Effect of the Ca2+-ATPase inhibitor, cyclopiazonic acid, on electromechanical coupling in the guinea-pig ureter

Abstract

1 We have investigated the effect of the sarcoplasmic reticulum (SR) Ca²⁺-ATPase inhibitor, cyclopiazonic acid (CPA), on electromechanical coupling in the guinea-pig ureter. All experiments were performed in capsaicin-pretreated (10 μm for 15 min) ureters to prevent the release of sensory neuropeptides from afferent nerves. 2 In organ bath experiments, electrical field stimulation (EFS, 10 Hz for 1 s, 5 ms pulse width, 60 V) produced tetrodotoxin- (1 μm) resistant phasic contractions which were enhanced by Bay K 8644 (1 μm) and abolished by nifedipine (10–30 μm). 3 CPA (10 μm) 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 μm. The response to 10 μm CPA was biphasic: the maximal enhancement (58 ± 3% increase) was observed within 10–20 min from CPA administration, followed by a decline to a new steady state (25 ± 5% increase over baseline) at 50–60 min. The effect of CPA was reversed by washout. 4 Ryanodine (100 μm) produced a prompt enhancement of the EFS-evoked contractions of the guinea-pig ureter, which peaked at 42 ± 3% increase over baseline; the co-administration of CPA (10 μm) and ryanodine (100 μm) 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, showing a time-course similar to that observed with CPA alone. Tetraethylammonium (10 mm) produced a significantly larger effect (93 ± 13% increase over baseline) and its effect was sustained throughout the 60 min observation period. 5 In the presence of Bay K 8644, superfusion for 30 min with a low Na⁺ medium (60% of extracellular Na⁺ replaced by Li⁺ or choline) reduced the amplitude of EFS-evoked contractions by 20–35%. In both Li⁺- and choline-substituted media, spontaneous activity developed during super-fusion with low Na⁺ Krebs solution which was suppressed by 10 μm nifedipine. CPA (10 μm) produced a marked enhancement of the EFS-evoked contractions in low-Na⁺ medium (both Li⁺- and choline-substituted) and this effect was sustained throughout the 60 min observation period. 6 In the absence of Bay K 8644, the response of the ureter smooth muscle to EFS is characterized by a refractory period: an interval of about 30 s was required between two applied stimuli to produce a second response comparable in size to that elicited by the first stimulus. CPA (10 μm, 10–20 min before) markedly reduced the refractory period of the guinea-pig ureter to EFS. 7 CPA (10 μm, 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 contraction was observed at 1–2 min from addition of KCl. 8 In sucrose gap experiments, 10 μm CPA produced a sustained depolarization of the membrane and reduced the latency between application of electrical stimuli and onset of the action potential; these effects were maintained throughout the 60 min superfusion with CPA. CPA also transiently prolonged the plateau phase of the action potential and increased the peak amplitude of contraction: these effects peaked at about 10–20 min from start of superfusion with CPA and then declined. At the peak of its enhancing 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 depolarization. In the presence of Bay K 8644 (1 μm), action potentials and phasic contractions were superimposed on this depolarization which were abolished by nifedipine (10 μm). 10 These findings indicate that CPA augments the excitability and affects the contraction-relaxation cycle of the smooth muscle of the guinea-pig ureter, implying a role for sarcoplasmic reticulum Ca²⁺-ATPase in the regulation of electromechanical coupling. The effects of CPA resemble those produced by ryanodine and the effect of the two agents on the amplitude of contractions is non-additive. It appears that following blockade of the CPA-sensitive SR Ca²⁺ pump, other mechanism(s) may come into action to reduce intracellular Ca²⁺. The Na⁺/Ca²⁺ exchanger could be involved in the compensatory changes responsible for the fading of the response to CPA.