maximal contractile capacity of the vas, tested through cumulative concentrations of barium chloride (3 x 10(-2) M) was progressively reduced from about 110 mm to about 63 mm after 72 h, without further decay after 96 h. Spontaneous rhythmic contractions were practically absent. 3. A loss of endogenous pools of catecholamines was indicated by four parameters: (a) a decline of about 80% after 24 h and of more than 95% after 48 h of the contractile effect of the indirect sympathomimetic agonist tyramine; (b) a fall of about 20%, 50% and 85% on the concentration of noradrenaline, respectively after 24, 48 and 72 h; (c) a fall of about 25% and 90% after respectively 24 and 48 h, of the activity of dopamine-beta-hydroxylase (DBH); (d) a decline of noradrenaline-induced histofluorescence on cross sections of the vas. 4. A loss of neuronal uptake capacity was indicated by: (a) a progressive variation of the apparent affinity for adrenaline, expressed as pD2 values, that increased by about 1.5 log units (corresponding to a 30 fold potentiation) after 72 h, and (b) a reduction of the ability of cocaine to potentiate the contractile effects of adrenaline. 5. The pD2 values for barium chloride, 5-hydroxytryptamine (5-HT) and histamine were not significantly changed, while the corresponding value for acetylcholine was slightly but significantly reduced by about 0.8 log units.(ABSTRACT TRUNCATED AT 250 WORDS)     

Noradrenergic supersensitivity in the rat vas deferens during barbital withdrawal

Three days' withdrawal from long-term barbital administration results in a shift to the left and increase in the maximum response of the concentration--effect curve to norepinephrine in the isolated rat vas deferens. The maximum response to carbachol and potassium was also increased without a leftward displacement of the concentration--effect curves. This specific increase in sensitivity to norpinephrine could indicate an adaptive alteration of the adrenoreceptors of the rat vas deferens (receptor supersentivity) as has been proposed for supersensitivity in the central nervous system during withdrawal from neuroleptics and barbiturates.     

Parasympathetic denervation supersensitivity in the rat iris sphincter muscle: an in vitro study

Supersensitivity in chronically sympathectomized smooth muscle has long been studied, but little is known about chronically parasympathectomized smooth muscles. Iris is one of the few smooth muscle organs which can be parasympathetically denervated. We used rats because the denervation supersensitivity in the vas deferens has extensively been studied in this species. The rat was unilaterally ciliary-ganglionectomized. After 1-13 days, strips of the iris sphincter muscle obtained from denervated eye were tested for the sensitivity to acetylcholine (ACh), 5-hydroxytryptamine (5-HT) and K+. A significant increase in sensitivity to ACh and 5-HT was recognized as early as 1 day after the operation, and the sensitivity reached a maximum after 5-6 days, when ED50 values of ACh and 5-HT were reduced to approximately 1/28 and 1/4, respectively. The sensitivity to K+ was unaffected. When the sensitivity reached the maximum, the maximum responses to ACh, 5-HT and K+ were increased by 50%, 140% and 50%, respectively. The shift of the dose-response curve for bethanechol (BeCh) after denervation was comparable to that for ACh. Physostigmine shifted the curve for ACh, but did not shift that for BeCh either before or after denervation. Specific binding of 3H-quinuclidinyl benzylate tended to decrease after denervation, but the change was statistically not significant. These results indicate that the parasympathetic denervation supersensitivity in the rat iris sphincter involves a non-specific component, in addition to one specific to ACh and BeCh. The specific component seems to involve postsynaptic mechanisms.     

Effects of denervation and reserpine on nexuses in the rat vas deferens

Permanganate-fixed vasa deferentia from rats were examined for the presence of nexal-like contacts by electron microscopy. There was a significantly greater incidence of nexuses (2×) in chronically denervated tissues (5–7 days) but not in tissues from reserpine-pretreated animals (1.0 mg/kg/day for 5–7 days). The results suggest that an increase in nexal regions may not be a general feature of postjunctional supersensitivity but rather may contribute to other denervation-induced changes in contractile response.     

Participation of protein synthesis in development of supersensitivity in cultured rat vas deferens

Organ culture of rat vas deferens produced supersensitivity to norepinephrine and acetylcholine in contractile response without change in alpha 1-adrenergic and muscarinic acetylcholine receptors. The development of supersensitivity was inhibited by low temperature and protein synthesis inhibitors. However, protein synthesis inhibitor had no significant effect on the receptors. These findings suggested that the supersensitivity may be induced by synthesis of protein(s) which have a stimulatory effect in a process(es) after activation of receptor to contraction.     

Effects of various denervation techniques on the ATP of the rat vas deferens.

Postganglionic denervation of the vas deferens of the rat by surgical means or pretreatment with 6-hydroxy-dopamine results in a substantial decrease in the concentration of ATP in the tissue. The loss of ATP cannot entirely be accounted for by the loss of neuronal ATP nor by a compromised blood supply. The results suggest that the postganglionic nerve plays an important role in the regulation of the ATP of the effector cells.     

The effect of denervation on the synchronization of contraction of the rat vas deferens

Denervation of the rat vas deferens results in both quantitative and qualitative changes in the contraction of the isolated smooth muscle. The maximum contraction induced by norepinephrine, acetylcholine or serotonin is enhanced by a denervation. The response of the tissue to 2 × 10^?4 M BaCl₂ is also larger after denervation and in addition the character of the contraction is changed. In the denervated muscle BaCl₂ initiates a contraction which is more synchronized than the response of the control tissue. By use of ¹⁴C-sorbitol and Co-EDTA for estimating extracellular space and from fluorescence histochemical observations, it appears that facilitation of drug diffusion through the extracellular space plays an unimportant role in the altered response to drugs. In experiments which utilized a double-compartment tissue bath it was found that denervation enhanced the propagation of the contraction in the longitudinal direction. From measurements of the longitudinal tissue impedance it was determined that the resistance between smooth muscle cells was significantly decreased by denervation. These results suggest that denervation improves the propagation of excitation in this smooth muscle.     

Nonspecific supersensitivity of the guinea-pig vas deferens produced by decentralization and reserpine treatment

1. The sensitivity of the guinea-pig vas deferens to noradrenaline, histamine, methylfurmethide and potassium was examined in vitro following decentralization and reserpine treatment.2. One day after decentralization or administration of reserpine (1.0 mg/kg daily) the sensitivity of the vas deferens was not increased. After 5 days' treatment the muscle was supersensitive to all four stimulants.3. The magnitude of the sensitivity increase to an individual drug was the same following both chronic reserpine treatment and decentralization. However, the degree of supersensitivity differed for the four stimulants. The order of potentiation was noradrenaline>histamine>methylfurmethide>potassium.4. The magnitude of the supersensitivity was inversely correlated with the slope of the dose-response curves to the four agonists. The dose-response curve to potassium had the steepest slope, followed in order by methylfurmethide, histamine and noradrenaline.5. A hypothesis is presented to account for the inverse relationship between the slope of the dose-response curve and the degree of supersensitivity which follows reserpine treatment or decentralization.     

Nitric oxide as neuromodulator of sympathetic transmission in rat vas deferens

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Autoradiographic study of the rat vas deferens incubated with 3H-noradrenaline

Summary Rat vasa deferentia were incubated with 0.2 mol/l ³H-noradrenaline for 60 min and then washed out with amine-free solution for 100 min. Autoradiography then revealed a preferential labelling of the varicosities in the immediate vicinity of the surface of the tissue. However, when tissues were obtained from reserpine-and pargyline-pretreated rats (to block vesicular uptake and monoamine oxidase), ³H-noradrenaline was able to penetrate more deeply into the tissue. These differences are in accordance with the view that the autoradiographs reflect the ³H-noradrenaline concentration gradient (within the extracellular space) generated by the neuronal uptake of the 3H-amine; the concentration gradient is steeper (and the heterogeneity of labelling is more pronounced) in tissues with intact vesicular uptake and monoamine oxidase than in tissues in which these mechanisms had been inhibited.Send offprint requests to I. Azevedo at the above adress     

Lack of evidence for epsilon-opioid receptors in the rat vas deferens

Experiments were performed to test the hypothesis that the field-stimulated rat vas deferens preparation contains opioid receptors, other than of mu-type, which mediate part of the inhibitory effect of beta-endorphin. The Piebald Viral Glaxo strain of rats was used. The reported finding that delta-opioid receptors are present in Sprague-Dawley rat vas deferens, the effects of which are greatly enhanced in reduced calcium concentrations, could not be replicated in the rat strain used. Reducing the calcium concentration from 2.5 to 1.25 mM improved the response to opioid drugs: all full agonists were about 10 times more potent, the partial agonist normorphine became able to inhibit the twitch completely, and morphine (which behaves as a competitive antagonist in 2.5 mM Ca2+) appeared to behave as a partial agonist. The pA2 values for antagonism by naloxone in low calcium of the mu-selective peptide [D-Ala2,MePhe4,Gly(ol)5]enkephalin and other mu- or delta-selective agonists were consistent with an action at mu-receptors only. The value for beta-endorphin was slightly but significantly lower. A similar small discrepancy was found with two other competitive antagonists. The discrepancy remained in the presence of the peptidase inhibitors thiorphan, bestatin and bacitracin. Responses to both [D-Ala2,MePhe4,Gly(ol)5]enkephalin and beta-endorphin were attenuated by the irreversible antagonists beta-funaltrexamine and beta-chlornaltrexamine.(ABSTRACT TRUNCATED AT 250 WORDS)