Membrane hyperpolarization, cyclic nucleotide levels and relaxation in the guinea-pig internal anal sphincter

1. Changes in membrane potential (measured with an intracellular microelectrode) and in cyclic nucleotide (adenosine 3':5'-cyclic monophosphate, cyclic AMP and guanosine 3':5'-cyclic monophosphate, cyclic GMP) levels (measured by radioimmunoassay) in response to inhibitory non-adrenergic non-cholinergic (NANC) field stimulation and drugs were investigated in the guinea-pig internal and anal sphincter (gpIAS) in the presence of phentolamine and atropine (each 10(-6) M). 2. Inhibitory NANC nerve stimulation (single pulse, 5 pulses at 5, 10 and 20 Hz, 0.5 ms supramaximal voltage) and adenosine triphosphate (ATP, 10(-7)-10(-3) M) inhibited spike discharge, hyperpolarized the membrane and relaxed the sphincter. The effects of inhibitory nerve stimulation were blocked by tetrodotoxin (TTX, 10(-6) M) and, with those of ATP, were blocked by apamin (5 x 10(-6) M). 3. Isoprenaline (10(-9)-10(-4) M), cromakalim (10(-9)-10(-5) M), sodium nitroprusside (NaNP 10(-5) M), M&B 22948 (10(-4) M) and 8-bromocyclic GMP (8-Br-cyclic GMP, 10(-4) M) also inhibited spike discharge, hyperpolarized the membrane and relaxed the sphincter. The effects of isoprenaline were blocked by propranolol (10(-6) M). However, forskolin (10(-9)-10(-7) M), M&B 22948 (10(-9)-10(-5) M) and lower concentrations of NaNP (10(-9)-10(-6) M) relaxed the sphincter without affecting the membrane potential. 4. The characteristics of the membrane potential changes in response to different inhibitory stimuli in the gpIAS differed. Hyperpolarization produced by inhibitory NANC nerve stimulation and ATP were rapid in onset, of brief duration and of comparable amplitude. Isoprenaline and direct electrical stimulation also hyperpolarized the membrane and relaxed the muscle although the extent of the relaxation in these two cases was much less than that with nerve stimulation and ATP. In each case, the membrane potential change preceded relaxation and probably accounted for it. 5. Both inhibitory NANC nerve stimulation (80 pulses 8Hz supramaximal voltage 0.5 ms) and ATP (10-aM) raised levels of cyclic GMP significantly and to a comparable degree and relaxed the sphincter. The effect of nerve stimulation was prevented by TTX (10- 6M) but not by apamin (5 x 10- 6M). Isoprenaline (10-s M), cromakalim (10 5 M) and forskolin (10 5M) were ineffective. 6. Inhibitory NANC nerve stimulation (80 pulses 8Hz 0.5ms supramaximal voltage) and ATP (10-4M) raised levels of cyclic AMP significantly to a comparable degree and relaxed the sphincter. The increase produced by nerve stimulation was abolished by TTX (10-6M) and apamin (5 x 10-6M). Isoprenaline (10-4M), cromakalim (10-5 M) and forskolin (10-5 M) raised levels of this nucleotide significantly.