EFFECTS OF NITRIC-OXIDE DONORS, S-NITROSO-L-CYSTEINE AND SODIUM-NITROPRUSSIDE, ON THE WHOLE-CELL AND SINGLE-CHANNEL CURRENTS IN SINGLE MYOCYTES OF THE GUINEA-PIG PROXIMAL COLON

1 The nature of the membrane channels underlying the membrane conducta nce changes induced by the nitric oxide (NO) donors, S-nitroso-L-cyste ine (NOCys) and sodium nitroprusside (SNP) were investigated in single myocytes isolated from the circular muscle layer of the guinea-pig pr oximal colon, by use of standard whole-cell and single channel recordi ng techniques. 2 Under voltage clamp, depolarizing steps from -60mV el icited a rapidly-developing, little-inactivating outward K+ current (I -K) at potentials positive to -40 mV (at 20-25 degrees C). The steady- state level (I-SS) of this K+ current increased in amplitude as the st ep potential was made to more positive potentials. If the depolarizing steps were made from a holding potential of -80 mV an additional rapi dly activating and inactivating outward K+ current was also elicited, superimposed on I-K. 3 At 20-25 degrees C, NOCys (2.5 mu M), SNP (100 mu M) and 8-bromo-cyclic GMP (500 mu M) increased the amplitude of I-S S of I-K elicited from a holding potential of -60 mV. In contrast, NOC ys (2-5 mu M) had little effect on I-SS at 35 degrees C. Higher concen trations (greater than or equal to 5 mu M at 20-25 degrees C and great er than or equal to 10 mu M at 35 degrees C) of NOCys decreased the pe ak amplitude (I-peak) and I-SS of I-K in a concentration-dependent man ner. This blockade of I-K with NOCys was always associated with an inc rease of the holding current (I-Hold), due to the activation of a memb rane conductance with a reversal potential between 0 and +30 mV and wh ich was reduced approximately 50% upon the addition of Cd2+ (1 mM). 4 NOCys (2.5 to 10 mu M) or SNP (100 mu M) increased the activity of lar ge conductance Ca2+-activated (BK) K+ channels in both cell-attached a nd excised inside-out patches, bathed in either a symmetrical high K(130 mM) or an asymmetrically K+ (6 mM(out): 130 mM(in)) physiological saline. Increases in BK channel activity in NOCys (10 mu M) or SNP (1 00 mu M) were associated with an increase in the probability of BM cha nnel opening (N.P-o), and with a negative shift of the plots of In (N. P-o) against the patch potential, with little change in the slopes of these plots. In cell-attached patches, the increase in N.P-o with NOCy s was often associated with a decrease in the BK single channel conduc tance. 5 In both cell-attached and excised patches, NOCys (2.5 to 10 m u M) also activated an additional population of channels which allowed inward current flow at potentials positive to E-K. In excised inside- out patches bathed in asymmetrical K+ physiological saline, these sing le channel currents were 2-3 pA in amplitude at -30 mV and reversed in direction near + 10 mV, even if the NaCl (126 mM) concentration in th e pipette solution had been replaced with an equimolar concentration o f Na gluconate. 6 Under current clamp, NOCys (2.5 mu M) and SNP (100 m u M) had variable effects on the membrane potential of colonic myocyte s, inducing either a small membrane hyperpolarization of <5 mV, or a s lowly-developing membrane depolarization of about 5 mV. In contrast, N OCys (5 mu M) produced a transient membrane hyperpolarization which wa s followed by a large depolarization of the membrane potential to posi tive potentials. The electrotonic potentials elicited in response to a n injection of constant hyperpolarizing current (10 pA for 400 ms) wer e little changed during the NOCys (5 mu M)-induced membrane hyperpolar ization, but significantly reduced (to 61% of control) during the peri ods of membrane depolarization. 7 It was concluded that NOCys and SNP, directly increased the number of active BK channels in the membrane o f colonic myocytes which leads to a small rapidly oscillating membrane hyperpolarization. The following rebound depolarization in NOCys aris es from both the direct opening of a population of cationic channels a nd the blockade of voltage-and Ca-activated K+ conductances. Finally, the apamin-sensitive K+ channels underlying the initial transient hype rpolarization recorded in the intact proximal colon, in response to ne rve-released or directly-applied NO, have yet to be identified at the single channel or whole-cell current level.