Guinea-pig Vas Deferens Research Articles

The mechanisms of enhancement and inhibition of field stimulation responses of guinea-pig vas deferens by prostacyclin analogues.

1. In the guinea-pig isolated vas deferens preparation bathed in Tyrode's solution, the prostacyclin analogues, cicaprost, TEI-9063, iloprost, taprostene and benzodioxane-prostacyclin, enhanced twitch responses to submaximal electrical field stimulation (20%-EFS). The high potency of cicaprost (EC150 = 1.3 nM) and the relative potencies of the analogues (equi-effective molar ratios = 1.0, 0.85, 1.6, 17 and 82, respectively) suggest the involvement of a prostacyclin (IP-) receptor. 2. Maximum enhancement induced by cicaprost in 2.5 mM K+ Krebs-Henseleit solution was similar to that in Tyrode solution (2.7 mM K+), but was progressively reduced as the K+ concentration was increased to 3.9, 5.9 and 11.9 mM. There was also a greater tendency for the other prostacyclin analogues to inhibit EFS responses in 5.9 mM standard K+ Krebs-Henseleit solution; this may be attributed to their agonist actions on presynaptic EP3-receptors resulting in inhibition of transmitter release. 3. The EFS enhancing action of cicaprost was not affected by the alpha1-adrenoceptor antagonist prazosin (100 and 1000 nM). Cicaprost (20 and 200 nM) did not affect contractile responses of the vas deferens to either ATP (5 microM) or alpha,beta-methylene ATP (1 microM) in the presence of tetrodotoxin (TTX, 100 nM). In addition, enhancement by cicaprost of responses to higher concentrations of ATP (30 and 300 microM) in the absence of TTX, as shown previously by others, was not seen. Prostaglandin E2 (PGE2, 10 nM) and another prostacyclin analogue TEI-3356 (20 nM) enhanced purinoceptor agonist responses. Unexpectedly, TTX (0.1 and 1 microM) partially inhibited contractions elicited by 10-1000 microM ATP; contractions elicited by 1-3 microM ATP were unaffected. Further studies are required to establish whether a pre- or post-synaptic mechanism is involved. 4. In a separate series of experiments, cicaprost (5-250 nM), TEI-9063 (3-300 nM), 4-aminopyridine (10-100 microM) and tetraethylammonium (100-1000 microM) enhanced both 20%-EFS responses and the accompanying overflow of noradrenaline to a similar extent. In further experiments with the EP1-receptor antagonist AH 6809, TEI-3356 (1.0-100 nM) and the EP3-receptor agonist, sulprostone (0.1-1.0 nM) inhibited both maximal EFS responses and noradrenaline overflow, thus confirming previous reports of the high activity of TEI-3356 at the EP3-receptor. Cicaprost had no significant effect on noradrenaline overflow at 10 and 100 nM, but produced a modest inhibition at 640 nM. 5. In conclusion, our studies show that prostacyclin analogues (particularly TEI-3356) can inhibit EFS responses of the guinea-pig vas deferens by acting as agonists at presynaptic EP3-receptors. Prostacyclin analogues (particularly cicaprost and TEI-9063) can also enhance EFS responses through activation of IP-receptors. The mechanism of the enhancement has not been rigorously established but from our results we favour a presynaptic action to increase transmitter release.

Neuronal release of soluble nucleotidases and their role in neurotransmitter inactivation.

Efficient control of synaptic transmission requires a rapid mechanism for terminating the actions of neurotransmitters. For amino acids and monoamines, this is achieved by their uptake into the cell by specific high-affinity transporters; acetylcholine is first broken down in the extracellular space and then choline is taken up by the cell. Because ATP is hydrolysed to adenosine by membrane-bound enzymes (ectonucleotidases) that are present in most tissues, it has been assumed that these enzymes terminate the neurotransmitter actions of ATP in the brain and in the periphery. We show here, however, that stimulation of sympathetic nerves innervating the guinea-pig vas deferens releases not only neuronal ATP, but also soluble nucleotidases that break down this ATP to adenosine, indicating that inactivation of ATP is increased by nerve activity. This release of specific nucleotidases together with ATP represents a new mechanism for terminating the actions of a neurotransmitter.

Effects of arachidonic acid on A-type potassium currents in smooth muscle cells of the guinea pig.

Effects of arachidonic acid (AA) and related fatty acids on Ca2+ -independent transient (A-type) K+ current (I(A)) were examined in single myocytes of guinea pig vas deferens, ureter, and proximal colon as well as in rabbit vas deferens. The peak amplitude of I(A) was reduced by external application of AA (half-maximal inhibitory concentration = approximately 1 microM). The blocking effect was not changed significantly by indomethacin, nordihydroguaiaretic acid, guanosine 5'-O-(2-thiodiphosphate), or guanosine 5'-O-(3-thiotriphosphate). Pharmacological studies suggested that the effect of AA was not mediated by activation of protein kinases A or C or tyrosine kinase. AA (20:4) was the most potent of the four types of cis-eicosanoic acids with two to five double bonds (20:2 to 20:5) that were tested. I(A)-like current in cardiac atrial myocytes of the rabbit was not affected significantly by 30 microM AA. These results indicate that AA itself directly blocks A-type K+ channels. A relationship between stereospecific chemical structure of fatty acids and their blockade of A-type K+ channels is suggested. A-type K+ channels in smooth muscle cells can be clearly resolved from those in atrial myocytes by the responses to AA.

Responses of the isolated sympathetic nerve‐ductus deferens preparation of the guinea‐pig

Responses of the isolated sympathetic nerve‐ductus deferens preparation of the guinea‐pig

Identification of the Components of Excitatory Junction Potentials in the Guinea Pig Vas Deferens

The contributions to the generation of excitatory junction potentials (EJPs) during neurotransmission in a smooth muscle organ have been investigated. The effects of nerve stimulation intensity on EJPs with complex time courses have been explored in the guinea pig vas deferens. It is shown that complex EJPs are resolvable into intermediate rapid and slow components, produced by the activity of separately recruitable innervating axons. The effects of the putative gap junctional uncoupling agent heptanol on EJPs have also been studied. Heptanol abolished the EJP reversibly. However, brief stimulus-locked depolarizations continued to occur intermittently at the same latency as the EJP even while the EJP was completely suppressed. These depolarizations had properties very similar to those of SEJPs, and reflect the fundamental quantal depolarizations underlying the EJP. They are termed quantal EJPs (QEJPs). Our results show that there are two principal contributions to the generation of the EJP, namely (i) QEJ...

Effects of heptanol on electrical activity in the guinea-pig vas deferens.

1. The effects of the putative intercellular uncoupling agent I-heptanol on electrical activity in the guinea-pig vas deferens were studied by use of intracellular and extracellular recording techniques. 2. At concentrations of 0.5, 1 and 2 mM, heptanol rapidly, monotonically and reversibly attenuated intracellularly recorded excitatory junction potential (e.j.p.) amplitude without affecting its time course, while spontaneous excitatory junction potentials (s.e.j.ps) were left unaffected. 3. Heptanol did not affect either the extracellularly recorded evoked excitatory junction current (e.j.c.), or the nerve terminal impulse that preceded it. These observations indicate that heptanol does not affect nerve impulse conduction, neurotransmitter release, or the postjunctional receptors involved in the production of the e.j.p. 4. E.j.ps appear to be suppressed by heptanol due to its intercellular uncoupling effects. Therefore, functional intercellular coupling may be necessary for the generation of the e.j.p. in smooth muscle.

P-658 - ATP release evoked by sitmuration of peptides-receptors froe taenia coli and vas deferens of guinea pig

P-658 - ATP release evoked by sitmuration of peptides-receptors froe taenia coli and vas deferens of guinea pig

P-674 - The mode of contraction and confocal measurement of intracellular concentration of Ca2+ and Mn2+ in dispersed smooth muscle cells from guinea pig vas deferens

P-674 - The mode of contraction and confocal measurement of intracellular concentration of Ca2+ and Mn2+ in dispersed smooth muscle cells from guinea pig vas deferens

Changes in the smooth muscle of the guinea-pig vas deferens by thermal stress

Changes in the smooth muscle of the guinea-pig vas deferens by thermal stress

2424 Changes in the smooth muscle of the guinea-pig vas deferens by thermal stress

Currently, it is not possible to prevent essential hypertension and this presents a challenge to the clinician, pharmacologist, and medicinal chemist to discover adequate therapies. This chapter discusses the beneficial effects of drug therapy in the management of hypertension, the significance of catecholamine levels in hypertension, role of the sympathoadrenal axis in hypertension, and the need for intact vascular endothelium for vascular responses to a number of agents. A cautionary note offered that the levels in mixed venous blood are unreliable indicators of sympathetic neural activity in hypertension. Current evidence supports the hypothesis that converting enzyme (CEI) exerts their hypotensive effects primarily by inhibition of circulating and tissue-bound converting enzyme. A summary of the pharmacology of enalapril (MK-421) has appeared along with an account of the clinical studies done with lisinopril (MK-521) that suggest this drug will be suitable for once-a-day anti-hypertensive therapy. Analogs of enalapril that have appeared include the perhydroindole derivative. New evidence suggests certain calcium entry blockers (CEB) affect neural regulation of blood vessels at several sites. Studies in anesthetized dogs indicate that nifedipine increases and verapamil decreases the sensitivity of carotidsinus baroreceptors apparently by interference with a calcium-dependent and a sodium-dependent mechanism, respectively. New studies have examined the interaction of various CEB with α-adrenoceptors and their effect on the processes mediated by a-adrenoceptors. The (+) and (-) antipodes of verapamil, nicardipine, and (+) D-600 showed similar inhibition of radioligand binding to al-adrenoceptors, whereas diltiazem and nifedipine showed a very low level of activity.

Peptidyl Conjugates of Adenosine 5′-Carboxylic Acid Synthesized and Evaluated as Ligands for P2 Purinoceptors

The role of extracellular ATPin vivoand the various cellular responses mediated by P2 purinoceptors have not yet been fully elucidated, in part depending on the lack of subtype-specific high affinity antagonists. Here we describe the synthesis of a new class of compounds, peptidyl derivatives of adenosine 5′-carboxylic acid, among which some have inhibitory effects in certain P2 purinoceptor-carrying biological systems, e.g., glioma and smooth muscle cell lines and isolated smooth muscle tissue preparations from guinea pig vas deferens and urinary bladder.

Developmental changes in sympathetic contraction of the circular muscle layer in the guinea-pig vas deferens

Contractile responses of the circular muscle of the isolated vas deferens to electrical stimulation (10–80 Hz) and to noradrenaline significantly decreased with increasing age in 3-week-, 10-week- and 18-month-old guinea pigs, observed by the cannula insertion method. There were no significant differences in the contractile responses induced by α,β-methylene ATP or BaCl 2 between 3 and 10 weeks old, but the responses to α,β-methylene ATP or BaCl 2 decreased in 18-month-old guinea pigs. The contractile response to electrical stimulation was monophasic in 3-week-old guinea pigs, a small portion of which remained after the treatment with prazosin. Desensitisation of P 2x-purinoceptors with α,β-methylene ATP significantly inhibited the contractile responses to stimulation with relatively low frequencies, and the combination of both prazosin and α,β-methylene ATP abolished the stimulation-induced contractions. In 10-week- and 18-month-old guinea pigs electrical stimulation evoked a transient contraction followed by a second contraction at the offset of the stimulation (the after-response). The after-responses were blocked by prazosin. These results show that the dominant component of sympathetic cotransmission is noradrenaline; a purinergic component also exists in the sympathetic contraction in the circular muscle of the vas deferens in young guinea pigs, but is virtually absent in the later stages of development. The sympathetic contractions of the circular muscles significantly decrease with increasing age and this appears to be due to changes in postjunctional, rather than prejunctional, mechanisms.

Evidence for the differential release of the cotransmitters ATP and noradrenaline from sympathetic nerves of the guinea-pig vas deferens.

1. Experiments were carried out to quantify the stimulation-evoked overflow of catecholamines and purines (ATP, ADP, AMP and adenosine) from an in vitro sympathetic nerve-smooth muscle preparation of the guinea-pig vas deferens and from isolated bovine adrenal chromaffin cells. The superfused preparations were stimulated for 60 s with electrical field stimulation (EFS; vas deferens), dimethylphenylpiperazinium (chromaffin cells) or KCl (both preparations). 2. Samples of superfusate were taken at 10 s intervals during the 60 s stimulation period for analysis of purines by HPLC-fluorescence detection and catecholamines by HPLC-electrochemical detection. 3. The evoked overflow of catecholamines and purines from chromaffin cells occurred with the same time course and in a constant ratio of approximately 4:1 (catecholamine to purine). These findings are compatible with the release of catecholamines and purines from a homogeneous population of exocytotic vesicles in the chromaffin cells. 4. The evoked overflow of purines and noradrenaline (NA) from the vas deferens preparation differed from the pattern of overflow from chromaffin cells and there was also some temporal disparity in the overflow of the two cotransmitters. The evoked overflow of ATP exceeded that of NA. In addition, the overflow of NA was tonic while the overflow of ATP and the other purines was phasic. 5. The EFS-evoked overflow of NA and the purines from the guniea-pig vas deferens preparation was examined after treatment with the neuronal amine-uptake inhibitors desipramine and cocaine, the alpha 1-adrenoceptor agonist methoxamine, the alpha 1-adrenoceptor antagonist prazosin, the alpha 2-adrenoceptor antagonists idazoxan and yohimbine, the noradrenaline-depleting drug reserpine and the adrenergic neuron-blocking agent guanethidine. The results of these studies, together with an analysis of the metabolic degradation of extracellular ATP, indicated that the temporal disparity in the overflow of NA and ATP is unlikely to be due to differences in the clearance of the cotransmitters or to the release of purines from non-neuronal sites. These results indicate that evoked overflow of the cotransmitters accurately reflects release from nerves. This pattern of release from nerves suggests that the two cotransmitters are released from two separate populations of exocytotic vesicles. 6. Superfusion of the vas deferens with exogenous epsilon-ATP, a fluorescent derivative of ATP, revealed that there was essentially no metabolism of the nucleotide over 60 s unless the tissue was subjected to EFS. Upon EFS, there was a rapid and nearly complete degradation of ATP with a corresponding increase in ADP, AMP and adenosine. This indicates the presence of a nerve stimulation-dependent metabolism of ATP.

Inhibition of purinergic transmission by prostaglandin E1 and E2 in the guinea-pig vas deferens: an electrophysiological study.

1. The effects of prostaglandin E1 (PGE1) and E2 (PGE2) on postjunctional electrical activity in the guinea-pig vas deferens evoked by sympathetic nerve stimulation were investigated using both intracellular and focal extracellular recording techniques in vitro. 2. Bath application of PGE1 (1-100 nM) or PGE2 (0.1-100 nM) concentration-dependently inhibited the amplitudes of all excitatory junction potentials (e.j.ps) evoked during short trains of stimuli (10 stimuli at 1 Hz). Increasing the duration of nerve stimulation (100 stimuli at 1 Hz) did not overcome this inhibitory effect. At these concentrations PGE1 and PGE2 were without any apparent inhibitory effect on the amplitudes of spontaneous e.j.ps. 3. Local application of PGE1 (10-100 nM) or PGE2 (10-30 nM) markedly reduced the frequency of occurrence of excitatory junction currents (e.j.cs) evoked by trains of 20-100 stimuli at 1 to 4 Hz without changing the amplitudes of spontaneous e.j.cs or the configuration of the nerve terminal impulse. 4. In the presence of PGE1 or PGE2, raising the frequency of stimulation (from 1 to 4 Hz), increased the likelihood of e.j.c. occurrence. 5. The postjunctional electrical activity recorded in the guinea-pig vas deferens is believed to be due to ATP released from the sympathetic nerve endings. Thus the present study demonstrates that both PGE1 and PGE2 powerfully inhibit quantal ATP release in the guinea-pig vas deferens.

Opposite modulation of noradrenaline and ATP release in guinea-pig vas deferens through prejunctional beta-adrenoceptors: evidence for the beta 2 subtype.

Activation of prejunctional beta-adrenoceptors has been suggested to increase the release of noradrenaline but to decrease the neural release of ATP in the guinea-pig vas deferens. Experiments were carried out to determine the subtype of beta-adrenoceptor involved. In [3H]-noradrenaline-preincubated tissues superfused with medium containing prazosin and suramin, isoprenaline (1-100 nM), salbutamol (0.01-1 microM) and terbutaline (0.1-10 microM) increased the overflow of tritium but reduced the overflow of ATP elicited by electrical stimulation (210 pulses/7 Hz). The effects of isoprenaline were blocked by the beta 2-selective antagonist 1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3- [(1-methylethyl)amino]-2-butanol (ICI 118,551; 100 nM). In prazosin- and suramin-free medium, isoprenaline (100 nM) did not change the overflow of ATP elicited by exogenous noradrenaline (10 microM). Isoprenaline (1-100 nM), salbutamol (0.01-1 microM) and terbutaline (0.1-10 microM) reduced the initial twitch contraction elicited by electrical stimulation (210 pulses/7 Hz) in prazosin- and suramin-free medium as well as the isolated purinergic neurogenic contraction obtained by exposure to prazosin. They increased or tended to increase the secondary sustained concentration elicited by electrical stimulation in prazosin- and suramin-free medium as well as the isolated adrenergic neurogenic contraction obtained in the presence of suramin. The inhibition by isoprenaline of the isolated purinergic contraction was attenuated by ICI 118,551 (100 nM) but not by the beta 1-selective antagonist 1-[2-((3-carbamoyl- 4-hydroxy)phenoxy)ethylamino]-3-[4-(1-methyl-4- trifluoromethyl-2-imidazolyl)phenoxy]-2-propanol (CGP 20712A; 100 nM). The results confirm the opposite beta-adrenoceptor-mediated modulation of noradrenaline and neural ATP release in the guinea-pig vas deferens. They show that the prejunctional beta-adrenoceptor is of the beta 2-subtype.

Differences in the responses to purinergic nerve stimulation and applied ATP in the guinea-pig vas deferens

The responses to sympathetic nerve stimulation and to applied ATP in the guinea-pig vas deferens were compared. Nifedipine (10 μ) markedly reduced the non-adrenergic neural contraction but only partially blocked the contractions produced by bath-applied ATP. Suramin (300 μM) also markedly reduced the contractile responses produced by nerve stimulation, but had no significant effect on the contractions produced by bath-applied ATP. Using intracellular recording techniques, nerve stimulation was shown to produce an excitatory junction potential which was abolished by suramin (1 μM). Ionophoretic application of ATP and bath-applied ATP also produced a depolarization. Suramin (1 μ) failed to abolish the response to bath-applied ATP and enhanced the ionophoretically induced depolarization. These results suggest either that ATP is not a transmitter in the vas deferens or that two classes of purinoceptor are present, one suramin-sensitive receptor which produces a contraction via the opening of voltage-dependent Ca z+ channels, and another which is suramin-resistant and produces a contraction by another means.

Inhibition of neural ATP release by atrial natriuretic peptide in guinea-pig vas deferens

Effects of atrial natriuretic peptide (ANP) and 8-bromoguanosine 3′:5′-cyclic monophosphate (8-BrcGMP) on contraction, overflow of tritium (after [ 3H]noradrenaline labelling) and overflow of ATP elicited by electrical field stimulation ( 210 pulses 7 Hz ) were studied in guinea-pig vas deferens. ANP (1–100 nM) slightly increased contractions, did not alter the overflow of tritium but decreased the overflow of ATP by up to 50%. 8-BrcGMP (3–300 μM) markedly reduced contractions and ATP overflow with no effect on tritium overflow. Contractions were suppressed in the presence of prazosin plus suramin, and evoked overflow of ATP declined to 11%. ANP now gradually increased tritium overflow but again decreased the overflow of ATP. 8-BrcGMP did not change tritium overflow, as before, but increased ATP overflow. The results indicate that ANP inhibits neural release of ATP by a mechanism independent of guanylyl cyclase activation with no major effect on noradrenaline release.

Cooperation of ATP and Norepinephrine in Inducing Contraction in Guinea Pig Vas Deferens Is Not Associated with Change in Intracellular Ca2+ Level

Contractile responses and fura-2 fluorescence signals were simultaneously recorded in fura-2 loaded longitudinal muscle strips of guinea pig vas deferens to examine the relationship between cooperation of ATP and norepinephrine (NE) in the contractile response and intracellular Ca2+ level. ATP or NE induced a rapid or delayed contraction, respectively, with concomitant changes in fura-2 signal. Addition of both agonists potentiated the rapid, but not delayed contraction, while there was no potentiation in the fura-2 signal. In another series of experiments, NE (> or = 10 microM) contracted the muscle without further concomitant increase in Ca2+ level, indicating "Ca2+ sensitization" of the contractile apparatus. These findings suggest that cooperation of ATP and NE in inducing a rapid contraction of guinea pig vas deferens is mainly due to the "Ca2+ sensitization" effect of NE.

Mechanisms of ionic currents involved in suppressive effect of isoprenaline on the action potential of guinea-pig vas deferens in normal Krebs solution.

The effects of beta-adrenoceptor stimulation by isoprenaline (ISO) on action potential and membrane current were studied in the guinea-pig vas deferens in normal Krebs solution by using a double sucrose gap method. In current-clamp experiments, ISO produced the membrane hyperpolarization by reducing the resistance and modified the pattern of multi-spike activity elicited by long depolarizing currents; an initial spike potential was reduced in the amplitude and the rate of rising phases and the slope of diastolic depolarization was slowered, leading to a prolongation of the spike discharge interval. In voltage-clamp experiments, ISO greatly enhanced the late outward K+ current (Ik2) by increasing the conductance, but hardly affected the peak outward K+ current (Ik1). ISO also reduced the inward Ca2+ current (Ica) by decreasing the conductance (ga) as well as by reducing the driving force for Ca2+. While it increased the leakage conductance (g1) due to K+ associated with hyperpolarizing voltage steps. In the preparations treated with Ca2+ channel blocker such as D-600, the enhanced effect of ISO on the Ik was still prominent, suggesting that Ca2+ and K+ channels appear to be independently regulated during beta-adrenoceptor stimulation. These results suggest that ISO exerts depressant actions on the vas deferens muscle by reduction of the Ica as well as by prominent enhancement of the Ik2 via beta-adrenoceptor activation.

Opposite modulation of cotransmitter release in guinea-pig vas deferens: increase of noradrenaline and decrease of ATP release by activation of prejunctional beta-adrenoceptors.

Effects of isoprenaline on contraction, release of noradrenaline and release of ATP elicited by electrical field stimulation (210 pulses, 7 Hz) as well as on contractions elicited by exogenous noradrenaline and ATP were studied in the isolated vas deferens of the guinea pig. Release of noradrenaline was assessed as overflow of total tritium after preincubation with [3H]-noradrenaline. ATP was measured by means of the luciferin-luciferase technique. In [3H]-noradrenaline-pretreated tissues, electrical stimulation elicited an overflow of tritium and ATP and a biphasic contraction. Isoprenaline (1-100 nM) reduced the contraction, mainly phase I, and enhanced the evoked overflow of tritium: evoked overflow of ATP was not changed significantly. No, or almost no, contraction remained in [3H]-noradrenaline-pretreated tissues exposed to both prazosin (0.3 microM) and suramin (300 microM), and the evoked overflow of ATP was reduced by about 82%. Under these conditions, isoprenaline (1-100 nM) again enhanced the evoked overflow of tritium, but it now decreased the evoked overflow of ATP. Propranolol (1 microM), when added on top of prazosin and suramin, prevented the effects of isoprenaline (1-100 nM). In some tissues not pretreated with [3H]-noradrenaline, purinergic and adrenergic components of the neurogenic contraction (again to 210 pulses, 7 Hz) were isolated by exposure to prazosin (0.3 microM) and suramin (300 microM), respectively. Isoprenaline (1-100 nM) decreased the isolated purinergic component but did not change significantly the isolated adrenergic component. Contractions elicited by ATP (1000 microM) were not changed and contractions elicited by noradrenaline (100 microM) were slightly increased by isoprenaline (1-100 nM). Isoprenaline (100 nM) did not change the degradation of ATP (100 microM) by pieces of the vas deferens. It is concluded that, in the guinea-pig vas deferens, activation of prejunctional beta-adrenoceptors modulates the neural release of noradrenaline and ATP in opposite directions: release of noradrenaline is enhanced, whereas release of ATP is decreased.