Nnaemeka Amobi

Effects of α,β-methylene ATP on biphasic responses of rat vas deferens

Abstract The epididymal region of isolated vas deferens of the rat was stimulated locally with field electrodes. Continuous perfusion with α,β-methylene-adenosine-5′-triphosphate (mATP, 10−5 M) desensitized the P2-purinoceptors and the effect of this on the two components of tetanic responses and the ‘non-adrenergic’ and adrenergic phases of single shock responses were examined. Exposure to mATP selectively prevented the contractions to adenosine-5′-triphosphate (ATP, 10−4-5 × 10−4 M) but not noradrenaline (NA, 10−5 M) and preferentially blocked the secondary but not the primary tetanic component. It also depressed markedly the non-adrenergic phase of single shocks and either reduced or abolished the adrenergic phase. Thus the secondary tension development depends in the rat vas deferens on both the activation of α1-adrenoceptors and the non-adrenergic twitch mechanism. The action of mATP involves more processes than the desensitization of the P2-purinoceptors so does not positively identify purinergic transmission. Direct NA-induced contraction contributes more to the primary than to the secondary tetanic component.

Discrimination by SZL49 between contractions evoked by noradrenaline in longitudinal and circular muscle of human vas deferens.

The effects of irreversible α1‐adrenoceptor antagonists, SZL‐49 (an alkylating analogue of prazosin), dibenamine and benextramine on contractions to noradrenaline (NA) in longitudinal and circular muscle of human epididymal vas deferens were investigated. Competitive α1‐adrenoceptor antagonists were also used to further characterize the α1‐adrenoceptor subtype stimulated by NA in longitudinal and circular muscle. NA evoked concentration‐dependent contractions of both muscle types (pD2; 5.4 and 5.2 respectively). The contraction of circular muscle was comparatively more sensitive than that of longitudinal muscle to pretreatment with SZL‐49. In contrast, dibenamine or benextramine produced comparable effects in both muscle types. The relationship between receptor occupancy and contraction in either longitudinal or circular muscle was nonlinear, with half‐maximal response requiring similar receptor occupancy (longitudinal muscle 14%, circular muscle 16%). Maximal response in both muscle types occurred with little or no receptor reserve (<10%). The competitive α1‐adrenoceptor antagonists produced dextral shifts of the dose‐response curves to NA in longitudinal and circular muscle. The inhibitory potencies, estimated from the apparent pKB values were significantly different in longitudinal and circular muscle respectively for either WB 4101 (pKB, 8.6 and 9.5) or RS‐17053 (pKB, 7.1 and 9.0) but not for Rec 15/2739 (pKB, 9.2 and 9.8) or HV 723 (pKB, 8.3 and 8.4). In conclusion, the potency profile of the competitive α1‐adrenoceptor antagonists and the lack of different receptor reserves for NA in the muscle types suggest that the discriminatory effects of SZL‐49 is primarily due to a predominance of the α1L‐adrenoceptor subtype in longitudinal muscle and α1A‐subtype in circular muscle.

Functional characterization of α1-adrenoceptor subtypes in longitudinal and circular muscle of human vas deferens

The alpha1-adrenoceptor subtype(s) mediating contraction to noradrenaline in longitudinal and circular muscle of human epididymal vas deferens was studied using competitive antagonists. The effects of the alkylating agents, phenoxybenzamine and chloroethylclonidine were also investigated. Noradrenaline evoked concentration-dependent contractions of longitudinal and circular muscle with comparable potencies (pD2; 5.6 and 5.5 respectively). The contractions in longitudinal and circular muscle respectively were inhibited by prazosin (pA2, 8.6 and pKB, 9.2), 5-methylurapidil (pKB, 8.7 and 9.1) and less potently by spiperone (pA2, 7.1) or BMY 7378 (pKB, 6.3 and 6.6). Contractions of the circular but not longitudinal muscle was comparatively insensitive to pretreatment with phenoxybenzamine. In contrast pretreatment with chloroethylclonidine reduced the contractions in both muscle types and also enhanced phenoxybenzamine-sensitivity in longitudinal but not circular muscle. The results suggest that contractions evoked by noradrenaline in both muscle types of human vas deferens is mediated via activation of alpha1-adrenoceptors with pharmacological profile of the alpha1A-subtype. However the involvement of alpha1A-adrenoceptor variants, such as the hypothesised alpha1L-subtype may underlie the differential effects of phenoxybenzamine in longitudinal and circular muscle. Factors contributing to chloroethylclonidine-sensitivity are discussed.

Perspective on the role of P2X-purinoceptor activation in human vas deferens contractility.

The contractile actions of α,β‐methylene ATP (α,β‐meATP) and ATP and the effects of K+ channel blockers in longitudinal and circular muscles of human vas deferens were investigated with a view to clarifying the functional importance of P2X1‐purinoceptor activation and K+ channels in modulating contractility of the tissues. The results provide an experiment‐based perspective for resolving differing reports on purinergic activation of the tissues and uncertain roles of large‐conductance Ca2+‐activated K+ (BKCa) and voltage‐gated delayed rectifier K+ (KV) channels. α,β‐Methylene ATP (3–100 μm) evoked suramin‐sensitive contractions of longitudinal muscle but rarely of circular muscle. ATP (0.1–3 mm) less reliably activated only longitudinal muscle contractions. These were enhanced by ARL 67156 (100 μm), but a different ectonucleotidase inhibitor, POM 1, was ineffective. Both muscle types were unresponsive to ADP‐βS (100 μm), a P2Y‐purinoceptor agonist. Longitudinal muscle contractions in response to α,β‐meATP were enhanced by FPL 64176 (1 μm), an L‐type Ca2+ agonist, TEA (1 mm), a non‐specific K+ channel blocker, 4‐aminopyridine (0.3 mm), a selective blocker of KV channels, and iberiotoxin (0.1 μm), a selective blocker of BKCa channels. Quiescent circular muscles responded to α,β‐meATP reliably in the presence of FPL 64176 or iberiotoxin. Apamin (0.1 μm), a selective blocker of small conductance Ca2+‐activated K+ (SKCa) channels had no effect in both muscle types. Y‐27632 (1–10 μm) reduced longitudinal muscle contractions in response to α,β‐meATP, suggesting involvement of Rho‐kinase‐dependent contractile mechanisms. The results indicate that P2X1‐purinoceptor stimulation elicits excitatory effects that: (a) lead to longitudinal muscle contraction and secondary activation of 4‐aminopyridine‐sensitive (KV) and iberiotoxin‐sensitive (BKCa) K+ channels; and (b) are subcontractile in circular muscle due to ancillary activation of BKCa channels. The novel finding of differential action by P2X1‐purinoceptor agonists in the muscle types has functional implication in terms of the purinergic contribution to overall contractile function of human vas deferens. The modulatory effects of KV and BKCa channels following P2X1‐purinoceptor activation may be pivotal in providing the crucial physiological mechanism that ensures temporal co‐ordination of longitudinal and circular muscle contractility.

The human vas deferens : correlation of response pattern to noradrenaline and histological structure

Specimens of human vas deferens were studied histologically and by measuring changes in response to adrenoceptor agonists. In intact tissues noradrenaline induced both lengthening and shortening responses which were identified with contractions of circular and longitudinal muscle respectively. The agonists phenylephrine or methoxamine evoked mainly shortening but little lengthening. The tissues were unresponsive to clonidine or isoprenaline. Longitudinal strips but not rings responded reliably to phenylephrine and to caffeine. The lengthening responses of intact specimens to noradrenaline were relatively insensitive to the antagonist phenoxybenzamine. In contrast both shortening and lengthening responses were inhibited by prazosin or phentolamine but not by idazoxan. A physiological function for the predominance of muscle types in different specimens is proposed. A pharmacological selectivity for the muscle types by alpha 1-selective adrenoceptor agonists and by phenoxybenzamine is discussed and the clinical implication considered.

Contractile actions of L-type Ca2+ agonists in human vas deferens and effects of structurally different Ca2+ antagonists

The actions of L-type Ca(2+) agonists, FPL 64176 and Bay K 8644 were investigated in human vas deferens in the presence of structurally different L-type Ca(2+) antagonists. The L-type Ca(2+) agonists (<or=3 microM, approximately 10 min) produced no detectable contractions but higher concentrations evoked intermittent rhythmic contractility of longitudinal and circular muscles. Exposure to the drugs (1 microM, >or=20 min) evoked only rhythmic contractility even in moderately depolarizing ([K(+)](o), 10mM) medium. These findings suggest low basal activity of L-type Ca(2+) channels (VOCs) in both muscle types. In the presence of L-type Ca(2+) agonists (1 microM), high [K(+)](o) (30 or 120 mM) evoked contractions with different profiles. Circular muscle had a predominance of rhythmic activity ([K(+)](o) 30 mM) and slow time to peak and decline ([K(+)](o) 120 mM). Longitudinal muscle was more tonic ([K(+)](o) 30 mM) with a rapid time to peak and decline ([K(+)](o) 120 mM). The contractions in both muscle types were blocked by nifedipine or methoxyverapamil; indicating the involvement of L-type VOCs and suggests that the distinct contractile profiles originate from differences in mechanisms that regulate contractility. In comparison to the conventional L-type Ca(2+) antagonists, fendiline, prenylamine and thioridazine were more effective against longitudinal than circular muscle contractions. Structurally similar diphenylalkylamines (cinnarizine, flunarizine, and pimozide) and phenothiazines (sulphoridazine, chlorpromazine, and trifluoperazine) inhibited the contractions comparably in both muscle types. These findings are discussed in relation to inhibition of muscle type-specific mechanisms that may contribute more to L-type VOC activation and contractility in longitudinal than in circular muscle.

Comparative effects of T-type and L-type Ca2+-antagonists against noradrenaline-induced contractions of human vas deferens

To investigate the effects of the relatively selective T‐type Ca2+‐antagonists, mibefradil and flunarizine, and the L‐type Ca2+‐antagonist, nifedipine, on the contractions of longitudinal and circular muscles of human vas deferens, to elucidate the possible involvement of T‐type voltage‐gated Ca2+ channels (VOCs) in the contractile function of the tissue.

Paradoxical effects of thioridazine on electromechanical coupling in the human and rat vas deferens

The effects of thioridazine on the responses of isolated human and rat vas deferens to high [K+]0, A23187 and caffeine were examined. In the presence of Ca2+ (2.5 mM), thioridazine (1-10 microM) induced spontaneous contractions but caused a dose-related inhibition of the phasic and secondary parts of the response to high [K+]0 (136 mM). The relaxation phase of the high [K+]0 response of the human vas deferens was unaffected by thioridazine (up to 10 microM). In Ca2(+)-free/EGTA (0.5 mM) media, thioridazine caused a dose-related potentiation, shortened the latency and prolonged the duration of high [K+]0 responses. Contractions to caffeine (20 mM) and A23187 (20-50 microM) were relatively unchanged by thioridazine (10 microM). The spontaneous activity caused by thioridazine (10 microM) was sensitive to the Ca2(+)-channel blockers nifedipine (10 microM) or verapamil (10 microM). These results indicate that the action of thioridazine during electromechanical coupling in the human and rat vas deferens may involve more than its blockade of voltage gated Ca2+ channels.