Alan R. Chipperfield

Occurrence of passive furosemide-sensitive transmembrane potassium transport in cultured cells

Furosemide (1 x 10(-4) M) inhibits a proportion of the total passive (ouabain-insensitive) K+ influx into primary chick heart cell cultures (85%), BC3H1 cells (75%), MDCK cells (40%) and HeLa cells (57%). This action of furosemide upon K+ influx is independent of (Na+ + K+)-pump inhibition since the furosemide-sensitive component of the K+ influx is identical in the presence and absence of ouabain (1 x 10(-3) M). For HeLa cells the passive, furosemide-sensitive component of K+ influx is markedly dependent upon the external K+, Na+ and Cl- content. Acetate, iodide and nitrate are ineffective as substitutes for Cl-, whereas Br- is partially effective. Partial Cl- replacement by NO3- gave an apparent affinity of 100 mM [Cl]. Na+ replacement by choline+ abolishes the furosemide-sensitive component, whereas Li+ replacement reduces this component by 48%. Partial Na+ replacement by choline+ gives an apparent affinity of 25 mM [Na+]. Variation in the external K+ content gives an affinity for the furosemide-sensitive component of approx. 1.0 mM. Furosemide inhibition of the passive K+ influx is of high affinity, half-maximal inhibition being observed at 5 x 10(-6) M furosemide. Piretanide (1 x 10(-4) M) and phloretin (1 x 10(-4) M) inhibit the same component of passive K+ influx as furosemide; ethacrynic acid and amiloride (both 1 x 10(-4) M) partially so. The stilbene, SITS (1 x 10(-6) M), was ineffective as an inhibitor for the furosemide-sensitive component.

Increased (Na+K+Cl) Cotransport in Rat Arterial Smooth Muscle in Deoxycorticosterone (DOCA)/Salt-Induced Hypertension

The inhibition by loop diuretics of K efflux (tracer 86Rb) from the rat femoral arterial smooth muscle was measured in normotension and in DOCA-salt hypertension. The sensitivity sequence (bumetanide > piretanide > furosemide) was the characteristic pharmacological profile of (Na+K+Cl) cotransport. In hypertension, cotransport activity was 46% greater than in normotension and the sensitivity to loop diuretics was threefold less. Intracellular [K] and the Na, K and Cl permeability ratios and electrogenic Na pump activity were assessed electrophysiologically in normotension and hypertension. [K]i was lower in hypertension (173 mM) than normotension (198 mM) but the other parameters (PNa/Cl = 0.14, PCl/PK = 0.19 and electrogenic pump = –8.3 mV in normotension) were not significantly different. Ionic permeabilities to Na, K and Cl were significantly lower in hypertension than normotension. Plasma [Na], but not [K], was higher in hypertension than normotension. The conclusion is that increased activation of (Na+K+Cl) cotransport in hypertension plays a major role in the elevation of [Cl]i and depolarisation of the membrane potential in vascular smooth muscle in DOCA-salt hypertension. The role of (Na+K+Cl) cotransport in vascular smooth muscle in this model of hypertension is discussed in relation to [Cl]i, depolarisation of the membrane potential and contraction and in relation to cell growth.

Stimulation of intracellular chloride accumulation by noradrenaline and hence potentiation of its depolarization of rat arterial smooth muscle in vitro

1 Double‐barrelled ion‐selective microelectrodes were used to examine the effects of exogenous noradrenaline upon the membrane potential (Em) and intracellular chloride concentration ([Cl]i) of arterial smooth muscle from the saphenous branch of the femoral artery of the rat. 2 After treatment with 0.6 mM 6‐hydroxydopamine (to functionally denervate the tissue), exogenous noradrenaline (5 nM) caused repeatable depolarization of Em from −63.7±2.4 mV (s.d., n=18) to −53.8±3.4 mV (P<0.0001) and increases in [Cl]i from 31.0±0.5 mM to 42.5±2.2 mM (P<0.0001). 3 In the presence of 10 μM bumetanide (an inhibitor of (Na‐K‐Cl) cotransport), 5 nM noradrenaline caused a depolarization of Em of 3.0±3.2 mV, and a rise in [Cl]i of 4.5±2.5 mM. 4 In the presence of bumetanide and 1 mM acetazolamide (used as an inhibitor of a Na‐independent inward Cl pump), noradrenaline had no effect on Em or [Cl]i. 5 In the absence of extracellular chloride, the rise in apparent [Cl]i in response to 5 nM noradrenaline was abolished but there was a depolarization of 2.0±3.9 mV. 6 These results are consistent with the stimulation of (Na‐K‐Cl) cotransport and a Na‐independent Cl pump by exogenous noradrenaline and with the consequent increase in [Cl]i and shift in ECl potentiating the depolarization caused by noradrenaline. The possibility that modulation of [Cl]i may be a general mechanism of Em regulation is discussed.

Loop diuretics may fail to inhibit (Na+, K+, Cl−) ‘cotransport’ in human red cells

The inhibition of passive K+ influx into human red blood ceils (RBC) by loop diuretics was found to be dependent on the external Na+ concentration, In the absence of external Na+, there was minimal inhibition but the influx remained dependent on CI- ions. Thus, raising the external Na+ concentration increased the affinity of the putative (Na+, K+, Cl− cotransport system in human RBC for loop diuretics.

Chronotropic actions of Na+,K+,Cl− cotransport inhibition in the isolated rat heart

Abstract The chronotropic actions of Na+,K+,Cl− cotransport were investigated by studying the effects of the loop diuretics bumetanide and furosemide, specific inhibitors of the cotransporter, on an isolated rat sino-atrial node preparation. Application of bumetanide decreased the cycle length from 0.334 s (±0.087 S.D.) to 0.279 s (±0.083, n = 16, P = 6.5 × 10−6) in Hepes-buffered physiological salt solution (PSS). Similar decreases were recorded in bicarbonate-buffered PSS. Chloride channel blockers indicate that the tachycardia evoked by loop diuretics is not due their blocking of chloride channels. Thus, 4,4′-dinitrostilbene-2,2′-disulphonic acid (DNDS) and 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) had a negative chronotropic action and 2-[(2-cyclopentyl-6,7-dichloro-2,3-dihydro-2-methyl-1-oxo-1 H-inden-5-yl) oxy] acetic acid (IAA-94) produced no change in cycle length. Pharmacological manoeuvres indicate that the positive chronotropic action of loop diuretics is associated with catecholamine release. The positive chronotropic action of bumetanide was inhibited by the β-adrenoceptor antagonists, propranolol and atenolol, but was unaffected by atropine.