| The roIe of calcium mobilization in sodiumnitroprusside-induced increase of caIcium-activatedpotassium currents in gastric antral circular myocytes of theguinea-pig.Stimulatoin of nonadrenergic, noncholinergic inhibitory (NANC) nerve evokesa membrane hyerpolarezation in circular smooth muscle cells of the gastrointestinal(GI) tract. The hyperpolarization of membrane potential is referred to as theinhibitory junction potential (IJP). These nerves mediated the majority of inhibitoryresponses in the GI tract and regulate many important physiologicaI reflexes, suchas relaxation of the lower esophageal sphincter after swallowing, receptiverelaxation of the proximal stomach during eating, and descending inhibition inresponse to distension. In the past ten years, many studies have suggested that nitricoxide (NO) may work as a NANC neurotransmitter, inhibiting motility of the GItract. The mechanism by Which NO relaxes smooth muscle has long been acontroversial topic. NO and NO donors, such as sodium nitroprusside (SNP) and3-morpholino-sydnodimine-hydroc (SIN-1), are generally thought to causecsmooth muscle relaxation by the activation of guanosine 3', 5'-cyclicmonophospate cGMP. Recent patch clamp studies demonstrated that NO-donorsenhanced calcium-activated potassium currents [IK(c.)1 via cGMP However, themechanism how NO and NO-donors increased IK(..) is not yet ful1y understood. Inthis work, we isolated gastric amral circular smooth muscle cells of the grinea-pigwith 0.l% type II collagenase. The majority of the freshly dissociated musclecelIs possessed an intact structure. In the present study, using the conventionalWhole-cell mode and perforated whole-cell patch claxnp mode, the role ofintracellular and extrce1lular calcium in SNP- induced increase of IK(..) wereinvestigated.l. Ca'+-activated potassium current was elicited by the step depolarization withthe pipette solution containing egtazic acid (EGTA) 0.1 mM. The membranepotential was clamped at to0mV and command pulse from wt0mV to 100mV for400ms wth a 20mV increment at 10 sec intervals. Under the conventional who1ecell and perforated whole cell patch clamp mode, the mean amplitude of IK(c,) at+60mV was 1.7 I l.0 nA (n=50) and 1.6i0.86 nd (n=50). Using the same pulseprotocol, l00 ll M, 300 ll M and l000 ll M SNP did not effect in IK(..) on theconventional whole cell patch clamp mode. Howevef, under the perforated wholecell patch clamp mode, l00 ll M SNP caused a efficient increase in IK(..) bY 20'2%t4.5% at 60mV 4-AP l0 mM, a kind of selective inhibiter of delayed rectifierpotassium current [IK(v)], can not inhibit the SNP-induced increase of IK(c.)'.2. The extent of change in IK(c.) was markedly dependent on the concentrationof perfusive SNP The normalized IK(c.) (at 60 mV) were l .0, l .2l f0.05, l .34I0. 1 l, 1 .68 f 0.09 at 30 u M, l00 u M, 300 u M and l000 n M SNP respectively.3' IK(..) began to increase after 4.2 l 2.9 seconds (the cel1s were superfused withl00 u M SNP bath solution) at 60 mV l0.6i4.3 seconds at peak, and resumedafter 272.3 I 87.0 seconds (wash out with physiological salt solution).4. Calcium channel current (IC. ) was recorded in extemal calcium 2.0 mM andperfOrate patch clamp mode. With holding potential of --80mV depolarizationbeyond --30 mV elicited an inward current, peak at +l0~+20mV and reversedbetWeen +50 mV and +60mV The peak current was --58.0i l3.4 PA (n=7).Replacement of 2.0mM of Ca2+ with l0mM Ba'+ increased the current amplitudesabout 3.9-folds (245.8 t40.6 pA, n=8). The IB. exhibited slower inactivation timecourse than the Ic. and did not completeIy inactivated during 400ms step pulses.With holding potential of --80 mV depolarization beyond --50 mV elicited aninward current, peaked at +l0mV and reversed between +40 mV and +60 mV 5 llM nicardipine, a potent blocker of L-type calcium channel, markedly blocked the' IB. at all test potelltials, but not affected l...
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