EFFECT OF BAY-K-8644 AND RYANODINE ON THE REFRACTORY PERIOD, ACTION-POTENTIAL AND MECHANICAL RESPONSE OF THE GUINEA-PIG URETER TO ELECTRICAL-STIMULATION

We have investigated the effect of the dihydropyridine calcium channel agonist, Bay K 8644, and of the plant alkaloid blocker of calcium-ind uced calcium release (CICR) from the sarcoplasmic reticulum, ryanodine , on the refractory period, action potential and mechanical response o f the guinea-pig isolated ureter to electrical stimulation. All experi ments were performed in ureters pre-exposed to 10 mu M capsaicin to el iminate the inhibitory influence exerted by local release of sensory n europeptides on ureteral excitability and contraction. In organ bath e xperiments, electrical field stimulation with parameters which produce direct excitation of ureteral smooth muscle (train of pulses at 10 Hz , 5 ms pulse width, 60 V for 1 s) produced tetrodotoxin- (1 mu M) resi stant phasic contractions. The response to EFS was abolished by nifedi pine (1 nM-3 mu M) and was enhanced by Bay K 8644 (1 nM-3 mu M). In th e presence of Bay K 8644 (1 mu M), nifedipine (30 mu M) abolished the evoked contractions. Ryanodine (10-100 mu M) had no significant effect on the amplitude of evoked contraction. The response of the guinea-pi g ureter to direct electrical stimulation of smooth muscle is characte rized by a refractory period: at least 40 s interstimulus interval was required to produce a second response in all preparations tested. Bay K 8644 (1 mu M) markedly reduced the refractory period of the ureter and a similar effect was observed with ryanodine (100 mu M). To furthe r analyze the effect of Bay K 8644 and ryanodine on the refractory per iod, the response of the ureter was investigated over a 10 s period of stimulation (other parameters as above). In control ureters, continuo us stimulation for 10 s produced only one phasic contraction just afte r the beginning of the train of stimuli. In the presence of Bay K 8644 or ryanodine, more than one phasic contraction developed during a 10 s stimulation, i.e. the refractory period became shorter than the trai n duration. When both Bay K 8644 and ryanodine were tested on the same preparations, an additive excitatory effect was observed on the mecha nical response to electrical stimulation. A slight elevation of KCl co ncentration (5 - 10 mM) reduced the refractory period of the ureter as observed with ryanodine or Bay K 8644. Application of KCl (80 mM) pro duced a biphasic contractile response of the ureter: a series of phasi c contractions occurred first, which were then replaced by a slowly de veloping tonic response. Bay K 8644 (1 mu M) enhanced both components of the response to KCl. Ryanodine (10 and 100 mu M) markedly prolonged the duration of phasic contractions evoked by KCl and, at 100 mu M, s lightly (about 25%) reduced the amplitude of tonic contraction. In suc rose gap experiments, electrical stimulation (single pulse, 40-130 V, 1-3 ms pulse duration) evoked an action potential and accompanying pha sic contraction which were abolished by 1 mu M nifedipine. Bay K 8644 (1 mu M) produced a marked prolongation of action potential duration, increased the number of spikes and enhanced contraction amplitude and duration. Ryanodine (100 mu M) depolarized the membrane, reduced the d elay between stimulus application and onset of the action potential, s hortened the action potential at 50% of repolarization and increased a fterhyperpolarization, without producing marked effects on the accompa nying mechanical response. KCl (5 mM) likewise produced a slight membr ane depolarization and decreased latency between stimulus application and onset of the action potential but did not affect action potential duration. The combined administration of ryanodine and Bay K 8644 prod uced additive effects on action potential and contractions: furthermor e, the contractile phase of the overall contraction-relaxation cycle w as significantly prolonged by the combined administration of the two a gents, an effect not observed with either drug alone. In the presence of both Bay K 8644 and ryanodine, multiple action potentials and contr actions were observed during a train of pulses delivered at a frequenc y of 1 Hz for 12 s: when a second action potential was triggered befor e relaxation of the preceding contraction, a summation of the contract ile response was observed. These findings demonstrate that availabilit y of voltage-dependent L-type calcium channels is a major mechanism in determining the refractory period. of the guinea-pig ureter and, cons equently, can be considered as a limiting step in regulating the maxim al frequency of ureteral peristalsis. Furthermore, a ryanodine-sensiti ve mechanism regulates the excitability and contraction-relaxation cyc le of ureteral smooth muscle. The increased electrical excitability of the ureter observed in the presence of ryanodine may involve blockade of transient outward currents triggered by spontaneous calcium releas e from the store and consequent membrane depolarization.