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The mechanisms of tumor promotion in liver by various xenobiotics of diverse structure are not well understood. However, these tumor promoters share the ability to exert growth-stimulatory effects on hepatocytes. Our laboratory has been utilizing normal rat hepatocytes under defined conditions of primary cultures, to investigate growth-stimulatory actions of liver tumor promoters. We have shown that most, if not all, of the liver tumor promoters tested stimulate hepatocyte DNA synthesis when added in combination with epidermal growth factor (EGF), insulin, and glucocorticoids. In the present study, we sought evidence for the role of the Na(+)/H(+) antiporter and cytoplasmic alkalinization in the direct growth-stimulatory actions of tumor promoters on hepatocytes. Hepatocytes cultured under conditions (bicarbonate-buffered medium) where intracellular pH (pH(i)) was independent of extracellular pH (pH(e)), EGF- and insulin-stimulated rates of DNA synthesis were unaffected by modest changes in pH(e). However, under conditions (HEPES-buffered medium) where pH(i) varied in a linear fashion with pH(e), rates of EGF- and insulin-stimulated DNA synthesis were highly dependent on pH(e). Similarly, 12-O-tetradecanoylphorbol-13-acetate (TPA) and alpha-hexachlorocyclohexane (HCH)-stimulated DNA synthesis were pH(e)-dependent but were stimulatory over different pH(e) ranges, suggesting that these promoters may act by distinct mechanisms. Chemicals that are capable of inducing rapid cytoplasmic alkalinization, ammonium chloride (1 and 15 mM) and monensin (0.5 microM), were found to stimulate hepatocyte DNA synthesis. The role of the Na(+)/H(+) antiport in controlling pH(i) of hepatocytes was demonstrated by artificially acidifying 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein acetoxymethyl (BCECF)-loaded isolated hepatocytes with 20 mM sodium acetate and the use of specific inhibitors. Amiloride and its analogues inhibited pH(i) recovery from the acid load in a dose dependent manner and the relative potency of these inhibitors paralleled their K(i) values for the Na(+)/H(+) antiport. At concentrations that stimulate hepatocyte DNA synthesis, some liver tumor promoters phenobarbital (PB) and HCH, were found to cause a rapid rise pH(i) in isolated hepatocytes which was sensitive to amiloride and its analogues. Taken together, our data suggest that activation of Na(+)/H(+) antiport activity may be one mechanism whereby some liver tumor promoters stimulate hepatocytes DNA synthesis. This study has implications for the mechanisms of tumor promotion in liver carcinogenesis., (Copyright 2003 Wiley-Liss, Inc.)

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., (Copyright 2000 Academic Press.)

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.

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.

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 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.

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.

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.

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.

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.

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.

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.

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.

A comprehensive examination of phenomena related to cancer is presented that is based on hydrogen ion dynamics, as viewed from the biological, biochemical, and biophysical perspective. A model is described that considers an array of cancer-associated events from oncogenesis to carcinogenesis from this perspective. The basic ideas are viewed from various aspects, ranging from the cellular level to the clinical situation. The novel types of therapeutic and prophylactic agents that result from applying these concepts are elucidated. Considerable insight into this modern approach is seen from some of the mechanisms that characterize the phenomenon of spontaneous regression of cancer.

In order to characterise the apically-located conductive cation pathway of the type II pneumocyte, apical plasma membranes were prepared from mature fetal guinea pig lung. The protocol yielded purified apical membranes that enriched 19-fold with the brush border enzyme marker alkaline phosphatase; there was no significant contamination with other cellular membranes. A technique for imposing an outwardly-directed electrochemical Na+ gradient was used to amplify conductive 22Na+ uptake into vesicles. Uptake of 22Na+ was time-dependent, proportional to the magnitude of the Na+ gradient, specific and sensitive to the amiloride analogues phenamil and EIPA (apparent minimum Ki values of 50 nM and 10 microM, respectively, with maximum uptake inhibition of 42% and 39% at 100 microM). Uptake experiments in which the outwardly-directed Na+ gradient was replaced by outwardly-directed gradients of small monovalent cations and molecular cations were performed. The Na+/K+ permeability ratio was 1.2:1, and over the extended range of small monovalent cations, a permeability sequence of Na+ > K+ > Li+ > Rb+ > Cs+ was observed, indicating the presence of fixed negative charge in or spatially close to the pore. The molecular cation permeability sequence of NH4+ > methylamine+ > dimethylamine+ > choline+ > N-methyl-D-glucamine+ > tetraethylammonium+ > tetramethylammonium+, after transformation, gives an estimate of 8 A for the conducting pore diameter. These data are consistent with the presence in the apical membrane of fetal type II pneumocytes of a cation specific channel with low Na+ selectivity and amiloride sensitivity.

Purpose: The major mechanisms that regulate the intracellular acidity of pHi in mammalian cells are the Na+/H+ exchange and HCO3-/Cl- exchange through the plasma membrane. The purpose of this study was to investigate the feasibility of increasing the thermosensitivity of tumors by increasing intracellular acidity with the use of drugs that inhibit the pHi regulatory mechanisms., Methods and Materials: The pHi of SCK tumor cells in vitro was determined with the fluorescence spectroscopy method. The thermosensitizing effects of the drugs on the cells in neutral (pH 7.2-7.5) and acidic (pH 6.6) media were determined by clonogenic assay. The thermosensitization of SCK tumors in vivo by the drugs was determined with the tumor growth delay and the in vivo-in vitro assay for clonogenic cells., Results: The pHi of SCK tumor cells in pH 7.2-7.5 media was similar to the media pH, while the pHi of the cells in pH 6.6 media was about 7.0. The pHi declined and the thermosensitivity of the tumor cells increased when the Na+/H+ exchange was inhibited with amiloride (3,5 diamino-6-chloro-N-(diaminomethylene) pyrazinecarboxamide) and its analogs, HMA (3-amino-6-chloro-5-(1-homopiperidyl)-N-(diaminomethylene) pyrazinecarboxamide) or EIPA (3-amino-6-chloro-5-(N-ethyl-N-isopropylamino)-N-diaminomethylene) pyrazinecarboxamide), especially in acidic medium. The potencies of HMA and EIPA to decrease the pHi and increase the thermosensitivity in vitro were more than 50 times greater than that of amiloride. DIDS (4,4-diiosothiocyanatostilbene-2,2'-disulfonic acid), an inhibitor of the Na(+)-dependent HCO3-/Cl- exchange, exerted little effect on the pHi and thermosensitivity of SCK cells in vitro, but it enhanced the effects of amiloride and its analogs. Amiloride and HMA also significantly enhanced the thermal effect on tumors in vivo, as judged by the tumor growth delay and also by the in vitro-in vivo assay for clonogenic cells. Combinations of DIDS with amiloride or HMA were more effective than either of them alone in increasing the thermal damage in vivo. As in vitro, HMA was far more potent than amiloride in increasing the thermosensitivity of tumor cells in vivo. However, EIPA was not effective in vivo, probably due to a rapid metabolic breakdown of the drug., Conclusion: The drugs that interfere with the pHi regulatory mechanism significantly thermosensitized the tumor cells in vitro, particularly those in acidic media. The drugs were also effective in increasing the thermosensitivity of tumors. Because the interstitial environment in tumors is acidic relative to that in normal tissues, the thermosensitization by the drugs may be greater in tumors than that in normal tissues.

Background: High salt intake is a risk factor for essential hypertension in man. There is evidence that, in hypertension, intracellular sodium content and univalent cation transport across erythrocyte membranes are changed. It has been proposed that a low-sodium diet has an antihypertensive effect; this may be related to changes in cation fluxes across plasma membranes., Methods: Sodium and potassium fluxes and the composition of fatty acids were studied in the erythrocytes of people who had eaten a low-sodium vegan diet for many years (n = 9) and in those of controls who had consumed a mixed diet (n = 11) to investigate the dependence of these variables on dietary factors. Both systolic and diastolic blood pressures were lower in vegans than in controls., Results: The passive permeability to sodium (P < 0.05) ,Na+,K+ cotransport (P < .001) and the intracellular content of exchangeable sodium (P = 0.076) were decreased in the erythrocytes of those who had consumed the low-sodium diet compared with the controls. The activity of the Na+-K+ pump, Na+-H+ exchange and the passive permeability to potassium were unaltered. Swelling-induced K+,C1- cotransport was increased in the erythrocytes of those who had eaten the low-sodium vegan diet compared with controls (P < 0.01). The proportion of linoleic acid was increased (P < 0.01) at the expense of eicosapentaenoic and docosahexaenoic acids (P < 0.001) in the erythrocyte membranes of the vegans., Conclusion: Our results show that levels of intracellular sodium and Na+,K+ cotransport activity, which increase in patients with hypertension, decreased in those consuming a low-sodium vegan diet. This suggests that the risk of essential hypertension was diminished in the vegan participants, confirming our observation that systolic and diastolic blood pressures were lower in the strict vegans than in the controls.

The current study was designed to determine the role of sodium-proton (Na+/H+) exchange in blood pressure regulation in sodium chloride (NaCl)-sensitive and NaCl-resistant spontaneously hypertensive rats and control Wistar-Kyoto rats (WKY) using 5-(N,N-hexamethylene)amiloride (HMA), a potent and selective inhibitor of Na+/H+ exchange. The response of mean arterial pressure to intravenous infusion of HMA was examined in conscious, unrestrained male rats maintained on normal (1%) or high (8%) NaCl diets for 3 weeks beginning at age 7 weeks. The HMA significantly increased mean arterial pressure in NaCl-sensitive spontaneously hypertensive rats and NaCl-resistant spontaneously hypertensive rats that were fed 1% NaCl, but not in WKY rats that were fed 1% NaCl; the 8% NaCl diet enhanced this pressor response in all 3 strains. The pressor response was accompanied by significant increases in plasma norepinephrine levels in NaCl-sensitive spontaneously hypertensive rats on both diets, but not in NaCl-resistant spontaneously hypertensive rats or WKY rats on either diet. There were no differences in steady-state levels (30-60 nM) of plasma HMA between diet groups in any strain. Therefore, administration of HMA in a dose at which it is highly selective for the Na+/H+ exchanger (Ki = 160 nM) caused a systemic pressor response in spontaneously hypertensive rats that was enhanced by dietary NaCl supplementation. With these data, it is suggested that inhibition of Na+/H+ exchange in vivo has a pressor effect greater in spontaneously hypertensive rats than in WKY rats and is further enhanced by NaCl supplementation.

Purpose: The effects of HMA (3-amino-6-chloro-5-(1-homopiperidyl)-N- (diaminomethylene)pyrazinecarboxamide), an analog of amiloride, on the intracellular pH (pHi) of SCK tumor cells in vitro and on the thermosensitivity of tumors in vivo were investigated., Methods and Materials: The pHi of SCK tumor cells in vitro was measured with the BCEC fluorescence spectroscopy method. The effect of HMA on the thermosensitivity of SCK tumors grown SC in the legs of A/J mice was assessed by the tumor growth delay method and the in vivo-in vitro excision assay method., Results: The pHi of SCK tumor cells in pH 7.5 and 6.6 medium was about 7.50 and 7.15, respectively. The presence of 10-50 microM of HMA lowered the pHi by 0.1-0.2 pH units both in pH 7.5 and 6.6 medium. Heating at 43 degrees C 120 min lowered the pHi by 0.2 and 0.3 pH units in pH 7.5 and 6.6 medium, respectively. When the cells were heated in the presence of 10-50 microM HMA, a marked decline in pHi occurred and and the decline in pHi resulting from the combination of heat and HMA was more pronounced in pH 6.6 medium than in pH 7.5 medium. Heating the SCK tumors grown SC in the legs of A/J mice at 43.5 degrees C for 1 h resulted in a growth delay of 3.7 days. When the host mice were i.v. injected with 0.1 mg/kg of HMA and the tumors were heated heated 20 min later, the tumor growth was delayed by 8.2 days, which was 4.5 days longer than that by heating alone. Heating the SCK tumor at 42.5 degrees C for 1 h caused a tumor growth delay of 0.9 days. An i.v. injection of 1 mg/kg or 10 mg/kg of HMA prior to heating at 42.5 degrees C for 1 h caused a tumor growth delay 2.1 and 3.1 days longer, respectively, than that by heating alone. Such an enhancement of heat-induced tumor growth delay by HMA was due to increased cell killing, as determined with the in vivo-in vitro excision assay of clonogenic cells in the tumors., Conclusion: HMA is a potent thermosensitizer, particularly in an acidic environment. Thermosensitization by HMA may occur preferentially in tumors relative to normal tissues since the intratumor environment is acidic.

Apical membrane vesicles purified from mucosal scrapings obtained from distal segments of organ donor colons and a 22Na-uptake technique were used to characterize the mechanism(s) of Na+ transport into these vesicles. An outwardly directed H+ gradient (pH 5.5in/7.5out) markedly increased uptake of 22Na into these vesicles. Osmolarity studies demonstrated that 22Na was taken up into the intravesicular space with minimal binding observed to the surface of the vesicles. Voltage clamping in the presence of K+/valinomycin reduced the H+ gradient-dependent 22Na uptake into these vesicles by approximately 45% and generation of an inside negative membrane potential significantly increased 22Na uptake. Under non voltage clamped conditions, H+ gradient-dependent 22Na uptake into these vesicles was significantly inhibited by specific inhibitors of Na(+)-H+ exchange (DMA, HMA and EIPA) as well as by inhibitor of epithelial Na+ channels (phenamil). Under voltage clamped conditions, H+ gradient-dependent 22Na uptake, however, was unaffected by phenamil (20 microM), but was almost completely inhibited by DMA, HMA and EIPA (20 microM each). The mechanism of amiloride inhibition of electroneutral Na(+)-H+ exchange was noncompetitive with a Ki for amiloride of 340 microM. Electroneutral 22Na uptake exhibited saturation kinetics with an apparent Km for Na+ of 8.7 +/- 1.7 mM and a Vmax of 2.02 +/- 0.45 nmol/mg per 5 s. The Na(+)-H+ exchange demonstrated cation specificity similar to the Na(+)-H+ exchangers described in other epithelia. These studies demonstrate for the first time that Na+ transport across the apical membranes of human distal colon involves both conductive Na+ uptake and an electroneutral Na(+)-H+ exchange process.

Epidermal growth factor (EGF) triggers rapid and delayed effects on gluconeogenesis, cytosolic (lactate/pyruvate ratio) and mitochondrial (3-hydroxybutyrate/acetoacetate ratio) redox states (Soler, C. and Soley, M., Biochem. J., 294 (1993) 865-872). This study attempts to determine whether the mechanism by which EGF modulates any of these parameters is dependent on the regulation of Na+/H+ exchange and/or Na+/K(+)-ATPase activities. The Na+/H+ exchange was inhibited by either amiloride or the analogue 5-(N,N-hexamethylene)amiloride (HMA), and the Na+/K(+)-ATPase activity was inhibited by ouabain. The delayed EGF inhibition of gluconeogenesis, increase of the lactate/pyruvate ratio and decrease in the 3-hydroxybutyrate/acetoacetate ratio were unaltered in the presence of amiloride, HMA or ouabain. The rapid EGF stimulation of gluconeogenesis was also observed in the presence of HMA or ouabain. Although Na+/H+ exchange and/or Na+/K(+)-ATPase are regulated by EGF, our results indicate that these activities are not required for the effects of EGF on gluconeogenesis and/or cytosolic and mitochondrial redox state.

The intracellular pH (pHi) indicator 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein and microfluorimetry were used to characterize several ion transport mechanisms in rat parotid striated ducts. The recovery of ductal pHi from an acute acid load was Na+ dependent and inhibited by the amiloride analogue ethylisopropylamiloride with 50% inhibitory concentration 4.7 +/- 0.8 microM, indicating the presence of a Na(+)-H+ exchanger of the amiloride-insensitive type. The rate of this recovery was stimulated approximately 20% in ducts pretreated with the muscarinic agonist carbachol (10(-5) M) and inhibited approximately 20% in ducts pretreated with the beta-adrenergic agonist isoproterenol (10(-6) M). Upon removal of extracellular K+, ductal pHi rapidly decreased (0.19 +/- 0.02 pH units/min), consistent with a coupling between K+ and H+ (or OH-) fluxes in this tissue. In HCO(3-)-containing medium, the acidification due to K+ removal was blunted, arguing against ductal K(+)-HCO3- cotransport. However, the effect of K+ removal was inhibited by the K+ channel blocker Ba2+ (1 mM) and by the H+ channel blocker Zn2+ (25 microM), consistent with the involvement of electrically coupled K+ and H+ channels. The effect of K+ removal was unaffected by pretreatment of ducts with isoproterenol (10(-6) M) but markedly inhibited (approximately 50%) by pretreatment with carbachol (10(-5) M). No evidence for a significant component of Cl(-)-HCO3- exchange was found in striated ducts. The properties of the Na(+)-H+ exchanger and K(+)-H+ exchange mechanism identified here are consistent with their involvement in ductal salt reabsorption and secretion.

Effects on ion transport of extracellular Na+ and Cl- ([Na+]o and [Cl-]o, respectively), HCO3(-)-CO2, 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP), epinephrine, and transport inhibitors were examined in short-circuited rabbit proximal colonic mucosa. Net Na+ flux was independent of Cl- but partially (60%) dependent on HCO3(-)-CO2. Net Cl- flux was partially (70%) dependent on Na+ and totally dependent on HCO3(-)-CO2. Both fluxes peaked between 25 and 60 mM and decreased at higher concentrations. Apical Na+ influx but not Cl- influx obeyed the same pattern. The inhibition resulted from increases in mucosal but not serosal [Na+]o and not from increases in [Cl-]o. Amiloride and benzamil (0.2-0.3 mM) partially inhibited net Na+ absorption, as did 8-BrcAMP, but these effects were independent of the inhibition seen at high [Na+]o. Net Cl- absorption was inhibited by 8-BrcAMP but not by 0.2 mM amiloride. At high [Na+]o and [Cl-]o, there were a residual ion flux suggesting HCO3- secretion and, in the presence of 8-BrcAMP and amiloride or benzamil, net secretions of Na+ and Cl-, the former larger than the latter. Epinephrine, via alpha 2-receptors, reversed the ion-transport effects of high [Na+]o but did not stimulate a mucosal-to-serosal unidirectional HCO3- flux (as shown in rabbit ileum). 4-Acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (0.5 mM) and bumetanide (10 microM) had no effect on ion transport. The results suggest 1) Na+ entry via two Na(+)-H+ exchangers, one inhibited by amiloride and cAMP and the other inhibited by high [Na+]o and stimulated by epinephrine; 2) Cl- entry via Cl(-)-HCO3- exchange; 3) HCO3- secretion at high [Na+]o and [Cl-]o; and 4) cAMP-induced secretion of Na+ and Cl- at high [Na+]o and [Cl-]o.

In this study we examined the mechanism whereby atrial natriuretic peptide secretion is increased when the frequency of contraction is raised from 2 to 5 Hz. We tested the hypothesis that calcium plays a significant role in the frequency-stimulated response. Using superfused rat left atria, we found that lowering the superfusate calcium concentration from 1.8 to 0.2 mmol/L abolished the frequency-stimulated atrial natriuretic peptide secretory response. Superfusion with ryanodine (1 mumol/L), an inhibitor of sarcoplasmic reticulum calcium release, resulted in a minimal inhibitory effect. Superfusion with 50 mumol/L nitrendipine or 10 mumol/L diltiazem inhibited the frequency-stimulated response by 46% to 48%. The lack of total inhibition suggested that an additional mechanism of calcium influx was involved, namely, inward calcium movement carried by Na(+)-Ca2+ exchange. As intracellular sodium has been reported to rise with an increase in beat frequency, a fall in the sodium gradient would favor inward calcium movement by Na(+)-Ca2+ exchange. Because we could not directly assess the role of Na(+)-Ca2+ exchange in this experimental paradigm, we examined the effect of lowering the transmembrane sodium gradient on atrial natriuretic peptide secretion by superfusion with the sodium channel activator veratridine or the sodium ionophore monensin. Superfusion with 1 mumol/L veratridine increased atrial natriuretic peptide secretion by 2.3-fold, and 1, 5, and 10 mumol/L monensin increased secretion by 1.1-, 2.1-, and 15.7-fold, respectively. In addition, we examined the possibility that the reported rise in intracellular sodium associated with increased beat frequency was due to enhanced Na(+)-H+ antiporter activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Earlier studies demonstrated swelling of endothelial cells in skeletal muscle capillaries during hemorrhagic shock with involvement of a pH- and amiloride-sensitive Na+/H+ antiport. The aim of this study was to determine the degree to which diminished capillary perfusion and metabolic acidosis are mechanisms for capillary narrowing. Capillary luminal diameters and microcirculatory blood flow were measured in the rabbit tenuissimus and gastrocnemius muscles, respectively. In seven experiments, occlusion of the distal aorta produced a low-flow state in the hind limbs as assessed by laser Doppler flowmetry (LDF). The 68.5 +/- 12.2% reduction in LDF flow in this local model of skeletal muscle ischemia was comparable to that in shock. After 1 h, systemic blood pH stayed the same, yet local tissue pH (venous effluent) became acidic. There was no capillary narrowing in contrast to an approximate 20% decrease in diameter found in shock. In additional experiments to simulate shock acidosis, the blood pH was reduced over 1 h by jugular vein infusion of hydrochloric acid (1.2 M, 4 ml.min-1.kg-1), with (n = 5) and without (n = 6) pretreatment with an amiloride analog [5-(N,N-hexamethylene)amiloride] specific to block Na+/H+ exchange. Mean arterial blood pressure and LDF flow were essentially unchanged in both groups. Diameters of treated capillaries did not change, whereas those of untreated capillaries decreased by 18.0 +/- 3.5% which would greatly elevate flow resistance. These results taken together suggest that systemic blood acidosis, and not low flow per se, induces a capillary narrowing which we contend is due to endothelial cell swelling.

Infection of permissive human embryo fibroblasts (MRC-5) with human cytomegalovirus (HCMV) increased the number of copies of the Na+,K(+)-ATPase (the Na+ pump) in the plasma membrane, measured as ouabain-binding sites. The increase was preceded by cell enlargement by about 24 hr, becoming significant between 48 and 72 hr after infection. Reduction in Na+ or Cl- concentration in the culture media immediately after infection partially prevented the increase in the number of ouabain-binding sites. The effect was reversible upon restoring Cl- or Na+ to the incubation medium, but withdrawal of either ion at 24 or 48 hr PE failed to prevent the increase in the number of binding sites. These results suggest that the processes that resulted in the increase of copies of the Na+,K(+)-ATPase required both Na+ and Cl- during the first 24 hr PE. Amiloride and ethylisopropylamiloride, two inhibitors of Na+ transport mechanisms of the plasma membrane have been previously shown to reduce the amount of virus yields and to prevent the onset of cytomegaly (Fons et al., 1991, Proc. Soc. Exp. Biol. Med. 196, 89-96). We show here that these agents partially block the increase in ouabain-binding sites caused by HCMV infection.

Like amiloride, 6-iodoamiloride (6-IA) competitively and reversibly inhibits rotation of the Na(+)-driven flagellar motors of alkalophilic Bacillus cells. However, when 6-IA-treated cells are irradiated with UV light, motility is irreversibly inhibited. This treatment does not alter the membrane potential or affect Na(+)-coupled alpha-aminoisobutyrate transport. An increase in the Na+ concentration during UV irradiation substantially protects the motors from irreversible inhibition. Thus, photoactivated 6-IA seems to bind specifically and covalently at or around the Na(+)-interaction site of the force-generating units of the motors to inhibit motor rotation irreversibly. Rotation of each motor, which is monitored using tethered alkalophilic Bacillus cells, is also inhibited by photoactivated 6-IA. In this case, however, the rotation rate during UV irradiation decreases stepwise, suggesting the presence of several independently functioning force-generating units in a motor. From the data of 14 tethered cells, the number of units/motor is estimated to be 5-9.

We examined the effects of 5-(N,N-hexamethylene)amiloride (one of the Na(+)-H+ exchange blockers, HMA) and amiloride (AM) on action potentials (APs), intracellular Na+ activity, and pH using conventional and double-barreled ion-selective microelectrodes in guinea pig papillary muscle in vitro. Papillary muscle preparations were superfused with HEPES-buffered solution, and intracellular Na+ (aiNa+) and H+ (intracellular pH, pHi) activities were measured in quiescent preparations without stimulation. HMA at a concentration of 1 microM began to induce prolongation of action potential duration (APD) and at concentrations > 10 microM induced a decrease in action potential amplitude (APA), depolarization of resting membrane potential (RMP), prolongation of APD and depression of the maximum upstroke velocity (Vmax). HMA exerted dose-, time-, and rate-dependent reduction in Vmax. AM began to prolong APD at a concentration of 10 microM and at 1 mM induced depolarization of RMP, decreased Vmax and induced significant prolongation of APD. HMA (100 microM) induced a decrease in aiNa+ by 2-3 mM, but exerted no effects on pHi in normal Tyrode's solution. Under conditions of intracellular acidosis induced by exposure to K(+)-free solution, HMA produced a further decrease in pHi. Our results provide direct evidence that HMA has a depressant action on cardiac Na+ channels and prolongs AP at concentrations that presumably affect Na(+)-H+ exchange in cardiac muscle.

LLC-PK1 cells are known to possess respective Na(+)-H+ exchangers (NHE) in apical and basolateral membranes. We examined the developmental difference between these NHEs. LLC-PK1 cells seeded on a filter membrane at a saturation density formed a confluent monolayer after 1 day. Intracellular pH (pHi) was measured 1-6 days after seeding using 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. The activities of apical and basolateral NHEs were estimated separately by the initial pHi responses to Na+ after NH3/NH4+ prepulses in the absence of HCO3- at 37 degrees C. Significant apical and basolateral NHE activities were detected at day 1 (1 day after seeding). Apical NHE activity increased 2.9-fold during days 1-3. By contrast, basolateral NHE activity remained unchanged up to day 6. At day 1, both apical and basolateral NHEs showed sensitivity to inhibition by ethylisopropyl amiloride (EPIA). Apical NHE acquired 4.5-fold resistance to EIPA during days 1-3, whereas the EIPA sensitivity of basolateral NHE was constant. As a result, apical NHE became 29-times more resistant to EPIA than basolateral NHE at day 3 or 4. Treatment with an antisense oligonucleotide targeting NHE-1 (inhibitor-sensitive NHE) mRNA decreased basolateral NHE activity at days 2 and 4, and apical NHE activity at day 2. These results suggest: (1) NHE-1 is distributed over the plasma membrane in early confluent LLC-PK1 monolayers; and (2) then, NHE-2 (inhibitor-resistant NHE) gradually begins to be expressed specifically in the apical membrane, and the distribution of NHE-1 becomes confined to basolateral membrane.

The aim of the present paper was to study the mechanisms of the inhibitory effect of atrial natriuretic peptide (ANP) on the sustained contraction phase of vascular smooth muscle cells (VSMC). Specifically, the potential role of ANP on the Na+/H+ antiporter and Na+ transport systems was investigated. Both ANP and 8-bromo cGMP inhibited 22Na+ uptake and decreased intracellular Na ([Na+]i) in VSMC, an effect that was mimicked by the specific Na+/H+ antiporter inhibitor, hexamethylen amiloride (HMA). The effect of ANP was not additive with HMA, therefore suggesting that both inhibit the same 22Na+ transport pathway. On the other hand, the inhibition of 22Na+ accumulation by ANP was additive with the inhibition by furosemide or bumetanide, thus suggesting that both drugs act on different Na+ exchange systems. In HEPES-buffered medium, ANP, cGMP, and HMA significantly inhibited the AVP-induced intracellular alkalinization, an effect which was associated with significant inhibition of the AVP-induced shape change. In bicarbonate buffered medium, ANP and cGMP decreased the pH level below the baseline after application of AVP, and an inhibition by ANP and cGMP of AVP-induced VSMC shape change was also observed. The recovery of cellular pH after three different types of acid load, namely, ammonium chloride pulse, nigericin clamp and lowering of extracellular pH, was significantly decreased by ANP and cGMP. Taken together, these results indicate that ANP/cGMP inhibit the activity of the Na+/H+ antiporter in VSMC, either in hormone- or pH-stimulated conditions.

Background/aims: The mechanisms of Na+ movement across colonocyte plasma membranes in the human colon are not well understood. Current studies were undertaken to investigate Na+ transport pathways in apical membranes of proximal organ donor colons., Methods: Purified apical membrane vesicles and rapid filtration 22Na-uptake techniques were used., Results: An outwardly directed H(+)-gradient (pH 5.5 in/7.5 out) increased 22Na uptake into these vesicles. H+ gradient-driven 22Na uptake was significantly reduced by voltage clamping with K+/valinomycin, but was significantly stimulated by creation of an inside-negative potential. Potential sensitive 22Na uptake was inhibited by Na+ channel inhibitors phenamil and benzamil. Electroneutral 22Na uptake was insensitive to phenamil and benzamil, but was inhibited by amiloride, 5-(N,N-dimethyl)amiloride, 5-(N,N-hexamethylene)amiloride, and 5-(N-ethyl-N-isopropyl)amiloride. Electroneutral 22Na uptake showed saturation kinetics with an apparent Michaelis constant for Na+ of 11.8 +/- 2.4 mmol/L and a maximal velocity of 2.5 +/- 0.6 nmol.mg protein-1 x 5 s-1. The mechanism of amiloride inhibition was noncompetitive with an inhibitor constant for amiloride of 325 mumol/L. Acetazolamide, furosemide, bumetanide, 4-acetamido-4'-isothiocyano-2,2'-disulfonic acid stibene, and 4,4'-di-isothiocyanatostilbene-2,2'-disulfonic acid (1 mmol/L each) failed to inhibit 22Na uptake. Li+ and NH4+ (but not Cs+, K+, or choline+) inhibited H(+)-gradient driven 22Na uptake., Conclusions: Na+ transport in human proximal colonic apical membrane vesicles involves both conductive Na+ transport and an electroneutral Na(+)-H+ exchange.

Interleaved 23Na- and 31P-nuclear magnetic resonance (NMR) spectra were continuously collected on perfused rat hearts subjected to low-flow ischemia (30 min, 10% flow) or zero-flow ischemia (21 min) followed by reperfusion. During untreated low-flow and zero-flow ischemia, intracellular Na+ (Nai+) increased by 53 +/- 11 (+/- SE) and 78 +/- 8%, respectively, and remained elevated for zero-flow hearts. However, during both low- and zero-flow ischemia, Nai+ did not increase in hearts treated with the Na(+)-H+ exchange inhibitor, 5-(N-ethyl-N-isopropyl)amiloride (EIPA). The pH decreases during ischemia were unchanged. EIPA treatment reduced ATP depletion during ischemia. During reperfusion from zero-flow ischemia, EIPA-treated hearts displayed more rapid and extensive recoveries of phosphocreatine and ATP. Recovery of left ventricular developed pressure was improved for zero-flow hearts treated with EIPA during the ischemic period exclusively (104 +/- 13%) compared with untreated hearts (36 +/- 21%). These data indicate that Na(+)-H+ exchange is an important mechanism for Nai+ accumulation, but not for pH regulation, during myocardial ischemia. Additionally, Nai+ homeostasis plays an important role in the postischemic recovery of cellular energy and ventricular function.

We studied the role of sodium transport and calcium channels in protein kinase C mediated signal-transduction pathways in bovine airway smooth muscle. 4-beta phorbol 12,13 dibutyrate (PDB), an activator of protein kinase C, caused dose-related slowly developing contraction in bovine bronchial rings with a peak effect at 60-90 min. 4-alpha PDB, an inactive analogue, was without effect. Mean peak PDB-induced contraction (measured as a percentage of the maximum response to methacholine) was reduced from 122% to 20% when experiments were carried out in calcium-free fluids +EDTA (10(-3) M). Similar reductions were seen in the presence of nifedipine and verapamil, inhibitors of voltage-dependent calcium channels. Amiloride at high concentrations (10(-3) M) reduced the contractile response to PDB from 87% to 20%, but at a concentration which inhibits the sodium entry channel (10(-6) M), amiloride was without effect. 5-N,N-hexamethylene amiloride (10(-5) M), a specific inhibitor of Na+/H+ exchange, did not alter the contraction produced by PDB. Frusemide (10(-5) M), an inhibitor of Na(+)-K(+)-Cl- cotransport, was without effect on PDB contractions. We conclude that phorbol ester-induced contraction of bovine airway smooth muscle is dependent on calcium entry via voltage-dependent calcium channels but is independent of Na+ entry, Na+/H+ exchange or Na(+)-K(+)-Cl- cotransport.

Angiogenesis is important to such processes as normal embryonic development and tissue growth, and is also a central feature of diseases such as diabetic retinopathy and the growth of solid tumors. Understanding the basic events governing angiogenesis has therefore attracted great interest. The ion channel blocking agent, amiloride, has been shown to inhibit angiogenesis in an in vivo model (Lansing et al., '91). This suggested a vital role for Na(+)-coupled transport processes in angiogenesis. A large number of structural analogues of amiloride have been synthesized (Kleyman and Cragoe, '88), and many of these are well characterized with respect to biological activity. These analogues present an opportunity to dissect the process of angiogenesis and identify potentially important physiological events. In this report we describe the effects of amiloride on an in vitro model for angiogenesis employing vascularized tissue explants. Amiloride inhibits capillary morphogenesis completely and reversibly at concentrations as low as 134 microM. It appears to act by blocking endothelial cell proliferation, but not migration. Inhibition is heightened by the introduction of hydrophobic groups on the terminal guanidino nitrogen atom, or on the 5-amino position. An analogue substituted at both of these positions is 30-fold more potent than the parent compound. Of amiloride's known biological activities, these results most closely correlate with the inhibition of Ca2+ transport processes, and thereby suggest an important role for Ca2+ transport in capillary morphogenesis.

Several of the pyrazine derivatives are widely used for inhibiting sodium flux via Na+/Ca2+ exchange or Na+/H+ exchangers or through the epithelial cation channels. These processes can profoundly affect cytosolic Ca2+. We found that the widely used fluorescent probes fura-2 and indo-1 could not be used to measure the effect of pyrazine analogs on the cytosolic free calcium ([Ca2+]i) of YAC-1 lymphoma cells treated with the pore-forming protein cytolysin/perforin. We show that the excitation spectra of pyrazine derivatives that specifically inhibit Na+/Ca2+ exchange [5-(N-4-chlorobenzyl)-2',4'-dimethylbenzamil], Na+/H+ exchange [5-(N-ethyl-N-isopropyl)-amiloride], and Na+ channels (phenamil) overlap with those of fura-2 and indo-1. In the presence of Ca2+, fluorescence readings for fura-2 plus drug are greater than those of fura-2 alone with the typically used 340- and 380-nm excitation light wavelengths; F380 readings were more affected than F340 readings. The effect was drug dose dependent. Hence, calculations that use F340 readings in the presence of pyrazine derivatives will result in overestimates of [Ca2+]i, while those that use the corresponding ratio readings, R340/380, will result in underestimates of [Ca2+]i. We found that the luminescent intracellular Ca2+ indicator aequorin could be used successfully with pyrazine derivatives, and that the ability of these compounds to enhance cytolysin/perforin-mediated increases in [Ca2+]i corresponded to their previously reported ability to inhibit Na+/Ca2+ exchange in pituitary cell plasma membrane vesicles. YAC-1 lymphoma cells are easy to culture and handle and may be a useful model for the studies of the Na+/Ca2+ exchanger in situ.

Background: For acid injury to occur in esophageal epithelium, extracellular HCl must lower intracellular pH. Therefore, we sought to define the mechanisms for translation of low extracellular pH (pHo) into low intracellular pH (pHi)., Methods: To define the mechanisms, primary cultures of rabbit esophageal epithelial cells were loaded with the pH-sensitive fluorescent dye, 2'7''-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF), which enabled pHi to be recorded by microfluorimetry., Results: Lowering pHo to 6.0 with HCl caused pHi to decline at 0.07-0.08 units/min and produced an average cellular H+ load of 3-6 mmol/L at 1 minute and of 11 mmol/L at 5 minutes. This degree of acidification was primarily attributable to increased H+ entry via a 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS)-sensitive Clo-dependent mechanism because DIDS or exposure to Clo-free solution blocked cell acidification. Furthermore a contribution to low pHi at low pHo of abolition of acid extrusion via Na+/H+ and Na(+)-dependent Cl-/HCO3- exchangers was sought but not established because exposure at low pHo to either 5-(N-ethyl-N-isopropyl)amiloride (EIPA) or Na(+)-free solution + EIPA increased the degree of acid loading over untreated cells., Conclusions: Low pHo results in low pHi in esophageal cells primarily because of increased H+ entry via a DIDS-sensitive, Clo-dependent mechanism consistent with the known acid-loading Na(+)-independent Cl-/HCO3- exchanger.

Changes in Na+ transport in rat alveolar type II (ATII) cells during culture were quantified and related to alterations in spatial distribution of proteins antigenically related to amiloride-sensitive Na+ channels. Adult rat ATII cells were cultured for periods ranging from 24 to 96 h. When patch clamped in the whole cell mode, both freshly isolated and cultured ATII cells exhibited outwardly rectified Na+ currents. At 0 and 24 h in culture, these currents were equally inhibited by amiloride, benzamil, and 5-(N-ethyl-N-isopropyl)-2',4'-amiloride (inhibitory constant approximately 1 microM). These conductive pathways were equally permeable to Na+ and K+. Immunocytochemical localization at 0 or 24 h in culture revealed the presence of plasma membrane antigenic sites; after 48 h, the appearance of intracellular antigenic sites increased significantly. A single band of molecular mass 135 kDa in membrane proteins of freshly isolated ATII cells was recognized in Western blots; at 48 h in culture, two lower bands with molecular masses of 75 and 65 kDa were detected in either membrane or cytoplasmic proteins. Photolabeling with 2'-methoxy-5'-nitrobenzamil showed that the 135-, 75-, and 65-kDa bands contained amiloride-binding sites. These results suggest the presence of low amiloride affinity conductive pathways in freshly isolated and cultured ATII cells. Culturing ATII cells resulted in internalization and possible breakdown of these pathways and decreased Na+ transport.

Both Na(+)-dependent HCO3- influx and the Na(+)-H+ antiport have been shown to contribute to recovery from intracellular acidosis in avian and mammalian cardiac tissue. We have investigated the participation of these mechanisms in the recovery of intracellular pH (pHi) after an acid load in the Langendorff-perfused ferret heart. pHi was measured from the phosphorus-31 nuclear magnetic resonance chemical shift of 2-deoxy-D-glucose 6-phosphate. Basal pHi was higher in HCO(3-)-buffered solution (7.05 +/- 0.01; n = 8) than in nominally HCO(3-)-free N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) solution (6.98 +/- 0.02; n = 9). Addition of 5-(N-ethyl-N-isopropyl)amiloride (EIPA) caused a significant fall in pHi in HEPES solution (6.91 +/- 0.02; n = 5) but not in HCO3- solution (7.02 +/- 0.02; n = 5). Intrinsic intracellular buffering capacity in 0 Na(+)-HEPES solution was 37 +/- 2 mmol/l (n = 4), and additional buffering due to HCO(3-)-CO2 was approximately 13 mmol/l in HCO3- solution. After an intracellular acidosis induced by an NH4Cl prepulse, the proton efflux rate (JH) at pHi 6.90 was 0.5 +/- 0.2 nmol.l-1.min-1 (n = 14) in HEPES solution and 1.2 +/- 0.4 mmol.l-1.min-1 (n = 13) in HCO3- solution. The addition of 1 microM EIPA effectively blocked proton efflux in HEPES solution (JH < 0.1 mmol.l-1.min-1; n = 8), whereas it slowed pHi recovery in HCO3- solution (JH = 0.6 +/- 0.2 mmol.l-1.min-1; n = 9). There was no recovery of pHi in Na(+)-free HCO3- solution (JH < 0.1 mmol.l-1.min-1; n = 3). The Na(+)-H+ antiport and a mechanism requiring both external Na+ and HCO3- each contribute approximately 50% to proton efflux at pHi 6.90 during the recovery from intracellular acidosis in the isolated perfused mammalian heart.

When subjected to hypotonic stress, human peripheral neutrophils initially swell due to rapid water entry and thereafter recover toward the normal cell size (approximately 330 microns 3). Neutrophils do not behave as perfect osmometers: when resuspended in half-isotonic medium (150 mosM), they swell by only approximately 40% rather than doubling in size as predicted for ideal behavior. As with lymphocytes, restoration to the normal cell size involves the net loss of K+ and Cl- from the cytosol through independent conductance pathways. Volume regulation is sensitive to 0.4-1 mM of quinine, UK-5099, 3,5-diiodosalicylate (DISA), MK-473 (an indanyloxyacetate derivative), and to MK-447 [a 2-(aminomethyl)phenol]. From correlation of drug effects on the time course of cell volume recovery and the associated volume-activated 86Rb+ and 36Cl- fluxes, it was evident that quinine blocked only K+ channels, whereas MK-447 acted as a selective inhibitor of Cl- channels. In contrast, UK-5099, DISA, and MK-473 were nonspecific in that the compounds displayed comparable suppressive effects on all three parameters. Structure-activity relationships in the MK-447 series revealed the critical elements of the molecule responsible for drug potency. In particular, the importance of the neighboring ionizable 1-hydroxyl and 2-aminomethyl groups and the formation of secondary ring structures for biological activity is emphasized. The most potent derivative thus far identified, termed analogue A [inhibitor constant (Ki) approximately 16 microM], had a potency approximately sixfold greater than that of the parent compound (Ki approximately 90 microM). These findings define the mechanism of action of a relatively new class of agents that behave as inhibitors of swelling-activated Cl- channels in these cells.

In primary cultures of striatal neurons, stimulation of N-methyl-D-aspartic acid (NMDA) receptors or associative activation (but not separate activation) of (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors and metabotropic glutamate receptors (mGluR) strongly increased arachidonic acid (AA) release via activation of phospholipase A2 (PLA2). Depolarizing agents, such as veratridine, were as potent as NMDA in stimulating AA release. However, increasing the intracellular Ca2+ concentration via voltage-sensitive Ca2+ channels did not result in a significant stimulation of PLA2. Substitution of sodium by lithium, a monovalent cation that does not participate in the Na+/Ca2+ exchanger activity but permeates ionotropic glutamate receptor channels, blocked AA release induced by veratridine or AMPA plus mGluR agonists. It also reduced the NMDA-induced AA release, to a lesser extent. The contribution of the Na+/Ca2+ exchanger to the activation of PLA2 after veratridine, NMDA receptor, or AMPA receptor plus mGluR stimulation was confirmed by using a selective inhibitor of the Na+/Ca2+ exchanger.

1. Hypotonic stress unmasked inward and outward K+ and Cl- movements in rat thymocytes. This KCl flux stimulation was reduced by DIOA (dihydroindenyl-oxy-alkanoic acid), but not by DIDS (4,4'-diisothiocyanostilbene-2,2'-disulphonate), quinidine, DPAC 144 (5-nitro-2-(2-phenylethyl-amino)-benzoic acid), bumetanide or ouabain. 2. In isotonic media (308 +/- 5 mosmol kg-1), the cells exhibited the following DIOA-sensitive fluxes: (i) a K+ efflux of 42.7 +/- 17.1 mmol (l cells.h)-1 (mean +/- S.D., n = 7), (ii) a Cl- efflux of 68 +/- 21 mmol (l cells.h)-1 (n = 3), (iii) a Rb+ influx of 9.7 +/- 3.9 mmol (l cells.h)-1 (n = 6) and (iv) a Cl- influx of 9.4 +/- 4.1 mmol (l cells.h)-1 (n = 6). 3. Hypotonic shock (183-200 mosmol kg-1) induced a sevenfold stimulation of DIOA-sensitive K+ and Cl- effluxes and a twofold stimulation of DIOA-sensitive Rb+ and Cl- influxes (with a Rb+ to Cl- stoichiometry of 1.04 +/- 0.31; mean +/- S.D., n = 6). 4. The DIOA-sensitive membrane carrier catalysed net outward KCl extrusion (the outward/inward flux ratio was 5-7 in isotonic media and 20 in hypotonic media at 189 mosmol kg-1). Inhibition of DIOA-sensitive 36Cl- efflux by cell K+ depletion suggested coupling of outward K+ and Cl- fluxes. Conversely, inward K+ and Cl- fluxes were found to be uncoupled in NO3- media and in K(+)-free media. 5. The results clearly show that rat thymocyte membranes possess a 1:1 K(+)-Cl- co-transport system which is strongly activated by hypotonic shock and catalyses net KCl extrusion.

Ouabain increases atrial natriuretic peptide (ANP) secretion. When isolated superfused rat left atria were paced at 2 Hz, ouabain at concentrations of 50, 100, and 200 microM increased ANP secretion by 2.0 +/- 0.3-, 3.2 +/- 0.5-, and 4.2 +/- 0.5-fold, respectively. In this study, we examine the mechanism of ouabain-stimulated ANP secretion using the dose of 100 microM. To determine whether calcium played a role, atria were superfused with the calcium antagonist lanthanum. Superfusion with 2 mM LaCl3 completely inhibited ouabain-stimulated secretion, suggesting that calcium influx and/or sarcoplasmic reticulum (SR) calcium release provide essential sources of calcium for the stimulatory pathway. To determine the contribution of calcium from the SR, atria were superfused with ryanodine, an agent that depletes the SR of calcium. Superfusion with 1 microM ryanodine inhibited ouabain-stimulated secretion by 47%. Inhibition of Na+,K(+)-ATPase allows sodium to accumulate in the cell. A rise in intracellular sodium alters Na(+)-Ca2+ exchange, leading to an increase in cytosolic calcium. To determine the mechanism of sodium entry, atria were superfused with 5-(N,N-hexamethylene)amiloride (HMA), an inhibitor of Na(+)-H+ exchange, or with bumetanide, an inhibitor of Na(+)-K(+)-Cl- cotransport. Superfusion with 25 microM HMA inhibited ouabain-stimulated secretion by 71%; however, 100 microM bumetanide had no significant effect on secretion. Ouabain failed to stimulate ANP secretion by nonpaced (nonbeating) atria. Likewise, superfusion with the combination of ryanodine (1 microM) and the calcium channel antagonist israpidine (10 microM) totally blocked ouabain-stimulated ANP secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

The thyroid epithelium possesses a bidirectional fluid transport system capable of absorbing Na+ and secreting Cl-. In the present studies we have examined its possible role in the regulation of thyroid follicular size. When exposed to hypotonic media (200 mosM) cultured porcine thyroid follicles first swelled and then displayed a regulatory volume decrease (RVD) over 60 min. This was associated with a transient depolarization of the transepithelial potential difference (TEP) and subsequent hyperpolarization with a time course similar to RVD. Phenamil (1 microM), an antagonist of epithelial Na+ channels, did not affect initial swelling but prevented the subsequent follicular RVD. Phenamil abolished hyperpolarization of TEP, but the loop diuretic bumetanide, which inhibits Cl- secretion in thyroid cells, did not prevent it. Exposure to hypotonic medium produced a slow hyperpolarization of the intracellular potential (basolateral membrane potential) consistent with an increase in basolateral membrane K+ conductance. Ba2+ and quinidine, which are known to inhibit K+ channels in epithelia, prevented RVD. Addition of the K+ ionophore valinomycin (1 microM) caused follicle shrinkage that was prevented by phenamil (1 microM). We conclude that cultured follicles respond to hypotonically induced stretch by activating outwardly directed Na+ transport through a mechanism which involves change in the basolateral K+ conductance. This response would be characteristic of a system that controlled follicle volume. However, it is not clear from these studies whether the cells responded primarily to the increase in follicle volume or to the change in cell volume that is expected to accompany hypotonic challenge.

Active Na+ transport by the alveolar epithelium plays a major role in reabsorption of the fetal lung fluid after birth. We characterized the biochemical and physiological characteristics of Na+ conductive pathways in distal fetal lung epithelial (FLE) cells isolated from 20-day-old rat fetuses. We demonstrated that a polyclonal antibody to Na+ channel protein (NaAb) binds to the plasma membranes of FLE cells. In Western blot studies, this NaAb and an anti-idiotypic monoclonal antibody to the amiloride-binding subunit of the Na+ channel protein recognized 150- and 90-kDa polypeptides in plasma membrane vesicles of FLE. 22Na+ flux measurements across plasma membrane vesicles of FLE revealed the existence of electrogenic Na+ transport, which was twice as high as the corresponding adult value. One hundred micromolars of amiloride, benzamil, and 5-(N-ethyl-N-isopropyl)-2'-4'-amiloride inhibited 30, 40, and 70% of the electrogenic Na+ transport across plasma membrane vesicles of FLE cells, respectively. The half-maximum inhibition of electrogenic Na+ transport by these substances occurred between 0.3 and 1 microM. [3H]benzamil equilibrium binding studies in membrane vesicles of FLE cells revealed the existence of two binding sites that had dissociation constant values of 19 and 1,525 nM, respectively. These data indicate the presence of both high- and low-amiloride affinity Na+ conductive pathways (channels) in FLE cells.

We previously reported that the thermosensitivity of tumor cells can be increased when the intracellular pH is lowered by inhibiting Na+/H+ exchange through the plasma membrane with amiloride (3,5-diamino-6-chloro-N-(diamino methylene)pyrazinecarboxamide) or its analogues and HCO3-/Cl-exchange with 4,4-diiothiocyanato-stilbene-2,2'-disulfonic acid. In this study, we investigated the effects of (3-amino-6-chloro-5- (1-homopiperidyl)-N-(diaminomethylene)pyrazine-carboxamide) (HMA), an analogue of amiloride and a potent inhibitor of Na+/H+ exchange, and R(+)-[(5,6-dichloro-2,3,9,9a-tetrahydro-3-oxo-9a-propyl-1H-fluoren -7- yl)oxy]acetic acid [B-3(+)], a potent inhibitor of HCO3-/Cl- exchange, on the thermosensitivity of SCK tumor cells in vitro. We observed that 10 microM of HMA could effectively increase the cell death by heating at 43 degrees in pH 6.6 medium but not in pH 7.5 medium. The B-3(+) at 50 microM alone had no effect on the thermosensitivity of cells, but it increased the thermosensitizing effect of HMA in acidic medium. Our results strongly suggested that a combination of HMA and B-3(+) may preferentially thermosensitize tumors in vivo since the interstitial environment in tumors is acidic relative to that in normal tissues.

One of the metabolic events that results from ligation of the CD3-TCR is an increase in [Ca2+]i. The mechanisms that generate this rise in [Ca2+]i are poorly understood, but involve mobilization of intracellular Ca2+ stores and the movement of extracellular Ca2+ into the cell. To examine the role of Na+/Ca2+ exchange in the increase in [Ca2+]i after CD3-TCR engagement, the effects of specific inhibitors of Na+/Ca2+ exchange, DCB, CBDMB, and bepridil, were examined. Inhibitors of Na+/Ca2+ exchange suppressed IL2 production and the rise in [Ca2+]i in Jurkat cells stimulated by anti-CD3 mAb. Mobilization of intracellular Ca2+ stores and mitogen-stimulated inositol phosphate production were not inhibited by these agents. Inhibitors of Na+/Ca2+ exchange also inhibited mitogen responses of normal T cells and the sustained increase in [Ca2+]i resulting from cross-linking class I MHC molecules, addition of PHA, or anti-CD3 mAb. Additional evidence for an important role of a Na+/Ca2+ exchanger in generating increases in [Ca2+]i after CD3 ligation was the finding that replacing extracellular Na+ with Li+, that cannot be transported by the Na+/Ca2+ exchanger, nearly abrogated the rise in [Ca2+]i induced by cross-linking CD3. These results are consistent with the conclusion that a Na+/Ca2+ exchanger is important in regulating changes in [Ca2+]i that are critical for T cell activation.

In a search for an organic analogue of K+ or Na+ ions that binds to the cation binding sites of Na+/K(+)-ATPase with high affinity, the potency of the diuretic amiloride and its derivatives in blocking Rb+ occlusion has been tested. Although amiloride itself has a low affinity (> 200 microM), insertion of short alkyl chains in position 5 of the pyrazine ring of the molecule dramatically increased the affinity of the compound. For example, 5-(N-ethyl-N-isopropyl)amiloride (EIPA) competes with a Ki approximately 10 microM. In derivatives lacking a halogen in position 6 of the ring, a 6-fold decrease in affinity was found. Substitutions in the guanidinium moiety did not produce high affinity inhibitors of Rb+ occlusion. Several derivatives at positions 5 and 6 of the pyrazine ring were found to be strictly competitive inhibitors with respect to Rb+ ions. The highest affinity was observed around pH 8.0-8.2, and low temperature. EIPA and 5-(N-methyl-N-isobutyl)amiloride (MIBA) stabilized the E1 form of FITC1-labelled Na+/K(+)-ATPase, behaving as Na+ analogues. The present findings are similar to our previous results, showing that alkyl- and arylguanidinium derivatives are competitive Na(+)-like antagonists in cation sites. Conclusions concerning the structural features of amiloride derivatives which are necessary to produce the highest binding affinity, are being exploited in synthesis of competitive cation analogues. Derivatives with sufficiently high affinity (0.1-1 microM) will be converted to affinity and photoaffinity reagents.

We have investigated the mechanism by which amiloride and 5-(N-ethyl-N-isopropyl)amiloride (EIPA) inhibit glucose-stimulated medium acidification in the fission yeast Schizosaccharomyces pombe. The addition of glucose to an unbuffered suspension of cells results in the extrusion of acid. This process was inhibited by diethylstilbestrol (DES), an inhibitor of the H(+)-ATPase (IC50 71 microM), and also by amiloride (IC50 824 microM) and EIPA (IC50 203 microM). The presence of 100 mM NaCl reduced the degree of inhibition observed for amiloride and EIPA, but had no effect on inhibition by DES. N-Methylglucosamine partially protected the cells against the effect of amiloride, but choline chloride did not, suggesting that sodium may be important in the action of amiloride. To establish the site of action of amiloride and EIPA, ATP hydrolysis assays were performed on isolated plasma membranes. H(+)-ATPase activity was inhibited by orthovanadate, but not by amiloride or EIPA. However, both amiloride and EIPA were found to inhibit the incorporation of radioactivity from labelled glucose in S. pombe, with IC50 values of 879 and 272 microM for amiloride and EIPA respectively. Again, 100 mM NaCl was found to reduce the effectiveness of inhibition. Amiloride had no effect on the uptake of 2-deoxyglucose under the same conditions, indicating that amiloride does not inhibit the glucose transporter. We propose that amiloride and EIPA disrupt glucose-induced acidification by inhibiting glucose metabolism.