Mechanisms underlying ATP-induced Ca2+ mobilization in human neutrophils

The effect of ATP on Ca2+ mobilization in human neutrophils was examined by using fura-2 as a Ca2+ indicator. ATP (0.1-100 microM) caused a significant [Ca2+]i increase in a concentration-dependent manner. The [Ca2+]i signal comprised an initial rise followed by a plateau. Removal of external Ca2+ diminished the peak value of the [Ca2+]i signal. In Ca2+-free medium, pretreatment with an endoplasmic reticulum Ca2+ pump inhibitor, thapsigargin, prevented ATP from releasing Ca2+. In contrast, thapsigargin still increased [Ca2+], after pretreatment with 10 microM ATP. These results indicate that 10 microM ATP released Ca2+ mainly from thapsigargin-sensitive stores. Adding 3 mM Ca2+ induced a concentration-dependent increase in [Ca2+]i after pretreatment with ATP or thapsigargin in Ca2+-free medium, suggesting ATP induced Ca2+ influx via capacitative Ca2+ entry. ATP (10 microM)-induced Ca2+ release was abolished by inhibiting phospholipase C with 2 microM U73122, indicating that inositol-1,4,5-trisphosphate (IP3) mediates ATP-induced Ca2+ release. Conversely, ATP-induced [Ca2+]i increase was abolished by activating protein kinase C (PKC) with 10 nM phorbol myristate acetate (PMA), but was not altered by inhibiting PKC with 2 microM GF 109203X. This implies ATP-induced [Ca2+]i increase is a PMA-linked event. Together, the results suggest ATP increases [Ca2+]i in human neutrophils by releasing Ca2+ from IP3-coupled, thapsigargin-sensitive Ca2+ stores, and inducing Ca2+ influx via the process of capacitative Ca2+ entry. The ATP-induced Ca2+ signal is a PMA-linked event.     

Release of Ca2+ from the endoplasmic reticulum is not the mechanism for bile acid-induced cholestasis and hepatotoxicity in the intact rat liver.

The hypothesis that monohydroxy bile acids exert their cholestatic and hepatotoxic effects via a sustained elevation of cytosolic [Ca2+] was tested in the isolated perfused rat liver. Infusion of the specific inhibitor of microsomal Ca2+ sequestration, 2,5-di(tert-butyl)-1,4-benzohydroquinone (tBuBHQ) (25 microM for 10 min) produced efflux of Ca2+ from the liver and a sustained (20 min) increase in cytosolic [Ca2+] as indicated by the threefold increase in hepatic glucose output. Release of the endoplasmic reticular Ca2+ pool was demonstrated by the complete abolition of vasopressin- and phenylephrine-induced Ca2+ exchange between the liver and perfusate. Despite the profound perturbation of intracellular Ca2+ homeostasis produced by tBuBHQ, there was no decrease in bile flow and no evidence of hepatocellular injury (for 60 min), as indicated by lactate dehydrogenase release. In contrast, lithocholic acid (25 microM for 10 or 30 min) or taurolithocholic acid (5 microM for 10 or 30 min) produced an 80-90% inhibition of bile flow and a progressive increase in perfusate lactate dehydrogenase activity. During and after bile acid infusion, there was no change in Ca2+ fluxes between liver and perfusate, no stimulation of glucose output from the liver, and hormone-stimulated Ca2+ responses were preserved. It is concluded that the mechanisms for bile acid-induced cholestasis and hepatotoxicity in the intact liver are not attributable to changes in intracellular Ca2+ homeostasis, and especially not to prolonged release or depletion of Ca2+ sequestered in the endoplasmic reticulum.     

Inhibition of apical Na+ channels in rabbit cortical collecting tubules by basolateral prostaglandin E2 is modulated by protein kinase C.

We used the cell-attached patch clamp technique to investigate the interaction of exogenous prostaglandins (PG), intracellular [Ca2+]i, and protein kinase C (PKC) on the high selectivity, 4 pS Na+ channel found in the principal cell apical membrane of rabbit cortical collecting tubule (CCT) cultures grown on collagen supports with 1.5 microM aldosterone. Application of 0.5 microM PGE2 to the basolateral membrane decreased mean NP0 (number of channels times the open probability) for apical Na+ channels by 46.5% (n = 9). There was no consistent change in NP0 after apical 0.5 microM PGE2 (n = 12) or after apical or basolateral 0.5 microM PGF2 alpha (n = 8). Release of [Ca2+]i stores with 0.25 microM thapsigargin (n = 7), or activation of apical membrane PKC with apical 0.1 microM 4 beta-phorbol-12-myristate-13-acetate (n = 5) or 10 microM 1-oleyl-2-acetylglycerol (n = 4) also decreased NP0. Depletion of [Ca2+]i stores (0.25 microM thapsigargin pretreatment) (n = 7) or inhibition of apical PKC (100 microM D-sphingosine pretreatment) (n = 8) abolished the inhibitory effects of basolateral PGE2. Conclusions: (a) apical Na+ transport in rabbit CCT principal cells is modulated by basolateral PGE2; (b) the mechanism involves release of IP3-sensitive, [Ca2+]i stores; and (c) Ca(2+)-dependent activation of apical membrane PKC, which then inhibits apical Na+ channels.     

Mechanism of chloride secretion induced by carbachol in a colonic epithelial cell line.

Serosal application of carbachol to T84 cell monolayers mounted in an Ussing chamber caused an immediate increase in short circuit current (Isc) that peaked within 5 min and declined rapidly thereafter, although a small increase in Isc persisted for approximately 30 min. The increase in Isc was detectable with 1 microM carbachol; half-maximal with 10 microM carbachol; and maximal with 100 microM carbachol. Unidirectional Na+ and Cl- flux measurements indicated that the increase in Isc was due to net Cl- secretion. Carbachol did not alter cellular cAMP, but caused a transient increase in free cytosolic Ca2+ ([Ca2+]i) from 117 +/- 7 nM to 160 +/- 15 nM. The carbachol-induced increase in Isc was potentiated by either prostaglandin E1 (PGE1) or vasoactive intestinal polypeptide (VIP), agents that act by increasing cAMP. Measurements of cAMP and [Ca2+]i indicated that the potentiated response was not due to changes in these second messengers. Studies of the effects of these agents on ion transport pathways indicated that carbachol, PGE1, or VIP each increased basolateral K+ efflux by activating two different K+ transport pathways on the basolateral membrane. The pathway activated by carbachol was not sensitive to barium, while that activated by PGE1 or VIP was; furthermore, their action on K+ efflux are additive. Our study indicates that carbachol causes Cl- secretion, and that this action may result from its ability to increase [Ca2+]i and basolateral K+ efflux. Carbachol's effect on Cl- secretion is greatly augmented in the presence of VIP or PGE1, which open a cAMP-sensitive Cl- channel on the apical membrane, accounting for a potentiated response.     

Mechanism of norepinephrine release elicited by Na+-K+ adenosine triphosphatase inhibition in the isolated rat kidney: involvement of voltage-dependent Ca++ channels

The mechanism by which ouabain and Na+ depletion enhance the release of norepinephrine (NE) was investigated in the isolated rat kidney prelabeled with [3H]NE by examining the efflux of tritium elicited by these stimuli during 1) Ca++ depletion and 2) administration of tetrodotoxin, amiloride and Ca++ channel blockers. In kidneys perfused with Tyrode's solution containing low K+ solution (0.54 mM), ouabain (10(-4) M) enhanced tritium efflux markedly by about 20-fold at 30 min. Depletion of Na+ from the perfusion medium also produced an increase in tritium overflow which peaked at 20 min. Administration of tetrodotoxin (0.3 microM) inhibited the effect of ouabain, but not that of Na+ depletion, to increase tritium efflux and perfusion pressure. In contrast, amiloride (180 microM) enhanced the overflow of tritium elicited by ouabain but failed to alter that elicited by Na+ depletion. The rise in perfusion pressure caused by both stimuli was attenuated by amiloride. Omission of Ca++ (1.8 mM) from the perfusion medium inhibited the increase in tritium efflux and perfusion pressure elicited by ouabain and Na+ depletion by 80 and 65%, respectively. The Ca++ channel blockers omega-conotoxin (50 nM), diltiazem (60 microM) and flunarizine (2 microM), but not nifedipine (1.4 microM), inhibited tritium overflow elicited by ouabain. However, nifedipine, diltiazem and flunarizine, but not omega-conotoxin attenuated the tritium overflow elicited by Na+ depletion. The rise in perfusion pressure elicited by ouabain in low K+ and Na+ depletion was inhibited by these Ca++ channel blockers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relationship between calcium loading and impaired energy metabolism during Na+, K+ pump inhibition and metabolic inhibition in cultured neonatal rat cardiac myocytes.

This study tested the hypothesis that the initiating mechanism is a major determinant of the response to calcium (Ca) accumulation in myocardium. Cultured neonatal rat ventriculocytes were exposed to Na+, K+ pump inhibition with 1 mM ouabain and metabolic inhibition with 20 mM 2-deoxy-D-glucose and 1 mM cyanide (DOG-CN) for up to 2 h. Microspectrofluorometry of myocytes loaded with fura-2 showed that ouabain resulted in a relatively rapid increase in [Ca2+]i up to 2-3 microM (two to threefold above peak systolic level) and that DOG-CN produced an initial decrease and then a relatively slow increase in [Ca2+]i up to peak systolic level. Electron probe x-ray microanalysis (EPMA) showed prominent increases in Na and Ca and decreases in K and Mg in cytoplasm and mitochondria with both interventions, although the increases in Ca were greater with ouabain than DOG-CN. ATP was reduced by 58% after 1 and 2 h of ouabain and by 70 and 90% after 1 and 2 h of DOG-CN, respectively. Thus, ouabain produced greater calcium accumulation and less ATP reduction than DOG-CN. Upon return to normal medium for 30 min, myocytes showed recovery of most electrolyte alterations and resumption of normal Ca2+ transients after 1 h exposure to either ouabain or DOG-CN; however, recovery was less after 2 h of either treatment, with elevated [Ca2+]i maintained in many myocytes. We conclude that the severity of myocyte injury is influenced by the magnitude and duration of both ATP reduction and calcium accumulation.     

Alterations in cation homeostasis in cultured chick ventricular cells during and after recovery from adenosine triphosphate depletion.

Alterations in cation homeostasis during and after recovery from myocardial ischemia may account for some of the reversible and irreversible components of myocardial cell injury. To investigate possible mechanisms involved, we exposed cultured layers of spontaneously contracting chick embryo ventricular cells to media containing 1 mM cyanide (CN) and 20 mM 2-deoxyglucose (2-DG), and zero glucose for up to 6 h, and then allowed cultured cells to recover in serum-free culture medium for 24 h. Changes in Na, K, and Ca contents, 42K uptake and efflux, ATP content, cell water content, and lactate dehydrogenase (LDH) release were measured, and compared with changes produced by exposure to 10(-3) M ouabain and severe hypoxia. Exposure to CN and 2-DG caused marked increase in cell Na (sevenfold) and Ca (fivefold) contents, and a decrease in K content (one-fifth normal), coincident with ATP depletion to one-tenth normal levels. This produced only slight cell injury, evidenced by increased LDH release. Recovery for 24 h resulted in return to near normal values (expressed in nanomoles per milligram of protein) of Na, Ca, and ATP contents. However, there was failure of cell K content to return to normal, associated with a persistent reduced net uptake of 42K, and an increase in the rate of 42K efflux. These abnormalities in K homeostasis were associated with a decrease in cell volume and water content per milligram of protein. More marked ATP depletion (to 1/100 normal values) was produced by hypoxia plus 2-DG and zero glucose, and was associated with much more severe cell injury manifested by LDH loss. Ouabain exposure resulted in a much greater Ca gain (20-30-fold), relative to increase in Na content, than did either CN and 2-DG or hypoxia; and ouabain effects were not reversible (after a 15-fold or greater increase in Ca content was produced) and were associated with significant LDH release. We conclude that these cells are resistant to cell injury caused by moderately severe Ca overload and ATP depletion produced by exposure to CN and 2-DG. However, metabolic inhibition of ATP production produces persistent abnormalities in K homeostasis, associated with functional abnormalities.     

Calcium- and CaMKII-dependent chloride secretion induced by the microsomal Ca(2+)-ATPase inhibitor 2,5-di-(tert-butyl)-1,4-hydroquinone in cystic fibrosis pancreatic epithelial cells.

Microsomal Ca(2+)-ATPase inhibitors such as thapsigargin (THG), cyclopiazonic acid (CPA) and 2,5-di-(tert-butyl)-1,4-hydroquinone (DBHQ) have been shown to inhibit Ca2+ reuptake by the intracellular stores and increase cytosolic free Ca2+ ([Ca2+]i). DBHQ is a commercially available non-toxic synthetic compound chemically unrelated to THG and CPA. In this study, we tested the feasibility of utilizing DBHQ to improve Cl- secretion via the Ca(2+)-dependent pathway, in the cystic fibrosis (CF)-derived pancreatic epithelial cell line CFPAC-1. DBHQ stimulated 125I efflux and mobilized intracellular free Ca2+ in a dose-dependent manner. The maximal effects were seen at concentrations of 25-50 microM. DBHQ (25 microM) caused a short-term rise in [Ca2+]i in the absence of ambient Ca2+, and a sustained elevation of [Ca2+]i in cell monolayers bathed in the efflux solution (1.2 mM Ca2+), which was largely attenuated by Ni2+ (5 mM). Bath-application of DBHQ induced an outwardly-rectifying whole-cell Cl- current, which was abolished by pipette addition of BAPTA (5 mM) or CaMK [273-302] (20 microM), an inhibitory peptide of multifunctional Ca2+/calmodulin-dependent protein kinase (CaMKII). Pretreatment of monolayers of CFPAC-1 cells with DBHQ for 4-5 min significantly increased the Ca(2+)-independent or autonomous activity of CaMKII assayed in the cell homogenates. Thus, DBHQ appears to enhance Cl- channel activity via a Ca(2+)-dependent mechanism involving CaMKII. Pretreatment of CFPAC-1 cells with up to 50 microM DBHQ for 6 h did not cause any detectable change in cell viability and did not significantly affect the cell proliferation rate. These results suggest that appropriate selective microsomal Ca(2+)-ATPase inhibitors may be therapeutically useful in improving Cl- secretion in CF epithelial cells.     

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.     

Extracellular adenosine triphosphate activates calcium mobilization in human phagocytic leukocytes and neutrophil/monocyte progenitor cells.

We have examined the ability of extracellular ATP to elicit intracellular Ca2+ mobilization in a broad range of human leukocytes at particular stages of hematopoietic differentiation. The average cytosolic [Ca2+] in various leukocyte populations was measured in Fura 2-loaded cell suspensions while the cytosolic [Ca2+] in individual, Indo 1-loaded leukocytes was assayed by flow cytometric methods. Utilizing normal blood- and marrow-derived cells, human leukemic cell lines, and mononuclear cell fractions derived from the blood of patients with various leukemias, we have found that ATP-induced Ca2+ mobilization appears restricted to leukocytes of neutrophil/monocyte ontogeny. Significant ATP-induced increases in cytosolic [Ca2+] were observed in neutrophils, monocytes, and myeloid progenitor cells as immature as myeloblasts, but not in lymphocytes. Extensive characterization of the ATP-induced changes in [Ca2+] observed in the HL-60 promyelocytic cell line have indicated these Ca2+-mobilizing effects of ATP can be correlated with an activation of inositol phospholipid breakdown via the occupation of P2-purinergic receptors Significantly, of the various agonists (FMLP, platelet-activating factor, LTB4, and ATP) which elicit equivalent and maximal Ca2+ mobilization in mature neutrophils and monocytes, ATP was the most efficacious stimulant of Ca2+ mobilization in immature neutrophil/monocyte precursors. Thus, expression of putative P2-purinergic receptors for ATP appears to precede expression of other receptor types known to activate the inositol phospholipid signaling cascades in terminally differentiated phagocytes.     

Glycochenodeoxycholate-induced lethal hepatocellular injury in rat hepatocytes. Role of ATP depletion and cytosolic free calcium.

Chenodeoxycholate is toxic to hepatocytes, and accumulation of chenodeoxycholate in the liver during cholestasis may potentiate hepatocellular injury. However, the mechanism of hepatocellular injury by chenodeoxycholate remains obscure. Our aim was to determine the mechanism of cytotoxicity by chenodeoxycholate in rat hepatocytes. At a concentration of 250 microM, glycochenodeoxycholate was more toxic than either chenodeoxycholate or taurochenodeoxycholate. Cellular ATP was 86% depleted within 30 min after addition of glycochenodeoxycholate. Fructose, a glycolytic substrate, maintained ATP concentrations at 50% of the initial value and protected against glycochenodeoxycholate cytotoxicity. ATP depletion in the absence of a glycolytic substrate suggested impairment of mitochondrial function. Indeed, glycochenodeoxycholate inhibited state 3 respiration in digitonin-permeabilized cells in a dose-dependent manner. After ATP depletion, a sustained rise in cytosolic free calcium (Cai2+) was observed. Removal of extracellular Ca2+ abolished the rise in Cai2+, decreased cellular proteolysis, and protected against cell killing by glycochenodeoxycholate. The results suggest that glycochenodeoxycholate cytotoxicity results from ATP depletion followed by a subsequent rise in Cai2+. The rise in Cai2+ leads to an increase in calcium-dependent degradative proteolysis and, ultimately, cell death. We conclude that glycochenodeoxycholate causes a bioenergetic form of lethal cell injury dependent on ATP depletion analogous to the lethal cell injury of anoxia.     

Involvement of Na+-Ca2+ exchanger in intracellular Ca2+ increase and neuronal injury induced by polychlorinated biphenyls in human neuroblastoma SH-SY5Y cells

In SH-SY5Y, a human neuroblastoma cell line, Aroclor 1254 (A1254), induced a dose-dependent (10-50 microg/ml) intracellular calcium concentration ([Ca2+]i) increase. Two rather specific sodium-calcium (Na+-Ca2+) exchanger (NCX) inhibitors, bepridil (10 microM) and KB-R7943 [2-[2-[4-(4-nitrobenzyloxy) phenyl]ethyl]isothiourea methanesulfonate] (10 microM), reduced A1254-induced [Ca2+]i increase. A 24-h exposure to 30 microg/ml A1254 caused remarkable SH-SY5Y neuroblastoma cell damage. It is noteworthy that both bepridil and KB-R7943 counteracted A1254-induced neuronal injury. These results indicate that NCX contributes to [Ca2+]i increase and neuronal injury induced by A1254. RT-PCR experiments revealed in SH-SY5Y neuroblastoma cells the expression of NCX1 and NCX3 isoforms. To investigate which isoform was involved in [Ca2+]i increase and neuronal damage induced by A1254, we used specific antisense oligodeoxynucleotides (ODNs) to reduce NCX1 or NCX3 protein expression. The results showed that only NCX1 ODN reduced [Ca2+]i increase and neuronal injury induced by A1254. In conclusion, these results indicate that NCX1 may participate to [Ca2+]i increase and neurotoxicity evoked by A1254 in SH-SY5Y neuroblastoma cells.     

Glucose-stimulated insulin secretion correlates with changes in mitochondrial and cytosolic Ca2+ in aequorin-expressing INS-1 cells.

Nutrient-stimulated insulin secretion is dependent upon the generation of metabolic coupling factors in the mitochondria of the pancreatic B cell. To investigate the role of Ca2+ in mitochondrial function, insulin secretion from INS-1 cells stably expressing the Ca2+-sensitive photoprotein aequorin in the appropriate compartments was correlated with changes in cytosolic calcium ([Ca2+]c) and mitochondrial calcium ([Ca2+]m). Glucose and KCl, which depolarize the cell membrane, as well as the Ca2+-mobilizing agonist, carbachol (CCh), cause substantial increases in [Ca2+]m which are associated with smaller rises in [Ca2+]c. The L-type Ca2+-channel blocker, SR7037, abolished the effects of glucose and KCl while attenuating the CCh response. Glucose-induced increases in [Ca2+]m, [Ca2+]c, and insulin secretion all demonstrate a pronounced initial peak followed by a sustained plateau. All three parameters are increased synergistically when glucose and CCh are combined. Finally, [Ca2+]m, [Ca2+]c, and insulin secretion also display desensitization phenomena following repeated additions of the three stimuli. The high sensitivity of [Ca2+]m to Ca2+ influx and the desensitization-resensitization effects can be explained by a model in which the mitochondria of INS-1 cells are strategically located to sense Ca2+ influx through plasma membrane Ca2+ channels. In conclusion, the correlation of [Ca2+]m and [Ca2+]c with insulin secretion may indicate a fundamental role for Ca2+ in the adaptation of oxidative metabolism to the generation of metabolic coupling factors and the energy requirements of exocytosis.     

Differential regulation of Ca2+ influx by fMLP and PAF in human neutrophils: possible involvement of store-operated Ca2+ channel

Calcium (Ca2+) influx into human polymorphonuclear cells (PMNs) in response to N-formyl-Met-Leu-Phe (fMLP) and platelet-activating factor (PAF) stimulation was studied. Whole blood was taken by venous puncture from healthy human volunteers. PMNs were isolated, diluted, and incubated with 2 microM fura-2 AM. The cytosolic free calcium concentration, [Ca2+]i, in human neutrophils was determined by microfluorometry. We found that the net area under the fMLP- or PAF-induced [Ca2+]i rise curve in Ca2+-free medium decreased to 75% or 30% of the area under the curve in Ca2+ medium. Treatment of PMNs with phorbol myristate acetate (PMA), a protein kinase C activator, completely abolished the intracellular Ca2+ level stimulated by PAF, but not the intracellular Ca2+ level stimulated by fMLP. Treatment of PMNs with PAF did not abolish the intracellular Ca2+ level elevation stimulated by fMLP. In addition, treatment of PMNs with fMLP did not abolish intracellular Ca2+ level elevation stimulated by PAF. Loperamide, a positive modulator for store-operated calcium (SOC) channels, elicited an increase in intracellular calcium after the activation of SOC channels stimulated by fMLP or PAF. After the addition of guanosine 3',5'-cyclic monophosphate, N2,2'-O-Dibutyryl-, sodium salt (db-cGMP), the initial increase of PAF- or fMLP-induced PMNs intracellular Ca2+ fluorescences was well preserved, but the slope and the peak height of fluorescence curves declined compared with the curves without db-cGMP. In conclusion, we found that PAF and fMLP regulate the Ca2+ influx of PMNs in different ways. Most of the PAF-induced [Ca2+]i rise resulted from Ca2+ influx, and most of the fMLP-induced [Ca2+]i elevation resulted from intracellular stores release. The initial mobilization of intracellular Ca2+ stores in PAF-stimulated signals is mediated by protein kinase C (PKC) phosphorylation, but not in fMLP-stimulated route. SOC channels are present and important in the fMLP- or PAF-induced PMNs Ca2+ influx. There was no apparent cross-regulation between PAF- and fMLP-stimulated intracellular Ca2+ influx.     

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.     

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.     

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.     

Propafenone modulates potassium channel activities of vascular smooth muscle from rat portal veins

We have studied the effects of the class Ic antiarrhythmic propafenone on K+ currents in freshly isolated smooth muscle cells from rat portal veins and on the spontaneous contractions in whole tissues. Under Ca2+-free conditions, when cells were clamped at -80 mV (whole-cell configuration) depolarizing steps from -80 to +50 mV induced a family of K+ currents (I(Ktotal)) that mainly comprised the delayed rectifier current [I(K(V))], whereas when held at -10 mV only small-amplitude, noninactivating, currents (I(NI)) were recorded. Propafenone (10 microM) markedly inhibited I(Ktotal), but at potentials positive to +30 mV it also induced a noisy outwardly rectifying current [I(BK(Ca))] that was abolished by iberiotoxin (0.1 microM). Inhibition of I(Ktotal) by propafenone was concentration-dependent (EC50 = 0.059 +/- 0.009 microM). Propafenone also inhibited the transient outward current [I(K(A))] and ATP-sensitive potassium current [I(K(ATP))] induced by levcromakalim (10 microM). Inhibition of I(K(V)), I(K(A)), and I(K(ATP)) by propafenone was voltage-independent. In Ca(2+)-containing conditions propafenone inhibited I(K(V)) and I(BK(Ca)) and immediately abolished spontaneous outward transient K+ currents. In whole veins, propafenone behaved as the K(V) inhibitor 4-aminopyridine, increasing the amplitude and duration of spontaneous contractions. Propafenone also inhibited the inhibitory effects of the K(ATP) channel opener levcromakalim on spontaneous contractions. These results indicate that in vascular smooth muscle cells, propafenone inhibits K(V), K(A), BK(Ca), and K(ATP) channels. These actions correlated with its effects on mechanical activity in whole portal veins.     

Temporal differences in actions of calcium channel blockers on K+ accumulation, cardiac function, and high-energy phosphate levels in ischemic guinea pig hearts

We investigated temporal differences in the protective action of three types of Ca2+ channel blockers in myocardial ischemia, focusing particularly on the blocking ability under depolarizing conditions. The effects of diltiazem, verapamil, and nifedipine on extracellular potassium concentration ([K+]e), acidosis, and level of metabolic markers were examined during 30-min global ischemia and postischemic left ventricular (LV) function in isolated guinea pig hearts. Diltiazem and verapamil, but not nifedipine, inhibited the late phase (15-30 min) of [K+]e elevation, whereas all three blockers delayed the onset of the early phase (0-8 min) of [K+]e elevation. Diltiazem and verapamil inhibited ischemic contracture and improved postischemic LV function to a greater extent. These differences appeared to be linked to preservation of ATP and creatine phosphate and delay of cessation of anaerobic glycolytic activity. Maneuvers to preserve energy sources during ischemia (decrease in external Ca2+ concentration or pacing at a lower frequency) attenuated the late phase of [K+]e elevation. Inhibition of LV pressure was potentiated 12- and 8.2-fold by diltiazem and verapamil, respectively, at 8.9 mM K+ as compared with 2.9 mM K+, whereas that by nifedipine was unchanged. These results indicate that the differential cardioprotection of Ca2+ channel blockers in the late period of ischemia correlates with preservation of high-energy phosphates as a result of different Ca2+ channel blocking abilities under high [K+]e conditions.     

Effect of inhibition of Na+/K(+)-adenosine triphosphatase on vascular action of vasopressin.

The present study was undertaken to examine the cellular interaction between a Na+/K(+)-ATPase inhibitor, ouabain, and arginine vasopressin (AVP) in rat vascular smooth muscle cells (VSMC) in culture. Preincubation with 10(-5) M ouabain for 60 min increased basal cytosolic free Ca2+ [( Ca2+]i) concentration and intracellular 45Ca2+ uptake. Ouabain, however, did not affect basal 45Ca2+ efflux or AVP-stimulated 45Ca2+ efflux. As assessed by cell shape change, preincubation with 10(-5) M ouabain for 60 min also enhanced the sustained cellular contractile effect of a submaximal (10(-8) M AVP, 21.5% vs. 30.5%, P less than 0.01) but not maximal dose of 10(-6) M AVP. Preincubation with 10(-5) M ouabain for 60 min did not change AVP-induced V1-specific surface receptor binding or AVP-induced inositol phosphate production but did however potentiate the mobilization of [Ca2+]i induced by a submaximal (10(-8) M AVP, 301 vs. 385 nM, P less than 0.01) but not a maximal dose of AVP. These effects of ouabain on the mobilization of [Ca2+]i were abolished by incubation in Ca2(+)-free buffer or 5 X 10(-5) M verapamil. Ouabain (10(-5) M) also enhanced the sustained cellular contractile effect of a direct protein kinase C activator, phorbol 12-myristate 13-acetate. The present results therefore indicate that the inhibition of Na+/K(+)-ATPase may enhance the vascular action of AVP, and perhaps other vasoconstrictors, by increasing the AVP-induced mobilization of [Ca2+]i and by potentiating the activity of protein kinase C stimulated by AVP through enhancing basal and AVP-stimulated cellular Ca2+ uptake.