Bicarbonate-dependent action of Bay K 8644 in the smooth muscle of the rat vas deferens.

Effects of Bay K 8644, partial depolarization with high potassium, and nifedipine on the dose-response curves of the rat vas deferens to norepinephrine, methacholine and KCI were investigated in HEPES-buffered physiological salt solution (PSS) with or without 20 mM sodium bicarbonate. In the bicarbonate-containing PSS, Bay K 8644 at 10(-6) M enhanced the maximal contractions in response to norepinephrine, methacholine and KCI by 31.4, 103.3 and 40.1%, respectively. In the bicarbonate-free PSS, where the maximal contractions induced by norepinephrine, methacholine and KCI were 77.5, 75.0 and 68.2% of those in the bicarbonate-containing PSS, respectively. Bay K 8644 did not enhance the maximal contractile response to any of the agonists, although the contractions induced by low concentrations of KCI were increased by Bay K 8644. Increasing the potassium concentration in the PSS from 6 to 20 mM enhanced the maximal contractions in response to norepinephrine and methacholine in the bicarbonate-containing PSS, whereas in the bicarbonate-free PSS, the treatment decreased the contractions. In the two PSSs, nifedipine similarly inhibited the contraction in response to either 10(-5) M norepinephrine or 68 mM KCI. These results suggest that bicarbonate ion modulates the function of the voltage-dependent calcium channel in the smooth muscle of the rat vas deferens.     

Pre- and postjunctional muscarinic receptor subtypes in the vas deferens of rat

• 1.1. A pharmacological study of the pre- and postjunctional muscarinic receptors of the isolated rat vas deferens was carried out using more selective agonists and antagonists.
• 2.2. The prejunctional receptor was characterized on electrically stimulated preparations, while the postjunctional receptor was studied on vasa deferentia without stimulation.
• 3.3. The results indicate that atropine exhibited a similar affinity for the two populations of muscarinic receptor subtypes of this tissue.
• 4.4. 4-DAMP was able to differentiate with high affinity a subtype located at postjunctional level which had pharmacological similarities with the M₃-ACh subtype and with low affinity a subtype located at prejunctional level.
• 5.5. The selective M₁-ACh agonist McN-A-343 was not able to activate the postjunctional receptor, but showed a similar affinity to ACh for the prejunctional one.
• 6.6. At present, the prejunctional receptor can be considered as an atypical M₁-ACh subtype based on the results obtained with the selective drugs available.

     

Involvement of postjunctional purinergic mechanisms in the facilitatory action of bradykinin in neurotransmission in the rat vas deferens

To investigate the nature of the bradykinin-induced potentiation of electrically driven muscle twitches in the isolated vas deferens, bradykinin, noradrenaline and adenosine 5'-triphosphate (ATP) concentration-response curves were made with control, reserpinized and chemically sympathectomized rats. Bradykinin potentiated the ATP- but not the noradrenaline-induced contractions in the epididymal and prostatic segments of the ductus. The epididymal segment of the ductus did not respond to transmural electrical stimulation following reserpine treatment. Bradykinin potentiated the muscular contractions caused by exogenous ATP but not by noradrenaline. In contrast, the transmurally evoked twitches of the prostatic portion of the ductus remained almost unaltered; bradykinin increased the motor effect of ATP without modifying the potency of noradrenaline. All neuronally induced contractile activity was absent in sympathectomized rats; bradykinin potentiated the contractile effect of ATP without altering the noradrenaline-induced contractions. These results suggest that bradykinin potentiates the ATP-evoked contractions by acting postjunctionally.     

Some pharmacological properties of the circular smooth muscle layer of the rat vas deferens

Vas deferens preparations were perfused in-vitro through the lumen and externally with a modified Tyrode solution alone or containing drugs. Contractions of the circular (internal) smooth muscle layer were recorded as changes in the pressure of internal perfusion. Contractions of the longitudinal (external) layer were simultaneously recorded through a tension transducer. When the organ was perfused through the lumen, the circular layer contracted after addition of methacholine (pD2 = 4.13), and noradrenaline (pD2 = 5.00), and relaxed after addition of isoprenaline (pD2 = 5.22). These effects were also observed when the drugs were perfused externally, although with lower values of pD2 for noradrenaline and methacholine. The circular fibres were less sensitive when compared with the longitudinal fibres perfused externally with the above agonists. Methacholine-induced contractions of the circular layer were competitively antagonized by atropine (pA2 = 8.53), indicating the presence of muscarinic receptors. The effects induced by noradrenaline and isoprenaline were antagonized by indoramin (pA2 = 7.78), and timolol (pA2 = 8.68), respectively, indicating the presence of alpha- and beta-adrenoceptors. The effect of noradrenaline was potentiated by cocaine and denervation, indicating the presence of neuronal uptake, and by corticosterone, indicating the presence of extraneuronal uptake in the circular layer.     

Inhibitory action of propranolol on the contractions induced by nerve stimulations or calcium in the smooth muscle of rat vas deferens

Inhibitory effects of propranolol on the contractions to various treatments were investigated in the epididymal half of the rat vas deferens. Reportedly, 10(-5)-3 X 10(-4) M propranolol inhibited 150 mM K-induced contractions dose-dependently; 3 X 10(-4) M propranolol abolished the contractions. The present results showed that propranolol at concentrations up to 10(-4) M did not inhibit the maximal contractions to 10(-3) M norepinephrine (NE) or 10(-2) M methacholine (MCh). Propranolol at 3 X 10(-4) M slightly inhibited contractions to NE and MCh by 11% and 12%, respectively. In contrast, propranolol inhibited twitch components of the contractions induced by nerve stimulations at similar doses to those reported for high K contractions. Propranolol also inhibited contractions to Ca in high K-containing solution and shifted the dose-response curve to the right. Propranolol did not affect the depolarizations by high K measured by microelectrodes. Propranolol at concentrations of 10(-5)-3 X 10(-5) M diminished the magnitude of spikes dose-dependently. Spikes were rarely observed in the presence of 10(-4) M propranolol in spite of generation of e.j.p.s with amplitudes that would be sufficient to induce spikes in the absence of propranolol. These results suggest that propranolol inhibits contractions by decreasing Ca-influx through the potential-operated Ca-channels in the smooth muscle cells of rat vas deferens.     

Partial agonistic action of morphine in the rat vas deferens

Effects of morphine on the force of contraction of rat vas deferens were investigated. Morphine and beta-endorphin decreased the electrically evoked twitch tension, in a dose dependent manner. The inhibitory effect of morphine, however, was much weaker than that of beta-endorphin. These effects of both morphine and beta-endorphin were completely antagonized by naloxone. In the presence of 30 microM morphine, the dose-response curve of beta-endorphin shifted to the right by about 10-fold. Moreover, morphine partly reversed the contraction depressed by 0.3 microM beta-endorphin, in a dose dependent manner. These findings suggest that morphine acts as a partial agonist on the rat vas deferens. Marked tolerance to beta-endorphin and change in the antagonist potency of morphine were not observed in the vas deferens isolated from morphine-dependent rats.     

The ontogenetic profiles of the pre- and postjunctional adenosine receptors in the rat vas deferens.

1. The ontogenetic profiles of the prejunctional A1 and postjunctional A1 and A2 receptors on the rat vas deferens were investigated, using a combination of functional and radioligand binding assays to follow the A1 receptors and functional assays alone to follow the development of the A2 receptors. 2. The prejunctional A1 receptor, assessed by the inhibitory action of N6-cyclopentyladenosine (CPA) (3 nM-3 microM) on nerve-mediated contractions, was present from day 15 onwards, day 15 being the earliest age at which nerve-mediated contractions could be detected. The potency of CPA was constant across the ages studied, with pD2 values ranging from 6.4-7.1, not significantly different from that previously observed in adult rat vas deferens. 3. The postjunctional A2 receptors, assessed by the inhibitory action of 5'-N-ethylcarboxamidoadenosine (NECA) (10 nM-30 microM) on KCl-induced contractions were present from day 10 onwards, day 10 being the earliest age at which responses to KCl could be observed. The potency of NECA remained constant with an increase in age, with potency values, expressed as pEC25 values, ranging from 6.5-7.0. 4. The postjunctional A1 receptor displayed a different development profile from that of the prejunctional A1 and postjunctional A2 receptors. Postjunctional A1 receptors were identified by the enhancement of KCl-induced contractions by CPA (10 nM-0.3 microM). At 10 and 15 days, CPA failed to enhance KCl-induced contractions. From day 20 to day 40, this enhancement increased with an increase in age and the level of enhancement achieved statistical significance from day 30. 5. Radioligand binding studies using 1,3-[3H]-dipropyl-8-cyclopentylxanthine ([3H]-DPCPX) revealed binding sites characteristic of A1 receptors on the vas deferens from rats aged 20 days onwards. The density (Bmax) of A1 receptors expressed relative to protein content was greatest at day 20 (153 +/- 33 fmol mg-1 protein) and declined at day 30 (43.9 +/- 3.7 fmol mg-1 protein) to a level commensurate with that previously determined in adult rat vas deferens (43.3 +/- 12 fmol mg-1 protein). However, when expressed relative to tissue wet weight little variation in receptor density was observed between these ages (Bmax 0.13 +/- 0.02 fmol mg-1 wet weight at 20 days; 0.17 +/- 0.01 fmol mg-1 wet weight at 30 days). The binding affinity (KD) remained constant with an increase in age and was similar to the KD value previously generated for adult rat vas deferens (approximately 1 nM). At ages 10 and 15 days no reproducible binding could be detected. 6. These results show the differential development of the adenosine receptors on the rat vas deferens with postjunctional A1 receptors demonstrating delayed development, while prejunctional A1 and postjunctional A2 receptors were present from the earliest ages studied. In addition, comparison of binding studies and functional studies suggests that the binding studies detect only the A1 receptors present on the smooth muscle and not those present on the nerve terminals.     

Pre- and postjunctional effects of N-ethylmaleimide in the isolated mouse vas deferens

The influence of N-ethylmaleimide (NEM) on contractions due to exogenously applied noradrenaline and bethanechol and on the inhibitory effects of clonidine, of the enkephalin derivative, FK 33-824, and 2-chloroadenosine (2-CLA) on field stimulation-response curves and [3H]noradrenaline [( 3H]NA) release was studied in the isolated mouse vas deferens. Exposure to NEM (60 microM: 10 min) caused a 30% reduction of the maximal contraction due to NA but nearly abolished the response to bethanechol. NEM partially reversed the depression of the pulse width-response curves by clonidine and FK 33-824 but was without effect with 2-CLA. The contractions evoked by stimulation frequencies above 20 Hz were depressed by NEM both in presence and absence of the agonists. NEM diminished the inhibition of the stimulation-evoked release of [3H]NA by the three agonists. The prejunctional effect of NEM was markedly influenced by the stimulation parameters. These findings support the suggestion that the inhibition mediated by alpha 2-adrenoceptors, mu- and P1-receptors in the mouse vas deferens is NEM-sensitive and possibly transmitted by a pertussis toxin-sensitive G-protein.     

Purinergic modulation of field stimulation responses of rat and human vas deferens smooth muscle.

1. Guanethidine at 5 x 10(-6) M strongly inhibited rat prostatic but not epididymal vas deferens, reflecting differences in innervation and the neurogenic field stimulation responses of these tissues. 2. Adenosine and ATP inhibited the field stimulation responses of rat prostatic vas deferens by 56 and 50% respectively. A 10-min pretreatment with 10(-4) M caffeine partly reversed this inhibition, by 55% in the case of adenosine and 60% for ATP. 3. Pretreatment for 10 min with 5 microM quinidine failed to significantly alter the extent of either adenosine or ATP inhibition of the field stimulation responses of rat prostatic vas deferens. 4. 8-Phenyltheophylline, the selective blocker of the A1 subtype of the P1 receptor, partly reversed adenosine-induced inhibition of the vas deferens FS responses. NECA, the selective agonist of the A2 subtype of the P1 receptor, very strongly inhibited vas deferens FS responses. 5. Field stimulation responses of human vas deferens were also inhibited by both adenosine and ATP but to a lesser extent and more variably than in rat tissue. 6. Adenosine and ATP inhibition was reversed by caffeine pretreatment, but far more variably than in rat tissue, and quinidine was without significant effect on inhibition of the responses. 7. It is concluded that in these tissues adenosine and ATP may operate via a P1 type receptor of both A1 and A2 subtypes and that a P2 type receptor may be lacking.     

Neurogenic contractile responses of the circular smooth muscle of the guinea-pig vas deferens

Contractile responses of the circular smooth muscle of the guinea-pig vas deferens to electrical stimulation were recorded isometrically using ring preparations of about 2 mm in breadth. Stimulation of low frequency (2.5 to 10 Hz, 0.05 msec duration) for 20 sec produced twitch contractions which occurred once or repetitively during the application of stimulation. Higher frequency stimulation (20 to 80 Hz) produced biphasic contractions, an initial phasic and a secondary tonic contraction. These contractions were abolished by 3 X 10(-6) M tetrodotoxin or 3 X 10(-5) M guanethidine; however, 3 X 10(-6) M atropine or hexamethonium did not affect the contractions. Prazosin at 10(-6) M, like 3 X 10(-7) M yohimbine, increased the amplitudes of twitch contractions to the low frequency stimulation and caused the twitch contractions to occur repetitively. On the other hand, prazosin suppressed the tonic contractions to the high frequency stimulation without substantially inhibiting the phasic contractions. Cocaine at 3 X 10(-6) M potentiated the twitch contractions in the presence of yohimbine. After in vivo reserpine treatments, the low and high frequency stimulation produced twitch and phasic contractions, respectively; however, tonic contractions were not induced. Prazosin at 10(-6) M did not qualitatively affect the contractions in preparations from the reserpine-treated animals. These results suggest that the neurogenic contractions of the circular muscle of the guinea-pig vas deferens are sympathetic in nature, but that they are not mediated solely by norepinephrine. Co-release of other transmitters was indicated to occur upon the electrical stimulation of wide frequency range.     

Blockade by calmodulin inhibitors of Ca2+ channels in smooth muscle from rat vas deferens.

1. Effects of three compounds which are used as calmodulin inhibitors (trifluoperazine, W-7 and calmidazolium) on Ca2+ channels were investigated in smooth muscle from rat vas deferens. 2. All three calmodulin inhibitors relaxed the smooth muscle precontracted by a high concentration of KCl (63.7 mM). The order of potency for the relaxation was trifluoperazine > W-7 > calmidazolium. 3. In binding studies using a microsomal fraction of vas deferens, all these calmodulin inhibitors displaced specific [3H]-nimodipine binding. Trifluoperazine and W-7 inhibited the binding at concentrations that relaxed the smooth muscle whereas calmidazolium inhibited at concentrations much lower than those necessary for muscle relaxation. 4. Ba2+ current flowing through voltage-gated Ca2+ channels was measured under whole-cell voltage-clamp conditions in isolated smooth muscle cells. The Ba2+ current was suppressed by the three calmodulin inhibitors in the concentration-range where inhibition of [3H]-nimodipine binding was observed. Neither voltage-dependence nor the inactivation time course of Ba2+ current were affected by these compounds. 5. The results suggest that the calmodulin inhibitors directly block Ca2+ channels in the smooth muscle cells. The channel inhibition by trifluoperazine and W-7, but perhaps not that by calmidazolium, may be responsible for the muscle relaxation observed with these compounds.     

Neurokinin A suppresses a voltage-gated K+ current in smooth muscle cells from rat vas deferens.

The effects of neurokinin A (NKA) on freshly isolated smooth muscle cells from the rat vas deferens were investigated electrophysiologically. NKA (10(-7) M) prolonged the duration of action potentials without affecting resting potentials. Under voltage-clamp conditions, NKA (10(-7) M) suppressed an outward K+ current activated by depolarizing voltage steps. Substance P (SP, 10(-7) M) did not affect the K+ current, suggesting that NKA is more potent in suppressing the K+ current than SP is, as is also true for the contractions of smooth muscles of the rat vas deferens. The suppression of the K+ conductance now reported may contribute to NKA-induced smooth muscle contraction.     

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.     

Post-receptor pathway of the ATP-induced relaxation in smooth muscle of the mouse vas deferens.

1. The post-receptor pathway of the ATP relaxant effect in K(+)-precontracted vas deferens smooth muscle (VD) was examined. 2. The relaxation to ATP was not antagonized either by 10 microM methylene blue, a cyclic GMP inhibitor, by 10 microM indomethacin, an inhibitor of prostaglandin synthesis or by 100 microM NG-nitro-L-arginine, an inhibitor of NO production. 3. The Rp-diastereomer of adenosine 3':5'-cyclic monophosphorothioate (Rp-cAMPS) 200 microM, a competitive inhibitor of cyclic AMP significantly diminished the relaxant response to ATP. 4. Isoprenaline 10 microM, a beta-adrenoceptor agonist, produced a sustained relaxation, inhibited by Rp-cAMPS, without a significant change in [Ca2+]i, thereby mimicking the ATP-induced relaxant effect. 5. The level of the phosphorylated myosin light chain in the precontracted VD was significantly lowered by 1000 microM ATP. 6. ATP (1000 microM) and isoprenaline (10 microM) produced the same increase (+ 50%) of [cyclic AMP] when applied to a resting VD. 7. The effect of simultaneous increases of [Ca2+]i and of [cyclic AMP] produced by externally applied ATP are discussed. 8. These results suggest that ATP-induced relaxation in K(+)-precontracted VD is mediated by the activation of adenylyl cyclase.     

Calcium mobilization and phosphatidylinositol turnover in vas deferens smooth muscle of diabetic rats

The study concerned Ca2+ channels that are receptor-operated by norepinephrine (NE) and mediate hyper-reactivity of vas deferens smooth muscle from rats with streptozotocin (STZ)-induced diabetes, and the mediatory responses of these channels, such as tension development, Ca2+ uptake and phosphatidylinositol (PI) turnover. The contractile responses induced by adrenoceptor agonists were significantly greater in diabetic rat vas deferens than in the controls. A greater Ca2+ uptake was induced by 10(-5) M NE in strips from diabetic rats than in the controls. The uptake of Ca2+ was completely inhibited by 10(-6) M prazosin but not by 10(-5) M verapamil. Enhancement of Ca2+ release by 10(-5) M NE was faster and greater in diabetic muscles than in the controls. The accumulation of [3H]inositol phosphates was increased 4-fold in the controls and 7-fold in diabetic muscles by 10(-5) M NE. This increase was completely inhibited by 10(-6) M prazosin but not by 10(-6) M yohimbine. The data suggest that vas deferens smooth muscle hyper-reactivity in diabetic rats is due to increased PI turnover mediated by alpha 1-adrenoceptors, to the release of intracellular bound Ca2+ and to an increase of Ca2+ uptake through receptor-operated Ca2+ channels.