PROTEIN-KINASE-A INHIBITORS SELECTIVELY INHIBIT THE TONIC CONTRACTIONOF THE GUINEA-PIG URETER TO HIGH POTASSIUM

1. We have investigated the effect of various protein kinase A (PKA) i nhibitors on the phasic and tonic components of the response to potass ium chloride (KCI) in the guinea pig ureter. All experiments were perf ormed in ureters pretreated with capsaicin (10 mu M for 15 min) to pre vent the release of sensory neuropeptides and in the presence of 1 mu M Bay K 8644 to maximize calcium (Ca) entry via voltage sensitive chan nels, The addition of 80 mM hypertonic KCl produced maximal shortening of the ureter with distinct phasic and tonic components, the latter f urther showing a transient and a sustained component. Nifedipine (30 m u M for 120 min) totally abolished all the responses to KCl. 2. The se lective PKA inhibitor, H89 (10 mu M), abolished the tonic response to KCI in about 30 min with minor inhibitory effect on the phasic contrac tion. This pattern was unchanged when extending the contact time to 12 0 min. When added 30 min before the next challenge, H89 (1-30 mu M) co ncentration-dependently inhibited the responses to KCl with a preferen tial inhibitory effect on the tonic contraction. Another PKA inhibitor , H8, produced similar effects at tenfold higher concentrations (10-30 0 mu M) than H89, consistent with the known potency ratio of these iso quinoline derivatives in inhibiting PKA. 3. The potent and nonselectiv e protein kinase inhibitor, staurosporine (10-100 nM) produced an even depression of the various phases of the response to KCl. The selectiv e protein kinase G inhibitor, KT 5823 (10 mu M for 60 min) produced on ly a slight reduction of the sustained tonic response to KCI. The sele ctive protein kinase C inhibitor GF 109,203X (1-3 mu M) and the cAMP a nalog, Rp-cAMPS (300 mu M for 60 min) had no effect on the three compo nents of the response to KCl. 4. In the presence of Bay K 8644, electr ical field stimulation (10 Hz for 1 sec, 60 V, pulse width 5 ms) produ ces direct myogenic phasic contractions (twitches) of the ureter which are suppressed by nifedipine (10-30 mu M), H89 (up to 30 mu M) and H8 9 (up to 300 mu M) had minor effect on the amplitude of twitches, cons istent with their poor inhibitory activity on the phasic responses to KCl. 5. In sucrose gap, superfusion with 80 mM hypertonic KCl produced action potentials followed by a sustained depolarization of the membr ane: the two electrical responses underlie the phasic and tonic compon ents of contraction to KCI, respectively. H89 (10 mu M for 30 min) did not affect the resting membrane potential nor the KCl-evoked action p otentials and sustained depolarization, H89 had no effect on the phasi c contraction to KCl but markedly depressed (about 65% inhibition) the tonic contraction. 6. The present findings are consistent with the vi ew that phosphorylation by PKA increases the availability of L-type Ca channels in the ureter smooth muscle, Blockade of PKA dissociates the electromechanical coupling between the sustained membrane depolarizat ion produced by KCI and the corresponding sustained increase in tensio n. The L-type Ca channel responsible for generating action potentials and phasic contractions to KCI are less sensitive to PKA inhibitors th an those responsible for the tonic contraction.