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A R Chipperfield

Publications and source records attributed to A R Chipperfield.

At least 19 recordsLinked to original sources
PubMed2000

The three mechanisms of intracellular chloride accumulation in vascular smooth muscle of human umbilical and placental arteries.

Recordings of membrane potential (Em) and intracellular [Cl-] ([Cl-]i) were made from the smooth muscle of human umbilical and placental arteries, using double-barrelled, ion-sensitive microelectrodes. In both arteries, [Cl-]i was above equilibrium with Em. In the umbilical artery, [Cl-]i was 33.8+/-0.9 mM (+/-SD, n=19) and Em -54.9+/-1.3 mV and in the placental artery respectively 35.1+/-0.7 mM (n=17) and -50.6+/-0.9 mV. In both arteries, [Cl-]i was reduced and Em hyperpolarised significantly by successive additions of 100 microM 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulphonic acid (DIDS), 10 microM bumetanide and 1 mM acetazolamide, thus revealing the presence of Cl-/HCO3- exchange, (Na+K+Cl) cotransport and "pump III". In the presence of all three inhibitors, [Cl-]i was in equilibrium with Em. As in earlier studies on rat arterial smooth and cardiac muscle, pump III was unaffected by DIDS, bumetanide, metolazone and the removal of Na+, partly inhibited by chlorothiazide and fully inhibited by ethacrynic acid. The results are discussed in terms of the possibility that of chloride accumulating systems may regulate vasomotor tone in the foetoplacental unit.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed2000

Chloride in smooth muscle.

Interest in the functions of intracellular chloride expanded about twenty years ago but mostly this referred to tissues other than smooth muscle. On the other hand, accumulation of chloride above equilibrium seems to have been recognised more readily in smooth muscle. Experimental data is used to show by calculation that the Donnan equilibrium cannot account for the chloride distribution in smooth muscle but it can in skeletal muscle. The evidence that chloride is normally above equilibrium in smooth muscle is discussed and comparisons are made with skeletal and cardiac muscle. The accent is on vascular smooth muscle and the mechanisms of accumulation and dissipation. The three mechanisms by which chloride can be accumulated are described with some emphasis on calculating the driving forces, where this is possible. The mechanisms are chloride/bicarbonate exchange, (Na+K+Cl) cotransport and a novel entity, "pump III", known only from own work. Their contributions to chloride accumulation vary and appear to be characteristic of individual smooth muscles. Thus, (Na+K+Cl) always drives chloride inwards, chloride/bicarbonate exchange is always present but does not always do it and "pump III" is not universal. Three quite different biophysical approaches to assessing chloride permeability are considered and the calculations underlying them are worked out fully. Comparisons with other tissues are made to illustrate that low chloride permeability is a feature of smooth muscle. Some of the functions of the high intracellular chloride concentrations are considered. This includes calculations to illustrate its depolarising influence on the membrane potential, a concept which, experience tells us, some people find confusing. The major topic is the role of chloride in the regulation of smooth muscle contractility. Whilst there is strong evidence that the opening of the calcium-dependent chloride channel leads to depolarisation, calcium entry and contraction in some smooth muscles, it appears that chloride serves a different function in others. Thus, although activation and inhibition of (Na+K+Cl) cotransport is associated with contraction and relaxation respectively, the converse association of inhibition and contraction has been seen. Nevertheless, inhibition of chloride/bicarbonate exchange and "pump III" and stimulation of (K+Cl) cotransport can all cause relaxation and this suggests that chloride is always involved in the contraction of smooth muscle. The evidence that (Na+K+Cl) cotransport more active in experimental hypertension is discussed. This is a common but not universal observation. The information comes almost exclusively from work on cultured cells, usually from rat aorta. Nevertheless, work on smooth muscle freshly isolated from hypertensive rats confirms that (Na+K+Cl) cotransport is activated in hypertension but there are several other differences, of which the depolarisation of the membrane potential may be the most important.Finally, a simple calculation is made which indicates as much as 40% of the energy put into the smooth muscle cell membrane by the sodium pump is necessary to drive (Na+K+Cl) cotransport. Notwithstanding the approximations in this calculation, this suggests that chloride accumulation is energetically expensive. Presumably, this is related to the apparently universal role of chloride in contraction.

Animals↗
PubMed1997

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 microM 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.

Acetazolamide↗
PubMed1997

Activation of two inward chloride transport systems in rat femoral arterial smooth muscle in deoxycorticosterone acetate/salt hypertension.

1. Intracellular [Cl-] ([Cl-]i) was measured with ion-selective microelectrodes in rat femoral arterial smooth muscle in normotensive controls and after the induction of deoxycorticosterone acetate/salt hypertension. 2. Linear regression of [Cl-]i and time after the induction of hypertension showed good correlation (r = 0.96) for 5-6 weeks, as [Cl-]i increased from 30 +/- 1 mmol/l (mean +/- SD, n = 16), to 49 +/- 2 mmol/l (n = 9, P < 0.0001). 3. Arterial systolic blood pressure also increased linearly (r = 0.97) for 5-6 weeks as hypertension developed from 122 +/- 1 mmHg (n = 20) to 187 +/- 7 mmHg (n = 14): there was consequently a linear relationship between [Cl-]i and arterial systolic blood pressure (r = 0.96). 4. The increase in [Cl-]i was partly because Na(+)-K(+)-Cl- co-transport activity, estimated from the fall in [Cl-]i caused by bumetanide, was greater in hypertension (18 mmol/l) than in normotension (10 mmol/l). This finding, and the depolarization of the membrane potential in hypertension (-56 +/- 3 mV compared with -64 +/- 4 mV in normotension; P < 0.0001), confirms previous studies. 5. The increase in [Cl-]i was also partly due to greater activity of an Na(+)- and HCO3(-)-independent, acetazolamide-sensitive inward Cl- transport system; thus acetazolamide reduced [Cl-]i by 7 mmol/l in normotension and by 16 mmol/l in hypertension. 6. In Cl(-)-free media, the membrane potential in normotension (-59 +/- 5 mV) was not significantly different from that in hypertension (-60 +/- 4 mV). 7. The role of [Cl-]i in the depolarization of the membrane potential in hypertension is discussed.

Acetazolamide↗
PubMed1997

Sodium-independent inward chloride pumping in rat cardiac ventricular cells.

The intracellular Cl concentration ([Cl]i) in rat cardiac ventricular muscle, measured with double-barreled microelectrodes in vitro, was 21.3 +/- 1.5 (SD) mM [number of observations (n) = 46]. With the Na-K-Cl cotransport inhibitor bumetanide (10 microM), it fell to 13.4 +/- 1.4 mM (n = 27), and with 1 mM acetazolamide, it fell further, to 7.2 +/- 1.5 mM (n = 5), close to equilibrium with the membrane potential. In the absence of Na, [Cl]i was 15.9 +/- 1.4 mM (n = 8), and with 1 mM acetazolamide, it fell to 6.5 +/- 0.6 mM (n = 4), again close to equilibrium. The bumetanide- and Na-insensitive components of inward Cl pumping were inhibited by chlorothiazide and ethacrynic acid but were unaffected by the Na-Cl cotransport inhibitor metolazone. There was inhibition of Na-K-Cl cotransport by chlorothiazide = acetazolamide > metolazone. The anion exchange inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and HCO3 had no effect on [Cl]i in any condition. Thus Cl accumulation in the rat ventricle is fully accounted for by two systems, namely, Na-K-Cl cotransport and an Na-independent, possibly primary active, process.

Acetazolamide↗
PubMed1995

Chronotropic action of Na+, K+, Cl- cotransport inhibition in the isolated rat heart.

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 x 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-1H-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 beta-adrenoceptor antagonists, propranolol and atenolol, but was unaffected by atropine.

Animals↗
PubMed1993

An acetazolamide-sensitive inward chloride pump in vascular smooth muscle.

In smooth muscle cells, which have a small but significant chloride ion permeability, Cl:HCO3 exchange and (Na+K+Cl) cotransport are known to act as inwardly directed chloride pumps. However, even allowing for overestimation of [Cl]i due to intracellular interference with the Cl-recording electrodes, there remained a residual accumulation of chloride in rat arterial smooth muscle under conditions in which neither of these processes is operative. Manipulation of [Cl]i and Cl permeability using pharmacological agents whilst monitoring both Em and [Cl]i showed that an acetazolamide-sensitive mechanism, perhaps a Cl-dependent ATPase, is the third pathway for Cl accumulation.

Acetazolamide↗
PubMed1993

Accumulation of intracellular chloride by (Na-K-Cl) co-transport in rat arterial smooth muscle is enhanced in deoxycorticosterone acetate (DOCA)/salt hypertension.

The accumulation of intracellular chloride above equilibrium by (Na-K-Cl) co-transport has been demonstrated in a variety of tissues, most recently in guinea-pig vas deferens. The depolarizing influence of the co-transporter on the membrane potential of arterial smooth muscle cells has been demonstrated in rat femoral artery. The inference is that the depolarisation of membrane potential seen in deoxycorticosterone acetate (DOCA)/salt hypertension is the result of increased chloride accumulation via (Na-K-Cl) co-transport, which is enhanced in hypertension. The questions addressed here are (i) whether chloride is accumulated above equilibrium in saphenous arterial smooth muscle cells from normotensive animals, and (ii) whether the accumulation is more pronounced in association with DOCA/salt hypertension. In arterial smooth muscle from DOCA/salt hypertensive animals, [Cl-]i was significantly elevated (P < 2 x 10(-9)) in comparison to arterial smooth muscle from normotensive control animals. The loop diuretic bumetanide caused a reversible hyperpolarization of Em and a fall in [Cl-]i and these effects were enhanced in hypertension. These results are consistent with an increase in the activity of the (Na-K-Cl) co-transporter in rat femoral arterial smooth muscle in DOCA/salt hypertension. A preliminary report of this work has been published.

Animals↗
PubMed1991

Comparison of the electrical properties of arterial smooth muscle in normotensive rats and rats with deoxycorticosterone acetate-salt-induced hypertension: possible involvement of (Na(+)-K(+)-Cl-) co-transport.

1. Hypertension was induced in male Sprague-Dawley rats by left unilateral nephrectomy and deoxycorticosterone acetate--salt administration. After 5 weeks, arterial systolic blood pressure was significantly elevated in these animals (191.5 +/- 6.0 mmHg, mean +/- SD, n = 17) compared with age-matched, unoperated control animals (134.0 +/- 4.2 mmHg, n = 8, P less than 0.001). 2. The membrane potential of femoral artery vascular smooth muscle measured in vitro was -55.1 +/- 6.3 mV (mean +/- SD, n = 154) for normotensive and -50.8 +/- 5.7 mV (n = 82) for hypertensive animals. The difference in membrane potential was significant (P less than 0.001). 3. The relationship between the log of the extracellular K+ concentration and membrane potential was nonlinear over the extracellular K+ concentration range 2.5-20 mmol/l, and showed a small positive shift with hypertension. 4. Tenfold reductions in the extracellular concentrations of Na+ or Cl- resulted in a membrane potential hyperpolarization in vascular smooth muscle from normotensive animals (4.9 +/- 2.0 mV, n = 13 and 12.1 +/- 1.3 mV, mean +/- SD, n = 14, respectively). In vascular smooth muscle from hypertensive animals, the hyperpolarization in low-Na+ media was significantly increased to 12.2 +/- 2.6 mV (mean +/- SD, n = 5), but that in low-Cl- media was unaffected (2.7 +/- 1.6 mV, n = 6). 5. The loop diuretic, bumetanide (10 mumol/l), hyperpolarized the membrane potential in vascular smooth muscle from both normotensive and hypertensive rats, but not in low-Na+ or low-Cl- media.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Sodium entry into human placental microvillous (maternal) plasma membrane vesicles.

The uptake of sodium into vesicles isolated from the microvillous, maternal-facing plasma membrane of human placenta was studied. In equilibrium exchange conditions, sodium entry increased with time towards an equilibrium value after 30-60 min. Over 90% of the uptake was into an osmotically active space and the initial rate of uptake was halved by amiloride but unaffected by loop diuretics and capnophorin inhibitors. The apparent inhibition constant (Ki) for amiloride was 3.5 x 10(-6) M and the Michaelis constant (Km) with respect to sodium of the amiloride-sensitive component was 7-11 mM. With an imposed outward H+ gradient, sodium was transiently accumulated within the vesicles. The overshoot was abolished by amiloride and shown, by experiments with FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone) and potassium and valonomycin, not to be dependent on any electrical potential generated by the H+ gradient. The evidence for Na+-H+ exchange at this surface and its possible functions are discussed.

Biological Transport, Active↗
PubMed1986

The influence of pH and membrane potential on passive Na+ and K+ fluxes in human red blood cells.

Passive (ouabain-insensitive) Na+ and K+ effluxes from human red blood cells were measured over the range pHo 6.2-8.5. On raising pHo, Na+ efflux increased and this was mainly attributable to the piretanide-sensitive component: K+ efflux likewise but attributable to both piretanide-sensitive and piretanide-insensitive components. On replacing Cl- with non-penetrating anions (mainly gluconate), Na+ and K+ effluxes increased, mostly attributable to the piretanide-insensitive components. On restoring pHi either by reducing pHo or by applying DIDS, the influence of pHo on Na+ and K+ effluxes was diminished. These results suggest that pHi rather than Em is the dominant influence. Passive Na+ and K+ effluxes and influxes in the presence of bumetanide were tested fro conformity to the Ussing independence relationship. For K+, the calculated and observed ratios agreed, indicating that the sodium pump, 'cotransport' and leak wholly account for K+ fluxes in human red blood cells. For Na+, the ratios did not agree and a 1:1 Na+/Na+ exchange did not account for the discrepancy. Pathways for Na+ appear to be more numerous than for K+.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1986

The dependence on chloride ions of the loop diuretic sensitive component of passive sodium efflux from human red cells.

A study has been made of the passive (ouabain-insensitive) Na efflux from human red cells. The inhibition by loop diuretics (furosemide, piretanide and bumetanide) was additive with respect to ouabain and non-additive with respect to each other. They inhibited with high affinity in the sequence bumetanide greater than piretanide greater than furosemide. Passive Na efflux was not inhibited by amiloride, DIDS or SITS. In Cl-free media, Na efflux was reduced and there was no diuretic-sensitive component with any of the six Cl substitutes tested, except Br. The chloride concentration dependence of the diuretic-sensitive efflux was generally linear but saturable with sulphate and concave with acetate. In Na-free media, efflux was reduced, the apparent affinity for loop diuretics was lower and there was no chloride dependence. The diuretic-insensitive efflux was unaffected by anions in all conditions.

Anions↗
PubMed1985

Loop diuretics may fail to inhibit (Na+, K+, Cl-) 'cotransport' in human red cells.

The inhibition of passive K+ influx into human red blood cells (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 Cl- ions. Thus, raising the external Na+ concentration increased the affinity of the putative (Na+, K+, Cl-) cotransport system in human RBC for loop diuretics.

Biological Transport, Active↗
PubMed1985

Influence of loop diuretics and anions on passive potassium influx into human red cells.

Passive K influx into human red cells was measured with and without Cl ions, Na ions and loop diuretics. Ouabain and loop diuretics appear to inhibit specifically and respectively the Na pump and (Na+K) 'co-transport'. Inhibitors of other pathways, e.g. 4,4'-diisothiocyantostilbene-2,2'-disulphonic acid or amiloride did not inhibit passive K influx. Loop diuretics inhibited with high apparent affinity in Na-containing media and with low apparent affinity in Na-free media where there was a substantial Cl-dependent component. The Cl concentration dependence was measured using six anion substitutions for Cl. With NO3, acetate and gluconate, the curves were sigmoidal and not fully saturable at 150 mM-Cl; with iodide and thiocyanate, the curves were convex; with sulphate, there was saturation at 120 mM-Cl. The half-maximal K influx as a function of [Na]0 was 40 mM for the Cl-dependent flux component and 12 mM for the diuretic-sensitive flux.

Anions↗
PubMed1984

Potassium influx into erythrocytes in essential hypertension.

Sodium plus potassium (Na+K) cotransport in erythrocytes of patients with essential hypertension has mainly been studied elsewhere by measuring net outward frusemide-sensitive Na and K movements. We compared K influx (tracer 86 Rubidium) in control subjects and hypertensives who had never previously been treated for hypertension. The cotransport K influx rates in controls and hypertensives were 0.44 +/- 0.02 (mumol/ml cells/h; mean +/- s.e.; n = 20) and 0.67 +/- 0.06 (mumol/ml cells/h; mean +/- s.e.; n = 23) respectively. The active influxes were 1.03 +/- 0.03 and 2.06 +/- 0.13 (mumol/ml cells/h; mean +/- s.e.) respectively. The variance of both parameters was significantly greater in the hypertensive group but, taking this into account, the differences were significant (P less than 0.01).

Adult↗
PubMed1981

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.

Animals↗