I.Christopher H. Smith

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.

Early induction by crotoxin of biphasic frequency changes and giant miniature endplate potentials in frog muscle.

1 Following the addition of crotoxin (250 nm) at the frog neuromuscular junction, there was an initial fall in frequency of miniature endplate potentials (m.e.p.ps), followed by a secondary rise which was characterized by the appearance of large spontaneous potentials (giants, g.m.e.p.ps) and an occasional large potential of the burst type. 2 In the presence of 2‐(4‐phenylpiperidino)cyclohexanol (AH5183, vesicamol), an inhibitor of vesicular acetylcholine uptake, the frequency of g.m.e.p.ps induced by crotoxin was reduced. 3 The characteristic changes in m.e.p.p. frequency and amplitude distribution were absent with crotoxin in Sr‐EGTA Ringer. In the presence of high concentrations of Mn (3.6 or 5.4 mM with 0.9 mM Ca), the crotoxin‐induced initial fall and the onset of the secondary rise in m.e.p.p. and g.m.e.p.p. frequencies were slower. The timing of these phases was unaffected by Ca concentrations ranging from 6.3 to 0.9 mM. 4 High concentrations of Mn ions partially inhibited the phospholipase A2 activity of crotoxin on artificial phospholipid membranes. This also supports the involvement of the Ca‐dependent phospholipase A2 subunit in both phases of the physiological action of the toxin. 5 G.m.e.p.ps were associated with a moderate increase in m.e.p.p. frequency (2–3 s−1) and were of a time‐course similar to that of m.e.p.ps. They persisted after washing with medium lacking Ca ions and in the presence of Ca‐Mn Ringer that blocked evoked responses. 6 It is concluded that crotoxin, acting through its phospholipase A2 subunit, produces specific disturbances of synaptic exocytosis and vesicle formation in the axolemma of the motor nerve terminal which lead to biphasic changes in m.e.p.p. frequency and the onset of large spontaneous potentials.

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.

The importance of phospholipase A2 in the early induction by crotoxin of biphasic changes in endplate potentials at the frog neuromuscular junction

In Ca2+-Ringer, crotoxin induced a rapid fall in amplitude of endplate potentials followed by a marked secondary rise but, in Ca2+-free, Sr2+-EGTA Ringer, crotoxin failed to elicit either response. In Ca2+-washout experiments, the onset of giant miniature endplate potentials indicated that binding of crotoxin occurred in Sr2+-EGTA Ringer. Facilitation of endplate potential amplitude due to a closely spaced twin impulse was increased to 162% of control near the peak of the crotoxin-induced secondary phase. These findings are consistent with the phospholipase A2 subunit acting specifically at the active zone.

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.

An electrophysiological method for measuring the potassium permeability of the nerve perineurium

An electrophysiological method is described for measuring the potassium permeability (PK) of the perineurium of the sciatic nerve of the frog. The method is based on the principle of grease-gap recording, in which an insulating compartment separates two surface recording electrodes. The sciatic nerves of frogs Rana temporaria and R. pipiens were isolated and mounted across a five compartment chamber, with Vaseline grease seals on the partitions between compartments. Compartments #1, #2 and #5 contained frog Ringer solution, #4 was filled with Vaseline and formed the grease gap, and #3 was the test compartment in which solutions could be changed. The nerve was stimulated via platinum electrodes in compartments #1 and #2, and DC potentials and compound action potentials (CAP) were recorded between Ag/AgCl electrodes connected through Ringer-agar bridges to compartments #3 and #5. In nerves with undamaged perineurium, changing from normal Ringer to high [K+] Ringer (100 mM, KCl replacing NaCl) for 2 min caused negligible change in DC potential or CAP, indicating that raised [K+] was not reaching the axon surface, and hence that the perineurium was exerting a diffusional restriction on K+ entry. In nerves damaged by stretching or drying, K+ pulses caused a depolarising change in DC potential (delta DC), and corresponding decline in CAP amplitude, consistent with a leaky perineurium allowing K+ entry and axonal depolarisation. Ringer made hypertonic by the addition of 2.5 M sucrose or 5 M NaCl caused increased perineurial permeability to K+. The method was calibrated by measuring the delta DC in response to raised [K+] in the range 5-100 mM [K+] in desheathed nerves; from this calibration curve relating delta DC to endoneurial [K+] it was possible to calculate the change in endoneurial [K+] occurring in intact preparations. The calculations showed that the undamaged perineurium had a PK of < 6.3 x 10(-7) cm.s-1, similar to the value calculated for in situ nerves using radioisotopic techniques, but less than the value reported for isolated perineurial cylinders. The method gives real-time information on the K+ permeability of the nerve perineurium and its modulation by experimental treatments.

Properties of the early phases of crotoxin poisoning at frog neuromuscular junctions.

Addition of crotoxin to the frog neuromuscular junction in either 0.9 mM Ca2+ plus tubocurarine or 0.5 mM Ca2+ Ringer solution produced a triphasic change in the amplitude of nerve-evoked endplate potentials (e.p.p.s) and, with 0.5 mM Ca2+, a biphasic change in miniature endplate potential (m.e.p.p.) frequency. The secondary phase of rising e.p.p. amplitude was associated with an increase in facilitation of e.p.p. amplitude with closely spaced twin impulses; the increase in spontaneous release lagged behind that of evoked release. When a calcium chelator, BAPTA, was loaded into presynaptic nerve terminals to buffer cytosolic free Ca2+, both e.p.p. amplitude and twin-impulse facilitation were increased by crotoxin to a similar extent relative to that in the control without BAPTA. The duration of the increase in twin-impulse facilitation was reduced but the duration of the increase in e.p.p. amplitude was unaffected by BAPTA loading. The presence of BAPTA did not alter the characteristic changes in spontaneous release in response to crotoxin. These results suggest that the augmentation of evoked and spontaneous transmitter release by crotoxin is not primarily due to changes in cytosolic Ca2+. The response time between stimulus and e.p.p. peak was lengthened in all phases due to prolongation of the interval between stimulus and e.p.p. onset and, in the secondary and tertiary phases, slowing of e.p.p. rise-time. The protein kinase C inhibitor, H-7, produced complex changes in e.p.p.s. under control conditions but did not alter the triphasic response characteristic of intoxication. These results suggest that crotoxin initiates a primary disturbance in the phasic release process leading to a series of time-gated changes which include transient facilitation then uncoupling of phasic release and generalized acceleration of spontaneous release.

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.