The effect of amiloride on hormonal regulation of amino acid transport in isolated and cultured adult rat hepatocytes

Insulin and glucagon stimulate amino acid transport in isolated rat hepatocytes. Amiloride, a specific Na+-influx inhibitor, completely inhibited the hormonal (glucagon or insulin) stimulation of alpha-aminoisobutyric acid influx by preventing the emergence of a high-affinity transport component. The drug also inhibited [14C]valine incorporation into hepatocyte protein. The half-maximal concentration of amiloride for inhibition of protein synthesis was similar to that required for inhibition of hormone-stimulated amino acid transport (approx. 0.1 mM). In primary cultured rat hepatocytes, amiloride markedly depressed the stimulation of alpha-aminoisobutyric acid transport by glucagon, or a mixture of glucagon, insulin and epidermal growth factor. These results suggest that amiloride inhibits the hormonal stimulation of hepatocyte amino acid transport by preventing the synthesis of high-affinity transport proteins. They also suggest that the hormonal stimulation of hepatocyte amino acid transport is dependent, at least partly, on Na+ influx.     

Insulin and glucagon stimulation of (Na+-K+)-ATPase transport activity in isolated rat hepatocytes

The effects of insulin and glucagon on the (Na+-K+)-ATPase transport activity in freshly isolated rat hepatocytes were investigated by measuring the ouabain-sensitive, active uptake of 86Rb+. The active uptake of 86Rb+ was increased by 18% (p less than 0.05) in the presence of 100 nM insulin, and by 28% (p less than 0.005) in the presence of nM glucagon. These effects were detected as early as 2 min after hepatocyte exposure to either hormone. Half-maximal stimulation was observed with about 0.5 nm insulin and 0.3 nM glucagon. The stimulation of 86Rb+ uptake by insulin occurred in direct proportion to the steady state occupancy of a high affinity receptor by the hormone (the predominant insulin-binding species in hepatocytes at 37 degrees C. For glucagon, half-maximal response was obtained with about 5% of the total receptors occupied by the hormone. Amiloride (a specific inhibitor of Na+ influx) abolished the insulin stimulation of 86Rb+ uptake while inhibiting that of glucagon only partially. Accordingly, insulin was found to rapidly enhance the initial rate of 22Na+ uptake, whereas glucagon had no detectable effect on 22Na+ influx. These results indicate that monovalent cation transport is influenced by insulin and glucagon in isolated rat hepatocytes. In contrast to glucagon, which appears to enhance 86Rb+ influx through the (Na+-K+)-ATPase without affecting Na+ influx, insulin stimulates Na+ entry which in turn may increase the pump activity by increasing the availability of Na+ ions to internal Na+ transport sites of the (Na+-K+)-ATPase.     

Role of microtubules in insulin and glucagon stimulation of amino acid transport in isolated rat hepatocytes

The effects of the microtubule inhibitor, colchicine, on insulin or glucagon stimulation of alpha-amino[1-14C]-isobutyric acid (AIB) transport were investigated in isolated hepatocytes from normal fed rats. Under all conditions tested, AIB uptake appeared to occur through two components of transport: a low affinity (Km approximately 50 mM) component and a high affinity (Km approximately 1 mM) component. Within 2 h of incubation, insulin and glucagon, at maximal concentrations, increase AIB (0.1 mM) uptake by 2- to 3-fold and 4- to 6-fold, respectively. Colchicine, at the low concentration of 5 X 10(-7) M, slightly reduces basal AIB transport, decreases by 80% the simulatory effect of insulin, and diminishes by 40% the stimulatory effect of either glucagon or dibutyryl cAMP. Kinetic analysis of AIB influx indicates that the drug inhibits the increase in Vmax of a high affinity (Km approximately 1 mM) component of transport stimulated by insulin or glucagon, without affecting the kinetic parameters of a low affinity component of transport (Km approximately 50 mM). Various short term hormonal effects of insulin and glucagon (changes in glucose, urea, and lactate production) were found not to be modified by the drug. Vinblastine elicits similar changes as colchicine on AIB uptake. Lumicolchicine, a colchicine analogue that does not bind to tubulin, has no effect. The concentration of colchicine (10(-7) M) required for half-maximal inhibition of hormone-stimulated AIB transport is in the appropriate range for specific microtubule disruption. These data suggest that microtubules are involved in the regulation of the insulin or glucagon stimulation of AIB transport in isolated rat hepatocytes.     

Ca2+-activated Na+ fluxes in human red cells. Amiloride sensitivity

The effect of Ca2+ on the ouabain- and bumetanide-resistant Na+ fluxes in intact red cells was studied at relatively constant internal Ca2+, membrane potential, and cell volume. The red cell calcium concentration was modified using the ionophore A23187. In fresh red cells, the Na+ influx and efflux (1.2 +/- 0.13 and 0.26 +/- 0.07 mmol/liter cells x h, respectively) were not affected by amiloride (1 mM). When external Ca2+ was raised from 0 to 150 microM, in the presence of A23187, both the Na+ influx and efflux were stimulated (about 3.5-fold). The Ca2+-activated Na+ efflux and influx had an apparent Km for activation by Ca2+o of about 25 microM. The Ca2+-dependent Na+ transport was inhibited 30-60% by amiloride (ID50 = 17.3 +/- 8 microM). Amiloride, however, had no effect on the Ca2+-dependent K+ influx. The amiloride-sensitive (AS) transport pathway was a linear function of the Na+o concentration in the range from 0 to 75 mM. The Ca2+i activation seems to depend on the metabolic integrity of red cells. 1) It does not take place in ATP-depleted red cells; 2) ATP-repletion of ATP-depleted red cells fully restored AS Na influx; and 3) ATP-enrichment (ATP-red cells) enhanced the AS Na influx by about 100%. The Ca2+-activated AS Na+ influx was not affected by either DIDS or trifluoperazine. The present results indicate that in human erythrocytes an increase in internal Ca2+ activates on otherwise silent AS Na+-transport system, which is dependent on the metabolic integrity of the red cells.     

Sodium transport in erythrocytes of the mollusc Anadara broughtonii: Evidence for inhibition by quinine

Sodium transport through the molluscan erythrocyte membrane was examined using ²²Na as a tracer. Incubation of the red cells in standard saline resulted in a rapid ²²Na uptake reaching steady state concentration (about 21.5 mmol/l cells) in the first 60 min. A similar pattern in the time course of ²²Na uptake was seen in the erythrocytes incubated in mantle fluid. The average value of unidirectional Na⁺ influx, measured as a 5-min ²²Na uptake, was 7.76 ± 0.36 mmol/1 cells/5 min or 93 ± 4.3 mmol/1 cells/hr. The initial rate of Na⁺ influx increased in a saturable fashion as a function of external Na⁺ concentration with apparent AT., of 380±12mM and V_max of 14.3 ± 2.4 mmol/1 cells/5 min. Amiloride (1 mM), furosemide (1 mM), and DIDS (0.1 mM) had no effect on either initial Na⁺ influx (5 min ²²Na uptake) or equilibrium Na⁺ concentration (60 min and 120min ²²Na uptake) in the molluscan red cells exposed to standard saline. Quinine (1 mM) caused a significant fall in the initial Na⁺ influx (by 48%) and in 60-min ²²Na uptake (by 32%) as compared with control levels. In the presence of 0.1 mM ouabain, ²²Na uptake into the red cells was enhanced by an average 27% and 44% during 60 min and 120 min of cell incubation, respectively. The ouabain-sensitive Na⁺ accumulation in the red cells reflected a contribution of the Na, K-pump to Na⁺ transport and the mean value was 5.6 ± 1.0 mmol/1 cells/hr.     

Circadian Dependent Effect of Epidermal Growth Factor,Insulin and Glucagon on Hepatic Pyruvate Kinase and Malic Enzyme of Mice

The influence of epidermal growth factor (EGF), 0.75 μg g^?1; insulin, 1.5 μg g^?1; glucagon, 1.25 ygg^?1 and their combinations on the activities of hepatic pyruvate kinase (PK) and malic enzymes (ME) was monitored. Male CD2F₁ mice were treated toward the end of the light or dark periods, 9 or 23 /tours after /ights on (9 or 23 HALO), and subgroups of six mice were killed at 4,8 or 12 hr post-treatment. PK and ME activities from control mice were well characterized by cosine curves. The PK activity was maximal when ME activity was minimal at the transition from light to dark (9 HALO plus 4 hr) and PK was at a minimum when ME was highest (23 HALO plus 4 hr). Both enzymes were influenced by at least one peptide hormone, and the effects were strongly circadian -stage dependent. The only effect attributed to EGF was an increase of PK activity (23%) 12 hr after injection at 23 HALO. PK activity was increased by insulin (23%) at 23 HALO (4 hr after injection), but not at 9 HALO, and decreased (17%) by glucagon 12 hr after injection at 9 HALO. Several reductions in PK activity in response to various combinations of peptides were observed, and appeared to be caused by glucagon but influenced by insulin. The activity of ME was decreased (33%) in response to insulin 4 hr after injection at 23 HALO but not at 9 HALO and increased (60-70%) by glucagon alone or in combinations with insulin or EGF, or both, at 4 hr after injection at 9 HALO but not at 23 HALO. In general, when ME activity was altered by either insulin or glucagon, PK activity was also altered in the opposite direction, and the effects of glucagon were opposed by insulin.     

Activation of sodium transport in rat erythrocytes by inhibition of protein phosphatases 1 and 2A

Four structurally different protein phosphatases (PPs) inhibitors - fluoride, calyculin A, okadaic acid and cantharidin--were tested for their ability to modulate unidirectional Na(+) influx in rat red blood cells. Erythrocytes were incubated at 37 degrees C in isotonic and hypertonic media containing 1 mM ouabain and (22)Na in the absence or presence of PP inhibitors. Exposure of the cells to 20 mM fluoride or 50 nM calyculin A for 1 h under isosmotic conditions caused a significant stimulation of Na(+) influx, whereas addition of 200 microM cantharidin or 100 nM okadaic acid had no effect. After 2 h of treatment, however, all these PPs blockers significantly enhanced Na(+) transport in rat erythrocytes. Selective inhibitors of PP-1 and PP-2A types, calyculin A, cantharidin and okadaic acid, produced similar ( approximately 1.2-1.4-fold) stimulatory effects on Na(+) influx in the cells. Activation of Na(+) influx was unchanged with increasing calyculin A concentration from 50 to 200 nM. No additive stimulation of Na(+) influx was observed when the cells were treated with combination of 20 mM fluoride and 50 nM calyculin A. Na(+) influx induced by PPs blockers was inhibited by 1 mM amiloride and 200 muM bumetanide approximately in the equal extent, indicating the involvement of Na(+)/H(+) exchange and Na-K-2Cl cotransport in sodium transport through rat erythrocytes membrane. Activation of Na(+) transport in the cells induced by calyculin A and fluoride was associated with increase of intracellular Na(+) content. Shrinkage of the rat erythrocytes resulted in 2-fold activation of Na(+) influx. All tested PPs inhibitors additionally activated the Na(+) influx by 70-100% above basal shrinkage-induced level. Amiloride and bumetanide have diminished both the shrinkage-induced and PPs-inhibitors-induced Na(+) influxes. Thus, our observations clearly indicate that activities of Na(+)/H(+) exchanger and Na-K-2Cl cotransporter in rat erythrocytes are regulated by protein phosphatases and stimulated when protein dephosphorylation is inhibited.     

Circadian Dependent Effect of Epidermal Growth Factor, Insulin and Glucagon on Hepatic Pyruvate Kinase and Malic Enzyme of Mice

The influence of epidermal growth factor (EGF), 0.75 μg g^-1; insulin, 1.5 μg g^-1; glucagon, 1.25 ygg^-1 and their combinations on the activities of hepatic pyruvate kinase (PK) and malic enzymes (ME) was monitored. Male CD2F₁ mice were treated toward the end of the light or dark periods, 9 or 23 /tours after /ights on (9 or 23 HALO), and subgroups of six mice were killed at 4,8 or 12 hr post-treatment. PK and ME activities from control mice were well characterized by cosine curves. The PK activity was maximal when ME activity was minimal at the transition from light to dark (9 HALO plus 4 hr) and PK was at a minimum when ME was highest (23 HALO plus 4 hr). Both enzymes were influenced by at least one peptide hormone, and the effects were strongly circadian -stage dependent. The only effect attributed to EGF was an increase of PK activity (23%) 12 hr after injection at 23 HALO. PK activity was increased by insulin (23%) at 23 HALO (4 hr after injection), but not at 9 HALO, and decreased (17%) by glucagon 12 hr after injection at 9 HALO. Several reductions in PK activity in response to various combinations of peptides were observed, and appeared to be caused by glucagon but influenced by insulin. The activity of ME was decreased (33%) in response to insulin 4 hr after injection at 23 HALO but not at 9 HALO and increased (60-70%) by glucagon alone or in combinations with insulin or EGF, or both, at 4 hr after injection at 9 HALO but not at 23 HALO. In general, when ME activity was altered by either insulin or glucagon, PK activity was also altered in the opposite direction, and the effects of glucagon were opposed by insulin.     

Dual effects of glucagon and cyclic AMP on DNA synthesis in cultured rat hepatocytes: stimulatory regulation in early G1 and inhibition shortly before the S phase entry

Although several lines of evidence implicate cyclic AMP in the humoral control of liver growth, its precise role is still not clear. To explore further the role of cyclic AMP in hepatocyte proliferation, we have examined the effects of glucagon and other cyclic AMP-elevating agents on the DNA synthesis in primary cultures of adult rat hepatocytes, with particular focus on the temporal aspects. The cells were cultured in a serum-free, defined medium and treated with epidermal growth factor (EGF), insulin, and dexamethasone. Exposure of the hepatocytes to low concentrations (10 pM-1 nM) of glucagon in the early stages of culturing (usually within 6 h from plating) enhanced the initial rate of S phase entry without affecting the lag time from the plating to the onset of DNA synthesis, whereas higher concentrations inhibited it. In contrast, glucagon addition at later stages (24-45 h after plating) produced only the inhibition. Thus, if glucagon was added at a time when there was a continuous EGF/insulin-induced recruitment of cells to S phase, the rate of G1-S transition was markedly decreased within 1-3 h. This inhibitory effect occurred at low glucagon concentrations (ID50 less than 1 nM) and was mimicked by cholera toxin, forskolin, isobutyl methylxanthine, and 8-bromo cyclic AMP. The results indicate that cyclic AMP has dual effects on hepatocyte proliferation with a stimulatory modulation early in the prereplicative period (G0 or early G1), and a marked inhibition exerted immediately before the transition from G1 to S phase.     

Inhibition of hepatic proteolysis by insulin. Role of hormone-induced alterations of the cellular K+ balance

1. Proteolysis was measured as [3H]leucine release from isolated perfused livers from rats, which had been labeled in vivo by an intraperitoneal injection of [3H]leucine about 16 h prior to the perfusion experiment. In livers from fed rats, insulin (35 nM) inhibited [3H]leucine release by 24.5 +/- 1.3% (n = 15) and led to an amiloride-sensitive, bumetanide-sensitive and furosemide-sensitive net K+ uptake of 5.53 +/- 0.31 mumol.g-1 (n = 15). Both the insulin effects on net K+ uptake and on [3H]leucine release were diminished by about 65% or 55% in presence of furosemide (0.1 mM) or bumetanide (5 microM), respectively. The insulin-induced net K+ uptake was virtually abolished in the presence of amiloride (1 mM) plus furosemide (0.1 mM). 2. In perfused livers from 24-h-starved rats, both the insulin-stimulated net K+ uptake and the insulin-induced inhibition of [3H]leucine release were about 80% lower than observed in experiments with livers from fed rats. The insulin effects on K+ balance and [3H]leucine release were not significantly influenced in the presence of glycine (2 mM), although glycine itself inhibited [3H]leucine release by 30.3 +/- 0.3% (n = 4) and 13.8 +/- 1.2% (n = 5) in livers from starved and fed rats, respectively. When livers from fed rats were preswollen by hypoosmotic perfusion (225 mOsmol.l-1), both the insulin-induced net K+ uptake and the inhibition of [3H]leucine release were diminished by 50-60%. 3. During inhibition of [3H]leucine release by insulin, further addition of glucagon (100 nM) led to a marked net K+ release from the liver (3.82 +/- 0.24 mumol.g-1), which was accompanied by stimulation of [3H]leucine release by 16.4 +/- 4.6% (n = 4). 4. Ba2+ (1 mM) infusion led to a net K+ uptake by the liver of 3.2 +/- 0.2 mumol.g-1 (n = 4) and simultaneously inhibited [3H]leucine release by 12.4 +/- 1.7% (n = 4). 5. There was a close relationship between the Ba2+ or insulin-induced net K+ uptake and the degree of inhibition of [3H]leucine release, even when the K+ response to insulin was modulated by bumetanide, furosemide, glucagon, hypotonic or glycine-induced cell swelling or the nutritional state. 6. The data suggest that the insulin-induced net K+ uptake involves activation of both NaCl/KCl cotransport and Na+/H+ exchange.(ABSTRACT TRUNCATED AT 400 WORDS)     

Initiation of cultured rat hepatocyte proliferation does not involve Na+-dependent plasma membrane Ca2+ fluxes

Adult rat hepatocytes in primary culture were examined to determine if Na+-dependent transmembrane Ca2+ fluxes precede reinitiation of DNA synthesis. Studies with 45Ca2+ and atomic absorption measurements of 40Ca2+ showed that hepatocytes lack plasma membrane Na+-Ca2+ exchange activity. Under chemically defined conditions, combinations of mitogens - EGF, insulin, and glucagon - failed to induce transmembrane Ca2+ fluxes early in the prereplicative phase. In addition, a Ca2+ ionophore, A23187, was non-mitogenic. Thus, plasma membrane Na+-Ca2+ exchange is not a mitogenic signal for hepatocytes. Elevated intracellular Ca2+ levels are thought to mediate early prereplicative events required for animal cell proliferation. These conclusions stem partly from findings that A23187, a Ca2+ ionophore, stimulates transmembrane Ca2+ fluxes and proliferation in several cell systems (reviewed in Boynton et al., 1982). Sodium ion fluxes also are implicated as "initiating" mitogenic signals (Koch and Leffert, 1979). In particular, amiloride-sensitive Na+ influxes, stimulated by growth factors, may be necessary to initiate DNA synthesis in rat hepatocytes, mouse and human fibroblasts, rat liver derived cell lines, mouse sympathetic neurons, human lymphocytes, and monkey kidney epithelial cells (reviewed in Leffert, 1982). Several investigators, using cells from electrically excitable tissues (Schellenberg and Swanson, 1981; Eckert and Grosse, 1982), have reported that plasma membrane Na+-Ca2+ exchange carriers regulate intracellular Na+ and Ca2+ concentration. It is unclear if this exchange system exists in non-electrically excitable membranes, especially with regard to hepatocytes (Judah and Ahmed, 1964; van Rossum, 1970). We have here investigated the possible association of Na+ influxes with transmembrane Ca2+ movement following reinitiation of hepatocyte growth.     

Na+ participates in loop diuretic-sensitive Cl(-)-cation co-transport in the pancreatic beta-cells

In order to investigate whether Na+ participates in loop diuretic-sensitive Cl(-)-cation co-transport in the beta-cells, we tested the interaction between the effects of Na+ deficiency, furosemide and D-glucose on 86Rb+ fluxes in beta-cell-rich mouse pancreatic islets. Removal of extracellular Na+ slightly reduced the ouabain-resistant 86Rb+ influx and the specific effect of 1 mM furosemide on this influx was significantly smaller in Na(+)-deficient medium. The capacity of 20 mM D-glucose to reduce the ouabain-resistant 86Rb+ influx was not changed by removal of extracellular Na+. The 86Rb+ efflux from preloaded islets was rapidly and reversibly reduced by Na+ deficiency. Furosemide (1 mM) reduced the 86Rb+ efflux and the effect of the combination of Na+ deficiency and 1 mM furosemide was not stronger than the effect of furosemide alone. 22Na+ efflux was reduced by both ouabain and furosemide and the effects appeared to be additive. The data suggest that Na+ participates in loop diuretic-sensitive Cl(-)-cation co-transport in the pancreatic beta-cells. This adds further support to the idea that beta-cells exhibit a Na+, K+, Cl- co-transport system. Since some of the furosemide effect on 86Rb+ efflux persisted in the Na(+)-deficient medium, it is likely that also loop diuretic-sensitive K+, Cl- co-transport exists in this cell type.     

Chemotactic factor-induced activation of Na+/H+ exchange in human neutrophils. I. Sodium fluxes

The nature of Na+ fluxes in resting and in chemotactic factor-activated human neutrophils was investigated. In resting cells, ouabain-insensitive unidirectional 22Na+ in- and effluxes represented passive electrodiffusional fluxes through ion channels: they were nonsaturable and voltage-dependent (PNa = 4.3 X 10(-9) cm/s). Amiloride (1 mM) had little effect on resting 22Na+ influx (approximately 0.8 meq/liter X min), thereby suggesting a minor contribution of Na+/H+ exchange and a lack of amiloride-sensitive Na+ channels. When neutrophils were exposed to the chemotactic tripeptide N-formyl-methionyl-leucyl-phenylalanine (FMLP, 0.1 microM), 22Na+ influx was stimulated approximately 30-fold (initial rate approximately 22 meq/liter X min). The FMLP-induced 22Na+ influx was saturable with respect to external Na+ (Km 26-35 mM, Vmax approximately 28 meq/liter X min), was electroneutral, and could be competitively inhibited by amiloride (Ki 10.6 microM). From a resting value of approximately 30 meq/liter of cell water, internal Na+ in FMLP-stimulated cells rose exponentially to reach a concentration of approximately 60 meq/liter by 10-15 min. This uptake was blocked by amiloride. FMLP also stimulated the efflux of 22Na+ which followed a single exponential time course (rate coefficient approximately 0.16 min-1). The FMLP-induced 22Na+ fluxes were similar to those observed with 10 microM monensin, a known Na+/H+ exchanging ionophore. The data indicate that FMLP activates an otherwise quiescent, amiloride-sensitive Na+/H+ exchange. Furthermore, all of the FMLP-induced 22Na+ fluxes can be satisfactorily accounted for by transport through the exchanger, leaving little room for an appreciable increase in Na+ conductance.     

Barium mimics the effect of D-glucose on 86Rb+ fluxes in mouse pancreatic beta-cells

The interaction between Ba2+, furosemide and D-glucose on 86Rb+ fluxes in ob/ob mouse islets was investigated. Ba2+ (2 mM) significantly reduced the ouabain-resistant 86Rb+ influx, without affecting the ouabain-sensitive influx. D-Glucose (20 mM) reduced the 86Rb+ influx in the absence of Ba2+ (2 mM) but not in the presence of the cation. Furosemide, an inhibitor of Na+, K+, Cl- co-transport, reduced the 86Rb+ influx and the effect was partly additive to the effect of 2 mM Ba2+. When the islets were preincubated with Ba2+ (2 mM) the specific effect of 1 mM furosemide on the 86Rb+ influx was reduced, whereas, in acute experiments, Ba2+ (2 mM) did not affect the specific effect of furosemide on 86Rb+ influx. 86Rb+ efflux from preloaded islets was significantly reduced by 2 mM Ba2+ and during the first 5 min of ion efflux the effect of the combination of 2 mM Ba2+ and 1 mM furosemide was stronger than the effect of Ba2+ alone. The data show that Ba2+ reduces 86Rb+ fluxes in the beta-cells and suggest that this is mainly mediated by inhibition of K+ channels in the beta-cell plasma membrane. Long-term exposure to Ba2+ may also reduce the activity of the Na+, K+, Cl- co-transport system. The effect of Ba2+ on K+ channels may help to explain the stimulatory effect on insulin release in the absence of nutrient secretagogues.     

Induction of DNA synthesis in adult rat hepatocytes cultured in a serum-free medium

Isolated hepatocytes from adult rats were cultured for 3 days in a serum-free synthetic medium. Supplementation with fibrinogen digests, glucagon and insulin remarkably increased DNA synthesis in hepatocytes. DNA synthesis began to increase at 35 h and reached a maximum at 41 to 54 h after plating. At this time, cells were morphologically identifiable as hepatocytes. Glucagon could be replaced by dibutyryl cyclic AMP or isobutyl-methyl-xanthine. Addition of amiloride (a Na⁺ influx inhibitor) during the initial 22 h completely inhibited DNA synthesis. These results suggest that influx of Na⁺ during early prereplicative period and increase in cellular cyclic AMP levels during late prereplicative period are necessary for the induction of DNA synthesis in hepatocytes.     

Kinetics and stoichiometry of coupled Na efflux and Ca influx (Na/Ca exchange) in barnacle muscle cells

Coupled Na+ exit/Ca2+ entry (Na/Ca exchange operating in the Ca2+ influx mode) was studied in giant barnacle muscle cells by measuring 22Na+ efflux and 45Ca2+ influx in internally perfused, ATP-fueled cells in which the Na+ pump was poisoned by 0.1 mM ouabain. Internal free Ca2+, [Ca2+]i, was controlled with a Ca-EGTA buffering system containing 8 mM EGTA and varying amounts of Ca2+. Ca2+ sequestration in internal stores was inhibited with caffeine and a mitochondrial uncoupler (FCCP). To maximize conditions for Ca2+ influx mode Na/Ca exchange, and to eliminate tracer Na/Na exchange, all of the external Na+ in the standard Na+ sea water (NaSW) was replaced by Tris or Li+ (Tris-SW or LiSW, respectively). In both Na-free solutions an external Ca2+ (Cao)-dependent Na+ efflux was observed when [Ca2+]i was increased above 10(-8) M; this efflux was half-maximally activated by [Ca2+]i = 0.3 microM (LiSW) to 0.7 microM (Tris-SW). The Cao-dependent Na+ efflux was half-maximally activated by [Ca2+]o = 2.0 mM in LiSW and 7.2 mM in Tris-SW; at saturating [Ca2+]o, [Ca2+]i, and [Na+]i the maximal (calculated) Cao-dependent Na+ efflux was approximately 75 pmol#cm2.s. This efflux was inhibited by external Na+ and La3+ with IC50's of approximately 125 and 0.4 mM, respectively. A Nai-dependent Ca2+ influx was also observed in Tris-SW. This Ca2+ influx also required [Ca2+]i greater than 10(-8) M. Internal Ca2+ activated a Nai-independent Ca2+ influx from LiSW (tracer Ca/Ca exchange), but in Tris-SW virtually all of the Cai-activated Ca2+ influx was Nai-dependent (Na/Ca exchange). Half-maximal activation was observed with [Na+]i = 30 mM. The fact that internal Ca2+ activates both a Cao-dependent Na+ efflux and a Nai-dependent Ca2+ influx in Tris-SW implies that these two fluxes are coupled; the activating (intracellular) Ca2+ does not appear to be transported by the exchanger. The maximal (calculated) Nai-dependent Ca2+ influx was -25 pmol/cm2.s. At various [Na+]i between 6 and 106 mM, the ratio of the Cao-dependent Na+ efflux to the Nai-dependent Ca2+ influx was 2.8-3.2:1 (mean = 3.1:1); this directly demonstrates that the stoichiometry (coupling ratio) of the Na/Ca exchange is 3:1. These observations on the coupling ratio and kinetics of the Na/Ca exchanger imply that in resting cells the exchanger turns over at a low rate because of the low [Ca2+]i; much of the Ca2+ extrusion at rest (approximately 1 pmol/cm2.s) is thus mediated by an ATP-driven Ca2+ pump.(ABSTRACT TRUNCATED AT 400 WORDS)     

Induction of DNA synthesis in dog thyrocytes in primary culture: synergistic effects of thyrotropin and cyclic AMP with epidermal growth factor and insulin

We have investigated the growth effects of thyrotropin (TSH) (mimicked by forskolin and acting through cyclic AMP), epidermal growth factor (EGF), serum (10%) and insulin on quiescent dog thyroid epithelial cells in primary culture in a serum-free defined medium. These cells were previously shown to retain the capacity to express major thyroid differentiation markers. In the presence of insulin and after a similar prereplicative phase of 18 +/- 2h, TSH, EGF, and serum promoted DNA synthesis in such quiescent cells only a minority of which had proliferated in vitro before stimulation. The combination of these factors induced more than 90% of the cells to enter S phase within 48 h and near exponetial proliferation. Analysis of the cell cycle parameters of the stimulated cells revealed that the G1 period duration was similar to the length of the prereplicative phase of quiescent thyroid cells; this might indicate that they were in fact in an early G1 stage rather than in G0 prior to stimulation. TSH and EGF action depended on or was potentiated by insulin. Strikingly, nanomolar concentrations of insulin were sufficient to support stimulation of DNA synthesis by TSH, while micromolar concentrations of insulin were required for the action of EGF. This suggests that insulin supported the action of TSH by acting on its own high affinity receptors, whereas its effect on EGF action would be related to its somatomedinlike effects at high supraphysiological concentrations. Insulin stimulated the progression in the prereplicative phase initiated by TSH or forskolin. In addition, in some primary cultures TSH must act together with insulin to stimulate early events of the prereplicative phase. In the presence of insulin, EGF, and forskolin, an adenylate cyclase activator, markedly synergized to induce DNA synthesis. Addition of forskolin 24 h after EGF or EGF 24 h after forskolin also resulted in amplification of the growth response but with a lag equal to the prereplicative period observed with the single compound. This indicates that events induced by the second factor can no longer be integrated during the prereplicative phase set by the first factor. These findings demonstrate the importance of synergistic cooperation between hormones and growth factors for the induction of DNA synthesis in epithelial thyroid cells and support the proposal that essentially different mitogenic pathways--cyclic AMP-dependent or independent--may coexist in one cell.     

Cation-coupled chloride influx in squid axon. Role of potassium and stoichiometry of the transport process

Evidence is presented showing that the Cl- uptake process in the squid giant axon is tightly coupled not only to Na+ uptake but also to K+ uptake. Thus, removal of external K+ causes both Cl- and Na+ influxes to be reduced, particularly when [Cl-]i is low, that is, under conditions previously shown to be optimal for Cl-/Na+-coupled influx. In addition, there exists a ouabain-insensitive K+ influx, which depends on the presence of external Cl- and Na+, is inversely proportional to [Cl-]i, and is blocked by furosemide/bumetanide. Finally, this ouabain-insensitive K+ influx appears to require the presence of cellular ATP. The stoichiometry of the coupled transport process was measured using a double-labeling technique combining in the same axon either 36Cl and 42K or 22Na and 42K. The stoichiometry of the flux changes occurring in response either to varying [Cl-]i between 150 and 0 mM or to treatment with 0.3 mM furosemide is, in both cases, approximately 3:2:1 (Cl-/Na+/K+). Although these fluxes require ATP, they are not inhibited by 3 mM vanadate. In addition, treatment with DIDS has no effect on the fluxes.     

Sodium fluxes in human fibroblasts: Effect of serum,Ca+2, and amiloride

Human fibroblasts that have been serum deprived for 4 hours have a digitoxin-insensitive Na influx of 9.5 ± 1.0 (n = 4) μmol/g prot/min which is not significantly different from the influx of 9.4 ± 0.6 (n = 3) μmol/g prot/min measured in cells arrested in the G₁/G₀ state by serum-deprivation for a period of four days. The Na influx in serum-deprived cells is rapidly stimulated (within one minute) simply by assaying the cells in medium containing 10% fetal bovine serum (FBS). The digitoxin-insensitive Na influx for cells in the presence of 10% FBS is 22.9 ± 1.1 (n = 6) μmol/g prot/min. the stimulation of Na influx in serumdeprived cells can also be achieved by the addition of the purified mitogen, epidermal growth factor (EGF). Addition of EGF to serum-deprived cells gives a maximal stimulation of Na influx of approximately 1.6-fold, with the concentration for half-maximal stimulation being 7.5 ng/ml. The stimulation of Na influx results from the activation of an amiloride-sensitive pathway, which appears to be minimally active in serum-deprived cells. Kinetic analysis of Na influx experiments in the presence of 10% FBS and varying concentrations of amiloride indicate that at infinite concentrations of amiloride the Na flux would be reduced to 8.9 μmol/g prot/min, which is comparable to the level of Na flux measured in serum-deprived cells in the presence of 5 mM amiloride. Thus, amiloride can totally inhibit the serum-stimulated component of Na influx while inhibiting less than 10% of the Na influx in serum-deprived cells. The Na influx in serum-deprived cells can also be stimulated 2.5-fold by preincubating cells in the presence of the Ca⁺ ionophore A23187 to elevate the intracellular Ca content. This stimulation of Na influx by intracellular Ca⁺² can be virtually eliminated by adding 1 mM amiloride.     

Ionic responses and growth stimulation induced by nerve growth factor and epidermal growth factor in rat pheochromocytoma (PC12) cells

Rat pheochromocytoma cells (clone PC12) respond to nerve growth factor (NGF) by the acquirement of a phenotype resembling neuronal cells. In an earlier study we showed that NGF causes an increase in Na+,K+ pump activity, as monitored by ouabain-sensitive Rb+ influx. Here we show that addition of epidermal growth factor (EGF) to PC12 cells resulted in a stimulation of Na+,K+ pump activity as well. The increase of Na+,K+ pump activity by NGF or EGF was due to increased Na+ influx. This increased Na+ influx was sensitive to amiloride, an inhibitor of Na+,H+ exchange. Furthermore, no changes in membrane potential were observed upon addition of NGF or EGF. Amiloride-sensitive Na+,H+ exchange in PC12 cells was demonstrated by H+ efflux measurements and the effects of weak acids on Na+ influx. These observations suggest that both NGF and EGF activate an amiloride-sensitive, electroneutral Na+,H+ exchange mechanism in PC12 cells. These findings were surprising in view of the opposite ultimate biological effects of NGF and EGF, e.g., growth arrest vs. growth stimulation. However, within 24 h after addition, NGF was found to stimulate growth of PC12 cells, comparable to EGF. In the presence of amiloride, this stimulated growth by NGF and EGF was abolished. In contrast, amiloride did not affect NGF-induced neurite outgrowth of PC12 cells. From these observations it is concluded that in PC12 cells: (a) NGF has an initial growth stimulating effect; (b) neurite outgrowth is independent of increased amiloride-sensitive Na+ influx; and (c) growth stimulation by NGF and EGF is associated with increased amiloride-sensitive Na+ influx.