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

Abstract

1. The nature of the membrane channels underlying the membrane conductance changes induced by the nitric oxide (NO) donors, S-nitroso-L-cysteine (NOCys) and sodium nitroprusside (SNP) were investigated in single myocytes isolated from the circular muscle layer of the guinea-pig proximal colon, by use of standard whole-cell and single channel recording techniques.

2. Under voltage clamp, depolarizing steps from −60 mV elicited a rapidly-developing, little-inactivating outward K⁺ current (I_K) at potentials positive to −40 mV (at 20–25°C). The steady-state level (I_SS) of this K⁺ current increased in amplitude as the step potential was made to more positive potentials. If the depolarizing steps were made from a holding potential of −80 mV an additional rapidly activating and inactivating outward K⁺ current was also elicited, superimposed on I_K.

3. At 20–25°C, NOCys (2.5 μM), SNP (100 μM) and 8-bromo-cyclic GMP (500 μM) increased the amplitude of I_SS of I_K elicited from a holding potential of −60 mV. In contrast, NOCys (2–5 μM) had little effect on I_SS at 35°C. Higher concentrations (⩾5 μM at 20–25°C and ⩾10 μM at 35°C) of NOCys decreased the peak amplitude (I_Peak) and I_SS of I_K in a concentration-dependent manner. This blockade of I_K with NOCys was always associated with an increase of the holding current (I_Hold), due to the activation of a membrane conductance with a reversal potential between 0 and +30 mV and which was reduced approximately 50% upon the addition of Cd²⁺ (1 mM).

4. NOCys (2.5 to 10 μM) or SNP (100 μM) increased the activity of large conductance Ca²⁺-activated (BK) K⁺ channels in both cell-attached and 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 μM) or SNP (100 μM) were associated with an increase in the probability of BK channel opening (N.P_o), and with a negative shift of the plots of ln(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 NOCys was often associated with a decrease in the BK single channel conductance.

5. In both cell-attached and excised patches, NOCys (2.5 to 10 μM) 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 single 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 the pipette solution had been replaced with an equimolar concentration of Na gluconate.

6. Under current clamp, NOCys (2.5 μM) and SNP (100 μM) had variable effects on the membrane potential of colonic myocytes, inducing either a small membrane hyperpolarization of <5 mV, or a slowly-developing membrane depolarization of about 5 mV. In contrast, NOCys (5 μM) produced a transient membrane hyperpolarization which was followed by a large depolarization of the membrane potential to positive potentials. The electrotonic potentials elicited in response to an injection of constant hyperpolarizing current (10 pA for 400 ms) were little changed during the NOCys (5 μM)-induced membrane hyperpolarization, but significantly reduced (to 61% of control) during the periods of membrane depolarization.

7. It was concluded that NOCys and SNP, directly increased the number of active BK channels in the membrane of colonic myocytes which leads to a small rapidly oscillating membrane hyperpolarization. The following rebound depolarization in NOCys arises from both the direct opening of a population of cationic channels and the blockade of voltage- and Ca-activated K⁺ conductances. Finally, the apamin-sensitive K⁺ channels underlying the initial transient hyperpolarization recorded in the intact proximal colon, in response to nerve-released or directly-applied NO, have yet to be identified at the single channel or whole-cell current level.

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