Cation-selective channels in amphibian epithelia: electrophysiological properties and activation

1. Na+ as well as Li+ move across the apical membrane through amiloride-sensitive ionic channels. 2. K+ movements across the apical membrane occur through Ba2+- and Cs+-sensitive channels which do not allow the passage of Na+ or Li+. 3. A third pathway in the apical membrane is permeable for Na+, K+, Cs+, Rb+, NH+4 and Ti+. The currents carried by these monovalent cations are blocked by Ca2+ and divalent cations as well as La3+. 4. In the urinary bladder, the Ca2+-sensitive currents are stimulated by oxytocin, activators of cytosolic cAMP and cAMP analogues. Also the oxytocin activated currents are blocked by divalent cations and La3+. 5. Nanomolar concentrations of mucosal Ag+ activate the third channel and open the pathway for movements of Ca2+, Ba2+ and Mg2+, which are known to permeate through Ca2+ channels in excitable tissues.     

Ca2(+)-sensitive K+ channel in aortic smooth muscle of rats

We measured K+ channel activity in inside-out patches of cell membrane from aortic vascular smooth muscle cultured (Passages 1-3) from Wistar, Wistar-Kyoto, and spontaneously hypertensive rats (SHR). With [Ca2+]i between 25 and 100 nm and 150 mm K+ on both sides of the membrane, the conductance of this channel was 55 +/- 7 pS (slope of current-voltage curve through 0 mV) and the current was outwardly rectified. There was no difference in single-channel conductance among the three rat strains. Increasing negative holding voltages or increasing [Ca2+]i, increased the probability of this type channel being open (Npo; P less than 0.01); SHR had a larger NPo (P less than 0.01). Compared with cells from Wistar and Wistar-Kyoto, cells from SHR also had the longest mean open time. The increased NPo and mean open time we observed in this K+ channel of cells from SHR could contribute, at least in part, to the increased membrane K+ permeability, reported previously.     

Ion transport in erythrocytes of Prague hypertensive rat

The Prague hypertensive rat is a unique strain exhibiting genetic hypertension in which a hypertensive line (PHR) was bred in parallel with a normotensive one (Prague normotensive rat--PNR) from the same parental pair. Sodium efflux from Na(+)-loaded erythrocytes into Mg2(+)-sucrose medium was measured in these two strains as well as in spontaneously hypertensive rats (Okamoto-Aoki, SHR) and in normotensive outbred Wistar rats. Kinetic parameters--maximal velocity and apparent dissociation constant (reflecting the affinity for internal sodium)--were calculated. It was found that PHR as well as SHR had a higher Na+ leak and a decreased activity of the ouabain-sensitive Na+ transport as compared to Wistar rats. Furosemide-sensitive Na+ transport was substantially lower in erythrocytes of both hypertensive strains (PHR and SHR) than in the respective normotensive strains (PNR and Wistar).     

Transport of univalent cations across the erythrocyte membrane of hypertensive rats of various ages

In the erythrocytes incubated at low temperature (3-6 degrees C), the uptake of Li+ in 6- and 16-week old spontaneously hypertensive rats (SHR) was significantly higher than in the normotensive rats (WKY) of the same age. During the incubation of cells at 37 degrees C no difference occurred in either ouabain-sensitive or ouabain-resistant fluxes of Rb+, Na+ and Li+ between the 16-week old SHR and the WKY. K+ efflux from the erythrocytes at 3 degrees C was consistently stimulated after addition to the incubation medium of 1 mmol/l Ca2+. The value of Ca2+-dependent K+-transport was significantly elevated in 16-week old SHR than in the WKY, but there was no difference in 6-week old rats. Propranolol-induced Ca2+-dependent K+ efflux from the cells at 22 degrees C was markedly higher in 6- and 16-week old SHR as compared with the WKY. The results provide a further evidence in favor of the hypothesis on the existence of a "membrane defect" in red blood cells in the SHR.     

Magnesium: Effects on reperfusion arrhythmias and membrane potential in isolated rat hearts

Myocardial Na+,K+-ATPase was studied in patients with aortic valve disease, and myocardial Na+,K+- and Ca2+-ATPase were assessed in spontaneously hypertensive rats (SHR) and hereditary cardiomyopathic hamsters using methods ensuring high enzyme recovery. Na+,K+-ATPase was quantified by [3H]ouabain binding to intact myocardial biopsies from patients with aortic valve disease. Aortic stenosis, regurgitation and a combination hereof were compared with normal human heart and were associated with reductions of left ventricular [3H]ouabain binding site concentration (pmol/g wet weight) of 56, 46 and 60%, respectively (p < 0.01). Na+,K+ and Ca2+-ATPases were quantified by K+- and Ca2+-dependent p-nitrophenyl phosphatase (pNPPase) activity determinations in crude myocardial homogenates from SHR and hereditary cardiomyopathic hamsters. When SHR were compared to age-matched Wistar Kyoto (WKY) rats an increase in heart-body weight ratio of 75% (p < 0.001) was associated with reductions of K+- and Ca2+-dependent pNPPase activities (mol/min/g wet weight) of 42 (p < 0.01) and 27% (p < 0.05), respectively. When hereditary cardiomyopathic hamsters were compared to age-matched Syrian hamsters an increase in heart-body weight ratio of 69% (p < 0.001) was found to be associated with reductions in K+- and Ca2+-dependent pNPPase activities of 50 (p < 0.001) and 26% (p = 0.05), respectively. The reductions in Na+,K+- and Ca2+-ATPases were selective in relation to overall protein content and were not merely the outcome of increased myocardial mass relative to Na+,K+- and Ca2+-pumps. In conclusion, myocardial hypertrophy is in patients associated with reduced Na+,K+-ATPase concentration and in rodents with reduced Na+,K+- and Ca2+-ATPase concentrations. This may be of importance for development of heart f in hypertrophic heart disease.     

Sodium intake is increased by social stress and the Y chromosome and reduced by clonidine

The objectives were to determine 1) if female rats have higher Na intake than males and if social stress increases Na intake, 2) if the sympathetic nervous system (SNS) mediates the stress effects and the gender effect, and 3) if the Y chromosome (Yc) from a hypertensive father increases Na intake. Four rat strains (n = 10/group) of both sexes were used: 1) Wistar Kyoto normotensive (WKY), 2) an F(16) backcross with a Yc from a hypertensive father (SHR/y), 3) spontaneously hypertensive rat (SHR), and 4) an F(16) backcross with a Yc from a normotensive father (SHR/a). Females showed greater baseline Na intake than males (hypertensive strains), intruder stress increased Na intake, and clonidine decreased Na intake, but not in WKY or SHR females. SHR/y males had higher baseline Na intake compared with WKY males. In conclusion, the higher Na intake in females during baseline and stress was partially mediated through the SNS in hypertensive strains and the SHR Yc was partially responsible for the increased Na intake in SHR/y and SHR males compared with WKY.     

Multinuclear NMR studies of intracellular cations in perfused hypertensive rat kidney.

We have investigated hypertension-associated alterations in intracellular cations in the kidney by measuring intracellular pH, free Mg2+, free Ca2+, and Na+ concentrations in perfused normotensive and hypertensive rat (8-14 weeks old) kidneys using 31P, 19F, and double quantum-filtered (DQ) 23Na NMR. The effects of both anoxia and ischemia on the 23Na DQ signal confirmed its ability to detect changes in intracellular Na+. However, there was a sizable contribution of the extracellular Na+ to the 23Na DQ signal of the kidney. The intracellular free Ca2+ concentration, measured using 19F NMR and 5,5'difluoro-1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid, also increased dramatically during ischemia; the increase could be partly reversed by reperfusion. No significant differences were found between normotensive and hypertensive kidneys in the ATP level, intracellular pH, intracellular free Mg2+, and the 23Na DQ signal or in the extent of the extracellular contribution to the 23Na DQ signal. Oxygen consumption rates were also similar for the normotensive (5.02 +/- 0.46 mumol of O2/min/g) and hypertensive (5.47 +/- 0.42 mumol O2/min/g) rat kidneys. The absence of a significant difference in intracellular pH, Na+ concentration, and oxygen consumption between normotensive and hypertensive rat kidneys suggests that an alteration in the luminal Na+/H+ antiport activity in hypertension is unlikely. However, a highly significant increase (64%, p less than 0.01) in free Ca2+ concentration was found in perfused kidneys from hypertensive rats (557 +/- 48 nM, blood pressure = 199 +/- 5 mmHg, n = 6) compared with normotensive rats (339 +/- 21 nM, blood pressure = 134 +/- 6, n = 4) indicating altered renal calcium homeostasis in essential hypertension. An increase in intracellular free Ca2+ concentration without an accompanying change in the intracellular Na+ suggests, among many possibilities, that the Ca2+/Mg(2+)-ATPase may be inhibited in the hypertensive renal tissue.     

Force-interval relationship and its response to ryanodine in streptozotocin-induced diabetic rats.

Post-quiescent potentiation (PQP), an enhanced contraction following a long pause that occurs as a result of increased Ca2+ release from intracellular stores, and post-stimulation potentiation (PSP), an enhanced contraction following a rapid series of contractions that is believed to be related to increased Ca2+ influx, were measured in streptozotocin-treated Wistar, spontaneously hypertensive (SHR), and Wistar-Kyoto (WKY) diabetic heart tissues. Decreased PQP values were found in Wistar and SHR diabetic papillary muscles (PM) in comparison with the same strain controls, which suggests a diminished degree of releasable Ca2+ from sarcoplasmic reticulum (SR) in these tissues. Decreased PSP was found in SHR diabetic PM, which may be related primarily to a depressed sarcolemmal (SL) Na(+)-Ca2+ exchange in this tissue. PSP was not decreased in diabetic Wistar or WKY cardiac preparations, indicating that Ca2+ entry via channels must be involved in the PSP mechanism. Ryanodine depressed PQP in Wistar and SHR PM, and SHR left atria in both control and diabetic tissues. It abolished PQP and SHR diabetic tissues but had no effect on WKY control and diabetic tissues. The data suggest that the ryanodine effect differs in the various strains of rat. These differences may be due to differences in the SR sensitivity to ryanodine among the strains. Diabetic SR with impaired Ca2+ uptake may contribute to these phenomena. Ryanodine depressed PSP of Wistar and SHR diabetic PM but had no effects on tissues from controls. The influence of ryanodine on diabetic SL Na(+)-Ca2+ exchange requires further investigation.     

Intestinal calcium transport in spontaneously hypertensive rats (SHR) and their genetically matched WKY rats

Calcium transport across the basolateral membranes of the enterocyte represents the active step in calcium translocation. This step occurs by two mechanisms, an ATP-dependent pump and a Ca2+/Na+ exchange process. These studies were designed to investigate these two processes in jejunal basolateral membrane vesicles (BLMV) of the spontaneously hypertensive rats (SHR) and their genetically matched controls, Wistar-Kyoto (WKY) rats. The ATP-dependent calcium uptake was stimulated several-fold compared with no ATP condition in both SHR and WKY, but no differences were noted between rate of calcium uptake in SHR and WKY. Kinetics of ATP-dependent calcium uptake at concentrations between 0.01 and 1.0 microM revealed a Vmax of 0.67 +/- 0.03 nmol/mg protein/20 sec and a Km of 0.2 +/- 0.03 microM in SHR and Vmax of 0.69 +/- 0.12 and a Km of 0.32 +/- 0.14 microM in WKY rats. Ca2+/Na+ exchange in jejunal BLMV of SHR and WKY was investigated in two ways. First, sodium was added to the incubation medium (cis-Na+). Second, Ca2+ efflux from BLMV was studied in the presence of extravesicular Na+ (trans-Na+). Both studies suggest a decreased exchange of calcium and Na+. Kinetic parameters of Na(+)-dependent Ca2+ uptake at concentrations between 0.01 and 1.0 microM exhibited Vmax of 0.05 +/- 0.01 nanmol/mg protein/5 sec and a Km of 0.21 +/- 0.13 microM in SHR and Vmax of 0.11 +/- 0.02 nanmol/mg protein/5 sec and a Km of 0.09 +/- 0.05 in WKY, respectively. These results confirm that the intestinal BLMV of SHR and WKY rats have two mechanisms for calcium extrusion, an ATP-dependent Ca2+ transport process and a Na+/Ca2+ exchange process. The ATP-dependent process appears to be functional in SHR; however, the Ca2+/Na+ exchange mechanism appears to have a marked decrease in its maximal capacity. These findings suggest that calcium extrusion via Ca2+/Na+ is impaired in the SHR, which may lead to an increase in intracellular calcium concentration. These findings may have relevance to the development of hypertension.     

ATP inactivates hydrolysis of the K+-sensitive phosphoenzyme of kidney Na+,K+-transport ATPase and activates that of muscle sarcoplasmic reticulum Ca2+-transport ATPase

In order to characterize low affinity ATP-binding sites of renal (Na+,K+) ATPase and sarcoplasmic reticulum (Ca2+)ATPase, the effects of ATP on the splitting of the K+-sensitive phosphoenzymes were compared. ATP inactivated the dephosphorylation in the case of (Na+,K+)ATPase at relatively high concentrations, while activating it in the case of (Ca2+)ATPase. When various nucleotides were tested in place of ATP, inactivators of (Na+,K+)ATPase were found to be activators in (Ca2+)ATPase, with a few exceptions. In the absence of Mg2+, the half-maximum concentration of ATP for the inhibition or for the activation was about 0.35 mM or 0.25 mM, respectively. These values are comparable to the previously reported Km or the dissociation constant of the low affinity ATP site estimated from the steady-state kinetics of the stimulation of ATP hydrolysis or from binding measurements. By increasing the concentration of Mg2+, but not Na+, the effect of ATP on the phosphoenzyme of (Na+,K+)ATPase was reduced. On the other hand, Mg2+ did not modify the effect of ATP on the phosphoenzyme of (Ca2+)ATPase. During (Na+,K+)ATPase turnover, the low affinity ATP site appeared to be exposed in the phosphorylated form of the enzyme, but the magnesium-complexed ATP interacted poorly with the reactive K+-sensitive phosphoenzyme, which has a tightly bound magnesium, probably because of interaction between the divalent cations. In the presence of physiological levels of Mg2+ and K+, ATP appeared to bind to the (Na+,K+)ATPase only after the dephosphorylation, while it binds to the (Ca2+)-ATPase before the dephosphorylation to activate the turnover.     

Cationic distribution in vessels, heart, skeletal muscle, and blood of normotensive (NWR) and spontaneously hypertensive rats (SHR)

Electrolytic analysis carried out with the atomic absorption spectrophotometer permitted to compare the cationic (Ca2+, Mg2+, Zn2+, K+ and Na+) tissue content of spontaneously hypertensive male rats (SHR) and normotensive male Wistar rats (NWR). In all SHR-tissues, Zn2+ is augmented, but mainly in the atria (left atrium), inferior vena cava, left ventricle, and skeletal muscle. The inferior cava vein and the right atrium have a similar, accentuated high bivalent (Ca2+, Mg2+, and Zn2+) cation content; Ca2+ and Mg2+ is present in a minor content in the right ventricle and type II "pale" skeletal muscle while only Mg2+ was also reduced in the left atrium, aorta, and the whole blood. A higher K+ concentration is seen in the right atrium, aorta, and type I "red" skeletal muscle. In the aortic wall and the whole blood a higher Na+- tissue content is found, confirming earlier observations. There is a discret water retention in the tissues from the left ventricle and skeletal muscle, simultaneously with a small depletion in the whole blood. We have concluded, that there is a specific cationic profile in SHR structural and functional different cardiovascular tissues, including skeletal muscle. The cationic tissue distribution is related to underlying genetic and adaptative factors and may be involved into specific drug effects.     

Electrophysiological demonstration of Na+/Ca2+ exchange in bovine articular chondrocytes

Altered fluxes of Ca2+ across the chondrocyte membrane have been proposed as one pathway by which mechanical load can modulate cartilage turnover. In many cells, Na+/Ca2+ exchange (NCX) plays a key role in Ca2+ homeostasis, and recent studies have suggested it is operative in articular chondrocytes. In this study, an electrophysiological characterisation of NCX in articular bovine chondrocytes has been performed, using the whole-cell patch clamp technique, and the effects of inhibitors and the transmembrane electrochemical gradients of Na+ and Ca2+ on NCX function have been assessed. A Ni2+-sensitive current (I(NCX)) which exhibited outward rectification, was elicited by a voltage ramp protocol. The current was also attenuated by the NCX inhibitors benzamil and KBR7943, without significant differences between the effect of these two compounds upon outward and inward currents. The Ni2+-sensitive current was modulated by changes in extracellular and pipette Na+ and Ca2+ in a manner characteristic of I(NCX). Measured values for the reversal potential differed significantly from those predicted for an exchanger stoichiometry of 3Na+ : 1Ca2+, implying that accumulation of intracellular Ca2+ (from influx or release from stores) or more than one transport mode is occurring. These results demonstrate the operation of NCX in articular chondrocytes and suggest that changes in its turnover rate, as might occur in response to mechanical load, may modify cell composition and thereby dictate cartilage turnover.     

Presence of a potential-sensitive Na+ transport across renal brush-border membrane vesicles from rats of the Milan hypertensive strain

Sodium transport was measured in brush-border membrane vesicles prepared from kidney cortex of the Milan hypertensive strain (MHS) rats and the corresponding normotensive controls. In the presence of an outwardly directed proton gradient, 22Na was transiently accumulated in the vesicles. When a transmembrane electrical potential was imposed across membrane vesicles, both the accumulation ratio and the initial uptake were increased, indicating the presence of an electrogenic pathway for sodium in these membranes. The potential-dependent sodium uptake was significantly higher in MHS rats. Kinetic analysis give simple Michaelis Menten curves in the presence and in the absence of a membrane potential. In both conditions Jmax was significantly increased in MHS rats, whereas Km was the same for the two rat strains. Sodium uptake was inhibited by amiloride at concentrations that inhibit Na+-H+ exchange. The presence of the higher, potential-sensitive, sodium uptake in MHS is in agreement with studies on renal physiology which support the hypothesis that an increase in tubular sodium reabsorption may be the primary cause for the development of hypertension in this rat strain.     

Protective effects of selenium, vitamin C and vitamin E against oxidative stress of cigarette smoke in rats

Cataractous lenses have been found to have an altered distribution of the intracellular ionic environment: the concentrations of potassium and magnesium being decreased and the concentrations of sodium and calcium increased. These changes arise as a result of changes to lens membrane characteristics causing an increase in lens membrane permeability. In this study flame atomic absorption spectroscopy (AAS) was used for calcium, magnesium, iron and zinc determination, and flame atomic emission spectroscopy (AES) was used for sodium and potassium contents in normal and cigarette smoke-exposed rat lenses. The methods are sensitive enough to detect quantitatively all six cations in a single rat lenses. In this work, six elements, including Ca2+, K+, Na+, Zn2+, Fe2+ and Mg2+ in experimental rat eye lenses and normal transparent lenses were determined. It was found that the concentrations of Ca2+, Na+, Zn2+, and Fe2+ were increased dramatically while K+ and Mg2+ decreased in smoke-exposed rat lenses when compared to the control rat lenses. There were no significant changes between 'smoked' rats supplied with vitamin C and control groups. A positive correlation was found also in the other two groups of 'cigarette smoked' animals supplemented with selenium plus vitamin E and selenium when compared with 'cigarette smoked' without any supplements. These data provide support for the hypothesis that cigarette smoking increases the risk of cataract formation. We investigated whether vitamin C is the most important antioxidant in the body. The roles of diet with optimum amounts of antioxidant vitamins C and vitamin E and the antioxidant mineral selenium are discussed.     

Calcium uptake kinetics into brush-border membrane vesicles: higher Vmax in the spontaneously hypertensive rat than in normotensive control

Arterial hypertension in the spontaneously hypertensive rat (SHR) is associated with an abnormal Ca2+ homeostasis, compared with its normotensive control, the Wistar Kyoto rat (WKY). In particular, epithelial Ca2+ transport is perturbed, with intestinal absorption and renal tubular reabsorption being decreased in the adolescent and adult SHR. In the present study we examined Ca2+ uptake into isolated duodenal brush-border membrane vesicles (BBMV) in 12-14 week-old male rats. This uptake can be separated in a nonsaturable and a saturable component. The latter follows Michaelis-Menten kinetics. Vmax of this component was found to be significantly higher (p less than 0.05) in SHR than in WKY (0.58 +/- 0.19 versus 0.35 +/- 0.06 nmol/mg protein x 10 sec, mean +/- SD) whereas Km did not differ. Thus, the defect in the intestinal Ca2+ absorption previously identified in the SHR of this age is not due to a decrease in Ca uptake at the level of the duodenal brush-border membrane, but is most likely located in the baso-lateral membrane.     

Effects of divalent cations and pH on amiloride-sensitive Na+ fluxes into luminal membrane vesicles from pars recta of rabbit proximal tubule.

The effect of Ca2+, Cd2+, Ba2+, Mg2+ and pH on the renal epithelial Na(+)-channel was investigated by measuring the amiloride-sensitive 22Na+ fluxes into luminal membrane vesicles from pars recta of rabbit proximal tubule. It was found that intravesicular Ca2+ as well as extravesicular Ca2+ substantially lowered the channel-mediated flux. Amiloride sensitive Na+ uptake was nearly completely blocked by 10 microM Ca2+ at pH 7.4. The inhibitory effect of Ca2+ was dependent on pH. Thus, 10 microM Ca2+ produced 90% inhibition of 22Na+ uptake at pH 7.4, and only 40% inhibition at pH 7.0. The tracer fluxes measured in the absence of Ca2+ were pH independent over the range from 7.0 to 7.4. All the cations Ca2+, Cd2+, Ba2+ except Mg2+ inhibited the 22Na+ influx drastically when added extravesicularly in millimolar concentrations. The cations Cd2+, Ba2+ and Mg2+ in the same concentrations intravesicularly inhibited the 22Na+ influx only slightly. A millimolar concentration of Ca2+ intravesicularly blocked the amiloride-sensitive 22Na+ flux completely. The data indicate that Ca2+ inhibits Na+ influx specifically by binding to sites composed of one or several deprotonated groups on the channel proteins.     

Renal Na+-K+-ATPase in weanling and adult spontaneously hypertensive rats

The interrelationships among plasma renin activity (PRA, ng AI/ml plasma/hr), aldosterone concentration (ng%), and renal Na+-K+-ATPase activity (mumole PO4/mg protein/hr) were studied in 9 weanling normotensive spontaneously hypertensive rats (SHR), 9 adult hypertensive SHR, and 9 weanling and 9 adult normotensive Wistar-Kyoto rats (WKY). All groups were placed on a normal (0.4% sodium) diet. PRA and plasma aldosterone, measured in samples drawn from the ether-anesthetized rat, were higher in weanling SHR (15.2 +/- 2.0, 37 +/- 4.2) than in WKY. PRA measured in samples collected from a separate group of unanesthetized weanling SHR was also greater than in age-matched WKY. In adult SHR, PRA (6.1 +/- 0.9) and plasma aldosterone (20.0 +/- 2.7) were decreased. During the weanling period Na+-K+-ATPase activity in SHR was not only greater than in age-matched WKY but was also increased compared to adult normotensive and hypertensive rats (137 +/- 9 weanling SHR, 89 +/- 7 weanling WKY, 73 +/- 11 adult SHR, 84 +/- 17 adult WKY). Thus, during the weanling period the renin-angiotensin-aldosterone (R-A-A) system and renal Na+-K+-ATPase activity are activated in SHR. The elevation of Na+-K+-ATPase activity may be due to increased aldosterone levels. It was noted, however, that plasma aldosterone was similar in adult WKY and weanling SHR, while Na+-K+-ATPase activity was higher in SHR. These findings involving R-A-A and renal Na+-K+-ATPase activity prior to the elevation of blood pressure suggest that the kidneys may play a role in the initiation of hypertension in SHR.     

Inhibition of Na(+)-independent H+ pump by Na(+)-induced changes in cell Ca2+

Apical membrane H+ extrusion in the renal outer medullary collecting duct, inner stripe, is mediated by a Na(+)-independent H+ pump. To examine the regulation of this transporter, cell pH and cell Ca2+ were measured microfluorometrically in in vitro perfused tubules using 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein and fura-2, respectively. Apical membrane H+ pump activity, assayed as cell pH recovery from a series of acid loads (NH3/NH+4 prepulse) in the total absence of ambient Na+, initially occurred at a slow rate (0.06 +/- 0.02 pH units/min), which was not sufficient to account for physiologic rates of H+ extrusion. Over 15-20 min after the initial acid load, the rate of Na(+)-independent cell pH recovery increased to 0.63 +/- 0.09 pH units/min, associated with a steady-state cell pH greater than the initial pre-acid load cell pH. This pattern suggested an initial suppression followed by a delayed activation of the apical membrane H+ pump. Replacement of peritubular Na+ with choline or N-methyl-D-glucosamine resulted in an initial spike increase in cell Ca2+ followed by a sustained increase in cell Ca2+. The initial rate of Na(+)-independent cell pH recovery could be increased by elimination of the Na+ removal-induced sustained cell Ca2+ elevation by: (a) performing studies in the presence of 135 mM peritubular Na+ (1 mM peritubular amiloride used to inhibit basolateral membrane Na+/H+ antiport); (b) clamping cell Ca2+ low with dimethyl-BAPTA, an intracellular Ca2+ chelating agent; or (c) removal of extracellular Ca2+. Cell acidification induced a spike increase in cell Ca2+. The late acceleration of Na(+)-independent cell pH recovery was independent of Na+ removal and of the method used to acidify the cell, but was eliminated by prevention of the cell Ca2+ spike and markedly delayed by the microfilament-disrupting agent, cytochalasin B. This study demonstrates that peritubular Na+ removal results in a sustained elevation in cell Ca2+, which inhibits the apical membrane H+ pump. In addition, rapid cell acidification associated with a spike increase in cell Ca2+ leads to a delayed activation of the H+ pump. Thus, cell Ca2+ per se, or a Ca(2+)-activated pathway, can modulate H+ pump activity.     

Effect of vanadate and of removal of extracellular Ca2+ and Na+ on tension development and 45Ca efflux in rat and frog myocardium

Vanadate in the range 0-5 mM has positive inotropic effects on myocardial strips of frog and to a lesser extent on those of rat. Inhibiting the sarcolemmal Na+, Ca2+ exchange by a solution free of Ca2+ and Na+ caused a drop in 45Ca efflux and a transient increase in resting tension. These effects were more expressed for the frog than for the rat myocardium, which suggests that the Na+ for Ca2+ exchange across the cell membrane is more important in the frog than in the rat myocardium. A subsequent addition of vanadate at 2 or 5 mM had no effect on 45Ca efflux, while it increased the resting tension. This increase was higher for the frog than for the rat myocardium. These results suggest that the inotropic effects of vanadate may be due to an effect on membrane-bound Ca2+-ATPase.     

Postnatal development of E-4031-sensitive potassium current in rat carotid chemoreceptor cells.

The O2 sensitivity of dissociated type I cells from rat carotid body increases with age until approximately 14-16 days. Hypoxia-induced depolarization appears to be mediated by an O2-sensitive K+ current, but other K+ currents may modulate depolarization. We hypothesized that membrane potential may be stabilized in newborn type I cells by human ether-a-go-go-related gene (HERG)-like K+ currents that inhibit hypoxia-induced depolarization and that a decrease in this current with age could underlie, in part, the developmental increase in type I cell depolarization response to hypoxia. In dissociated type I cells from 0- to 1- and 11- to 16-day-old rats, using perforated patch-clamp and 70 mM K+ extracellular solution, we measured repolarization-induced inward K+ tail currents in the absence and presence of E-4031, a specific HERG channel blocker. This allowed isolation of the E-4031-sensitive HERG-like current. E-4031-sensitive peak currents in type I cells from 0- to- 1-day-old rats were 2.5-fold larger than in cells from 11- to 16-day-old rats. E-4031-sensitive current density in newborn type I cells was twofold greater than in cells from 11- to 16-day-old rats. Under current clamp conditions, E-4031 enhanced hypoxia-induced depolarization in type I cells from 0- to- 1-day-old but not 11- to 16-day-old rats. With use of fura 2 to measure intracellular Ca2+, E-4031 increased the cytosolic Ca2+ concentration response to anoxia in cells from 0- to- 1-day-old but not cells from 11- to 16-day-old rats. E-4031-sensitive K+ currents are present in newborn carotid body type I cells and decline with age. These findings are consistent with a role for E-4031-sensitive K+ current, and possibly HERG-like K+ currents, in the type I cell hypoxia response maturation.