Actions of external hypertonic urea, ADH, and theophylline on transcellular and extracellular solute permeabilities in frog skin

Increases in transepithelial solute permeability were elicited in the frog skin with external hypertonic urea, theophylline, and vasopressin (ADH). In external hypertonic urea, which is known to increase the permeability of the extracellular (paracellular) pathway, the unidirectional transepithelial fluxes of Na (passive), K, Cl, and urea increased substantially while preserving a linear relationship to each other. The same linear relationship was also observed for the passive Na and urea fluxes in regular Ringer and under stimulation with ADH or 10 mM theophylline, indicating that their permeation pathway was extracellular. A linear relationship between Cl and urea fluxes could be demonstrated if the skins were separated according to their open circuit potentials; parallel lines were obtained with increasing intercepts on the Cl axis as the open circuit potential decreased. The slopes of the Cl vs. urea lines were not different from that obtained in external hypertonic urea, indicating that this relationship described the extracellular movement of Cl. The intercept on the ordinate was interpreted as the contribution from the transcellular Cl movement. In the presence of 0.5 mM theophylline or 10 mU/ml of ADH, mainly the transcellular movement of Cl increased, whereas 10 mM theophylline caused increases in both transcellular and extracellular Cl fluxes. These and other data were interpreted in terms of a possible intracellular control of the theophylline-induced increase in extracellular fluxes. The changes in passive solute permeability were shown to be independent of active transport. The responses of the active transport system, the transcellular and paracellular pathways to theophylline and ADH could be explained in terms of the different resulting concentrations of cyclic 3'-5'-AMP produced by each of these substances in the tissue.     

The Effect of Ca and Antidiuretic Hormone on Na Transport across Frog Skin : II. Sites and mechanisms of action

A method has been developed for determining unidirectional Na fluxes across the two faces of the transporting cells in the frog skin. The method has been used to investigate the location of the sites at which Ca and anti-diuretic hormone act to alter the rate of active Na transport across the skin. The results have indicated that the primary effect of both agents is on the Na permeability of the outward facing membrane of the cells. Ca decreases and the hormone increases permeability of this barrier. Neither agent appears to have a direct effect on the active transport system itself assuming that it is located at the inner membrane of the cells. The rate of active Na transport is altered as a result of changes in the size of the Na pool in the cells which occur because of changes in the rate of Na entry through the outer membrane. Thus, the results indicate that the Na permeability of the outer membrane plays an important role in controlling the rate of net active Na transport across the skin.     

Possible role of Ca2+-binding sites in the regulation of Na+ transport in toad urinary bladder

La3+ was used to assess the role of membrane-bound Ca2+ in the regulation of basal and antidiuretic hormone (ADH)-induced Na+ transport by the isolated toad urinary bladder. Na+ transport was monitored by means of a short-circuit current (Isc) device. Mucosal La3+ (0.5-5 mM) increased Isc, while serosal La3+ (5 mM) produced a biphasic response (stimulation followed by inhibition). The stimulatory effects of La3+ were additive when present on both sides and were suppressed by mucosal amiloride or serosal ouabain. The action of mucosal La+ was reversible but the inhibition produced by serosal La3+ was not. In the presence of serosal La3+ the natriferic effect of ADH was abolished, but Theophylline, dibutyryl-cAMP, Amphotericin B, mucosal La3+, mucosal low pH, and phospho(enol) pyruvate, were able to increase Isc. These results suggest that Ca2+ binding sites in apical and basolateral membranes may play a key role in the modulation of both basal and ADH-induced Na+ transport. Serosal La3+ apparently inactivates the hormone-receptor interaction and/or the link between the ADH-receptor complex and the activation of adenylate cyclase, but does not interfere with the operation of the Na+ "pump", the basal activity of adenylate cyclase or any of the intracellular events that mediate the effect of ADH on Na+ transport.     

Beta-adrenergic effects on cellular Na, Mg, Ca, K and Cl in vascular smooth muscle: electron probe analysis of rabbit pulmonary artery.

The effects of beta-adrenergic stimulation on the cellular content and subcellular distribution of Na, Mg, Ca, K and Cl were determined by electron probe X-ray microanalysis of muscles stimulated with 5-hydroxytryptamine. Isoproterenol caused a significant decrease in cytoplasmic and mitochondrial Na and Cl, and an increase in cytoplasmic Mg. Isoproterenol also significantly decreased total cytoplasmic Ca measured with small diameter probes, without affecting cellular Ca measured with large probes that included the sarcoplasmic reticulum (SR). The decrease in cytoplasmic Na and the effects on cytoplasmic and cellular Ca are consistent with, respectively, beta-adrenergic stimulation of the Na-pump and of Ca-uptake into the SR, but the beta-adrenergic increase in cytoplasmic Mg also raises the possibility of stimulated Na/Mg exchange.     

Na(+)-dependent Ca(2+) transport modulates the secretory response to the Fcepsilon receptor stimulus of mast cells

Immunological stimulation of rat mucosal-type mast cells (RBL-2H3 line) by clustering of their Fcepsilon receptors (FcepsilonRI) causes a rapid and transient increase in free cytoplasmic Ca(2+) ion concentration ([Ca(2+)](i)) because of its release from intracellular stores. This is followed by a sustained elevated [Ca(2+)](i), which is attained by Ca(2+) influx. Because an FcepsilonRI-induced increase in the membrane permeability for Na(+) ions has also been observed, and secretion is at least partially inhibited by lowering of extracellular sodium ion concentrations ([Na(+)](o)), the operation of a Na(+)/Ca(2+) exchanger has been considered. We found significant coupling between the Ca(2+) and Na(+) ion gradients across plasma membranes of RBL-2H3 cells, which we investigated employing (23)Na-NMR, (45)Ca(2+), (85)Sr(2+), and the Ca(2+)-sensitive fluorescent probe indo-1. The reduction in extracellular Ca(2+) concentrations ([Ca(2+)](o)) provoked a [Na(+)](i) increase, and a decrease in [Na(+)](o) results in a Ca(2+) influx as well as an increase in [Ca(2+)](i). Mediator secretion assays, monitoring the released beta-hexosaminidase activity, showed in the presence of extracellular sodium a sigmoidal dependence on [Ca(2+)](o). However, the secretion was not affected by varying [Ca(2+)](o) as [Na(+)](o) was lowered to 0.4 mM, while it was almost completely inhibited at [Na(+)](o) = 136 mM and [Ca(2+)](o) < 0.05 mM. Increasing [Na(+)](o) caused the secretion to reach a minimum at [Na(+)](o) = 20 mM, followed by a steady increase to its maximum value at 136 mM. A parallel [Na(+)](o) dependence of the Ca(2+) fluxes was observed: Antigen stimulation at [Na(+)](o) = 136 mM caused a pronounced Ca(2+) influx. At [Na(+)](o) = 17 mM only a slight Ca(2+) efflux was detected, whereas at [Na(+)](o) = 0.4 mM no Ca(2+) transport across the cell membrane could be observed. Our results clearly indicate that the [Na(+)](o) dependence of the secretory response to FcepsilonRI stimulation is due to its influence on the [Ca(2+)](i), which is mediated by a Na(+)-dependent Ca(2+) transport.     

The relationship between mitogen-induced changes in the cytoplasmic pH and free Ca2+ concentration in rat thymocytes

The relationship between pHi and [Ca]i signals generated in rat thymocytes by the mitogen Con A has been investigated. It is shown that the mitogen-induced [Ca]i rise is dependent on Na+/H+ exchange or some other Na(+)-sensitive process. This conclusion is based on the following findings: (i) [Ca]i response to Con A weakens upon decreasing the concentration of extracellular Na+, or inhibiting Na+/H+ exchange; (ii) agents that alkalinize the cytoplasm (the phorbol ester TPA, the Na+/H+ ionophore monensin and NH4Cl) cause an increase in [Ca]i (Klip, A., Rothstein, A. and Mack, E. (1984) Biochem. Biophys. Res. Commun. 124, 14-22; Grinstein, S. and Goetz, J.D. (1985) Biochim. Biophys. Acta 819, 267-270); (iii) The effects of Con A, TPA and monensin on [Ca]i are not additive. The last observation suggests that all these agents activate the same Na+/H+ (Na+ and/or H+)-dependent system of Ca2+ transport. It is found that the pH i and [Ca]i responses in rat thymocytes are sensitive to changes in the intracellular levels of cyclic nucleotides, ATP and in temperature. These regulatory effects on the ionic signals are different for Con A, TPA and monensin. In particular, both the stimulation of Na+/H+ antiport and the [Ca]i rise brought about by Con A or TPA are inhibited upon elevating the cellular cAMP. In contrast, the monensin-induced [Ca]i signal is almost independent of cAMP but is highly sensitive to changes in cGMP and temperature. Reducing the ATP level eliminates both the pHi and [Ca]i responses to Con A but not to monensin. These different characteristics of [Ca]i signals elicited by the mitogen and the Na+/H+ ionophore indicate that these agents use different mechanisms to activate the Na+/H(+)-dependent Ca2+ transporting system. A [Ca]i response to monensin has been obtained in some other cell types, namely, in lymphoblastoid Raji cells, Ehrlich ascites tumor cells and also in platelets.     

A Na(+)-dependent Ca2+ exchanger generates the sustained increase in intracellular Ca2+ required for T cell activation.

Movement of extracellular Ca2+ is required for the sustained increase in [Ca2+]i necessary for T cell activation. However, the mechanisms mediating mitogen-stimulated Ca2+ movement into T cells have not been completely delineated. To explore the possibility that a Na(+)-dependent Ca2+ (Na+/Ca2+) exchanger might play a role in the mitogen-induced increases in [Ca2+]i required for T cell activation, the effects of inhibitors of this exchanger were examined. Inhibitors of Na+/Ca2+ exchange suppressed the sustained increase in [Ca2+]i stimulated by ligation of the CD3-TCR complex, but did not affect mobilization of intracellular Ca2+ stores. Consistent with the importance of this prolonged increase in [Ca2+]i in T cell activation, Na+/Ca2+ exchange inhibitors, but not inhibitors of the Na+/H+ antiporter, inhibited DNA synthesis stimulated by immobilized anti-CD3 mAb. Inhibition only occurred when the agents were present during the first hours after stimulation. These agents also inhibited IL-2 production, but not expression of the IL-2R or of an early activation Ag, 4F2. Inhibition of IL-2 production did not account for the inhibition of T cell proliferation as addition of exogenous IL-2 or phorbol ester (PDB) did not overcome the inhibition. In contrast, activation pathways that are not thought to require an increase in [Ca2+]i such as IL-1 + PDB or engagement of CD28 in the presence of PDB were less sensitive to the suppressive effects of inhibitors of Na+/Ca2+ exchange. Thus, proliferation induced by these stimuli was not suppressed by low concentrations of these inhibitors and IL-2 production induced by mAb to CD28 + PDB was not inhibited by any concentration of inhibitors of Na+/Ca2+ exchange. These results suggest that stimulation of a Ca2+ transporter with the same spectrum of inhibition as the Na+/Ca2+ exchanger in other tissues mediates the sustained increase in [Ca2+]i required for T cell activation after CD3 ligation.     

Regulation of protein phosphorylation and sodium transport in toad bladder

It is well established that active sodium-ion transport and water flow across isolated toad bladder are increased by antidiuretic hormone (ADH) and by cAMP. These agents were also observed in previous studies to cause changes in the amount of radioactive phosphate in a specific protein in the toad bladder. This protein, found by SDS-polyacrylamide gel electrophoresis of toad bladder epithelial preparations, had an apparent molecular weight of 49,000 daltons. In the present study, a correlation was found between the ability of a variety of substances to affect the amount of radioactive phosphate in this 40,000-dalton protein and their ability to alter the rate of sodium transport. Thus several agents (ADH, cAMP, theophylline, adenine, prostaglandin E1, and Mn Cl-2) caused a decrease in the amount of radioactive phosphate in the 49,000-dalton protein and also stimulated active sodium transport across the bladder. Conversely, ZnCl-2 produced an increase in the amount of radioactive phosphate in this protein and an inhibition of sodium transport. With each of these agents, the time-course of change in phosphorylation of this protein was, in general, similar to that for sodium transport. A second phosphoprotein, with an apparent molecular weight of about 42,000 daltons, showed changes in parallel with, but less extensive than, those observed in the 49,000 dalton protein. There was no consistent relationship between changes in level of phosphorylation of either in the 49,000- or 42,000- dalton protein and changes in osmotic water permeability. The results are compatible with the possibility that regulation by ADH and by cAMP of sodium transport in the toad bladder epithelium may be mediated through regulation of the amount of phosphate in a specific protein.     

Na+-Ca2+ exchange in mouse oocytes: modifications in the regulation of intracellular free Ca2+ during oocyte maturation

Increases in intracellular free Ca(2+)+ concentration (Ca(2+)+ oscillations) occur during meiotic maturation and fertilization of mammalian oocytes but little is known about the mechanisms of Ca(2+) homeostasis in these cells. Cells extrude Ca(2+) from the cytosol using two main transport processes, the Ca(2+)-ATPase and the Na(+)-Ca(2+) exchanger. The aim of this study was to determine whether Na(+)-Ca(2+) exchange activity is present in immature and mature mouse oocytes. Na(+)-Ca(2+) exchange can be revealed by altering the Na(+) concentration gradient across the plasma membrane and recording intracellular free Ca(2+) concentrations using Ca(2+)-sensitive fluorescent dyes. Depletion of extracellular Na(+) caused an immediate increase in Ca(2+) concentration in immature oocytes and a delayed increase in mature oocytes. The Na(+) ionophore, monensin, caused an increase in intracellular Ca(2+) in immature oocytes similar to that induced by Na(+)-depleted medium. In mature oocytes, monensin had no effect on intracellular Ca(2+) but the time taken for Ca(2+) to reach a peak value on removal of extracellular Na(+) was significantly decreased. Finally, addition of Ca(2+) to immature oocytes incubated in Ca(2+)-free medium caused an increase in the concentration of intracellular Ca(2+) that was dependent upon the presence of extracellular Na(+). This effect was not seen in mature oocytes. The data show that Na(+)-Ca(2+) exchange occurs in immature and mature mouse oocytes and that Ca(2+) homeostasis in immature oocytes is more sensitive to manipulations that activate Na(+)-Ca(2+) exchange.     

Serosal Na/Ca exchange and H+ and Na+ transport by the turtle and toad bladders

Summary A Na/Ca exchange system has been described in the plasma membrane of several tissues and seems to regulate the concentration of calcium in cytosol. Replacement of extracellular Na by sucrose increases calcium uptake into and decreases calcium efflux from the cell, leading to an increase in cytosolic calcium. The effect of an increase in cytosolic calcium mediated by the Na/Ca exchange system on H⁺ and Na transport in the turtle and toad bladder was investigated by replacing serosal Na isosmotically by sucrose or choline. Replacement of serosal by sucrose was associated with a significant inhibition of H⁺ secretion or Na transport which was reversible by addition of NaCl. Replacement of mucosal Na by sucrose failed to alter H⁺ secretion. Removal of serosal Na was associated with a significant increase in⁴⁵Ca uptake which could be blocked by pretreatment with lanthanum chloride. Pretreatment with lanthanum chloride blunted the inhibitory effect of replacement of serosal Na by sucrose on H⁺ and Na transport, thus suggesting that the increase in calcium uptake and the inhibition of transport are causally related. Under anaerobic conditions the rate of H⁺ or Na transport are linked to the rate of lactate production. The inhibition of Na or H⁺ transport by removal of serosal Na was accompanied by a proportional decrease in lactate production, thus suggesting that an increase in cytosolic calcium does not inhibit transport by uncoupling glycolysis from transport. Replacement of serosal Na by sucrose did not alter the force of the H⁺ or Na pump but led to an increase in resistance of the active pathway of H⁺ and Na transport. The inhibition of Na transport by replacement of serosal Na with sucrose could be reversed by addition of amphotericin B, an agent which increases luminal permeability to Na, thus suggesting that decreased Na entry across the apical membrane is the mechanism responsible for the inhibition of Na transport. The results of the present studies strongly suggest that an increase in cytosolic calcium through the serosal Na/Ca exchange system inhibits H⁺ and Na transport in the turtle and toad bladder probably by increasing the resistance of the luminal membrane.     

Cell volume regulation by Amphiuma red blood cells. The role of Ca+2 as a modulator of alkali metal/H+ exchange

In response to osmotic perturbation, the Amphiuma red blood cell regulates volume back to "normal" levels. After osmotic swelling, the cells lose K, Cl, and osmotically obliged H2O (regulatory volume decrease [RVD] ). After osmotic shrinkage, cell volume is regulated as a result of Na, Cl, and H2O uptake (regulatory volume increase [RVI] ). As previously shown (Cala, 1980 alpha), ion fluxes responsible for volume regulation are electroneutral, with alkali metal ions obligatorily counter-coupled to H, whereas net Cl flux is in exchange for HCO3. When they were exposed to the Ca ionophore A23187, Amphiuma red blood cells lost K, Cl, and H2O with kinetics (time course) similar to those observed during RVD. In contrast, when cells were osmotically swollen in Ca-free media, net K loss during RVD was inhibited by approximately 60%. A role for Ca in the activation of K/H exchange during RVD was suggested from these experiments, but interpretation was complicated by the fact that an increase in cellular Ca resulted in an increase in the membrane conductance to K (GK). To determine the relative contributions of conductive K flux and K/H exchange to total K flux, electrical studies were performed and the correspondence of net K flux to thermodynamic models for conductive vs. K/H exchange was evaluated. These studies led to the conclusion that although Ca activates both conductive and electroneutral K flux pathways, only the latter pathways contribute significantly to net K flux. On the basis of observations that A23187 did not activate K loss from cells during RVI (when the Na/H exchange was functioning) and that amiloride inhibited K/H exchange by swollen cells only when cells had previously been shrunk in the presence of amiloride, I concluded that Na/H and K/H exchange are mediated by the same membrane transport moiety.     

Na+/Ca2+ exchangers: three mammalian gene families control Ca2+ transport

Mammalian Na+/Ca2+ exchangers are members of three branches of a much larger family of transport proteins [the CaCA (Ca2+/cation antiporter) superfamily] whose main role is to provide control of Ca2+ flux across the plasma membranes or intracellular compartments. Since cytosolic levels of Ca2+ are much lower than those found extracellularly or in sequestered stores, the major function of Na+/Ca2+ exchangers is to extrude Ca2+ from the cytoplasm. The exchangers are, however, fully reversible and thus, under special conditions of subcellular localization and compartmentalized ion gradients, Na+/Ca2+ exchangers may allow Ca2+ entry and may play more specialized roles in Ca2+ movement between compartments. The NCX (Na+/Ca2+ exchanger) [SLC (solute carrier) 8] branch of Na+/Ca2+ exchangers comprises three members: NCX1 has been most extensively studied, and is broadly expressed with particular abundance in heart, brain and kidney, NCX2 is expressed in brain, and NCX3 is expressed in brain and skeletal muscle. The NCX proteins subserve a variety of roles, depending upon the site of expression. These include cardiac excitation-contraction coupling, neuronal signalling and Ca2+ reabsorption in the kidney. The NCKX (Na2+/Ca2+-K+ exchanger) (SLC24) branch of Na+/Ca2+ exchangers transport K+ and Ca2+ in exchange for Na+, and comprises five members: NCKX1 is expressed in retinal rod photoreceptors, NCKX2 is expressed in cone photoreceptors and in neurons throughout the brain, NCKX3 and NCKX4 are abundant in brain, but have a broader tissue distribution, and NCKX5 is expressed in skin, retinal epithelium and brain. The NCKX proteins probably play a particularly prominent role in regulating Ca2+ flux in environments which experience wide and frequent fluctuations in Na+ concentration. Until recently, the range of functions that NCKX proteins play was generally underappreciated. This situation is now changing rapidly as evidence emerges for roles including photoreceptor adaptation, synaptic plasticity and skin pigmentation. The CCX (Ca2+/cation exchanger) branch has only one mammalian member, NCKX6 or NCLX (Na+/Ca2+-Li+ exchanger), whose physiological function remains unclear, despite a broad pattern of expression.     

Bumetanide inhibition of NaCl transport byNecturus gallbladder

Salt transport by the Necturus gallbladder epithelium is the result of the coupled entry of NaCl into the cells across the apical membrane and the active transport of Na out of the cells across the basolateral membrane. The NaCl entry step was studied by measuring the rate of cell volume increase accompanying ouabain inhibition of the Na--K-ATPase in the basolateral membrane. When bumetanide, a diuretic analog of furosemide, was added to the mucosal bathing solution it reversibly blocked the entry of NaCl into the cells and abolished fluid transport. A dose-response relationship showed half-maximal inhibition of NaCl entry at a bumetanide concentration of 10(-9) M; complete inhibition of coupled NaCl movement occurred with as little as 10(-7) M bumetanide. Partial substitution of Na or Cl in the mucosal solution failed to demonstrate competition between bumetanide and either of the ions. The drug was also effective in blocking NaCl entry in the absence of ouabain; addition of the diuretic to the mucosal bathing solution resulted in prompt cell shrinkage and a decrease in intracellular NaCl. Cell volume decrease followed bumetanide addition to the mucosal bath because NaCl entry was blocked but active Na transport continued for several minutes until the intracellular Na transport pool was depleted.     

Effects of Na readmission on cellular45Ca fluxes in Na-Depleted guinea pig taenia coli

Summary The removal of Na from the medium causes a cellular Ca uptake in the smooth muscle of the guinea pig taenia coli which is rapidly reversed if medium Na is readmitted. This net extrusion was characterized in tissues which were first Na-depleted in a zero-Na (sucrose) solution. Li was able to substitute for Na in mediating this effect. K was also able to mimic Na in this respect if the depolarization-mediated Ca influx caused by the isotonic K solution was blocked with 10^–5 m D-600. The net Ca extrusion upon Na readmission was due to a small decrease in Ca influx, as well as a marked increase in the transmembrane Ca efflux rate, as revealed by⁴⁵Ca washout experiments. The increased⁴⁵Ca efflux upon Na readmission could be mimicked by Li, K, choline and tris. We conclude that the Na/Ca-exchange hypothesis is insufficient to explain these data, in that both Ca extrusion and⁴⁵Ca efflux can be stimulated in the absence of a Na gradient, or in the absence of any monovalent cationic gradient. These observations are discussed in terms of a possible intracellular competition of Ca and monovalent cations for anionic binding sites, as well as with regard to a possible direct stimulation of a plasmalemmal CaATPase by monovalent cations.     

Side specific effect of 5-hydroxytryptamine on NaCl transport in the apical and basolateral membrane of rat tracheal epithelia

5-Hydroxytryptamine (5-HT) can be released from mast cells and platelets through an IgE-dependent mechanism and may play a role in the pathogenesis of allergic bronchoconstriction. However, the effect of 5-HT on ion transport by the airway epithelium is still controversial. The objective of this study was to determine whether 5-hydroxytryptamine (5-HT) regulates NaCl transport by different mechanisms in the apical and basolateral membrane of tracheal epithelia. We studied the rat tracheal epithelium under short-circuit conditions in vitro. Short-circuit current (I(sc)) was measured in rat tracheal epithelial monolayers cultured on porous filters. 5-HT inhibited Na(+) absorption [measured via Na(+) short-circuit current (I(Na)(sc))] in the apical membrane and stimulated Cl(-) secretion [measured via Cl(-) short-circuit current (I(Cl)(sc))] in the basolateral membrane. Functional localization using selective 5-HT agonists and antagonists suggest that I(Cl)(sc)is stimulated by the basolateral membrane-resident 5-HT receptors, whereas I(Na)(sc) is inhibited by the apical membrane-resident 5-HT2 receptors. The basolateral addition of 5-HT increases intracellular cAMP content, but its apical addition does not. The addition of BAPTA/AM blocked the decrease of I(Na)(sc)which was induced by the apical addition of 5-HT, and 5-HT increased intracellular Ca concentrations. These results indicate that 5-HT differentially affects I(Na)(sc)and I(Cl)(sc)across rat tracheal monolayers through interactions with distinct receptors in the apical and the basolateral membrane. These effects may result in an increase of water movement towards the airway lumen.     

Ethacrynic acid and furosemide alter Cl,K, and Na distribution between blood,choroid plexus,CSF, and brain

Can loop diuretics like ethacrynic acid and furosemide, when administered intravenously, significantly alter ion transport and fluid dynamics in CNS? To shed light on this unresolved issue, we tested the ability of these agents to effect redistribution of Na, K and Cl in adult rat brain. Cl penetration into various CNS regions was assessed as the volume of distribution, i.e., uptake, of³⁶Cl from blood. Ethacrynic acid and furosemide (50 mg/kg IV) reduced by 20–30% the rate of permeation of³⁶Cl across the blood-CSF barrier, and they elevated [K] and [Cl] in choroid plexus (CP) by 15–25%. The loop diuretic-induced buildup of K and Cl in CP (lateral and 4th ventricle) was likely a reflection of decreased movement of these ions across the apical membrane into CSF.³⁶Cl activity in parietal cortex and pons-medulla decreased in treatment with furosemide and ethacrynic acid, due to slowing of Cl transport across blood-brain and/or blood-CSF barriers. Our inhibitory findings in intact rats are consistent with those from previous in vitro experiments demonstrating diminution by loop diuretics of Na, K and Cl transport across isolated CP membranes.     

Intracellular ion concentrations in the frog cornea epithelium during stimulation and inhibition of Cl secretion

Summary The intracellular electrolyte concentrations in the isolated cornea of the American bullfrog were determined in thin freeze-dried cryosections using energy-dispersive X-ray microanalysis. Stimulation of Cl secretion by isoproterenol resulted in a significant increase in the intracellular Na concentration but did not change the intracellular Cl concentration. Similar results were obtained when Cl secretion was stimulated by the Ca ionophore A23187. Inhibition of Cl secretion by ouabain produced a large increase in the intracellular Na concentration and an equivalent fall in the K concentration. Again, no increase or decrease in the intracellular Cl concentration was detectable. Clamping of the transepithelial potential to ±50 mV resulted in parallel changes in the transepithelial current and intracellular Na concentration, but, with the exception of the outermost cell layer, in no changes of the Cl concentration. Only when Cl secretion was inhibited by bumetanide or furosemide, together with a decrease in the Na concentration, was a large fall in the Cl concentration observed. Application of loop diuretics also produced significant increases in the P concentration and dry weight, consistent with some shrinkage of the epithelial cells. The results suggest the existence of a potent regulatory mechanism which maintains a constant intracellular Cl concentration and, thereby, a constant epithelial cell volume. Through the operation of this system any variation in the apical Cl efflux is compensated for by an equal change in the rate of Cl uptake across the basolateral membrane. Cl uptake is sensitive to loop diuretics, directly coupled to an uptake of Na, and dependent on the Na and K concentration gradients across the basolateral membrane. Isoproterenol and A23187 seem to increase the Cl permeability of the apical membrane and thus stimulate Cl efflux. Ouabain inhibits Cl secretion by abolishing the driving Na concentration gradient for Cl uptake across the basolateral membrane.     

Arginine vasopressin stimulation of cerebral microvascular endothelial cell Na-K-Cl cotransporter activity is V1 receptor and [Ca] dependent

Ischemia-induced brain edema formation is mediated by increased transport of Na and Cl across an intact blood-brain barrier (BBB). Our previous studies have provided evidence that a luminally located BBB Na-K-Cl cotransporter is stimulated during cerebral ischemia to increase transport of Na and Cl into the brain. The main focus of the present study was to evaluate the effects of arginine vasopressin (AVP), previously shown to be increased in the brain during ischemia and to promote edema formation, on activity of the BBB cotransporter. Cerebral microvascular endothelial cell (CMEC) monolayers were cultured in astroglial cell conditioned medium, and Na-K-Cl cotransporter activity was assessed as bumetanide-sensitive ⁸⁶Rb influx. In both human and bovine CMECs, as well as in freshly isolated microvessels, AVP stimulated cotransport activity. This stimulatory effect was mimicked by V₁ but not V₂ vasopressin agonists and was blocked by V₁ but not V₂ vasopressin antagonists. Consistent with a V₁ vasopressin receptor mechanism of action, AVP caused an increase in CMEC intracellular [Ca] that was blocked by a V₁ antagonist. Exposing the cells to [Ca]-free media and/or reducing intracellular [Ca] by BAPTA also blocked AVP stimulation of CMEC cotransporter activity, as did the phospholipase C inhibitor U-73122. Finally, we found that while stimulation of CMEC cotransporter activity by AVP occurred within minutes, it was also sustained for hours in the continued presence of AVP. These findings support the hypothesis that AVP, through a V₁ receptor- and [Ca]-dependent mechanism, stimulates the BBB Na-K-Cl cotransporter to participate in ischemia-induced edema formation. blood-brain barrier; stroke; cerebral ischemia; brain edema     

Papaverine reduces the sodium permeability of the apical membrane and the Potassium permeability of the basolateral membrane in isolated frog skin

Summary The effect of papaverine, an inhibitor of the phosphodiesterase responsible for breakdown of cAMP, on the transepithelial sodium transport across the isolated frog skin was investigated.Serosal addition of papaverine caused initially an increase in the short-circuit current (SCC), a doubling of the cellular cAMP content and a depolarization of the intracellular potential under SCC conditions (V _scc).The initial increase in the SCC was followed by a pronounced decrease both in the SCC and in the natriferic action of antidiuretic hormone (ADH), but papaverine had no inhibitory effect on the ability of ADH to increase the cellular cAMP content. As SCC declines, no hyperpolarization was observed.The I/V relationship across the apical membrane during the inhibitory phase, revealed that papaverine reduces the sodium permeability of the apical membrane (P _Na ^a )as well as intracellular sodium concentration. These observations and the previously noted effect of papaverine on V _scc indicates that papaverine must have an effect on the cellular Cl or K permeability.The basolateral Na,K,2Cl cotransporter was blocked with bumetanide, which should bring the cellular chloride in equilibrium. Bumetanide had no effect on basal SCC and V _scc. When papaverine was added to skins preincubated with bumetanide, the effect of papaverine on SCC and V _scc was unchanged. Therefore, the depolarization of V _scc, observed during the papaverine induced inhibition of the SCC, must be due to a reduction in the cellular K permeability.In conclusion, it is suggested that papaverine reduces the sodium permeability of the apical membrane and the potassium permeability of the basolateral membrane of the frog skin epithelium.     

The renal Na+/Ca2+ exchange system is located exclusively in the distal tubule.

The movement of Ca2+ across the basolateral plasma membrane was determined in purified preparations of this membrane isolated from rabbit proximal and distal convoluted tubules. The ATP-dependent Ca2+ uptake was present in basolateral membranes from both these tubular segments, but the activity was higher in the distal tubules. A very active Na+/Ca2+ exchange system was also demonstrated in the distal-tubular membranes, but in proximal-tubular membranes this exchange system was not demonstrable. The presence of Na+ outside the vesicles gradually inhibited the ATP-dependent Ca2+ uptake in the distal-tubular-membrane preparations, but remained without effect in those from the proximal tubules. The activity of the Na+/Ca2+ exchange system in the distal-tubular membranes was a function of the imposed Na+ gradient. These results suggest that the major differences in the characteristics of Ca2+ transport in the proximal and in the distal tubules are due to the high activity of a Na+/Ca2+ exchange system in the distal tubule and its virtual absence in the proximal tubule.