Guinea-pig Isolated Vas Deferens Research Articles

Inhibitory effect of the methanol extract and fractions of <i>Garcinia kola</i> seeds on KCl-induced contractions of isolated guinea pig vas deferens

The objective of the study was to investigate the phytochemical constituents and pharmacological effects of fractions of a methanol crude extract of Garcinia kola seed on chemically induced contractions of isolated guinea pig vas deferens. The extract was obtained by Soxhlet extraction with methanol (80% v/v) at 65 ℃ for 25 h. It was then successively fractionated with solvents of different polarities including n-hexane, chloroform, ethyl acetate and acetone respectively. In the same vein, the fractions were tested individually at a pre-determined dose of 0.4 mg/mL alone and in the presence of KCl-induced contraction of an isolated guinea pig vas deferens at a tension of 0.5 g. Each of the fractions was also screened to determine its phytochemical constituents. The results showed that none of the fractions induced contraction of the isolated guinea pig vas deferens at the dose used. All the fractions demonstrated inhibitory action to KCl-induced contraction of the isolated guinea pig vas deferens, with the residual methanol fraction demonstrating the strongest inhibitory action. Nifedipine (8×10-3 mg/mL), a known calcium channel blocker, expectedly and completely blocked the KCl-induced contraction. The phytochemical screening revealed the presence of cardiac glycosides in all the fractions but the absence of anthraquinones in all. The presence of other phytochemicals including alkaloids, saponins, tannins, flavonoids, steroids and terpenes were separately present in all the fractions. Fractions of a methanol crude extract of Garcinia kola seeds show inhibitory action on KCl-induced contraction of isolated guinea pig vas deferens.Keywords: KCl; Methanol extract; Vas deferens; Guinea pig; Phytochemical

Effect of naloxone on the sensitivity of the vas deferens to various agonists.

The effect of naloxone in vitro, on noradrenaline (NA)-induced responses in guinea-pig isolated vas deferens was studied. The responses of NA were potentiated by naloxone (3 microM). It potentiated the responses to methoxamine which has least affinity for uptake sites. Responses induced by acetylcholine and potassium chloride were also potentiated by naloxone. Low calcium (0.8 mM) in the medium antagonized the potentiating action of naloxone. It is suggested that naloxone produces non-specific potentiation, by promoting calcium influx at the post-synaptic site.

Pre- and postjunctional effects of diadenosine polyphosphates in the guinea-pig vas deferens.

The pre- and postjunctional activities of a number of diadenosine polyphosphates were examined in the guinea-pig isolated vas deferens at the level of the membrane potential, using a modified sucrose-gap technique. P1,P3-Di(adenosine 5')triphosphate (Ap3A), P1,P4-di(adenosine 5')tetraphosphate (Ap4A) and P1,P5-di(adenosine 5')pentaphosphate (Ap5A) all caused concentration-dependent depolarization of the smooth muscle membrane. The potency order was: Ap5A > Ap4A > or = Ap3A. P1,P2-Di(adenosine 5')pyrophosphate (Ap2A) did not evoke depolarization even at the highest concentration tested (1 mM). All the dinucleotides caused a reduction in the amplitude of evoked excitatory junction potentials (e.j.ps). The potency order was: Ap5A = Ap4A > Ap3A > Ap2A. The depolarizations evoked by the dinucleotides were markedly reduced by the selective P2X-purinoceptor antagonist, pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, 10 microM), as was the amplitude of the fully facilitated e.j.p. The inhibition of the e.j.p. evoked by Ap3A and Ap2A was reduced by the P1-purinoceptor antagonist, 8-p-sulphophenyltheophylline (8-pSPT, 50 microM), but that evoked by Ap5A and Ap4A was not. Thus, Ap3A, Ap4A and Ap5A evoke depolarization of the guinea-pig vas deferens via P2X-purinoceptors, and additionally Ap2A and Ap3A exert a prejunctional effect via P1-purinoceptors. The prejunctional activity of Ap4A and Ap5A is mediated via an undefined purinoceptor, which is neither P1 nor P2X.

The guinea-pig isolated vas deferens: a method for increasing sensitivity to drugs.

Abstract A preparation of the guinea-pig vas deferens was developed in which it was opened along its length. It was sufficiently sensitive to obtain reproducible dose-response curves to acetylcholine, noradrenaline, histamine, nicotine, 5-hydroxytryptamine and potassium chloride; it was more sensitive than the unopened vas deferens.

Anti-acetylcholine, and adrenaline-potentiating activity of oxotremorine on guinea-pig isolated vas deferens.

Anti-acetylcholine, and adrenaline-potentiating activity of oxotremorine on guinea-pig isolated vas deferens.

Effect of amphetamine on the uptake, release and effectiveness of xylocholine in the guinea-pig vas deferens.

Abstract Amphetamine sulphate will both reverse and prevent the adrenergic neuron blocking action of xylocholine (TM10 bromide) on the response of the guinea-pig isolated vas deferens to transmural electrical stimulation. A concentration of amphetamine sulphate capable of reversing the effect of xylocholine does not produce a significant reduction in the tissue concentration of 14C-TM10 iodide in the vas deferens. Although amphetamine reduces the rate of uptake of xylocholine, it does not prevent uptake. Comparisons of tissue concentrations with the degree of blockade produced in the normal and the amphetamine-treated vas deferens suggest that if the actions of amphetamine are to be accounted for entirely by displacement of xylocholine or by changes in uptake of xylocholine, only a very small percentage of the total tissue content of xylocholine can be involved in the production of its effects.

Some effects of pilocarpine and acetylcholine on the responses of the guinea-pig vas deferens to hypogastric nerve stimulation and transmural stimulation and applied (–)-noradrenaline

Enhancement by acetylcholine and by pilocarpine of the contractile responses of the guinea-pig isolated vas deferens to hypogastric nerve and transmural stimulation and to exogenous (minus)-noradrenaline was examined. At high concentration (600 nM) acetylcholine enhanced responses in the order:hypogastric nerve greater than transmural greater than exogenous (minus)-noradrenaline. Pilocarpine produced a similar maximal degree of enchancement with all three stimuli. All these effects were reversibly inhibited by 1-4 mum atropine, a concentration which alone did not affected responses to the stimuli. Pilocarpine (50-400 muM) gave a concentration-related inhibition of the enhancement of responses to hypogastric nerve stimulation by 600 nM acetylcholine.

Influence of the length of the stimulus period and frequency of sympathetic stimulation on the response of the guinea-pig isolated vas deferens to bretylium, guanethidine and amphetamine.

Abstract The height of contraction of guinea-pig isolated vas deferens preparations in response to pre- or postganglionic sympathetic nerve stimulation at various stimulus frequencies was shown to vary with varying length of stimulation period. Short stimulation periods, as used by some workers, produced suboptimal contractions especially at the lower frequencies. It is suggested that variations in the length of the stimulation period could account for some of the contradictory results reported by various workers using this preparation. Thus the effect of low concentrations of dexamphetamine was qualitatively changed as the length of the stimulation period was increased; with short stimulation periods dexamphetamine impaired the responses to sympathetic stimulation but enhanced them if stimulation was continued until contractions had reached the maximum height obtainable. Higher concentrations of dexamphetamine impaired the contractions, the effect being most marked against the higher stimulus frequencies. Using stimulation to maximal effect a qualitative difference in the blocking actions of bretylium and guanethidine was demonstrated. Bretylium reduced the responses to the higher stimulus frequencies to a greater extent than it reduced the responses to the lower frequencies. Guanethidine reduced the responses to all frequencies so that there was a parallel shift of the frequency/response curve to the right. The possible significance of these findings is discussed.

Sodium nitroprusside enhances contractions of the guinea-pig isolated vas deferens.

The effects of sodium nitroprusside on the electrical and mechanical properties of the smooth muscle of the guinea-pig vas deferens, and its responses to transmitter substances, have been investigated by use of the sucrose-gap technique. Isolated longitudinal segments of guinea-pig vas deferens contracted in response to electrical field stimulation (100 V, 0.04-0.1 ms, 1-5 Hz, 10 s train every 60 s) and application of ATP (1 mM) or noradrenaline (10 microM). Sodium nitroprusside (0.1 mM) did not affect resting tension but did enhance contractions evoked by electric-field stimulation but not by ATP or noradrenaline. The sodium nitroprusside-induced enhancement was unaffected by the nitric oxide synthase inhibitor, Nomega-nitro-L-arginine methyl ester (L-NAME) (0.1 mM). Conversely, electrically evoked contractions were unaffected by the nitric oxide precursor L-arginine (1 mM) or the nitric oxide donor S-nitroso-N-acetyl-DL-penicillamine (SNAP) (0.1 mM). The amplitudes of electrically evoked excitatory junction potentials (EJPs) were not affected by application of sodium nitroprusside, although it caused a small depolarization of 0.7+/-0.3 mV. Similarly, the depolarization caused by exogenous application of ATP or noradrenaline was unaffected by the presence of sodium nitroprusside. L-NAME, L-arginine and SNAP did not affect EJP amplitude or baseline membrane potential. It is concluded that sodium nitroprusside enhances electrically evoked contractions of the guinea-pig vas deferens by reducing the threshold voltage for action potential firing in smooth-muscle cells.

THE EFFECTS OF BLOCKING AGENTS UPON THE ISOLATED VAS DEFERENS OF THE GUINEA-PIG STIMULATED BY THE HYPOGASTRIC NERVE.

Abstract When the guinea-pig isolated vas deferens preparation was stimulated by its hypogastric nerve, the responses obtained at different frequencies of stimulation were all reduced proportionately by guanethidine, bretylium, reserpine and pentamethonium. The difference between guanethidine and bretylium in affecting the responses to nervous stimulation at different frequencies of the cat nictitating membrane reported by Boura & Green was not observed with this preparation. Papaverine reduced responses to high frequencies of stimulation more than those to low frequencies. Amphetamine also reduced responses to all frequencies uniformly except in the smallest doses used, which potentiated the responses to high frequencies; these doses reversed the reduction in response by guanethidine, bretylium and reserpine. Preparations stimulated between parallel electrodes responded similarly to those stimulated by the hypogastric nerve.

Superoxide Anion Stress Attenuates the Contractile Response of the Guinea Pig Vas Deferens to ATP and Diadenosine Tetraphosphate

Induction of endogenous superoxide anion stress by the use of the superoxide dismutase inhibitor diethylthiocarbamate (DETCA; 10 mmol/l) produced a potent inhibition of the ATP (0.3–10 mmol/l) and diadenosine tetraphosphate (AP₄A) contractile activity in the isolated vas deferens by 29–92 and 24–90%, respectively. Pyrogallol (0.1 mmol/l), the exogenous superoxide anion generator, produced a significant inhibition on the contractile activity of the vas deferens induced by ATP and AP₄A by 33–89 and 25–82%, respectively. DETCA (10 mmol/l) and pyrogallol (0.1 mmol/l) attenuated the contractile response of isolated guinea pig vas deferens strips to the selective P2X agonist α,β-methyleneATP (α,β-meATP; 50 µmol/l) by 25 and 47%, respectively. In Ca²⁺-free high-K⁺ (80 mmol/l) Krebs solution, pyrogallol and DETCA produced inhibition of the contractile response to α,β-meATP (50 µmol/l) in similar way to that in normal Krebs solution. The further addition of CaCl₂ (1 mmol/l) abolished the inhibitory effects exerted by pyrogallol and DETCA. The control contractile response to α,β-meATP (50 µmol/l) was not affected in Ca²⁺-free high-K⁺ (80 mmol/l) Krebs solution. It may be concluded that superoxide anion stress produces a significant inhibitory effect on both mono- and di-nucleotide purinergic contraction of the vas deferens. Superoxide anion appears to interrupt the P2X₁-mediated transduction cascade at some step(s) of intracellular calcium handling.

Cholinergic innervation of the guinea-pig isolated vas deferens

Cholinergic innervation of the guinea-pig isolated vas deferens

Cholinergic innervation of the guinea-pig isolated vas deferens.

Recently, a cholinergic neurogenic component of contraction has been characterised in the aganglionic mouse vas deferens. In this paper, a cholinergic component of contraction in the guinea-pig vas deferens is characterised pharmacologically. A residual, tetrodotoxin-sensitive (TTX, 0.3 microM), neurogenic contraction was revealed after prolonged exposure (5 h) to the adrenergic neurone blocker bretylium (20 microM) or in the presence of prazosin (100 nM) and alpha,beta-methylene ATP (1 microM), a purinergic agonist which desensitizes P2X receptors. The bretylium-resistant component was potentiated by the acetylcholinesterase (AChE) inhibitor neostigmine (10 microM) and inhibited by the muscarinic-receptor (mAChR) antagonist cyclopentolate (1 microM). Nicotine (30 microM) enhanced the bretylium-resistant component. Neostigmine increased the second component of contraction in the presence of prazosin and alpha,beta-methylene ATP, whilst yohimbine (1 microM), an alpha(2) adrenergic receptor antagonist, enhanced both the first and second components of the electrically evoked contraction. These enhanced contractions were blocked by cyclopentolate in both cases. Nicotine enhanced the cholinergic component of contraction revealed by neostigmine but failed to have any detectable effects in the presence of cyclopentolate. Neostigmine alone increased the slow component of contraction which was reversed by cyclopentolate to control levels. The M(3) receptor-antagonist 4-DAMP (10 nM) markedly inhibited the cholinergic component of contraction to a level comparable with cyclopentolate. Laser microscopy has shown that neostigmine also increased the frequency of spontaneous Ca(2+) transients remaining in smooth muscle cells after perfusion with prazosin and alpha,beta-methylene ATP, an effect blocked by 4-DAMP. These experimental data show that there is a functional cholinergic innervation in the guinea-pig vas deferens whose action is limited by acetylcholinesterase, blocked by cyclopentolate and mediated through M3 receptors. Moreover, by blocking the cholinesterase, the increased amount of ACh generates spontaneous Ca(2+) transients in smooth muscle cells.

Postjunctional Synergism of Norepinephrine with ATP and Diadenosine Tetraphosphate in Guinea Pig Vas Deferens

In isolated guinea pig vas deferens, prior addition of norepinephrine (NE) significantly potentiated the contractile responses to adenosine-5′-triphosphate (ATP) and diadenosine tetraphosphate (AP₄A) in a dose-dependent manner up to 240% of the control purine dose. The myosin light chain phosphatase (MLCP) inhibitor cantharidin at a dose of 10 µmol/l caused significant enhancement of ATP at concentrations of 1 and 3 mmol/l by 91 and 95% respectively. Similarly, cantharidin enhanced the contraction to AP₄A, 30 and 100 µmol/l by 92 and 100% respectively. Inhibition of protein kinase C (PKC) by the use of chelerythrine (10 µmol/l), incubated at the vas deferens for 60 min, inhibited the NE-induced enhancement of purine-induced contraction. Chelerythrine reversed the NE-ATP and NE-AP₄A synergism back close to control ATP and AP₄A contraction values respectively. It can be concluded that postjunctional synergism becomes evident not only for adenine mononucleotides and NE but also for diadenosine polyphosphates presented here by AP₄A in the guinea pig vas deferens. This synergism involves receptor-mediated activation of PKC and possibly PKC-induced inhibition of MLCP.

The effect of epibatidine on spontaneous and evoked neurotransmitter release in the mouse and guinea pig isolated vas deferens

Nicotinic agonists increase sympathetic field-stimulus-evoked contraction of the rodent vas deferens, presumably by increasing evoked neurotransmitter release. This presumption was tested in two species. The effect of the nicotinic acetylcholine receptor (nAChR) agonist epibatidine on neurotransmitter release in mouse and guinea pig isolated vas deferens was investigated using contraction studies and conventional intracellular recording techniques. In 12 of 14 mouse vasa deferentia, slow bath application of epibatidine (100 nM) had no significant effect on excitatory junction potential (EJP) amplitude and spontaneous EJP (SEJP) frequency. However, rapid application of epibatidine to the mouse vas deferens caused an increase in SEJP frequency (by 530%), with no effect on EJP amplitude. Despite the absence of an effect on EJPs, electrically-evoked contractions of the mouse vas deferens were significantly increased in the presence of epibatidine (by 50%). A transient contraction was reliably induced by a higher epibatidine concentration (1 microM). This contraction was significantly reduced in the presence of prazosin, tetrodotoxin, or alpha,beta-methyleneATP. Epibatidine did not induce a contraction in the presence of a combination of prazosin, alpha,beta-methyleneATP and cyclopentolate. In guinea pig vasa deferentia, bath-applied epibatidine potentiated EJP amplitude in a biphasic pattern, lasting for at least 30 minutes. The nAChR-mediated augmentation of neurogenic contraction is indeed prejunctional, but in the mouse arises from an increase in spontaneous neurotransmitter release that primes smooth muscle for subsequent contraction, while in the guinea pig there is a direct augmentation of evoked neurotransmitter (ATP) release.

NITRIC OXIDE MODULATES BIPHASIC CONTRACTILE RESPONSE OF GUINEA PIG VAS DEFERENS TO ELECTRICAL FIELD STIMULATION

Electrical field stimulation (EFS) produced a biphasic contractile response; viz. initial rapid phasic contraction and second slow tonic contraction, in isolated guinea pig vas deferens. Pretreatment with the substrate of nitric oxide (NO) synthase (NOS), 1 mM L-arginine (L-ARG), augmented both the initial rapid and the second slow contractile responses to EFS (5 Hz, 0.5 msec, 30 V, for 30 sec). The increase of stimulation frequency from 5 Hz to 10 Hz or 20 Hz tended to attenuate the augmented responses. On the contrary, pretreatment with an inhibitor of NOS, 0.1 mM NG-nitro-L-arginine (L-NNA) suppressed both the initial rapid and the second slow contractile responses to EFS. The suppressive effect on the initial rapid contraction was also attenuated by the increase of stimulation frequency from 5 Hz or 10 Hz to 20 Hz. Contractile response to exogenously administered 1 mM adenosine triphosphate (ATP) tended to be slightly increased and decreased by the treatment with 1 mM L-ARG and 0.1 mM L-NNA, respectively. Contractile response to exogenously administered 10 microM noradrenaline (NA) was almost unaffected by the treatment with 1 mM L-ARG, while the treatment with 0.1 mM L-NNA slightly depressed the response. Potentiated contractile response to 1 mM ATP in the presence of 10 microM NA was further potentiated by the treatment with 1 mM L-ARG, while the response was almost unaffected by the treatment with 0.1 mM L-NNA. These findings may indicate that NO acts mainly on presynaptic site and increases the release of chemical transmitter, ATP or prevents the inactivation of ATP. Also, NO may act, at least in part, on postsynaptic site and potentiates the contractile response to ATP in the presence of NA.

Potentiation of sympathetic neuromuscular transmission mediated by muscarinic receptors in guinea pig isolated vas deferens

In guinea-pig isolated vasa deferentia, purinergic neurogenic contractions and responses to applied adenosine 5'-triphosphate (ATP) were potentiated by carbachol; responses to adrenergic transmission and applied noradrenaline were not. Following blockade of P2 receptors and alpha-adrenoceptors, the residual neurogenic response was massively potentiated by carbachol, suggesting the presence of a non-purinergic, non-adrenergic component. In the presence of guanethidine, carbachol had no significant effect, indicating that sympathetic transmission was the only element involved. Use of oxotremorine and selective muscarinic receptor antagonists suggested that the potentiating effect of carbachol and oxotremorine was mediated via M3 muscarinic receptors without involvement of nicotinic receptors.

Mechanisms underlying postjunctional synergism between responses of the vas deferens to noradrenaline and ATP

Mechanisms of postjunctional synergism between adenosine 5′-triphosphate (ATP) and noradrenaline were studied in isolated guinea pig vas deferens. Whereas prior exposure to ATP had no significant effect on noradrenaline-mediated contractions, noradrenaline concentration-dependently enhanced ATP-induced contractions. Similarly to noradrenaline, histamine, which also acts via phospholipase-coupled receptors, induced contractions of the vas deferens and enhanced subsequent responses to ATP. Although phorbol-12, 13-dibutyrate (PDBu), a stimulant of protein kinase C (PKC), failed to induce contractions, it significantly potentiated ATP-induced contractions. The PKC inhibitor, Calphostin C, prevented this effect and the noradrenaline-mediated enhancement of ATP-induced contractions. The phosphatase inhibitor cantharidin induced a time- and concentration-dependent tonic contraction and markedly increased subsequent contractions to ATP. It is suggested that noradrenaline potentiates the contractile response of the vas deferens to ATP via a PKC-mediated mechanism. This may involve the inhibition of myosin light chain phosphatase (MLCP) and subsequent calcium sensitisation.

Enzyme kinetics and pharmacological characterization of nucleotidases released from the guinea pig isolated vas deferens during nerve stimulation: evidence for a soluble ecto-nucleoside triphosphate diphosphohydrolase-like ATPase and a soluble ecto-5'-nucleotidase-like AMPase.

Previously, we have demonstrated that stimulation of the sympathetic nerves of the guinea pig vas deferens evokes release not only of the cotransmitters ATP and norepinephrine but also of soluble nucleotidases that break down extracellular ATP, ADP, and AMP into adenosine. In this study we show that the apparent K(m) values of the releasable enzyme activity vary depending on which of these adenine nucleotides is used as initial substrate. The K(m) value for ATP was 33.6 +/- 2.3 microM, 21.0 +/- 2.3 microM for ADP, and 10.0 +/- 1.1 microM for AMP. The ratios of the V(max) values for each enzyme reaction were 4:2:3. We have also found a different sensitivity of the metabolism of ATP and AMP by releasable nucleotidases to known nucleotidase inhibitors. Suramin inhibited the breakdown of ATP by releasable nucleotidases in a noncompetitive manner and with a K(i) value of 53 microM, but had no effect on the breakdown of AMP. The 5'-nucleotidase inhibitor alpha,beta-methylene ADP inhibited the breakdown of AMP but not that of ATP. Concanavalin A inhibited the breakdown of AMP but had neither inhibitory nor facilitatory effects on the breakdown of ATP. 6-N,N-Diethyl-beta,gamma-dibromomethylene-D-ATP (ARL67156), an ecto-ATPase inhibitor, suppressed ATPase and AMPase activities, whereas NaN(3) (10 mM) affected neither reaction, but inhibited the ADP metabolism. Phosphatase- and phosphodiesterase inhibitors did not affect the activity of the releasable nucleotidases. This evidence suggests that the soluble nucleotidases released during neurogenic stimulation of the guinea pig vas deferens combine an ecto-5'-nucleotidase-like and an ecto-nucleoside triphosphate diphosphohydrolase-like activity.

Myosin light chain phosphorylation and Mn2+-dependent acetylcholine-induced contractions in guinea pig vas deferens.

We have reported that noradrenaline but not K+ induced a sustained and dose-dependent contraction without extracellular Ca2+ and Mn2+ in Ca2+-depleted Mn2+loaded vas deferens from the guinea pig. The Mn2+dependent noradrenaline-induced contractions developed without an increase in phosphorylation of 20-kDa myosin light chain. To clarify whether such an unusual Mn2+dependent contraction was induced only by noradrenaline or not, we examined effects of acetylcholine on Ca2+-depleted Mn2+-loaded guinea pig vas deferens. Acetylcholine (1 microM(-1) mM) induced a sustained dose-dependent contraction without extracellular Ca2+ or Mn2+. W-7 (10 microM or 100 microM) and wortmannin (1 microM) both reduced the Mn2+dependent acetylcholine-induced contractions similarly to Ca2+-dependent acetylcholine-induced contractions in isolated vas deferens without either Ca2+ depletion or Mn2+ loading. However, the Mn2+-dependent acetylcholine-induced contractions developed without a significant increase in the phosphorylation of the myosin light chain determined by urea-glycerol polyacrylamide gel electrophoresis and immunoblotting. These results indicate that acetylcholine as well as noradrenaline induces Mn2+-dependent contraction and are consistent with our previous assumption that Mn2+ may preferentially support receptor-mediated contractions in the guinea pig isolated vas deferens. The results also suggest that the activation of myosin light chain kinase is essential for the development of Mn2+-dependent acetylcholine-induced contractions, and that Mn2+ may accelerate formation of non-phosphorylated attached cross-bridges during receptor activation.