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H Murer

Publications and source records attributed to H Murer.

At least 253 records · Page 14Linked to original sources
PubMed1989

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.

Animals↗
PubMed1989

Sodium-hydrogen exchange system in brush border membranes from cortical and medullary regions of the proximal tubule.

The Na+/H+ exchange system was studied in brush border membrane vesicles isolated from cortical and medullary regions of the proximal tubule of rabbit kidney. The activity of the exchanger was assessed by measuring hydrogen influx (monitored by acridine orange fluorescence), 22 Na influx and the sensitivity of these fluxes to amiloride and its analogue ethylisopropyl amiloride. In contrast to previously published data (indicating the absence of pH-gradient driven and amiloride sensitive 22Na-influx in medullary site vesicles (13, 15], Na+/H+ exchange activity could be detected in both membrane preparations by sodium tracer and fluorescence detection of hydrogen influx. Amiloride inhibition of 22Na influx was more effectively protected by increasing sodium concentration in cortical than in medullary vesicles, suggesting differences in the action of amiloride in these preparations.

Amiloride↗
PubMed1989

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.

Animals↗
PubMed1989

Parathyroid hormone inhibition of Na+/phosphate cotransport in OK cells: generation of second messengers in the regulatory cascade.

Dose-dependent inhibition of Na/phosphate cotransport by parathyroid hormone (PTH) was correlated with the generation of hormone-mediated second messengers, cAMP, 1,2-diacylglycerol and inositol 1,4,5 trisphosphate in an established epithelial cell line (opossum kidney (OK) cells). PTH results in a dose-dependent decline in Na/phosphate cotransport with a half-maximal response at about 10(-11) M. This hormone concentration is commensurate with the levels required to increase 1,2-diacylglycerol and inositol 1,4,5-trisphosphate concentrations by about half maximal but not with those needed for cAMP generation (10(-9) to 10(-8) M PTH). Accordingly, activation of phospholipase C may be physiologically more important than stimulation of adenylate cyclase at normal PTH levels.

1-Methyl-3-isobutylxanthine↗
PubMed1989

Separation of hepatocyte plasma membrane domains by free flow electrophoresis.

We have applied free flow electrophoresis to separate the canalicular and basolateral (sinusoidal and lateral) domains of rat hepatocyte plasma membranes. Hepatocyte plasma membranes were prepurified by rat zonal and discontinous sucrose gradient centrifugation. In electrophoretic separation, the canalicular membranes were more deflected toward the anode than the basolateral membranes. Na+-dependent taurocholate uptake could be measured in both membrane fractions, transport activity being highest in fractions containing the highest specific activity in the basolateral marker enzyme Na+-K+-ATPase. Thus, differences in electrophoretic mobility permit the separation of functional intact plasma membrane vesicles derived from basolateral and canalicular plasma membrane domains of rat hepatocyte.

Absorption↗
PubMed1989

Simultaneous recording of cell volume, membrane current and membrane potential: effect of hypotonic shock.

Using the slow whole-cell recording technique (Horn & Marty 1988), we measured the change of membrane current and potential induced by a hypotonic shock, while simultaneously monitoring the cell size. A transient depolarization could be measured which was accompanied by a transient change of the membrane current. The maximal cell size was reached before the depolarization and the current adopted their maximal values. Volume regulatory decrease could also be observed in Ca2(+)-free hypotonic bath solutions, but the time course of cell shrinking as well as the change of membrane current and potential was slowed down. In contrast, a regulatory volume decrease could not be observed in whole-cell records and the cell size steadily increased. The data show that the slow whole-cell recording technique is suitable for analysis of ion transport systems involved in volume regulation.

Animals↗
PubMed1989

Flow cytometric analysis of intracellular pH in cultured opossum kidney (OK) cells.

Suspensions of OK cells (a continuous renal epithelial cell line originating from the opossum kidney) were examined by flow cytometry. Three parameters were evaluated simultaneously; cell integrity as assayed by propidium iodide fluorescence, cell size as measured by time-of-flight, and intracellular pH as measured by fluorescence of 2',7'-bis-(2-carboxyethyl)-5,6 carboxyfluorescein (BCECF). The suspension was shown to be composed of both intact singlets and doublets of cells, and no difference was noted in the behavior of these two populations with respect to the resting intracellular pH, or of the response of intracellular BCECF to changes in pH. Evidence suggests that using NH4 prepulses to create an acid load broadens the intracellular pH distribution. The population of OK cells demonstrates a recovery from this acid load which is very homogeneous with respect to its sensitivity to Na+ removal of EIPA (ethylisopropyl-amiloride), suggesting that virtually all cells utilize Na+/H+ exchange for this recovery. The data also suggest heterogeneity in the cellular pH recovery from an acid load with respect to the observed rates of Na+/H+ exchange. Despite this heterogeneity, the Na+/H+ exchanger is observed to focus the resting intracellular pH of the population to approximately pH 7.4-7.5. The response of the population to PTH suggests that the majority of cells respond to the hormone, and that the total Na+/H+ exchange in individual cells is only partially inhibited even in the presence of saturating PTH concentrations.

Animals↗
PubMed1989

Role of calcium and calmodulin in the regulation of the rabbit ileal brush-border membrane Na+/H+ antiporter.

In rabbit ileum, Ca2+/calmodulin (CaM) appears to be involved in physiologically inhibiting the linked NaCl absorptive process, since inhibitors of Ca2+/CaM stimulate linked Na+ and Cl- absorption. The role of Ca2+/CaM-dependent phosphorylation in regulation of the brush-border Na+/H+ antiporter, which is believed to be part of the neutral linked NaCl absorptive process, was studied using purified brush-border membrane vesicles, which contain both the Na+/H+ antiporter and Ca2+/CaM-dependent protein kinase(s) and its phosphorprotein substrates. Rabbit ileal villus cell brush-border membrane vesicles were prepared by Mg precipitation and depleted of ATP. Using a freezethaw technique, the ATP-depleted vesicles were loaded with Ca2+, CaM, ATP and an ATP-regenerating system consisting of creatine kinase and creatine phosphate. The combination of Ca2+/CaM and ATP inhibited Na+/H+ exchange by 45 +/- 13%. This effect was specific since Ca2+/CaM and ATP did not alter diffusive Na+ uptake, Na+-dependent glucose entry, or Na+ or glucose equilibrium volumes. The inhibition of the Na+/H+ exchanger by Ca2+/CaM/ATP was due to an effect on the Vmax and not on the Km for Na+. In the presence of CaM and ATP, Ca2+ caused a concentration-dependent inhibition of Na+ uptake, with an effect 50% of maximum occurring at 120 nM. This Ca2+ concentration dependence was similar to the Ca2+ concentration dependence of Ca2+/CaM-dependent phosphorylation of specific proteins in the vesicles. The Ca2+/CaM/ATP-inhibition of Na+/H+ exchange was reversed by W13, a Ca2+/CaM antagonist, but not by a hydrophobic control, W12, or by H-7, a protein kinase C antagonist. We conclude that Ca2+, acting through CaM, regulates ileal brush-border Na+/H+ exchange, and that this may be involved in the regulation of neutral linked NaCl absorption.

Adenosine Triphosphate↗
PubMed1989

Regulatory volume decrease by cultured renal cells.

Volume regulatory responses of OK cells (a continuous epithelioid cell line from opossum kidney) are examined by electronic cell sizing and measurements of intracellular pH in cell suspensions. In response to a 40% reduction in osmolality, the cells swell and then subsequently shrink toward their starting volume. This regulatory volume decrease (RVD) is reduced by replacement of Cl- in the medium with acetate. Replacement of Cl- with NO3- accelerates the RVD. The RVD response is inhibited by 1 mM quinine or 100 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) in the medium. The inhibitory effect of 100 microM DIDS (but not 1 mM quinine) is altered by replacement of Cl- by NO3- in the medium. Hypotonic challenge does not induce a DIDS-sensitive net flux of acid-base equivalents. Addition of (9 microM) valinomycin also inhibits the RVD response. It is suggested that the RVD response of OK cells involves activation of separate K+ and Cl- channels.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1989

Carrier-mediated transport of tetraethylammonium across rabbit renal basolateral membrane.

Mechanisms of tetraethylammonium (TEA) transport were investigated in basolateral membrane (BLM) vesicles from rabbit renal cortex. Preloading vesicles with 10 mM TEA or 1 mM mepiperphenidol stimulated the 15-s uptake of [14C]-TEA compared with control vesicles (258 and 200%, respectively) and produced an overshoot of the equilibrium value (3 and 1.6 times, respectively). In the presence of a K+ gradient, net TEA uptake was also increased (and showed an overshoot of 2-fold) when the membrane potential of vesicles was made interior negative by adding valinomycin. Both TEA-TEA exchange and the voltage-driven net TEA transport were cis-inhibited by other organic cations, and a similar affinity order was found for both transport mechanisms (quinine greater than amiloride greater than morphine greater than mepiperphenidol greater than choline = N1-methylnicotinamide). This data suggests that the same transport protein might be responsible for both phenomena. Other experiments determined that the BLM vesicles had no TEA-H+ exchange, and that contamination of the vesicle population by brush-border membranes was negligible in terms of their contribution to TEA transport. These results demonstrate the presence of an exchange reaction for TEA in the rabbit renal BLM and thus imply carrier-mediated transport of TEA by these membranes.

Acridine Orange↗
PubMed1989

Sodium-phosphate cotransport in OK cells: inhibition by PTH and "adaptation" to low phosphate.

Sodium-phosphate (Na-Pi) cotransport is principally regulated by parathyroid hormone (PTH) and by the intrinsic ability to "adapt" to ambient phosphate concentration. In the present study, these two control mechanisms were examined in a cloned opossum kidney (OK) cell line. PTH inhibited Na-Pi cotransport, half-maximal inhibition at 5 x 10(-12) M, by fractionally similar amounts irrespective of the initial transport rates predetermined by "adaptation" to media phosphate concentration. At maximal concentrations of PTH (10(-8) M), the residual Na-Pi cotransport activity was higher in cells exposed to low-phosphate media. Cells preexposed to PTH (10(-8) M) or dibutyryl adenosine 3'-5'-cyclic monophosphate (DBcAMP) (10(-5) M) and forskolin (10(-5) M) increase transport (adaptation) by fractionally similar amounts as control cells for any given external phosphate concentration. The protein kinase C inhibitor, staurosporine, prevented PTH action but did not alter the ability to adapt Na-Pi cotransport in response to low media phosphate concentration. These data support the notion that regulation of Na-Pi cotransport by PTH and the adaptive response to available media phosphate concentration are distinct regulatory control mechanisms.

Alkaloids↗
PubMed1988

Calcium transport in basolateral plasma membranes from kidney cortex of Milan hypertensive rats.

Ca2+ transport was investigated in basolateral plasma membranes (BLM) isolated from kidney cortex of the Milan strain of genetically hypertensive rats (MHS) and their normotensive controls (MNS) during a pre-hypertensive stage (age 3-4 weeks). It was found that the Vmax of ATP-dependent Ca2+ transport (in the presence of calmodulin) was about 16% lower in MHS than in control rats. In membranes from MNS rats which had been isolated in the presence of EGTA, the ATP-dependent Ca2+ transport showed a hyperbolic Ca2+ concentration dependence, a high Km (Ca2+) and a low Vmax; upon addition of exogenous calmodulin, the kinetics became sigmoidal, the Km (Ca2+) was decreased and the Vmax was increased. In membranes from MHS rats, the Ca2+ concentration dependence of ATP-driven Ca2+ transport was sigmoidal and the Ca2+ affinity was high in the absence of added calmodulin. Addition of exogenous calmodulin to these membranes resulted in an increase in Vmax, but no change in other kinetic parameters. Low-affinity hyperbolic kinetics of Ca2+ transport could only be obtained in MHS rats if the membranes were extracted with hypotonic EDTA and hypertonic KCl. These data suggest that the plasma membrane Ca2+-ATPase, which catalyses the ATP-dependent Ca2+ transport, exists in BLM of pre-hypertensive MHS rats predominantly in an activated, high-affinity form.

Adenosine Triphosphate↗
PubMed1988

ATP-dependent Ca2+ uptake and Ca2+-dependent protein phosphorylation in basolateral liver plasma membranes.

ATP-dependent Ca2+ uptake was measured in vesicles of rat liver cell basolateral plasma membranes. Nucleotide-dependent uptake was specific for ATP and observed at pH 7.0 and 7.4/7.5 but not at pH 8.0. ATP-dependent Ca2+ transport was only observed in the presence of Mg2+. Kinetic analysis of ATP-dependent transport revealed an apparent Km in the submicromolar region. Addition of calmodulin and trifluoperazine had no effect on ATP-dependent uptake. A Ca2+-dependent, phosphorylated intermediate with the apparent molecular weight of 135,000 could be demonstrated in the basolateral plasma membranes. Phosphorylated intermediates with apparent molecular weights of 200,000 and 110,000 were demonstrated in microsomes and appeared to contaminate 'basolateral' membrane protein phosphorylation. The results suggest that a 135,000 molecular weight protein is a Ca2+-ATPase and the enzymatic expression of the liver cell basolateral membrane Ca2+ pump.

Animals↗
PubMed1988

Regulation of Na+-H+ exchange in cultured opossum kidney cells by parathyroid hormone, atrial natriuretic peptide and cyclic nucleotides.

The activity of Na+-H+, exchange was studied in a cultured cell line derived from opossum kidney (OK cells). The activity of the exchanger was measured either as the amiloride (2 mM) inhibitable 22Na flux in acid-loaded cells, or as the Na+-dependent and amiloride-sensitive recovery of intracellular pH (pHi) from an acid load. Initial rates of tracer flux were analyzed in confluent monolayers while changes in pHi were evaluated in suspensions of trypsinized cells which had been loaded with 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein. Both 8-bromo-cAMP and 8-bromo-cGMP inhibit the activity of the exchanger in a dose-dependent manner. Maximal inhibition due to 8-bromo-cAMP was about 50% and was attained with 0.75 mM of the cyclic nucleotide. Parathyroid hormone (10(-9)-10(-7) M) and atrial natriuretic peptide (10(-7) M) also inhibit the activity of the exchanger. By measuring the rate of Na+-dependent pHi recovery from different starting pHi values, evidence was obtained for a cyclic nucleotide-dependent decrease in the response of Na+-H+ exchange to intracellular acidification. We conclude that cAMP and cGMP are intracellular messengers in the hormone-dependent regulation of Na+-H+ exchange activity in renal epithelial cells.

Amiloride↗
PubMed1988

Intracellular cascades in the parathyroid-hormone-dependent regulation of Na+/phosphate cotransport in OK cells.

Parathyroid hormone (PTH) increased intracellular cyclic AMP and reduces Na+/phosphate cotransport activity in OK cells [Malmström & Murer (1986) Am. J. Physiol. 251, C23-C31; Caverzasio, Rizzoli & Bonjour (1986) J. Biol. Chem. 261, 3233-3237]. It was also shown that PTH activates phosphoinositide metabolism in OK cells [Hruska, Moskowitz, Esprit, Civitelli, Westbrook & Huskey (1987) J. Clin. Invest. 79, 230-239]. In the present paper we show that tumour-promoting phorbol esters are effective in reducing Na+/phosphate cotransport. The Ca2+ ionophores A23187 and ionomycin had only a small effect on Na+/phosphate cotransport; added together, A23187 and phorbol esters showed a synergistic action. Phorbol esters and phorbol esters plus ionomycin stimulated prostaglandin synthesis as well as cyclic AMP production; acetylsalicylic acid prevented phorbol-ester-induced prostaglandin synthesis and cyclic AMP production, but had no effect on inhibition of Na+/phosphate cotransport. In suspensions of OK cells, PTH and thrombin produced a rise in intracellular Ca2+. In contrast with PTH, thrombin did not elevate cellular cyclic AMP in suspended OK cells. PTH and thrombin reduced Na+/phosphate cotransport in suspended OK cells. It is suggested that two regulatory cascades are involved in PTH action on Na+/phosphate cotransport: cyclic AMP/kinase A and Ca2+/diacylglycerol/kinase C.

Biological Transport↗
PubMed1988

Hypotonic shock evokes opening of Ca2+-activated K channels in opossum kidney cells.

Using the patch clamp technique we show that exposure of opossum kidney cells to hypotonic shock evokes an outward rectifying potassium current. The corresponding single channel slope conductance approaches 15 pS at depolarizing voltages. The K current also becomes activated after addition of the ionophore A23187 to an isotonic bath medium containing Ca2+. We therefore conclude that the K selective channels are modulated by an elevation of cytoplasmic Ca2+. Evidence is presented that release of Ca2+ from internal stores is involved.

Animals↗
PubMed1988

Bile acids increase cellular free calcium in cultured kidney cells (LLC-PK).

Suspensions of LLC-PK1 cells were used to determine the effect of bile acids on the cellular homeostasis of inorganic ions. It is determined that bile acids alter cellular free calcium (Cai) levels in LLC-PK1 cells. A series of bile acids were compared and found to produce increases in Cai in the order: lithocholate sulfate (LCS) greater than deoxycholate greater than chenodeoxycholate greater than lithocholate glucuronide greater than cholate. LCS (300 microM) produces changes in Cai (measured using Fura-2) qualitatively similar to those produced by 1 microM ionomycin, except that only ionomycin is able to release calcium from intracellular stores. The effect on Cai is roughly proportional to LCS concentration between 50 and 300 microM. The presence of 40 mM Na in the extracellular medium reduces the LCS-induced rise in Cai to 20% of that observed in the absence of Na. This effect is specific for Na versus 150 mM extracellular K, Li, or TMA. The effect is not dependent on the Na gradient across the membrane. At concentrations of LCS which induce changes in Cai, no significant effect of LCS is observed on either cellular Na or K levels, or intracellular pH.

Animals↗