Intracellular [Na+], Na+ pathways, and fluid transport in cultured bovine corneal endothelial cells.

The mechanism of fluid transport across corneal endothelium remains unclear. We examine here the relative contributions of cellular mechanisms of Na+ transport and the homeostasis of intracellular [Na+] in cultured bovine corneal endothelial cells, and the influence of ambient Na+ and HCO3- on the deturgescence of rabbit cornea. Bovine corneal endothelial cells plated on glass coverslips were incubated for 60 min with 10 microm of the fluorescent Na+ indicator SBFI precursor in HCO3- HEPES (BH) Ringer's solution. After loading, cells were placed in a perfusion chamber. Indicator fluorescence (490 nm) was determined with a Chance-Legallais time-sharing fluorometer. Its voltage output was the ratio of the emissions excited at 340 and 380 nm. For calibration, cells were treated with gramicidin D. For fluid transport measurements, rabbit corneas were mounted in a Dikstein-Maurice chamber, and stromal thickness was measured with a specular microscope. The steady-state [Na+]i in BH was 14.36+/-0.38 mM (n = mean+/-s.e.). Upon exposure to Na+ -free BH solution (choline substituted), [Na+]i decreased to 1.81+/-0.20mM (n = 19). When going from Na+ -free plus 100 microm ouabain to BH plus ouabain, [Na+]i increased to 46.17+/-2.50 (n = 6) with a half time of 1.26+/-0.04 min; if 0.1 microm phenamil plus ouabain were present, it reached only 21.78+/-1.50mm. The exponential time constants (min-1) were: 0.56+/-0.04 for the Na+ pump; 0.39+/-0.01 for the phenamil sensitive Na+ channel; and 0.17+/-0.02 for the ouabain-phenamil-insensitive pathways. In HCO3- free medium (gluconate substituted), [Na+]i was 14.03+/-0.11mM; upon changing to BH medium, it increased to 30.77+/-0.74 mm. This last [Na+]i increase was inhibited 66% by 100 microm DIDS. Using BH medium, corneal thickness remained nearly constant, increasing at a rate of only 2.9+/-0.9 microm hr-1 during 3 hr. However, stromal thickness increased drastically (swelling rate 36.1+/-2.6 microm hr-1) in corneas superfused with BH plus 100 microm ouabain. Na+ -free, HCO3- free solution and 100 microm DIDS also led to increased corneal swelling rates (17.7+/-3.6, 14.4+/-1.6 and 14.9+/-1.2 microm hr-1, respectively). The present results are explained by the presence of a DIDS-inhibitable Na+-HCO3- cotransporter and an epithelial Na+ channel, both previously found in these cells. On the other hand, the quantitative picture presented here appears a novelty. The changes we observe are consistent with pump-driven rapid exchange of intracellular Na+, and recirculation of fully 70% of the Na+ pump flux via apical Na+ channels.

[Mechanisms of action of hypersodium medium on contractile activity of isolated rat heart]. / Mekhanizmy vliianiia gipernatrievoi sredy na sokratitel'nuiu aktivnost' izolirovannogo serdtsa krys.

Despite the high efficiency of elevated concentrations of sodium ions during myocardial ischemia and calcium paradox, the molecular mechanism of action of hypersodium media on heart contractions remains unknown. The purpose of the investigation was to study mechanisms by which raised concentrations of sodium ions alter cardiac contractility. Subsequent to initially developed reduced pressure in the left ventricle, elevated concentrations of sodium ions (200 mM instead of 140 mM NaCl, 3 mM KCl) produced an increased force of contractions of about 50%. The first stage of decrease in developed pressure did not relate to elevated tonicity of extracellular ionic millieu because lithium chloride (60 mM) did not produce the same effect. This action of elevated concentrations of sodium ions has been shown to be independent of blockers of ion-transporting systems (caffeine, verapamile, ethmozine, HMA or lidocaine). Raising the contractions by elevating the concentration of sodium ions (second stage) has been shown to be susceptible to sodium channel blockers (6-IA, benzamil, of phenamil) and to caffeine. Decreasing of potassium concentration (from 3 mM to 1-2 mM amplified, and increasing of K+ level (from 3 mM to 6 mM) attenuated the positive inotropic action of the elevated concentration of sodium ions. The positive inotropic effect due to elevated concentrations of sodium ions remains even after heart arrest by high concentrations of verapamile (2 mcM). Lithium chloride (60 mM) failed to elevate left ventricle developed pressure which was raised by elevated concentrations of sodium ions. These data suggest that the elevated concentration of sodium ions could effect Na+/Ca2+ exchange and provoke Ca2+ release from sarcoplasmic reticulum by changing the sodium gradient and resulting in Ca2+ entry via Na+/Ca2+ exchange. These observations are consistent with the hypothesis of Leblanc N., Hume J.R. (1990) regarding sodium-induced calcium ion release from sarcoplasmic reticulum.

Participation of a K(+) channel modulated directly by cGMP in the speract-induced signaling cascade of strongylocentrotus purpuratus sea urchin sperm.

Speract, a decapeptide from Strongylocentrotus purpuratus sea urchin eggs, transiently stimulates a membrane guanylyl cyclase and activates a K(+)-selective channel that hyperpolarizes sperm. However, previous studies of sperm and of sperm membrane vesicles reached conflicting conclusions about the mechanisms that open these channels. We find that speract hyperpolarizes and increases the cGMP content of flagellar vesicles. We confirm previous findings that intravesicular GTPgammaS and GTP enhance this hyperpolarization, but not GDPbetaS. The G protein activators AlF(-)(4) and mastoparan also are ineffective. Thus, it is unlikely that a G protein participates in the speract response. In contrast, hyperpolarization responses to speract are increased by 3-isobutyl-1-methylxanthine, which preferentially inhibits cGMP-selective phosphodiesterases of sperm, and the 8Br-cGMP derivative hyperpolarizes vesicles in the absence of speract. The responses to speract and to 8Br-cGMP have similar ionic selectivities (K(+) > Rb(+) > > Li(+) > Na(+)) and sensitivities to the channel blockers 4-aminopiridine and 3, 4-dichlorobenzamil, indicating that they likely result from opening of the same K(+) channel. Inhibitors that preferentially inhibit cAMP-selective phosphodiesterases do not alter responses to speract, and permeant cAMP analogs do not hyperpolarize vesicles. In addition, inhibitors of protein kinases and phosphatases fail to alter vesicle hyperpolarization by speract. The increase in vesicular cGMP content produced by speract therefore may directly mediate opening of the channel that hyperpolarizes sperm membrane vesicles. Similar mechanisms presumably operate in intact sperm.

[Effect of sarcolemmal ion-transporting system blockers on the intensity of heart damage during "calcium paradox"]. / Vliianie blokatorov nekotorykh ionotransportnykh sistem sarkolemmy na intensivnost' povrezhdenii serdtsa pri "kal'tsievom paradokse".

The aim of present study was to investigate a role of different anions in calcium paradox development. It is accepted point of view that development of calcium paradox is depend on cation composition and activity of Na/Ca exchange. However, role of anion composition remain unknown. It is not studied role of some aniontransporting systems in development of calcium paradox. Experiments were carried out on isolated Langendorff perfused rat hearts. Hearts were perfused with calcium-containing solution for 15 minutes, calcium-free medium for 10 minutes and reperfused by initial calcium-containing solution with [Ca2+ = 2 mM]. Release of myoglobin was used as a marker of membrane damage. It has been shown that addition of 5-20 mM HCO3 exacerbated calcium paradox of the heart, elevated myoglobin release from 4.92 +/- 0.57 mcg/g dry weight to 11.3 +/- 1.6 mcg/g dry weight. An inhibitor of HCO3/Cl exchange, 10 mcM L-644,711 depressed elevation of myoglobin release to 4.8 +/- 1.05 mcg/g dry weight. An inhibitor of Cl- channels, 5 mcM DIOA caused raising of myoglobin loss to 7.3 +/- 0.8 mcg/g dry weight during calcium paradox. These data show dependence of calcium paradox on anion composition. A possible reason for exacerbation of calcium paradox by HCO3- rich medium could be consistence of HCO3/Cl and Na/Ca exchange. The results discover new perspectives in myocardial protection of calcium overload.

[The importance of the ion-transport systems of the sarcolemma and sarcoplasmic reticulum in changing rat cardiac contractile function under a hypersodium medium]. / Znachenie nekotorykh ionotransportnykh sistem sarkolemmy i sarkoplazmaticheskogo retikuluma v izmenenii sokratitel'noi funktsii serdtsa gipernatrievoi sredoi u krys.

Following a reduced pressure in the left ventricle, elevated concentrations of sodium ions enhanced by half the contraction force of the rat isolated heart. This effect was shown to be independent of the Na-channels blockers or Na/H exchange of caffeine but quite susceptible to sodium channel blockers, caffeine, and the blocking agent for Na-Ca exchange Ni2+. A decrease in potassium concentration amplified, and elevation of K+ level attenuated the positive inotropic effect of the elevated concentration of sodium ions. The effect was preserved even after heart arrest induced by verapamil. The findings suggest that elevated concentration of sodium ions may affect the Na+/Ca2+ exchange and provoke Ca2+ release from sarcoplasmic reticulum by means of changing the sodium gradient. These data corroborate the Leblanc and Hume hypothesis of the sodium-induced calcium ions release from sarcoplasmic reticulum.

[The role of ion transport system of sarcolemma in amino acid loss during myocardial perfusion with calcium-free medium and myocardium injury during "calcium paradox"]. / Rol' ionotransportnykh sistem sarkolemmy v potere aminokislot pri perfuzii serdtsa beskal'tsievoi sredoi i povrezhdenii miokarda pri "kal'tsievom paradokse".

The loss of myocardial amino acids is known to depend on sodium gradient across sarcolemma. This is regulated by changes of cellular volume as well. It is suggested that loss of amino acids can be regulated by blocking of anion-transporting systems during Ca-free perfusion. It have been found that calcium depletion from extracellular medium exacerbates release of amino acids two- four fold. Sodium lowering (from 140 mM to 30 mM) accelerates and sodium elevation (from 140 mM to 200 mM) attenuates loss of taurine, glutamine, glycine, glutamate, aspartate, alanine and asparagine, but does not hydrophobic amino acids. Inhibition of CI- channels by IAA94 or K-Cl cotransport with DIOA increases the loss of taurine, glutamine, glycine, glutamate, aspartate, alanine and asparagine during Ca-free perfusion. The release of amino acids during Ca-free perfusion is negatively correlated with recovery of oxidative phosphorylation during the second phase the calcium paradox-Ca-readmission.

Taurine indirectly increases [Ca]i by inducing Ca2+ influx through the Na(+)-Ca2+ exchanger.

Recent studies in heart cells have shown taurine to induce a sustained increase of both intracellular Ca2+ and Na+. These results led us to believe that the increase in Na+ by taurine could be due to Na+ entry through the taurine-Na+ cotransporter which in turn favours transarcolemmal Ca2+ influx through Na(+)-Ca2+ exchange. Therefore, we investigated the effect of beta-alanine, a blocker of the taurine-Na+ cotransporter and low concentrations of CBDMB (a pyrazine derivative, 5-(N-4chlorobenzyl)-2',4'-dimethylbenzamil), a Na(+)-Ca2+ exchanger blocker on taurine-induced [Ca]i increase in embryonic chick heart cells. Using Fura-2 Ca2+ imaging and Fluo-3Ca2+ confocal microscopy techniques, taurine (20 mM) as expected, induced a sustained increase in [Ca]i at both the cytosolic and the nuclear levels. Preexposure to 500 microM of the blocker of the taurine-Na+ cotransporter, beta-alanine, prevented the amino acid-induced increase of total [Ca]i. On the other hand, application of beta-alanine did not reverse the action of taurine on total [Ca]i. However, low concentrations of the Na(+)-Ca2+ exchanger blocker, CBDMB, reversed the taurine-induced sustained increase of cytosolic and nuclear free calcium (in presence or absence of beta-alanine). Thus, the effect of taurine on [Ca]i in heart cells appears to be due to Na+ entry through the taurine-Na+ cotransporter which in turn favours transarcolemmal Ca2+ influx through the Na(+)-Ca2+ exchanger.

Inhibition by 5-N-(4-chlorobenzyl)-2',4'-dimethylbenzamil of Na+/Ca2+ exchange and L-type Ca2+ channels in isolated cardiomyocytes.

The inhibitory effect of the amiloride derivative 5-N-(4-chlorobenzyl)-2',4'-dimethylbenzamil (CBDMB) on calcium (Ca2+) uptake via sarcolemmal sodium-calcium (Na+/Ca2+) exchange and L-type Ca2+ channels was investigated in isolated adult rat ventricular cardiomyocytes under depolarizing conditions in cells preincubated with 1 mM ouabain or 137 mM lithium (Li+), respectively. Fifteen or 120 min. preincubation with CBDMB inhibited Ca2+ uptake via Na+/ Ca2+ exchange in Na(+)-loaded depolarized cells completely at 100 microM with an IC50 of 21 microM. After 120 min. preincubation, CBDMB inhibited Ca2+ uptake via L-type Ca2+ channels by 75.1 +/- 8.1% (mean and S.E.M.) and IC50 of 4 microM, whereas no significant inhibition was observed after 15 min. preincubation. (+)-Isradipine (10 microM) inhibited high potassium (K+) induced Ca2+ uptake via L-type Ca2+ channels by 35% after 15 min. and by 70% after 120 min. preincubation. Inhibition by CBDMB of specific (+)-[3H]isradipine binding to L-type Ca2+ channels showed similar concentration dependency as inhibition of Ca2+ uptake via L-type Ca2+ channels. In conclusion, CBDMB inhibits sarcolemmal Na+/Ca2+ exchange in rat ventricular cardiomyocytes rapidly. However, after longer preincubation periods, L-type Ca2+ channels are inhibited as well and with higher potency than Na+/Ca2+ exchange.

Sodium-calcium exchanger in cultured human retinal pigment epithelium.

Regulation of intracellular free Ca2+ concentration ([Ca2+]i) by an Na+/Ca2+ exchanger was studied in cultures of human retinal pigment epithelial cells using Ca(2+)-indicator dyes (fura-2 and fluo-3) and digital fluorescence imaging. Mean resting [Ca2+]i of cultured RPE in a control Ringer solution was 189 +/- 16 nM. Replacing extracellular Na+ with N-methyl-D-glucamine elicited a two-fold rise in [Ca2+]i; the magnitude of the [Na+]o-free-induced rise in [Ca2+]i varied as a function of extracellular [Ca2+]. The [Na+]o-free response was not significantly affected by the Ca2+ channel blocker nifedipine, or by pretreatment with thapsigargin which depletes intracellular Ca2+ stores. By contrast, the [Na+]o-free-induced rise in [Ca2+]i was significantly reduced by CBDMB, an amiloride derivative that is highly selective for Na+/Ca2+ exchange inhibition. These findings indicate that removal of extracellular Na+ promotes net [Ca2+]i gain via Na+/Ca2+ exchange. Western and Northern blot analyses, respectively, confirmed the presence of Na+/Ca2+ exchanger protein and mRNA in cultures of human RPE. Specifically, Western blot analysis of whole cell lysates of cultured RPE using a polyclonal antibody made against the canine cardiac exchanger identified a major band at approximately 126 kD. Northern blot analysis of total human RPE RNA using a restriction fragment cRNA probe coding for the canine cardiac Na+/Ca2+ exchanger showed that the major exchanger-related transcript was approximately 6.8 kb. In sum, our findings demonstrate the presence of a cardiac-exchanger-related transcript was approximately 6.8 kb. In sum, our findings demonstrate the presence of a cardiac-type Na+/Ca2+ exchanger in cultures of human RPE.

[The mechanisms of the effect of a high extracellular sodium concentration on the contractile activity of the isolated rat heart]. / Mekhanizmy vliianiia vysokoi vnekletochnoi kontsentratsii natriia na sokratitel'nuiu aktivnost' izolirovannogo serdtsa krys.

Increase in the extracellular concentration of sodium (from 140 mM to 200 mM) causes an initial increase in the developing left-ventricular pressure which is replaced by increase of the amplitude of contractions by 40-50% in comparison with the initial condition. Lithium chloride (60 mM) did not cause similar changes. The initial diminution of myocardial contractility under the effect of a hypersodium medium was not sensitive to verapamil, ethmosin, lidocaine, caffeine or the Na-H exchange blocker hexamethylenamyloride (HMA). Verapamil (0.1 microM) or HMA (1 microM) did not influence the increase in the developing pressure induced by the hypersodium medium. The favorable inotropic effect of the hypersodium medium was manifested after arrest of the heart with verapamil (2 microM). Caffeine (2 microM), ethmosin (1 microM) or lidocaine (100 microM) weakened the favorable effect of the hypernatrium medium.

Effects of anion exchange on intracellular pH and tension in basilar artery of dogs.

We simultaneously measured intracellular pH (pHi) and isometric tension in canine basilar arteries. pHi was calculated from the ratio of fluorescence intensities at 540 nm of exciting wavelengths of 500 and 440 nm in the presence of 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). pHi was 7.33 +/- 0.02 in Krebs-Henseleit solution (pH of 7.4 at 37 degrees C). Application of the anion exchange blocker 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) decreased the resting pHi by 0.25 pH units. Increasing extracellular pH (pHo) [by decreasing CO2 tension (PCO2)] from 7.4 to 7.8 increased pHi by 0.38 pH units and increased tension by 2.28 +/- 0.21 mN. Decreasing pHo from 7.8 to 7.4 (by increasing PCO2) restored the pHi and muscle tension to their baseline levels. SITS inhibited the increase in pHi and isometric tension in response to the increase in pHo in an endothelium-independent fashion. The Na+/H+ exchange blockers, amiloride or 5-(N-methyl-N-guanidinocarbonylmethyl)-amiloride, did not affect the pHo or tension changes. The results suggest that in the range of pH tested, anion exchange is more important than Na+/H+ exchange in the regulation of pHi and mechanical tone in the basilar artery.

Differential regulation of cell cycle machinery by various antiproliferative agents is linked to macrophage arrest at distinct G1 checkpoints.

There is currently much interest in the mechanisms of action of antiproliferative agents and their effects on cell cycle machinery. In the present study we examined the mechanisms of action of four unrelated agents known to inhibit proliferation of CSF-1-stimulated bone marrow-derived macrophages (BMM). We report that 8-bromo-cAMP (8Br-cAMP) and lipopolysaccharide (LPS) potently reduced CSF-1-stimulated cyclin D1 protein, and cyclin-dependent kinase (cdk) 4 mRNA and protein levels, while the inhibitory effects of the Na+/ H+ antiport inhibitor 5-(N',N'-dimethyl) amiloride (DMA) and interferon gamma (IFN gamma ) were only weak. All agents repressed CSF-1-stimulated retinoblastoma protein phosphorylation. Furthermore, 8Br-cAMP and to a lesser extent IFN gamma, also reduced CSF-1-stimulated levels of E2F DNA binding activity in a macrophage cell line, BAC1.2F5. An explanation for the different effects of the agents is that 8Br-cAMP and LPS were found to arrest BMM in early/mid-G1, while IFN gamma and DMA arrested cells in late G1 or early S phase. These data indicate that (1) different antiproliferative agents can arrest the same cell type at distinct checkpoints in G1 and (2) effects of antiproliferative agents on cell cycle machinery is linked to the position at which they arrest cells in G1.

Electric field strength and epithelization in the newt (Notophthalmus viridescens).

There is convincing evidence that endogenous electric fields are necessary for normal wound epithelization, but it is unclear whether normal epithelization rates can be accelerated increasing normal field strengths. Although we confirmed that normal lateral fields are required for normal Notophthalmus viridescens epithelization rates, significant increases in epithelization were not achieved by increasing normal field strengths with direct current. With increases of less than 50%, epithelization rates were slightly increased. When the field strengths were augmented by 50%, epithelization was significantly retarded. This pattern was also observed when we applied direct current to wounds whose fields were nullified with benzamil. Epithelization was more rapid in wounds with field strengths raised to 20, 40, and 60 mV/mm than in benzamil-treated wounds without field augmentation. Epithelization was most rapid at 40 mV/mm, the normal value. When fields were augmented to 80 and 100 mV/mm, the epithelization rate diminished significantly. We also augmented field strengths by increasing the Na+ concentration in the medium surrounding the digits. Wounds healed more slowly in 10 mM Na+ than in 1.5 mM Na+, the normal pond water concentration. When field strengths in 10 mM Na+ were diminished to levels found in contralateral digits at 1.5 mM Na+ by applying direct current, the epithelization rate was restored to normal. We conclude that newt wound epithelization rates are nearly maximal at normal wound field strengths. Field strengths significantly higher than normal (50% more) result in significantly diminished rates of epithelization.

HCl-induced cell edema in rabbit esophageal epithelium: a bumetanide-sensitive process.

BACKGROUND & AIMS: The morphology of acid damage to esophageal epithelium is characterized by marked cell (swelling) edema. This observation suggests that, in the process of acid damage, an increase in osmotic forces develops within the cell that accounts for the increase in cell water. The aim of this study was to document that esophageal cells swell at acidic pH and to explore the nature of the osmolytes and mechanisms responsible for it. METHODS: Cell edema was assessed in sections of rabbit esophageal epithelium by correlating morphological change with change in tissue wet weight after immersion in acidic solutions for up to 4 hours. RESULTS: At pH < or = 2 for 2 hours, tissues gained weight and showed cell edema on both light and electron microscopy. In addition to being time- and pH-dependent, cell edema was dependent on bathing solution osmolytes, specifically Na+, K+, and Cl-, and could be inhibited by tissue pretreatment with bumetanide (or ethacrynic acid). CONCLUSIONS: HCl exposure can spontaneously produce cell edema in esophageal epithelium. The phenomenon is pH- and time-dependent and requires acid stimulation of osmolyte absorption through a bumetanide-sensitive process compatible with an NaK2Cl cotransporter in the epithelial cell membrane.

Microtubule integrity is necessary for the epithelial barrier function of cultured thyroid cell monolayers.

Vectorial transport in the thyroid epithelium requires an efficient barrier against passive paracellular flux, a role which is principally performed by the tight junction (zonula occludens). There is increasing evidence that tight junction integrity is determined by integral and peripheral membrane proteins which interact with the cell cytoskeleton. Although the contribution of the actin cytoskeleton to tight junction physiology has been intensively studied, less is known about possible interactions with microtubules. In the present study we used electrophysiological and immunohistochemical approaches to investigate the contribution of microtubules to the paracellular barrier in cultured thyroid cell monolayers which displayed a high transepithelial electrical resistance (6000-9000 ohm.cm2). Colchicine (1 microM) caused a progressive fall in electrical resistance to < 10% of baseline after 6 h and depolarization of the transepithelial electrical potential difference consistent with a significant increase in paracellular permeability. The effect of colchicine on TER was not affected by agents which inhibit the major apical conductances of thyroid cells but was reversed upon removal of the drug. Immunofluorescent staining for tubulin combined with confocal laser scanning microscopy demonstrated that thyroid cells possessed a dense microtubule network extending throughout the cytoplasm which was destroyed by colchicine. Colchicine also produced changes in the localization of the tight junction-associated protein, ZO-1: its normally continuous junctional distribution was disrupted by striking discontinuities and the appearance of many fine strands which extended into the cytoplasm. A similar disruption in E-cadherin staining was also observed, but colchicine did not affect the distribution of vinculin associated with adherens junctions nor the integrity of the perijunctional actin ring. We conclude that microtubules are necessary for the functional and structural integrity of tight junctions in this electrically tight, transporting epithelium.

The Na+/H+ antiport in renal mitochondria.

In isolated renal mitochondria, Na+ and Li+ stimulated H+ efflux from the mitochondrial matrix. In submitochondrial particles (SMP), Na+ flux was also coupled to H+ transport in the opposite direction. The overshoot of Na+ uptake in SMP with an outwardly directed H+ gradient indicated that downhill efflux of H+ through the mitochondrial membrane induced uphill transport of Na+. Similar to the Na+/H+ antiport in other types of mitochondria, the antiport in renal mitochondria was more sensitive to amiloride derivatives than to amiloride itself. Benzamil and ethylisopropylamiloride (EIPA), but not amiloride, inhibited the antiport, with 50% inhibition of 10(-4) M for both benzamil in mitochondria and EIPA in SMP. The Na+/H+ antiport in renal mitochondria had simple saturation kinetics for external Na+ [Michaelis-Menten constant (Km) = 3.27 +/- 0.63 mM; maximal velocity (Vmax) = 0.022 +/- 0.002 pH units/s] and Li+ (Km = 3.62 +/- 0.75 mM; Vmax = 0.022 +/- 0.002 pH units/s). NH4Cl and NH4 acetate stimulated Na+ efflux and inhibited Na+ uptake in SMP. Comparable results with NH4 acetate and chloride suggested that NH4+ modified Na+ transport through its direct interaction with the Na+/H+ antiport, rather than through the alkalinization of intra-SMP space from non-ionic diffusion of NH3. These results suggested that the Na+/H+ antiport may be a factor in the exit of NH4+ from renal mitochondria.

Mechanism of hyperosmolality stimulation of ANP secretion: its dependency on calcium and sodium.

The calcium dependency of hyperosmolality stimulation of atrial natriuretic peptide (ANP) secretion was determined using isolated superfused nonbeating rat left atrium. Increasing osmolality by 65, 85, and 100 mosmol/kgH2O by superfusion with sucrose produced a peak rise in ANP secretion of 1.8-, 2.0-, and 2.7-fold. To determine whether calcium influx played a role in osmolality (osm)-stimulated ANP secretion, atria were superfused with 2 mM lanthanum, a calcium antagonist. Lanthanum inhibited by 85% the response to a 100 mosmol/kgH2O increase in osm. The voltage-dependent calcium channel blocker isradipine had no effect on osm-stimulated ANP secretion, suggesting that calcium influx via voltage-dependent calcium channels was not playing a significant role. Likewise, depleting sarcoplasmic reticulum calcium with 1 microM ryanodine did not block the response to osm, suggesting that calcium influx was not adequate to induce consequential release of calcium from the sarcoplasmic reticulum. To determine whether calcium influx was via Na(+)-Ca2+ exchange, we determined the sodium dependency of osm-stimulated ANP secretion. Replacement of sodium with lithium or choline blocked the secretory response to 100 mosmol/kgH2O. We conclude that osm-stimulated ANP secretion is calcium and sodium dependent. Calcium influx via Na(+)-Ca2+ exchange is highly implicated as the mechanism of cellular calcium entry.

Chloride transport activation by plasma osmolarity during rapid adaptation to high salinity of Fundulus heteroclitus.

Transition from low salt water to sea water of the euryhaline fish, Fundulus heteroclitus, involves a rapid signal that induces salt secretion by the gill chloride cells. An increase of 65 mOsm in plasma osmolarity was found during the transition. The isolated, chloride-cell-rich opercular epithelium of sea-water-adapted Fundulus exposed to 50 mOsm mannitol on the basolateral side showed a 100% increase in chloride secretion, which was inhibited by bumetanide 10(-4) M and 10(-4) M DPC (N-Phenylanthranilic acid). No effect of these drugs was found on apical side exposure. A Na+/H+ exchanger, demonstrated by NH4Cl exposure, was inhibited by amiloride and its analogues and stimulated by IBMX, phorbol esters, and epithelial growth factor (EGF). Inhibition of the Na+/H+ exchanger blocks the chloride secretion increase due to basolateral hypertonicity. A Cl-/HCO3- exchanger was also found in the chloride cells, inhibited by 10(-4) M DIDS but not involved in the hyperosmotic response. Ca2+ concentration in the medium was critical for the stimulation of Cl- secretion to occur. Chloride cell volume shrinks in response to hypertonicity of the basolateral side in sea-water-adapted operculi; no effect was found on the apical side. Fresh-water-adapted fish chloride cells show increased water permeability of the apical side. It is concluded that the rapid signal for adaptation to higher salinities is an increased tonicity of the plasma that induces chloride cell shrinkage, increased chloride secretion with activation of the Na+K+2Cl- cotransporter, the Na+/H+ exchanger and opening of Cl- channels.

Intracellular pH in vascular smooth muscle: regulation by sodium-hydrogen exchange and multiple sodium dependent HCO3- mechanisms.

OBJECTIVES: The aim was to determine the mechanisms, particularly bicarbonate dependent mechanisms, of intracellular pH (pHi) recovery from various acidoses in vascular smooth muscle and to explore the ATP dependency of the respective mechanisms. METHODS: Experiments were conducted in rat aortic smooth muscle cells grown in primary culture and synchronised in a non-growing state by serum deprivation. pHi was measured in cells loaded with the pH sensitive fluorescent dye, 2',7'-bis-(2-carboxyethyl)-5-(and 6)-carboxyfluorescein (BCECF). Chloride efflux was studied by determination of the rate of efflux of 36Cl over 5 min. Cells were ATP depleted by substitution of glucose in the medium by 2-deoxyglucose. Acidoses were induced by CO2 influx and NH3 efflux techniques. RESULTS: In the absence of HCO3-, the 5-(N-ethyl-N-isopropyl) amiloride (EIPA) sensitive Na+/H+ exchange accounted for the recovery from intracellular acidosis. In the presence of HCO3- ions the response to respiratory acidosis (CO2 influx) was predominantly via activation of Na+/H+ exchange and an EIPA sensitive Na+ and HCO3- dependent mechanism. A 4-acetamido-4'-isothiocyanostilbene-2',2'-sulphonic acids (SITS) sensitive Na+ dependent Cl-/HCO3- mechanism which is also sensitive to EIPA makes a small contribution during severe intracellular acidosis. Under such conditions HCO3- dependent mechanisms contributed about 40% to the overall pHi regulating capacity of vascular smooth muscle cells. However, under conditions which deplete cellular ATP these pHi regulating mechanisms account for virtually all of theses cells' ability to regulate pHi. The inability of Na+/H+ exchange to participate in pHi recovery under these circumstances, reduces the ability of vascular smooth muscle cells to recover pHi by approximately 50-60%. Chloride efflux was approximately linear over 5 min and was increased by 36% in the presence of extracellular HCO3-. Efflux in the presence of HCO3- was inhibited similarly by both SITS and EIPA. CONCLUSIONS: At least three transporters contribute to recovery from acidosis in vascular smooth muscle: Na+/H+ exchange, an Na(+)-HCO3- cotransporter which is sensitive to EIPA, and an Na+ dependent HCO3-/Cl- exchange sensitive to both SITS and EIPA. The Na(+)-HCO3- cotransporter appears to be similar to that described in human vascular smooth muscle. When the Na+/H+ exchanger is attenuated by cellular ATP depletion, the alternative pathways, particularly the Na(+)-HCO3- cotransporter, ensure that substantial pHi regulatory capacity is maintained.