Parathyroid hormone inhibition of Na+/phosphate cotransport in OK cells: intracellular [Ca2+] as a second messenger

Parathyroid hormone increases cellular cAMP, 1,2-diacylglycerol, inositol 1,4,5-trisphosphate and cytosolic Ca2+ concentration ([Ca2+]i) in OK cells. In the present study, we determined the importance of the PTH-dependent increase in [Ca2+]i in the control of sodium-dependent phosphate (Na+/Pi) cotransport. PTH (10(-7) M) results in a transient increase in [Ca2+]i from basal levels of 67 +/- 4 nM to maximal concentrations of 190 +/- 9 nM. The increase in [Ca2+]i was dose-dependent with half-maximal increases at about 5.10(-8) M PTH. These hormone levels were 10(3)-fold higher than that required for half-maximal inhibition of Na+/Pi cotransport. Clamping [Ca2+]i with either intracellular Ca2+ chelators or by ionomycin in the presence of high concentrations of extracellular Ca2+ did not alter PTH-dependent inhibition of Na/Pi cotransport. Nor did indomethacin, an inhibitor of the cyclooxygenase pathway, influence the hormonal inhibition of cotransport. Accordingly, these data suggest that changes in [Ca2+]i and/or activation of the phospholipase A2 and the cyclooxygenase pathways are not involved in signal induction of the PTH-mediated control of Na+/Pi cotransport.     

Stimulation of Na+/phosphate cotransport in LLC-PK1 cells by 12-O-tetradecanoylphorbol 13-acetate (TPA)

We have tested for the effect of the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) on Na+/phosphate cotransport in an established epithelial cell line of renal origin (LLC-PK1). Incubation of LLC-PK1 cells with TPA produced an increase in Na+/phosphate (Pi) cotransport. The maximal response was reached at a TPA concentration of 10 ng/ml. Other phorbol esters which have no potency or a smaller one to activate protein kinase C had no effect on Na+/Pi cotransport. Incubation of LLC-PK1 cells with 10 ng/ml TPA for 8 h led to a 300% increase in Na+/Pi cotransport; in the presence of cycloheximide the increase amounted only to a 100% and was reached within 2 h. Kinetic analysis of Na+/Pi cotransport indicated an increase in the apparent Vmax without an effect on the apparent Km. The increased Pi transport was retained in isolated apical vesicles. Na+-dependent alanine transport into LLC-PK1 monolayers was affected by TPA administration in a similar manner. TPA had under the chosen experimental conditions no effect on [3H]thymidine incorporation into DNA excluding a general proliferative effect. We conclude that TPA via activation of protein kinase C regulates the number of operating transport systems. As also other Na+-coupled transport systems are influenced, the TPA effect appears to be related to the expression of a general 'adaptive' alteration of membrane transport in LLC-PK1 cells.     

The Na+/K+-ATPase reaction of human erythrocytes is not near equilibrium. A 31P-NMR study

We have addressed the question of whether the Na/K+-ATPase in the human erythrocyte is in a state of near-equilibrium by varying the extracellular ratio of Na+ and K+ and following the cytosolic phosphorylation potential by 31P-NMR and by combined enzymatic colorimetric measurements. There was no correlation at room temperature between the extracellular Na+/K+ ratio and the cytosolic phosphorylation potential measured either by NMR or alternative methods. The cytosolic phosphorylation potential measured by NMR was 4100 +/- 1300 (S.E.) M-1 at an extracellular K+ concentration of 5.9 mM (Na+/K+ ratio of 24.3) and 2800 +/- 700 (S.E.) M-1 at 75 mM extracellular K+ (Na+/K+ ratio of 0.99). The chemically determined phosphorylation potential was 6400 +/- 1200 (S.E.) and 5000 +/- 700 (S.E.) M-1 at 5.9 and 75 mM extracellular K+, respectively. Omission of Ca2+ from the buffer solutions did not affect the results. A consistent finding in this study was that the NMR-determined value of ATP was about 10-20% lower than the value determined enzymatically on perchloric acid extracts. The inorganic phosphate (Pi) was fully NMR visible.     

Expression of rat renal sodium/phosphate cotransporter in Xenopus laevis oocytes.

In an attempt to identify the renal Na+/Pi cotransporter, Xenopus laevis oocytes were used to express mRNA isolated from the renal cortex of rat kidney. Na(+)-dependent uptake of Pi in oocytes, injected with mRNA, resulted in an increase of 2-4-fold as compared to oocytes injected with water. Both the new expressed and endogenous Na(+)-dependent Pi uptake activity were inhibited with 2 mM phosphonoformic acid (PFA). Expression of Pi uptake into oocytes was dose-dependent with the amount of mRNA injected. When mRNA was fractionated on a sucrose gradient, a mRNA fraction of 2.5 kilobases expressed the Na+/Pi cotransport activity in oocytes. This fraction resulted in a 6-fold stimulation of Na(+)-dependent Pi transport when compared to oocytes injected with water. The Km and Vmax for Na(+)-dependent Pi uptake were 0.18 mM and 118 pmol/oocyte per 30 min, respectively.     

Effect of adenosine triphosphate on phosphate uptake in renal proximal tubule cells: involvement of PKC and p38 MAPK

ATP has been known to act as an extracellular signal and to be involved in various functions of kidney. Renal proximal tubular reabsorption of phosphate (Pi) contributes to the maintenance of phosphate homeostasis, which is regulated by Na+/Pi cotransporter. However, the effects of ATP on Na+/Pi cotransporters were not elucidated in proximal tubule cells (PTCs). Thus, the effects of ATP on Na+/Pi cotransporter and its related signal pathways are examined in the primary cultured renal PTCs. In the present study, ATP inhibited Pi uptake in a time (> 1 h) and dose (>10(-6)M) dependent manner. ATP-induced inhibition of Pi uptake was correlated with the decrease of type II Na+/Pi cotransporter mRNA. ATP-induced inhibition of Pi uptake may be mediated by P2Y receptor activation, since suramin (non-specific P2 receptor antagonist) and RB-2 (P2Y receptor antagonist) blocked it. ATP-induced inhibition of Pi uptake was blocked by neomycin, U73122 (phospholipase C (PLC) inhibitors), bisindolylmaleimide I, H-7, and staurosporine (protein kinase C (PKC) inhibitors), suggesting the role of PLC/PKC pathway. ATP also increased inositol phosphates (IPs) formation and induced PKC translocation from cytosolic fraction to membrane fraction. In addition, ATP-induced inhibition of Pi uptake was blocked by SB 203580 [a p38 mitogen activated protein kinase (MAPK) inhibitor], but not by PD 98059 (a p44/42 MAPK inhibitor). Indeed, ATP induced phosphorylation of p38 MAPK, which was not blocked by PKC inhibitor. In conclusion, ATP inhibited Pi uptake via PLC/PKC as well as p38 MAPK in renal PTCs.     

Effect of ischemia-reperfusion on the renal brush-border membrane sodium-dependent phosphate cotransporter NaPi-2

To understand the mechanisms underlying ischemia-reperfusion-induced renal proximal tubule damage, we analyzed the expression of the Na+-dependent phosphate (Na+/Pi) cotransporter NaPi-2 in brush border membranes (BBM) isolated from rats which had been subjected to 30 min renal ischemia and 60 min reperfusion. Na+/Pi cotransport activities of the BBM vesicles were also determined. Ischemia caused a significant decrease (about 40%, P < 0.05) in all forms of NaPi-2 in the BBM, despite a significant increase (31+/-3%, P < 0.05) in the Na+/Pi cotransport activity. After reperfusion, both NaPi-2 expression and Na+/Pi cotransport activity returned to control levels. In contrast with Na+/Pi cotransport, ischemia significantly decreased Na+-dependent glucose cotransport but did not affect Na+-dependent proline cotransport. Reperfusion caused further decreases in both Na+/glucose (by 60%) and Na+/proline (by 33%) cotransport. Levels of NaPi-2 were more reduced in the BBM than in cortex homogenates, suggesting a relocalization of NaPi-2 as a result of ischemia. After reperfusion, NaPi-2 levels returned to control values in both BBM and homogenates. These data indicate that the NaPi-2 protein and BBM Na+/Pi cotransport activity respond uniquely to reversible renal ischemia and reperfusion, and thus may play an important role in maintaining and restoring the structure and function of the proximal tubule.     

Charge translocation by the Na+/K+ pump under Na+/Na+ exchange conditions: intracellular Na+ dependence

The effect of intracellular (i) and extracellular (o) Na+ on pre-steady-state transient current associated with Na+/Na+ exchange by the Na+/K+ pump was investigated in the vegetal pole of Xenopus oocytes. Current records in response to 40-ms voltage pulses from -180 to +100 mV in the absence of external Na+ were subtracted from current records obtained under Na+/Na+ exchange conditions. Na+-sensitive transient current and dihydroouabain-sensitive current were equivalent. The quantity of charge moved (Q) and the relaxation rate coefficient (ktot) of the slow component of the Nao+-sensitive transient current were measured for steps to various voltages (V). The data were analyzed using a four-state kinetic model describing the Na+ binding, occlusion, conformational change, and release steps of the transport cycle. The apparent valence of the Q vs. V relationship was near 1.0 for all experimental conditions. When extracellular Na+ was halved, the midpoint voltage of the charge distribution (Vq) shifted -25.3+/-0.4 mV, which can be accounted for by the presence of an extracellular ion-well having a dielectric distance delta=0.69+/-0.01. The effect of changes of Nai+ on Nao+-sensitive transient current was investigated. The midpoint voltage (Vq) of the charge distribution curve was not affected over the Nao+ concentration range 3.13-50 mM. As Nai+ was decreased, the amount of charge measured and its relaxation rate coefficient decreased with an apparent Km of 3.2+/-0.2 mM. The effects of lowering Nai+ on pre-steady-state transient current can be accounted for by decreasing the charge available to participate in the fast extracellular Na+ release steps, by a slowly equilibrating (phosphorylation/occlusion) step intervening between intracellular Na+ binding and extracellular Na+ release.     

Calcium- Versus G Protein-Mediated Phosphoinositide Hydrolysis in Rat Cerebral Cortical Synaptoneurosomes

The role of calcium and sodium in stimulating phosphoinositide hydrolysis in brain was investigated in rat cerebral cortical synaptoneurosomes. In buffer containing 136 mM sodium and various concentrations of added calcium (0-1.0 mM), basal, potassium-stimulated, and norepinephrine-stimulated formation of 3H-inositol phosphates decreased with decreasing extracellular calcium. Potassium- and norepinephrine-stimulated formation of 3H-inositol phosphates was reduced to basal levels by addition of EGTA. Isosmotically replacing sodium with choline chloride or N-methyl-D-glucamine to disrupt Na+/Ca2+ exchange resulted in a large increase in the formation of 3H-inositol phosphates. Measurement of cytosolic calcium with fura-2 revealed that the cytosolic calcium concentration was sensitive to changes in the extracellular calcium concentration and increased on resuspension of synaptoneurosomes in sodium-free rather than sodium-containing medium. In the absence of sodium, potassium-stimulated formation of 3H-inositol phosphates was reduced or eliminated, depending on the extracellular calcium concentration. Subtraction of basal formation of 3H-inositol phosphates from that in the presence of 1 mM carbachol or 100 microM norepinephrine revealed that the carbachol-stimulated component was the same in the presence and absence of sodium, whereas the norepinephrine-stimulated component was reduced in the absence of sodium. Addition of the protein kinase C activator 12-O-tetradecanoylphorbol 13-acetate inhibited norepinephrine- and, to a lesser extent, carbachol but not basal or aluminum fluoride-stimulated formation of 3H-inositol phosphates in sodium-free medium. These results suggest that an increase in intracellular calcium, via disruption of Na+/Ca2+ exchange or depolarization-induced calcium influx, may explain previous demonstrations that agents that stimulate Na+ influx can also stimulate phosphoinositide hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of Na+/K+/Cl- cotransport and [3H]bumetanide binding site density by phorbol esters in HT29 cells

The involvement of protein kinase C in the regulation of Na+/K+/Cl- cotransport was investigated in cultured HT29 human colonic adenocarcinoma cells. We have demonstrated previously the presence of a Na+/K+/Cl- cotransport pathway in HT29 cells (Kim, H.D., Tsai, Y-S., Franklin, C.C., and Turner, J.T. (1989) Biochim. Biophys. Acta 946, 397-404). Treatment of cells with the phorbol esters phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate (PDBu) caused an increase in membrane-associated protein kinase C activity that was accompanied by a concomitant decrease in cytosolic protein kinase C activity. PMA also produced a rapid transient increase in cotransport to 137% of control values by 5 min followed by a progressive decrease to 19% of control values by 2 h. To determine the underlying mechanism for the reduction in Na+/K+/Cl- cotransport, changes in cotransporter number and/or affinity were determined in radioligand binding studies using [3H]bumetanide. PMA and PDBu produced essentially identical time- and dose-dependent decreases in specific [3H]bumetanide binding that were similar to the observed decreases in cotransport. Analysis of saturation and competition binding data indicated that the decrease in binding was due to a lowered Bmax with no change in affinity. Both the decrease in binding and the changes in cotransport elicited by PMA were prevented by the protein kinase inhibitor H7. These findings suggest that phorbol esters cause a decrease in the number of cotransporters in HT29 cells, resulting in a reduction in Na+/K+/Cl- cotransport activity.     

Interaction of acetyl phosphate and carbamyl phosphate with plant phosphoenolpyruvate carboxylase.

Acetyl phosphate produced an increase in the maximum velocity (Vmax. for the carboxylation of phosphoenolpyruvate catalysed by phosphoenolpyruvate carboxylase. The limiting Vmax. was 22.2 mumol X min-1 X mg-1 (185% of the value without acetyl phosphate). This compound also decreased the Km for phosphoenolpyruvate to 0.18 mM. The apparent activation constants for acetyl phosphate were 1.6 mM and 0.62 mM in the presence of 0.5 and 4 mM-phosphoenolpyruvate respectively. Carbamyl phosphate produced an increase in Vmax. and Km for phosphoenolpyruvate. The variation of Vmax./Km with carbamyl phosphate concentration could be described by a model in which this compound interacts with the carboxylase at two different types of sites: an allosteric activator site(s) and the substrate-binding site(s). Carbamyl phosphate was hydrolysed by the action of phosphoenolpyruvate carboxylase. The hydrolysis produced Pi and NH4+ in a 1:1 relationship. Values of Vmax. and Km were 0.11 +/- 0.01 mumol of Pi X min-1 X mg-1 and 1.4 +/- 0.1 mM, respectively, in the presence of 10 mM-NaHCO3. If HCO3- was not added, these values were 0.075 +/- 0.014 mumol of Pi X min-1 X mg-1 and 0.76 +/- 0.06 mM. Vmax./Km showed no variation between pH 6.5 and 8.5. The reaction required Mg2+; the activation constants were 0.77 and 0.31 mM at pH 6.5 and 8.5 respectively. Presumably, carbamyl phosphate is hydrolysed by phosphoenolpyruvate carboxylase by a reaction the mechanism of which is related to that of the carboxylation of phosphoenolpyruvate.     

Endocytosis and Na+/solute cotransport in renal epithelial cells

Endocytic uptake of [3H]sucrose and lucifer yellow, markers for fluid-phase endocytosis, was studied in cultures of the renal epithelial cell lines LLC-PK1 and OK. Endocytosis in LLC-PK1 cells was inhibited when the cells were grown in the presence of gentamicin (1 mg/ml) for 4 days or when the cells were treated with concanavalin A (1 mg/ml) for 5 h. These changes occurred without perturbation of intracellular Na+ and K+ content, indicating that the cells maintained normal ion gradients. The inhibition of endocytosis was accompanied by marked increases in the apparent Vmax for Na+-dependent cell uptake of solutes such as Pi and L-alanine. The apparent Km was unchanged. In contrast, treatment of OK cells with concanavalin A produced marked stimulation of endocytosis and inhibition of the Na+-dependent uptake of Pi and L-glutamate. These changes occurred in the absence of changes in intracellular Na+ and K+ content. Neither gentamicin nor concanavalin A had a direct effect on Na+/solute cotransport in these cell lines. The changes in Na+/Pi cotransport induced by concanavalin A in both LLC-PK1 and OK cells were blocked by keeping the cells at 4 degrees C during exposure to the lectin, suggesting that endocytosis may be part of the mechanism which mediates the changes in solute uptake. The reciprocal relationship between the changes in endocytosis and the changes in Na+/solute cotransport is consistent with the possibility that the number of Na+/solute cotransporters present in the plasma membrane may be altered by an increase or decrease in the rate of membrane internalization by endocytosis. The Vmax changes in Na+/solute cotransport provide indirect support for this conclusion.     

Parathyroid hormone inhibition of Na+/phosphate cotransport in OK cells: requirement of protein kinase C-dependent pathway

Parathyroid hormone (PTH) inhibits sodium/phosphate (Na+/Pi) cotransport across the apical membrane of opossum kidney (OK) cells principally through two pathways. First, cAMP stimulation and activation of protein kinase A; second, diacylglycerol release and stimulation of protein kinase C. Studies were designed to determine the importance of these regulatory cascades. Down-regulation of protein kinase C with prolonged phorbol ester (12-O-tetradecanoylphorbol 13-acetate (TPA] treatment leads to a refractory state in which the cells do not respond to PTH (10(-8) M), cAMP (10(-4) M) or rechallenge of TPA (200 nM) even though Na+/Pi cotransport is similar to control cells (8.1 +/- 0.1 nmol.mg-1 protein.5 min-1). Staurosporine, an inhibitor of protein kinase C, resulted in the complete inhibition of PTH, cAMP and TPA action in a dose-dependent manner. PTH, cAMP and TPA were additive below maximal concentrations, but had no further effect at maximal agonist concentrations. These results suggest that protein kinase C activity is important in PTH-mediated inhibition of Na+/phosphate cotransport in OK cells.     

The Na+/Pi-cotransporter of OK cells: reaction and tentative identification with N-acetylimidazole

Using an established renal epithelial cell line (OK cells) the effect of the amino-acid side-chain modifying reagent N-acetylimidazole (NAI) upon the sodium-dependent transport of phosphate (Pi) was investigated. After an incubation with 10 mM NAI for 20 min, cellular Na+/Pi uptake was inhibited by 70%. The presence of 5 mM Pi protected this transport function from being affected by NAI by 80 to 100%. Since the presence of sulfate was unable to protect the Na+/Pi transport inactivation by NAI and since the presence of Pi did not affect NAI inhibition of other transport systems, it is suggested that NAI interacts with the Pi transporter directly. The protective effect of Pi was used as a criterion to identify Pi-protectable [3H]NAI labelling of OK cell plasma membrane proteins. Pi protection was observed in four molecular mass regions: 31, 53, 104 and 176 kDa. Since the incorporation of [3H]NAI into these proteins was also affected by parathyroid hormone at 10(-10) M, it is concluded that the identified proteins represent possible candidates for the renal Na+/Pi cotransporter.     

Modes of operation and variable stoichiometry of the furosemide- sensitive Na and K fluxes in human red cells

We report in this paper different modes of Na and K transport in human red cells, which can be inhibited by furosemide in the presence of ouabain. Experimental evidence is provided for inward and outward coupled transport of Na and K, Ki/Ko and Nai/Nao exchange, and uncoupled Na or K efflux. The outward cotransport of Na and K was defined as the furosemide-sensitive (FS) component of Na and K effluxes into choline medium and as the Cl-dependent or cis-stimulated component of the ouabain-resistant (OR) Na and K effluxes. Inward cotransport of Na and K was defined by the stimulation by external Na (Nao) of the K influx and the stimulation by external K (Ko) of the Na influx in the presence of ouabain. Both effects were FS and Cl dependent. Experimental evidence for an FS Ki/Ko exchange pathway of the Na/K cotransport was provided by (a) the stimulation by external K of FS K influx and efflux, and (b) the stimulation by internal Na or K of FS K influx in the absence of external Na. Evidence for an FS Nai/Nao exchange pathway was provided by the stimulation of FS Na influx by internal Na from a K-free medium (130 mM NaCl). This pathway was four to six times smaller than the Ki/Ko exchange. In cells containing only Na or K, incubated in media containing only Na or K, respectively, there was FS efflux of the cation without simultaneous inward transport (FS uncoupled Na and K efflux). The stoichiometric ratio of FS outward cotransport of Na and K into choline medium varied with the ratio of Nai-to-Ki concentrations, and when Nai/Ki was close to 1, the ratio of FS outward Na to K flux was also 1. In choline media, FS Na efflux was inhibited by external K (noncompetitively), whereas FS k efflux was stimulated. The stimulation of FS K efflux was due to the stimulation by Ko of the Ki/Ko exchange pathway. Thus, the stoichiometry of FS Na and K effluxes also varied in the presence of external K. A minimal model for a reaction scheme of FS Na and K transport accounts for cis stimulation, trans inhibition, and trans stimulation, and for variable stoichiometry of the FS cation fluxes.     

Phosphate free perfusion prevents washout of tissue creatine in Langendorff perfused rabbit heart.

Rabbit hearts were perfused with Krebs-Henseleit bicarbonate buffer supplemented with 15 mM glucose and 10 mU/ml of insulin +/- Pi. At the end of 60 min the hearts were freeze-clamped and the content of ATP, creatine phosphate, creatine, lactate, pyruvate, DHAP and 3-P glycerate were determined enzymatically in neutralized perchloric acid tissue extracts. The free cytosolic ADP and Pi and the cytosolic NAD+ redox and phosphorylation potentials were calculated from the measured metabolite concentrations. Pi free perfusion resulted in increased creatine, free cytosolic ADP and cytosolic phosphorylation potential, decreased calculated free Pi and no change in cardiac ATP and creatine phosphate content. The increase in the cytosolic phosphorylation potential was due to the lowering of cytosolic free Pi. The increase in ADP was due to the increase in creatine. The increase in creatine appeared to be due to an inhibition of creatine efflux from the heart during Pi free perfusion which was mediated by an enhanced Na+ electrochemical gradient.     

Na+,K+ pump and Na+-coupled ion carriers in isolated mammalian kidney epithelial cells: regulation by protein kinase C.

This review updates our current knowledge on the regulation of Na+/H+ exchanger, Na+,K+,Cl- cotransporter, Na+,Pi cotransporter, and Na+,K+ pump in isolated epithelial cells from mammalian kidney by protein kinase C (PKC). In cells derived from different tubule segments, an activator of PKC, 4beta-phorbol 12-myristate 13-acetate (PMA), inhibits apical Na+/H+ exchanger (NHE3), Na+,Pi cotransport, and basolateral Na+,K+ cotransport (NKCCl) and augments Na+,K+ pump. In PMA-treated proximal tubules, activation of Na+,K+ pump probably plays a major role in increased reabsorption of salt and osmotically obliged water. In Madin-Darby canine kidney (MDCK) cells, which are highly abundant with intercalated cells from the collecting duct, PMA completely blocks Na+,K+,Cl- cotransport and decreases the activity of Na+,Pi cotransport by 30-40%. In these cells, agonists of P2 purinoceptors inhibit Na+,K+,Cl- and Na+,Pi cotransport by 50-70% via a PKC-independent pathway. In contrast with MDCK cells, in epithelial cells derived from proximal and distal tubules of the rabbit kidney, Na+,K+,Cl- cotransport is inhibited by PMA but is insensitive to P2 receptor activation. In proximal tubules, PKC-induced inhibition of NHE3 and Na+,Pi cotransporter can be triggered by parathyroid hormone. Both PKC and cAMP signaling contribute to dopaminergic inhibition of NHE3 and Na+,K+ pump. The receptors triggering PKC-mediated activation of Na+,K+ pump remain unknown. Recent data suggest that the PKC signaling system is involved in abnormalities of dopaminergic regulation of renal ion transport in hypertension and in the development of diabetic complications. The physiological and pathophysiological implications of PKC-independent regulation of renal ion transporters by P2 purinoceptors has not yet been examined.     

Increase in membrane fluidity modulates sodium-coupled uptakes and cyclic AMP synthesis by renal proximal tubular cells in primary culture.

In order to evaluate the influence of membrane fluidization on three apical transport systems and on a basolateral enzyme, and to analyse the mechanisms involved, we studied, in cultured rabbit proximal tubular cells, the effect of increasing concentrations of the local anesthetic drug benzyl alcohol on Na(+)-dependent uptakes of phosphate (Pi), methyl alpha-D-glucopyranoside (MGP), and L-alanine, as well as on basal and stimulated cyclic AMP content. At 10 mM, benzyl alcohol increased the Vmax of Pi uptake by 31%, decreased that of MGP uptake by 24%, and did not affect alanine uptake. Km values were not affected. Benzyl alcohol, up to 40 mM, increased in a concentration-dependent manner basal, PTH-stimulated, and cholera toxin-stimulated, but not forskolin-stimulated cyclic AMP accumulation. In the presence of 40 mM benzyl alcohol, the magnitude of PTH-induced inhibition of Pi uptake was enhanced from 11% to 24%. It is concluded that: (i) fluidization of apical membranes affected differently Na+/Pi, Na+/MGP, and Na+/alanine cotransports, reflecting differences in the lipidic environments of these transport system; (ii) fluidization of basolateral membranes enhanced PTH-stimulated cyclic AMP generation through improved coupling between the receptor-GS complex and the catalytic subunit of adenylate cyclase; (iii) these variations may result in physiological and pathophysiological modulation of the renal handling of solutes and of the phosphaturic effect of PTH.     

Fluorescence measurements of cytosolic free Na concentration, influx and efflux in gastric cells.

Regulation of cytosolic free Na (Nai) was measured in isolated rabbit gastric glands with the use of a recently developed fluorescent indicator for sodium, SBFI. Intracellular loading of the indicator was achieved by incubation with an acetoxymethyl ester of the dye. Digital imaging of fluorescence was used to monitor Nai in both acid-secreting parietal cells and enzyme-secreting chief cells within intact glands. In situ calibration of Nai with ionophores indicated that SBFI fluorescence (345/385 nm excitation ratio) could resolve 2 mM changes in Nai and was relatively insensitive to changes in K or pH. Measurements on intact glands showed that basal Nai was 8.5 +/- 2.2 mM in parietal cells and 9.2 +/- 3 mM in chief cells. Estimates of Na influx and efflux were made by measuring rates of Nai change after inactivation or reactivation of the Na/K ATPase in a rapid perfusion system. Na/K ATPase inhibition resulting from the removal of extracellular K (Ko) caused Nai to increase at 3.2 +/- 1.5 mM/min and 3.5 +/- 2.7 mM/min in parietal and chief cells, respectively. Na buffering was found to be negligible. Addition of 5 mM Ko and removal of extracellular Na (Nao) caused Nai to decrease rapidly toward 0 mM Na. By subtracting passive Na efflux under these conditions (the rate at which Nai decreased in Na-free solution containing ouabain), an activation curve (dNai/Nai) for the Na/K ATPase was calculated. The pump demonstrated the greatest sensitivity between 5 and 20 mM Nai. At 37 degrees C the pump rate was less than 3 mM/min at 5 mM Nai and 26 mM/min at 25 mM Nai, indicating that the pump has a great ability to respond to changes in Nai in this range. Carbachol, which stimulates secretion from both cell types, was found to stimulate Na influx in both cell types, but did not have detectable effects on Na efflux. dbcAMP+IBMX, potent stimulants of acid secretion, had no effect on Na metabolism.     

Intracellular pH-regulating mechanism of the squid axon. Interaction between DNDS and extracellular Na+ and HCO3-

Intracellular pH (pHi) of the squid axon is regulated by a stilbenesensitive transporter that couples the influx of Na+ and HCO3- (or the equivalent) to the efflux of Cl-. According to one model, the extracellular ion pair NaCO3- exchanges for intracellular Cl-. In the present study, the ion-pair model was tested by examining the interaction of the reversible stilbene derivative 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS) with extracellular Na+ and HCO3-. Axons (initial pHi approximately 7.4) were internally dialyzed with a pH 6.5 solution containing 400 mM Cl- but no Na+. After pHi, as measured with a glass microelectrode, had fallen to approximately 6.6, dialysis was halted. In the presence of both external Na+ and HCO3- (pHo = 8.0, 22 degrees C), pHi increased due to the pHi-regulating mechanism. At a fixed [Na+]o of 425 mM and [HCO3-]o of 12 mM, DNDS reversibly reduced the equivalent acid-extrusion rate (JH) calculated from the rate of pHi recovery. The best-fit value for maximal inhibition was 104%, and for the [DNDS]o at half-maximal inhibition, 0.3 mM. At a [Na+]o of 425 mM, the [HCO3-]o dependence of JH was examined at 0, 0.1, and 0.25 mM DNDS. Although Jmax was always approximately 20 pmol cm-2 s-1, Km(HCO3-) was 2.6, 5.7, and 12.7 mM, respectively. Thus, DNDS is competitive with HCO3-. At a [HCO3-]o of 12 mM, the [Na+]o dependence of JH was examined at 0 and 0.1 mM DNDS. Although Jmax was approximately 20 pmol cm-2 s-1 in both cases, Km(Na+) was 71 and 179 mM, respectively. At a [HCO3-]o of 48 mM, Jmax was approximately 20 pmol cm-2 s-1 at [DNDS]o levels of 0, 0.1, and 0.25 mM. However, Km(Na+) was 22, 45, and 90 mM, respectively. Thus, DNDS (an anion) is also competitive with Na+. The results are consistent with simple competition between DNDS and NaCO3-, and place severe restrictions on other kinetic models.     

Ouabain-insensitive Na-ATPase activity in the basolateral membrane from rat jejunum

1. In the basolateral membrane preparation of the rat enterocyte (jejunal tract) there is not only the well-known (Na,K)-ATPase activity, but also a ouabain-insensitive Na-ATPase. 2. The Na-ATPase is not activated by anions or other monovalent cations. As a substrate, ATP cannot be replaced by other nucleotides. 3. The Na-ATPase is insensitive to ouabain and bumetanide, inhibited partially by furosemide and totally by ethacrynate. 4. The activation of Na-ATPase at different Na concentrations shows an hyperbolic curve (Km = 15.7 +/- 2.3 mM and Vmax = 204 +/- 19 nmoles Pi/mg protein per min) different from the sigmoidal curve (Km = 9.8 +/- 1.2 mM and Vmax = 640 +/- 15 nmoles Pi/mg protein per min) shown by (Na,K)-ATPase. 5. These results are compared with the corresponding ones found in other animals and tissues in which the Na-ATPase was found. 6. The Na-ATPase activity can be interpreted as the enzymatic correspondent of a ouabain-insensitive Na pump, present in the basolateral membrane of the enterocyte different in behaviour with respect to the known Na pump.