EFFECT OF THE CA2-ATPASE INHIBITOR, CYCLOPIAZONIC ACID, ON ELECTROMECHANICAL COUPLING IN THE GUINEA-PIG URETER()

1 We have investigated the effect of the sarcoplasmic reticulum (SR) C a2+-ATPase inhibitor, cyclopiazonic acid (CPA), on electromechanical c oupling in the guinea-pig ureter. All experiments were performed in ca psaicin-pretreated (10 mu M for 15 min) ureters to prevent the release of sensory neuropeptides from afferent nerves. 2 In organ bath experi ments, electrical field stimulation (EFS, 10 Hz for 1 s, 5 ms pulse wi dth, 60 V) produced tetrodotoxin- (1 mu M) resistant phasic contractio ns which were enhanced by Bay K 8644 (1 mu M) and abolished by nifedip ine (10-30 mu M). 3 CPA (10 mu M) enhanced the EFS-evoked contractions both in the absence and presence of Bay K 8644. The effect of CPA was concentration-dependent between 1 and 30 mu M. The response to 10 mu M CPA was biphasic: the maximal enhancement (58 +/- 3% increase) was o bserved within 10-20 min from CPA administration, followed by a declin e to a new steady state (25 +/- 5% increase over baseline) at 50-60 mi n. The effect of CPA was reversed by washout. 4 Ryanodine (100 mu M) p roduced a prompt enhancement of the EFS-evoked contractions of the gui nea-pig ureter, which peaked at 42 +/- 3% increase over baseline; the co-administration of CPA (10 mu M) and ryanodine (100 mu M) produced a peak effect (60 +/- 8% enhancement) which was not different from that produced by CPA alone. With either ryanodine alone or ryanodine plus CPA, the enhancement of the EFS-induced contractions was biphasic, sho wing a time-course similar to that observed with CPA alone. Tetraethyl ammonium (10 mM) produced a significantly larger effect (93 +/- 13% in crease over baseline) and its effect was sustained throughout the 60 m in observation period. 5 In the presence of Bay K 8644, superfusion fo r 30 min with a low Na+ medium (60% of extracellular Na+ replaced by L i+ or choline) reduced the amplitude of EFS-evoked contractions by 20- 35%. In both Li+- and choline-substituted media, spontaneous activity developed during superfusion with low Na+ Krebs solution which was sup pressed by 10 mu M nifedipine. CPA (10 mu M) produced a marked enhance ment of the EFS-evoked contractions in low-Na+ medium (both Li+- and c holine-substituted) and this effect was sustained throughout the 60 mi n observation period. 6 In the absence of Bay K 8644, the response of the ureter smooth muscle to EFS is characterized by a refractory perio d: an interval of about 30 s was required between two applied stimuli to produce a second response comparable in size to that elicited by th e first stimulus. CPA (10 mu M, 10-20 min before) markedly reduced the refractory period of the guinea-pig ureter to EFS. 7 CPA (10 mu M, 30 -60 min before) increased the phasic component of contraction produced by 80 mM KCl. The tonic component of the response to KCl was slightly but not significantly reduced by CPA, and a 'hump' in the tonic contr action was observed at 1-2 min from addition of KCl. 8 In sucrose gap experiments, 10 mu M CPA produced a sustained depolarization of the me mbrane and reduced the latency between application of electrical stimu li and onset of the action potential; these effects were maintained th roughout the 60 min superfusion with CPA. CPA also transiently prolong ed the plateau phase of the action potential and increased the peak am plitude of contraction: these effects peaked at about 10-20 min from s tart of superfusion with CPA and then declined. At the peak of its enh ancing effect on contraction amplitude, CPA prolonged the contractile phase of the contraction-relaxation cycle. 9 Superfusion with a low-Na , choline-substituted Krebs solution produced a reversible membrane de polarization. In the presence of Bay K 8644 (1 mu M), action potential s and phasic contractions were superimposed on this depolarization whi ch were abolished by nifedipine (10 mu M). 10 These findings indicate that CPA augments the excitability and affects the contraction-relaxat ion cycle of the smooth muscle of the guinea-pig ureter, implying a ro le for sarcoplasmic reticulum Ca2+-ATPase in the regulation of electro mechanical coupling. The effects of CPA resemble those produced by rya nodine and the effect of the two agents on the amplitude of contractio ns is non-additive. It appears that following blockade of the CPA-sens itive SR Ca2+ pump, other mechanism(s) may come into action to reduce intracellular Ca2+. The Na+/Ca2+ exchanger could be involved in the co mpensatory changes responsible for the fading of the response to CPA.