Endothelin and angiotensin II stimulation of Na+-H+ exchange is impaired in cardiac hypertrophy.

We compared the effects of endothelin-1 (ET-1) on intracellular pH, intracellular [Ca2+]i, and cell contraction in hypertrophied adult ventricular myocytes from ascending aortic banded rats and age-matched controls. Intracellular pH (pH(i)) was measured in individual myocytes with SNARF-1, and [Ca2+]i was measured with indo-1, simultaneous with cell motion. Experiments were performed at 36 degrees C in myocytes paced at 0.5 Hz in Hepes-buffered solution (pH(o) 7.40) containing 1.2 mM CaCl2. At baseline, calibrated pH(i), diastolic and systolic [Ca2+]i values, and the amplitude of cell contraction were similar in hypertrophied and control myocytes. Exposure of the control myocytes to 10 nM ET-1 caused an increase in the amplitude of cell contraction to 163+/-22% of baseline (P < 0.05), associated with intracellular alkalinization (pH(i) + 0.08+/-0.02 U, P < 0.05) and a slight increase in peak systolic [Ca2+]i (104+/-11% of baseline, P < 0.05). In contrast, in the hypertrophied myocytes, exposure to ET-1 did not increase the amplitude of cell contraction or cause intracellular alkalinization (-0.01+/-0.02 U, NS). Similar effects were observed in the hypertrophied and control myocytes in response to exposure to 10 nM angiotensin II. ET-1 also increased the rate of recovery from intracellular acidosis induced by the washout of NH4Cl in the control cells, but did not do so in the hypertrophied cells. In the presence of 10 microM 5-(N-ethyl-N-isopropyl)-amiloride, which inhibits Na+-H+ exchange, ET-1 did not cause a positive inotropic effect or intracellular alkalinization in control cells. The activation of protein kinase C by exposure to phorbol ester caused intracellular alkalinization and it increased the rate of recovery from intracellular acidification induced by an NH4Cl pulse in control cells but not in hypertrophied cells. ET-1, as well as angiotensin II, and phorbol ester, fail to stimulate forward Na+-H+ exchange in adult hypertrophied myocytes. These data suggest a defect in the coupling of protein kinase C signaling with Na+-H+ exchange in adult hypertrophied myocytes.     

Na+/H+ exchange in human lymphocytes and platelets in chronic and subacute metabolic acidosis.

The effect of acid-base disturbances on sodium/proton (Na+/H+) exchange has been examined in animal models; however, few data are available from human studies. To test the effect of metabolic acidosis on Na+/H+ exchange in man, as well as to examine the relationship between Na+/H+ exchange and cytosolic calcium ([Ca2+]i), we measured both variables in patients with decreased renal function with mild metabolic acidosis (pH 7.34 +/- 0.06), in normal control subjects (pH 7.41 +/- 0.02), and in subjects before (pH 7.40 +/- 0.01), and after (pH 7.26 +/- 0.04) ammonium chloride (NH4Cl) 15 g for 5 d. Lymphocytes and platelets were loaded with the cytosolic pH (pHi) indicator 2'-7'-bis(carboxyethyl)-5,6-carboxyfluorescein and acidified to pH approximately 6.6 with propionic acid. To quantitate Na+/H+ exchange, dpHi/dt was determined at 1 min. [Ca2+]i was measured with fura-2. Na+/H+ exchange was significantly increased only in lymphocytes of patients with renal insufficiency. Neither intracellular pH (pHi) nor [Ca2+]i was different from controls. NH4Cl resulted in a significant increase in Na+/H+ exchange in lymphocytes, but not in platelets of normal subjects. Values of pHi and [Ca2+]i in either cell type remained unaffected. Since metabolic acidosis influenced Na+/H+ only in lymphocytes, but not in platelets, it is possible that protein synthesis may be involved in increasing Na+/H+ exchange.     

Intracellular acidification associated with changes in free cytosolic calcium. Evidence for Ca2+/H+ exchange via a plasma membrane Ca(2+)-ATPase in vascular smooth muscle cells.

The purpose of this study was to define the mechanism whereby agonists that increase free cytosolic calcium (Cai2+) affect intracellular pH (pHi) in smooth muscle. Rat aortic vascular smooth muscle cells grown on coverslips were loaded with BCECF/AM or fura-2/AM for continuous monitoring of pHi or Cai2+, respectively, in a HCO3-/CO2- containing medium. Recovery from rapid increases in Cai2+ produced by 1 microM angiotensin (Ang) II (delta Cai2+ -229 +/- 43 nM) or 1 microM ionomycin (delta Cai2+ -148 +/- 19 nM) was accompanied by a fall in pHi (delta pHi, -0.064 +/- 0.0085 P < 0.01, and -0.05 +/- 0.012 pH units, P < 0.01, respectively). Neither the fall in pHi nor the rise in Cai2+ elicited by Ang II was prevented by pretreatment with agents which block the action of this agonist on pHi via the stimulation of the Cl/HCo3 exchangers (DIDS, 50 microM) or the Na+/H+ antiporter (EIPA, 50 microM). In the presence of DIDS and EIPA, Ang II produced a fall in pHi (delta pHi, -0.050 +/- 0.014, P < 0.01) and a rise in Cai2+ (delta Ca2+ 252 +/- 157 nM, P < 0.01). That the change in pHi was secondary to changes in Cai2+ was inferred from the finding that, when the rise in Cai2+ elicited by Ang II was prevented by preincubation with a Ca2+ buffer, BAPTA (60 microM), the fall in pHi was abolished as well (delta pHi, 0.0014 +/- 0.0046). The pHi fall produced by Ang II and ionomycin was prevented by cadmium at a very low concentration (20 nM) which is known to inhibit plasma membrane Ca(2+)-ATPase activity (delta pHi -0.002 +/- 0.0006 and -0.0016 pH units, respectively). Cadmium also blunted Cai2+ recovery after Ang II and ionomycin. These findings suggest that the fall in pHi produced by these agents is due to H+ entry coupled to Ca2+ extrusion via the plasma membrane Ca(2+)-ATPase. Our results indicate that agonists that increase Cai2+ cause intracellular acidification as a result of Ca2+/H+ exchange across the plasma membrane. This process appears to be mediated by a plasma membrane Ca(2+)-ATPase which, in the process of extruding Ca2+ from the cell, brings in [H+] and thus acidifies the cell.     

Regulation of intracellular pH by Na+/H+ exchange in human lymphocytes

Spectrofluorimetry and the pH-sensitive fluorescent dye 2',7'-bis(carboxyethyl)-5(6)carboxyfluorescein (BCECF) was used to measure the intracellular pH (pHi) of suspended human lymphocytes. A linear relationship exists between pHi and the fluorescent spinal-ratio (I490nm/I435nm, emission 526 nm) between pH 6.5 and pH 7.8. At the end of each experiment the ratio was calibrated using the high [K+] nigericin technique. All solutions were HEPES buffered. The pHi in resting cells was 7.27 +/- 0.02 (n = 37) at 25 degrees C. Na+ free solution caused a pHi decrease to 6.81 +/- 0.08. The ammonium prepulse technique (25 mM NH4Cl) dropped the pHi to pH 6.80. A rapid recovery of the pHi after this acidification was observed in NaCl Ringer solution. Na+ free solution completely blocked the recovery. 1 mM amiloride led to a partial block of recovery. pHi was restored to the basal value after readdition of Na+. We conclude that in HEPES buffered solutions human lymphocytes recover pHi via a mechanism dependent on extracellular Na+ and largely accomplished by an amiloride inhibitory Na+/H+ exchanger.     

Spontaneously hypertensive rat vascular smooth muscle cells in culture exhibit increased growth and Na+/H+ exchange.

The cellular mechanisms responsible for abnormalities in spontaneously hypertensive rat (SHR) vascular smooth muscle cell (VSMC) growth and vasoreactivity are not defined. Because Na+/H+ exchange, which we have previously demonstrated in cultured VSMC, plays an essential role in mediating growth factor responses, we hypothesized that abnormalities in SHR growth regulation might be reflected in the activity of this transporter. To test this hypothesis, we studied DNA synthesis and Na+/H+ exchange (measured as the rate of amiloride-sensitive intracellular alkalinization or Na+ influx) in early subcultures (less than 6) of aortic VSMC from 12-wk-old SHR and Wistar Kyoto (WKY) animals. Serum-deprived SHR VSMC grew more rapidly in response to 10% serum with an increase in [3H]thymidine incorporation of 439% compared with 191% in WKY controls. Basal intracellular pH (pHi) values determined by fluorescent pH measurements were 7.37 +/- 0.04 and 7.27 +/- 0.03 (P less than 0.05) in early passage SHR and WKY, respectively. Acid recovery (initial pHi = 6.8) by SHR VSMC was faster than by WKY VSMC as measured by alkalinization (1.8 +/- 0.6 vs. 0.8 +/- 0.2 mmol H+/liter.min, P less than 0.05) or by amiloride-sensitive 22Na+ influx (14.5 +/- 1.2 vs. 4.0 +/- 0.5 nmol Na+/mg protein.min, P less than 0.05). In comparison to WKY cells early passage SHR VSMC exhibited 2.5-fold greater alkalinization and amiloride-sensitive 22Na+ influx in response to 100 nM angiotensin II. During serial passage, WKY cells acquired enhanced Na+/H+ exchange and growth rates so that by passage 6, these differences were no longer present. These findings in early cultures of SHR VSMC, removed from the in vivo neurohumoral milieu, suggest that increased Na+/H+ exchange in SHR may reflect alterations in Na+ homeostasis that might contribute to altered SHR VSMC function such as enhanced growth and vasoreactivity.     

Bicarbonate-dependent and -independent intracellular pH regulatory mechanisms in rat hepatocytes. Evidence for Na+-HCO3- cotransport.

Using the pH-sensitive dye 2,7-bis(carboxyethyl)-5(6)-carboxy-fluorescein and a continuously perfused subconfluent hepatocyte monolayer cell culture system, we studied rat hepatocyte intracellular pH (pHi) regulation in the presence (+HCO3-) and absence (-HCO3-) of bicarbonate. Baseline pHi was higher (7.28 +/- 09) in +HCO3- than in -HCO3- (7.16 +/- 0.14). Blocking Na+/H+ exchange with amiloride had no effect on pHi in +HCO3- but caused reversible 0.1-0.2-U acidification in -HCO3- or in +HCO3- after preincubation in the anion transport inhibitor 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS). Acute Na+ replacement in +HCO3- alos caused acidification which was amiloride independent but DIDS inhibitible. The recovery of pHi from an intracellular acid load (maximum H+ efflux rate) was 50% higher in +HCO3- than in -HCO3-. Amiloride inhibited H+ effluxmax by 75% in -HCO3- but by only 27% in +HCO3-. The amiloride-independent pHi recovery in +HCO3- was inhibited 50-63% by DIDS and 79% by Na+ replacement but was unaffected by depletion of intracellular Cl-, suggesting that Cl-/HCO3- exchange is not involved. Depolarization of hepatocytes (raising external K+ from 5 to 25 mM) caused reversible 0.05-0.1-U alkalinization, which, however, was neither Na+ nor HCO3- dependent, nor DIDS inhibitible, findings consistent with electroneutral HCO3- transport. We conclude that Na+-HCO3- cotransport, in addition to Na+/H+ exchange, is an important regulator of pHi in rat hepatocytes.     

Effects of FK506 on [Ca2+]i differ in mouse and rabbit ventricular myocytes

FK506 binding proteins (FKBPs 12 and 12.6) interact with ryanodine receptor (RyR) and modulate its functions. FK506 binds to and reverses effects of FKBP on RyR, thus increasing RyR sensitivity to Ca2+, decreasing RyR cooperativity, and increasing RyR open probability. FK506 would thus be expected to have an effect on excitation-contraction coupling, but which of these FK506 effects predominates and how the [Ca2+]i transient would be altered are difficult to predict. FK506 has been reported to increase the [Ca2+]i transient in rat myocytes, but effects in other species have not been described. We compared the effects of FK506 on [Ca2+]i transients, L-type Ca2+ channel and Na/Ca exchange currents, membrane potential, and sarcoplasmic reticulum (SR) Ca2+ content in adult mouse and rabbit ventricular myocytes (VM). FK506 (10 microM) increased the [Ca2+]i transient in mouse VM (656 +/- 116 to 945 +/- 144 nM, p < 0.001) but decreased the amplitude of [Ca2+]i transients in rabbit VM (627 +/- 61 to 401 +/- 37 nM, p < 0.001). Similar effects were observed with rapamycin. The effects of FK506 and rapamycin on [Ca2+]i transients in VM of both species were reversible upon washout. FK506 did not alter SR Ca2+ content in mouse VM (0.79 +/- 0.1 versus 0.78 +/- 0.1 pC/pF) but reduced the SR Ca2+ content in rabbit VM (0.43 +/- 0.05 versus 0.30 +/- 0.04 pC/pF, P < 0.05) [pC = the integral (pA. s) of the caffeine-induced inward I(Na/Ca) normalized by cell capacitance (pF)]. FK506 had no effects on membrane potential, I(Ca,L) and outward I(Na/Ca) in either mouse or rabbit VM. These results indicate that alteration of the functions of RyR by FK506-mediated dissociation of FKBP from RyR has different species-dependent effects on SR Ca2+ load and thus [Ca2+]i transients. This difference may result from the fact that [Na+]i is low in rabbit myocytes, allowing extrusion by Na+/Ca2+ exchange of Ca2+ released by FK506-induced dissociation of FKBP12.6 from SR RyR.     

Intracellular pH modulates the generation of superoxide radicals by human neutrophils.

The relationship of intracellular pH (pHi) to superoxide radical (O2-) generation was investigated in chemotactic factor-stimulated human neutrophils. Exposure of cells to 100 nM N-formylmethionyl-leucyl-phenylalanine (FMLP) caused activation of Na/H exchange which, in 140 mM Na medium (pH0 7.40), led to a rise in pHi from 7.22 to 7.80. This pHi change was sensitive to amiloride (apparent Ki 78 microM), an inhibitor of Na/H countertransport. The time course of the alkalinization was similar to that of FMLP-stimulated O2- production, which was complete by 5 min. In the presence of 1 mM amiloride, which nearly blocked the pHi transient elicited by FMLP, or in the absence of external Na, where intracellular acidification was observed in FMLP-stimulated cells, O2- release was still roughly 25-45% of normal. Thus, an alkalinization cannot be an obligatory requirement for O2- generation. By independently varying either pH0, pHi, or the internal or external concentrations of Na, both the direction and magnitude of the FMLP-induced pHi transients could be altered. In each instance, the amount of O2- release correlated directly with pHi and was enhanced by intracellular alkalinization. In the absence of FMLP, a rise in pHi to 7.7-7.8 by exposure of cells to 30 mM NH4Cl, 10 microM monensin (a Na/H exchanging ionophore), or after a prepulse with 18% CO2 did not result in O2- generation. Thus, these results imply that an alkalinization per se is not a sufficient trigger. Neutrophils exposed to 4 nM FMLP exhibited a threefold slower rate of alkalinization (reaching pHi approximately 7.80 by 20-30 min) as compared to that obtained with 100 nM FMLP and did not release significant amounts of O2- under normal incubation conditions. However, these cells could be induced to generate O2- when the degree of alkalinization was enhanced by internal Na depletion or by pretreatment with 18% CO2. Together, these results indicate a modulating effect of pHi on O2- production and suggest that other functional responses of neutrophils may be regulated by their pHi.     

Phospholipase C activation by Na+/Ca2+ exchange is essential for monensin-induced Ca2+ influx and arachidonic acid release in FRTL-5 thyroid cells.

BACKGROUND: Monensin, a Na+ ionophore, can increase cytosolic Ca2+ ([Ca2+]i) by reversing the Na+/Ca2+ exchange mechanism. This study provided additional insights into the mechanism of this Na+ ionophore-induced increase in [Ca2+]i, and emphasized the critical role of phospholipase C (PLC) in amplifying Na+/Ca2+ exchange-induced Ca2+ influx and subsequent arachidonic acid (AA) release in FRTL-5 thyroid cells. The possible involvement of protein kinase C (PKC), mitogen-activated protein kinase (MAPK), and GTP-binding (G) protein in mediating monensin-induced AA release was also explored. METHODS: FRTL-5 thyroid cells were maintained in Coon's modified Ham's F-12 medium supplemented with a 6-hormone (6H) mixture. Cytosolic Ca2+ was measured by using indo-1 AM and a dual-wave-length spectrofluorometer. Release of 3H-labeled inositol trisphosphates and arachidonic acid were determined by a scintillation counter. RESULTS: In Hank's balanced salt solution with Ca2+ (HBSS+), monensin (100 mumol/L) induced a 2.3-fold sustained Ca2+ increase associated with IP3 generation and a 6-fold increase in AA release. Deletion of extracellular Ca2+, or replacement of Na+ by choline chloride in the medium, reduced the [Ca2+]i increase by 77% and completely prevented the monensin-induced rise in AA release. Similar inhibitory effects were observed in cells pretreated with a Na+ channel blocker, or Na+/Ca2+ exchange inhibitors. In HBSS without Ca2+ (HBSS-), monensin induced a 1-fold transient [Ca2+]i increase but did not increase the AA. This Ca2+ increase was not suppressed by U-73122, a PLC inhibitor. In HBSS+, U-73122 did not affect the monensin-induced initial transient peak increase of [Ca2+]i, but reduced the sustained second phase of [Ca2+]i from 400 nmol/L to 250 nmol/L, and completely blocked AA release. A phospholipase A2 (PLA2) inhibitor blocked the monensin-induced AA release without affecting the [Ca2+]i increase. Inhibition of PKC prevented 87% to 94% of the monesin-stimulated AA release. The monensin-induced AA release was also inhibited 94% by pertussis and 51% by a MAP kinase cascade inhibitor. CONCLUSIONS: The results suggest that monensin initiates an increase in [Ca2+]i via a Na+/Ca2+ exchange mechanism that triggers more pronounced and sustained [Ca2+]i increase via activation of PLC and Ca2+ influx. The PLC activation, followed by sustained Ca2+ influx and PKC activation, is a prerequisite for PLA2-mediated processes in monensin-challenged FRTL-5 thyroid cells.     

Endothelin receptor-coupling mechanisms in vascular smooth muscle: a role for protein kinase C

Endothelin (ET), a peptide that is released from cultured endothelial cells, is a potent vasoconstrictor that induces characteristically long-lasting contractions. We used the A10 vascular smooth muscle cell (VSMC) line to probe mechanisms underlying ET-induced contractions. Intracellular Ca2+ ([Ca2+]i) and pH were monitored in A10 monolayers using the fluorescent dyes Fura-2 and 2,7-bis-carboxyethyl-5,6-carboxyfluorescein, respectively. Synthetic porcine ET induced rapid and transient increases in [Ca2+]i (EC50 value, 0.75 nM; maximum, approximately 6-fold above basal). External Ca2+ removal did not block the ability of ET (0.5 or 50 nM) to increase initial [Ca2+]i, although [Ca2+]i returned to prestimulus levels faster as compared with that seen in the presence of external Ca2+. Total cell 45Ca2+ content decreased within 30 sec and remained below prestimulus values for at least 20 min (34 +/- 2% decrease after 5 min, n = 3) in ET-stimulated VSMC. ET stimulated a transient rise in inositol trisphosphate formation in [3H]myo-inositol labeled VSMC, peaking in 30 sec (62 +/- 20% increase, n = 3). In contrast, ET-stimulated diacylglycerol formation in [3H]arachidonic acid-labeled VSMC was sustained and biphasic, exhibiting two peaks at 15 sec (41 +/- 16% increase) and at 5 min (75 +/- 7% increase, n = 3). ET (50 nM) also induced an intracellular alkalinization of 0.17 +/- 0.02 (n = 10) pH units above basal.(ABSTRACT TRUNCATED AT 250 WORDS)     

Low density lipoprotein enhances the cellular action of arginine vasopressin in rat glomerular mesangial cells in culture.

The present study was undertaken to determine whether low density lipoprotein (LDL) modulates the cellular action of arginine vasopressin (AVP) in rat glomerular mesangial cells in culture. AVP increased cellular free calcium ([Ca2+]i) in a dose-dependent manner. When cells were preincubated for 24 h with 10 microgram/ml LDL, the 1 x 10(-7) M AVP-mobilized [Ca2+]i was 874 nM, a value significantly greater than that of 375 nM in the intact cells. AVP caused a biphasic change in cellular pH (pHi), namely, an early acidification followed by a sustained alkalinization, and the change in pHi produced by AVP was also enhanced by LDL. AVP stimulated a 2.2-fold increase in [3H]thymidine incorporation, an effect significantly greater in the presence of 10 micrograms/ml LDL. Furthermore, 1 x 10(-7) M AVP significantly activated mitogen-activated protein kinase from 14.0 to 24.5 pmol/mg protein. Such an activation was significantly enhanced by the LDL pretreatment. Both [3H]thymide incorporation and mitogen-activated protein kinase were not altered by 10 micrograms/ml LDL. [3H]AVP receptor binding was not affected by the LDL pretreatment. 1 x 10(-7) M AVP increased inositol trisphosphate production by 1.9-fold, an effect significantly greater in the presence of LDL. These results indicate that LDL enhances the cellular action of AVP and the AVP-stimulated cellular proliferation in glomerular mesangial cells. A site of action of LDL is the hydrolysis of phosphatidylinositol.     

Endothelial cell Ca2+ increases upon tumor cell contact and modulates cell-cell adhesion.

The signal transduction mechanisms involved in tumor cell adhesion to endothelial cells are still largely undefined. The effect of metastatic murine melanoma cell and human prostate carcinoma cell contact on cytosolic [Ca2+] of bovine artery endothelial cells was examined in indo-1-loaded endothelial cell monolayers. A rapid increase in endothelial cell [Ca2+] occurred on contact with tumor cells, but not on contact with 8-microns inert beads. A similar increase in endothelial cell [Ca2+] was observed with human neutrophils or monocyte-like lymphoma cells, but not with endothelial cells, red blood cells, and melanoma cell-conditioned medium. The increase in endothelial cell [Ca2+] was not inhibited by extracellular Ca2+ removal. In contrast, endothelial cell pretreatment with thapsigargin, which releases endoplasmic reticulum Ca2+ into the cytosol and depletes this Ca2+ store site, abolished the cytosolic [Ca2+] rise upon melanoma cell contact. Endothelial cell pretreatment with the membrane-permeant form of the Ca2+ chelator bis-(O-aminophenoxyl)ethane-N,N,N',N'-tetraacetic acid blocked the increase in cytosolic [Ca2+]. Under static and dynamic flow conditions (0.46 dyn/cm2) bis-(O-aminophenoxyl)ethane-N,N,N',N'-tetraacetic acid pretreatment of bovine pulmonary artery endothelial cell monolayers inhibited melanoma cell adhesion to the endothelial cells. Thus, tumor cell contact with endothelial cells induces a rapid Ca2+ release from endothelial intracellular stores, which has a functional role in enhancing cell-cell adhesion.     

Regulation of rabbit medullary collecting duct cell pH by basolateral Na+/H+ and Cl-/base exchange.

The collecting duct of the inner stripe outer medulla (OMCDi) is a major site of distal nephron acidification. Using the pH sensitive fluorescent dye 2'-7'-bis(carboxyethyl)-5,6,-carboxyfluorescein (BCECF) and quantitative spectrofluorometry to measure intracellular pH in isolated perfused OMCDi, we have characterized basolateral transport processes responsible for regulation of intracellular pH. Experiments suggesting the existence of basolateral Cl-/base exchange were performed. In HCO3- containing buffers, bath Cl- replacement resulted in reversible alkalinization of the OMCDi from 7.22 +/- 0.05 to 7.57 +/- 0.12. Similarly 0.1 mM bath 4',4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS) alkalinized the OMCDi from 7.14 +/- 0.09 to 7.34 +/- 0.09 and blocked further alkalinization by bath Cl- removal (delta = + 0.02 pH units). The concentration dependence kinetics of Cl-/base exchange revealed a K1/2 of 10 mM for external Cl- with a Vmax of 0.50 pH U/min. Experiments suggesting the existence of basolateral Na+/H+ exchange were also performed. Replacement of bath Na+ by tetramethylammonium resulted in reversible cell acidification (7.14 +/- 0.09 to 6.85 +/- 0.1). Tubules that were acidified by a brief exposure to NH4Cl displayed recovery of cell pH back to baseline at a rate that was highly dependent on bath Na+ concentration. Half maximal recovery rate was achieved at 7 mM bath Na+ and Vmax was 0.605 pH U/min. The Na+-dependent rate of cell pH recovery after acidification was blocked by 0.2 mM bath amiloride. These results suggest that intracellular pH in the OMCDi is regulated by parallel basolateral Na+/H+ exchange and Cl-/base exchange.     

Effect of anti-inflammatory analgesic drugs on the regulation of cytosolic pH by anion antiport

Four nonsteroidal anti-inflammatory analgesic drugs acetylsalicylic acid (aspirin), salicylic acid, indomethacin and piroxicam were tested for their action on chloride/bicarbonate exchange by anion antiport and on the regulation of cytosolic pH (pHi) in Vero cells. The Na+-independent Cl-/HCO3- exchange regulates pHi back to normal after alkalinization and is therefore in a state of high activity at alkaline pHi, whereas it is in a state of low activity when pHi is below neutrality. Preincubation with the drugs at low pH strongly increased the activity of the Na+-independent Cl-/HCO3- exchange, whereas at high pH the antiport was inhibited. The Na+-dependent Cl-/HCO3- exchange, which is most active at low pHi and normalize pHi after acidification, was inhibited after preincubation with the drugs. In cells that had been incubated with the drugs, pHi was 0.1 to 0.4 pH units lower than in cells that had been incubated in the absence of drugs. The prostaglandins E2, F2 and I2 had little effect on anion antiport, and did not counteract the effects of the anti-inflammatory drugs on Cl-/HCO3- exchange, indicating that these actions of the drugs are not due to the inhibition of prostaglandin synthesis. The relevance of our findings to the clinical effects of these drugs is discussed.     

Evidence for role of cytosolic free calcium in hypoxia-induced proximal tubule injury.

The role of cytosolic free Ca2+ ([Ca2+]i) in hypoxic injury was investigated in rat proximal tubules. [Ca2+]i was measured using fura-2 and cell injury was estimated with propidium iodide (PI) in individual tubules using video imaging fluorescence microscopy. [Ca2+]i increased from approximately 170 to approximately 390 nM during 5 min of hypoxia. This increase preceded detectable cell injury as assessed by PI and was reversible with reoxygenation. 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA; 100 microM) reduced [Ca2+]i under basal conditions (approximately 80 nM) and during hypoxia (approximately 120 nM) and significantly attenuated hypoxic injury. When [Ca2+]i and hypoxic cell injury were studied concurrently in the same individual tubules, the 10 min [Ca2+]i rise correlated significantly with subsequent cell damage observed at 20 min. 2 mM glycine did not block the rise in [Ca2+]i, yet protected the tubules from hypoxic injury. These results indicate that in rat proximal tubules, hypoxia induces an increase of [Ca2+]i which occurs before cell damage. The protective effect of BAPTA supports a role for [Ca2+]i in the initiation of hypoxic proximal tubule injury. The glycine results, however, implicate calcium-independent mechanisms of injury and/or blockade of calcium-mediated processes of injury such as activation of phospholipases or proteases.     

Feedback inhibition of cyclic adenosine monophosphate-stimulated Na+ transport in the rabbit cortical collecting duct via Na(+)-dependent basolateral Ca++ entry.

Arginine vasopressin (AVP) transiently stimulates Na+ transport in the rabbit cortical collecting duct (CCD). However, the sustained effect of both AVP and its putative second messenger, cyclic adenosine monophosphate (cAMP), on Na+ transport in the rabbit CCD is inhibitory. Because maneuvers that increase [Ca++]i inhibit Na+ transport, the effects of AVP and cell-permeable cAMP analogues, on [Ca++]i were investigated in fura-2-loaded in vitro microperfused rabbit CCDs. Low-dose AVP (23-230 pM) selectively stimulated Ca++ influx, whereas 23 nM AVP additionally released calcium from intracellular stores. 8-chlorophenylthio-cAMP (8CPTcAMP) and 8-bromo-cAMP (8-Br-cAMP) also increased CCD [Ca++]i. The 8CPTcAMP-stimulated [Ca++]i increase was totally dependent on basolateral [Ca++]. In the absence of cAMP, peritubular Na+ removal produced a marked increase in [Ca++]i, which was also dependent on bath [Ca++], suggesting the existence of basolateral Na+/Ca++ exchange. Luminal Na+ removal in the absence of cAMP did not alter CCD [Ca++]i, but it completely blocked the cAMP-stimulated [Ca++]i increase. Thus the cAMP-dependent Ca++ increase is totally dependent on both luminal Na+ and basolateral Ca++, suggesting the [Ca++]i increase is secondary to cAMP effects on luminal Na+ entry and its coupling to basolateral Na+/Ca++ exchange. 8CPTcAMP inhibits lumen-to-bath 22Na flux [JNa(l-b)] in CCDs bathed in a normal Ca++ bath (2.4 mM). However, when bath Ca++ was lowered to 100 nM, a maneuver that also blocks the 8CPTcAMP [Ca++]i increase, 8CPTcAMP stimulated, rather than inhibited JNa(l-b). These results suggest that cAMP formation initially stimulates CCD Na+ transport, and that increased apical Na+ entry secondarily activates basolateral Ca++ entry. The cAMP-dependent [Ca++]i increase leads to inhibition Na+ transport in the rabbit CCD.     

Cytoplasmic pH regulation in monocytes and macrophages: mechanisms and functional implications.

Maintenance of cytoplasmic pH (pHi) within a narrow physiological range is critical to optimal cell function. Monocytes and macrophages (M?s) actively regulate their pHi through three distinct plasma membrane ion transport systems: (1) Na+/H+ exchange; (2) Na(+)-dependent anion exchange; and (3) vacuolar-type H+ ATPases. Alterations in the functional state of monocytes and M?s have been linked to changes in pHi and/or its regulation by these ion transport systems. Differentiation, proliferation, and activation of M?s in response to a variety of agents are associated with increased Na+/H+ exchange. The resultant cytoplasmic alkalinization typically observed in HCO3(-)-free media likely plays a permissive, rather than a triggering, role in mediating M? response to most of these agents. Prevention of cytoplasmic acidification is essential during M? activation, when production of metabolic acid increases. This is of particular importance within the in vivo microenvironment of an abscess or tumour, where pHi is further threatened by the low extracellular pH (pHo) which typically prevails. At low pHo, H+ ATPase-mediated H+ extrusion plays a critical role in maintenance of pHi, preserving the ability of M?s to generate a respiratory burst. The requirement for maintenance of pHi within a range conducive to efficient M? function may explain why M?s have acquired a variety of parallel systems for pHi regulation.     

Intracellular Mg2+ and magnesium depletion in isolated renal thick ascending limb cells.

Magnesium reabsorption and regulation within the kidney occur principally within the cortical thick ascending limb (cTAL) cells of the loop of Henle. Fluorometry with the dye, mag-fura-2, was used to characterize intracellular Mg2+ concentration ([Mg2+]i) in single cTAL cells. Primary cell cultures were prepared from porcine kidneys using a double antibody technique (goat anti-human Tamm-Horsfall and rabbit anti-goat IgG antibodies). Basal [Mg2+]i was 0.52 +/- 0.02 mM, which was approximately 2% of the total cellular Mg. Cells cultured (16 h) in high magnesium media (5 mM) maintained basal [Mg2+]i, 0.48 +/- 0.02, in the normal range. However, cells cultured in nominally magnesium-free media possessed [Mg2+]i, 0.27 +/- 0.01 mM, which was associated with a significant increase in net Mg transport, (control, 0.19 +/- 0.03 and low Mg, 0.35 +/- 0.01 nmol.mg-1 protein.min-1) as assessed by 28Mg uptake. Mg(2+)-depleted cells were subsequently placed in high Mg solution (5 mM) and the Mg2+ refill rate was assessed by fluorescence. [Mg2+]i returned to normal basal levels, 0.53 +/- 0.03 mM, with a refill rate of 257 +/- 37 nM/s. Mg2+ entry was not changed by 5.0 mM Ca2+ or 2 mM Sr2+, Cd2+, Co2+, nor Ba2+ but was inhibited by Mn2+ approximately La3+ approximately Gd3+ approximately Zn2+ approximately Be2+ at 2 mM. Intracellular Ca2+ and 45Ca uptake was not altered by Mg depletion or Mg2+ refill, indicating that the entry is relatively specific to Mg2+. Mg2+ uptake was inhibited by nifedipine (117 +/- 20 nM/s), verapamil (165 +/- 34 nM/s), and diltiazem (194 +/- 19 nM/s) but enhanced by the dihydropyridine analogue, Bay K 8644 (366 +/- 71 nM/s). These antagonists and agonists were reversible with removal and [Mg2+]i subsequently returned to normal basal levels. Mg2+ entry rate was concentration and voltage dependent and maximally stimulated after 4 h in magnesium-free media. Cellular magnesium depletion results in increases in a Mg2+ refill rate which is dependent, in part, on de novo protein synthesis. These data provide evidence for novel Mg2+ entry pathways in cTAL cells which are specific for Mg2+ and highly regulated. These entry pathways are likely involved with renal Mg2+ homeostasis.     

Intracellular pH regulation in rabbit renal medullary collecting duct cells. Role of chloride-bicarbonate exchange.

The renal medullary collecting duct (MCD) secretes protons into its lumen and HCO3 into its basolateral space. Basolateral HCO3 transport is thought to occur via Cl/HCO3 exchange. To further characterize this Cl/HCO3 exchange process, intracellular pH (pHi) regulation was monitored in freshly prepared rabbit outer MCD cells. Cells were separated by protease digestion and purified by Ficoll gradient centrifugation. pHi was estimated fluorometrically using the entrapped intracytoplasmic pH indicator, 6-carboxyfluorescein. Cells were preincubated in bicarbonate-containing solutions and then abruptly diluted into bicarbonate-free media. The MCD cell pHi response to abrupt removal of CO2/HCO3 included an initial alkalinization due to rapid CO2 efflux, followed by an acidification due to HCO3 efflux and a gradual recovery to the resting pHi of 7.24 +/- 0.06 partly due to the action of a plasma membrane H+-ATPase. The initial alkalinization required a CO2/HCO3 gradient and did not occur in the presence of acetazolamide. The acidification phase required intracellular HCO3 and extracellular Cl, which was consistent with a Cl/HCO3 exchange. MCD HCO3 efflux exhibited saturable kinetics for extracellular Cl, with a Michaelis constant (Km) of 29.9 +/- 7.7 mM. HCO3 efflux also exhibited preference for halides over NO3, SCN, and gluconate, and striking sensitivity to disulfonic stilbene and acetazolamide inhibition, with an apparent K1 of 5 X 10(-7) M for DIDS. The final pHi recovery required intracellular ATP, which indicated that Cl/HCO3 and H+-ATPase activities are present in the same cells in these suspensions. The results provide direct evidence for MCD Cl/HCO3 exchange and describe some of the properties of this transport process.     

Effect of secretion on intracellular pH regulation in isolated rat bile duct epithelial cells.

The effects of secretin on ion transport mechanisms involved in regulation of intracellular pH (pHi) and HCO3- excretion were characterized in bile duct epithelial (BDE) cells isolated from normal rat liver. pHi was measured with 2,7-bis(carboxy-ethyl)-5(6)-carboxy-fluorescein-acetomethylester (BCECF-AM) using a microfluorimetric method. Basal pHi of BDE was 7.04 +/- 0.06 in Hepes and 7.16 +/- 0.10 in KRB and was unaffected by secretin (50-200 nM). Recovery rates from an acid load in Hepes or in KRB media (with and without amiloride) were also not altered by secretin, indicating that Na+/H+ exchange and Na+/HCO3- cotransport were not affected by this hormone. After acute Cl- removal, pHi rose 0.24 +/- 0.08 pHU at a maximal rate of 0.125 +/- 0.06 pHU/min (H+ flux rates = 6.02 +/- 3.27 mM/min) and recovered after Cl- readmission (0.188 +/- 0.08 pHU/min; H+ flux rates = 11.82 +/- 5.34 mM/min). Pretreatment with 1 mM DIDS inhibited the effects of Cl- removal, while valinomycin, which induces cell depolarization, enhanced these effects, probably by stimulating electrogenic HCO3- influx. Secretin significantly increased both the maximal rate of alkalinization after Cl- removal (P < 0.012) and of pHi recovery after Cl- readmission (P < 0.025), indicating stimulation of Cl-/HCO3- exchange activity. These findings were reproduced with N6,2'-O-Dibutyryladenosine-3',5'-cyclic monophosphate (DBcAMP). The Cl- channel blocker 5-nitro-2'-(3-phenylpropylamino)-benzoate (NPPB, 10 microM) significantly decreased the effects of secretin and DBcAMP on the pHi changes promoted by acute Cl- removal/readmission. These findings establish that secretin stimulates the activity of the Cl-/HCO3- exchanger in BDE cells, probably by activating Cl- channels via the intracellular messenger cAMP. This in turn depolarizes the cell, stimulating electrogenic Na+/HCO3- symport. The cell depolarization induced by Cl- channel activation should enhance HCO3- entrance through electrogenic Na+/HCO3- symport, which in turn stimulates the Cl-/HCO3- exchange. These mechanisms could account for secretin stimulated bicarbonate secretion in bile.