Evidence that Na+/H+ exchange regulates angiotensin II-stimulated diacylglycerol accumulation in vascular smooth muscle cells

Angiotensin II stimulation of vascular smooth muscle cells results in initial, rapid diacylglycerol (DG) formation from the polyphosphoinositides accompanied by intracellular acidification, as well as a more sustained DG accumulation which is accompanied by a prolonged intracellular alkalinization. To determine whether intracellular pH (pHi) modulates DG accumulation, NH4Cl and potassium acetate were used to alter pHi and DG formation was measured. NH4Cl (10 mM) increased pHi from 7.15 +/- 0.05 to 7.34 +/- 0.02 pH units and markedly enhanced the sustained (5 min), but not the initial (15 s), phase of DG formation in response to 100 nM angiotensin II (65 +/- 13% increase). Conversely, intracellular acidification with Na+-free buffer and potassium acetate (20 mM) decreased pHi to 6.93 +/- 0.08 and reduced subsequent angiotensin II-induced sustained DG formation by 82 +/- 9%. In intact cells, inhibition of angiotensin II-stimulated alkalinization by incubation in Na+-free buffer or by addition of the Na+/H+ exchange inhibitor dimethylamiloride (10 microM) decreased the ability of the cell to sustain DG formation, suggesting that active Na+/H+ exchange is necessary for continued DG formation. Thus, it seems that sustained, angiotensin II-induced diacylglycerol accumulation is regulated by intracellular alkalinization secondary to Na+/H+ exchange in cultured vascular smooth muscle cells.     

CCK(B)/gastrin receptor mediates synergistic stimulation of DNA synthesis and cyclin D1, D3, and E expression in Swiss 3T3 cells.

In order to develop a model system for identifying signaling pathways and cell cycle events involved in gastrin-mediated mitogenesis, we have used high efficiency retroviral-mediated transfection of cholecystokinin (CCK)(B)/gastrin receptor into Swiss 3T3 cells. The retrovirally-transfected CCK(B)/gastrin receptor binds 125I-CCK-8 with high affinity (Kd = 1.1 nM) and is functionally coupled to intracellular signaling pathways including rapid and transient increase in Ca2+ fluxes, protein kinase C-dependent protein kinase D activation, and MEK-dependent ERK1/2 activation. In the presence of insulin, CCK-8 or gastrin induced a 66.5 +/- 8.8-fold (mean +/- SEM, n = 24 in eight independent experiments) increase in cellular DNA synthesis, reaching a level similar to that achieved by stimulation with a saturating concentration of fresh serum, and much greater than the response to each agonist added alone. CCK-8 also induced a striking increase in the expression of cyclins D1, D3, and E and hyperphosphorylation of Rb acting synergistically with insulin. Similar effects were observed when CCK(B)/gastrin receptor was activated in the presence of EGF or bombesin. Our results demonstrate that activation of CCK(B)/gastrin receptor retrovirally-transfected into Swiss 3T3 induces a potent synergistic effect on DNA synthesis, accumulation of cyclins D1, D3, and E and hyperphosphorylation of Rb in combination with insulin, EGF, or bombesin. Thus, the CCK(B)/gastrin receptor transfected into Swiss 3T3 cells provides a novel model system to elucidate mitogenic signal transduction pathways and cell cycle events activated via this receptor.     

Ca2+ mobilization can occur independent of acceleration of Na+/H+ exchange in thrombin-stimulated human platelets

Intracellular free Ca2+ [( Ca2+]i) and pH (pHi) were measured simultaneously by dual wavelength excitation in thrombin-stimulated human platelets double-labeled with the fluorescent probes fura2 and 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein to determine the relationship between changes in [Ca2+]i and pHi, respectively. At 37 degrees C, thrombin (0.5 or 0.1 units/ml) increased [Ca2+]i with no detectable lag period to maximum levels within 13 s followed by a slow return to resting levels. There was a transient decrease in pHi within 9 s that was immediately followed by an alkalinization response, attributable to activation of Na+/H+ exchange, that raised pHi above resting levels within 22 s. At 10-15 degrees C, thrombin-induced changes in [Ca2+]i and pHi were delayed and therefore better resolved, although no differences in the magnitude of changes in [Ca2+]i and pHi were observed. However, the increase in [Ca2+]i had peaked or was declining before the alkalinization response was detected, suggesting that Ca2+ mobilization occurs before activation of Na+/H+ exchange. In platelets preincubated with 5-(N-ethyl-N-isopropyl)amiloride or gel-filtered in Na+-free buffer (Na+ replaced with N-methyl-D-glutamine) to inhibit Na+/H+ exchange, thrombin stimulation caused a rapid, sustained decrease in pHi. Under these conditions there was complete inhibition of the alkalinization response, whereas Ca2+ mobilization was only partially inhibited. Nigericin (a K+/H+ ionophore) caused a rapid acidification of more than 0.3 pH unit that was sustained in the presence of 5-(N-ethyl-N-isopropyl)amiloride. Subsequent stimulation with thrombin resulted in slight inhibition of Ca2+ mobilization. These data show that, in human platelets stimulated with high or low concentrations of thrombin, Ca2+ mobilization can occur without a functional Na+/H+ exchanger and in an acidified cytoplasm. We conclude that Ca2+ mobilization does not require activation of Na+/H+ exchange or preliminary cytoplasmic alkalinization.     

EGF raises cytosolic Ca2+ in A431 and Swiss 3T3 cells by a dual mechanism. Redistribution from intracellular stores and stimulated influx

The changes in Ca2+ homeostasis and phosphoinositide hydrolysis induced by EGF were studied in human epidermoid carcinoma A431 cells both when attached to a substratum and after detachment and suspension. The cytosolic Ca2+ concentration was measured by the conventional fluorimetric technique, using the specific probe, quin2, as well as by a new microscopic technique in which single cells are investigated after loading with another probe, fura-2. EGF applied in the complete, Ca2+-containing medium caused a rapid rise in the cytosolic Ca2+ concentration, that remained elevated for several minutes. In Ca2+-free, EGTA-containing medium, part of this response persisted, as revealed by quin2 results in suspended cells and microscopic results with fura-2. The lack of Ca2+ rise seen in attached cells loaded with quin2 and treated with EGF in Ca2+-free medium was probably the result of a Ca2+ buffer artifact. Concomitantly to the Ca2+ signal, EGF induced phosphoinositide hydrolysis, with stimulated accumulation of inositol 1,3,4,trisphosphate and -1,3,4,5-tetrakisphosphate. These results, as well as additional microscopic fura-2 results in Swiss 3T3 fibroblasts, demonstrate that the Ca2+ signal elicited by EGF is due to two components: redistribution from an intracellular store (possibly mediated by generation of inositol trisphosphate) and stimulated influx across the plasmalemma. This latter process was not detected in 3T3 cells treated with either PDGF or bombesin (growth factors that cause much greater phosphoinositide hydrolysis and Ca2+ redistribution responses than EGF). It is therefore suggested that the Ca2+ influx effect of EGF is under the control of a separate, as yet unidentified mechanism.     

Bradykinin transiently activates protein kinase C in Swiss 3T3 cells. Distinction from activation by bombesin and vasopressin

The results presented here demonstrate that bradykinin, acting through a B2 subtype receptor, induces a unique pattern of early signals in quiescent Swiss 3T3 cells. Bradykinin caused a rapid mobilization of calcium from internal stores, as judged by measurements of intracellular Ca2+ concentration in fura-2-loaded cells and by 45Ca2+ efflux from radiolabeled cells. Analysis of phosphoproteins from 32P-labeled Swiss 3T3 cells by one- and two-dimensional gel electrophoresis revealed that bradykinin stimulated transient phosphorylation of an acidic cellular protein migrating with an apparent Mr = 80,000 (termed 80K), identified as a major and specific substrate of protein kinase C. Down-regulation of protein kinase C by pretreatment with phorbol 12,13-dibutyrate (PDBu) completely abolished the increase in 80K phosphorylation. In contrast to the sustained effect induced by bombesin, vasopressin, or PDBu, the stimulation of 80K phosphorylation by bradykinin reached a maximum after 1 min of incubation, and then it rapidly decreased to almost basal levels. Furthermore, bradykinin did not induce protein kinase C-mediated events such as inhibition of 125I-epidermal growth factor binding or enhancement of cAMP accumulation. Bombesin and vasopressin elicited both responses in parallel cultures. Bradykinin induced rapid accumulation of total inositol phosphates in cells labeled with myo-[3H]inositol. In contrast to bombesin and vasopressin which stimulated a linear increase in inositol phosphate accumulation over a 10-min period, the effect of bradykinin reached a plateau after 2.5 min of incubation with no further increase up to 10 min. The results demonstrate that the early signaling events triggered by bradykinin can be distinguished from those elicited by bombesin and vasopressin in Swiss 3T3 cells.     

Interleukin 2 induces a rapid increase in intracellular pH through activation of a Na+/H+ antiport. Cytoplasmic alkalinization is not required for lymphocyte proliferation

In several cell types, proliferation initiated by growth factors is associated with a rapid increase in cytoplasmic pH (pHi). This cytoplasmic alkalinization is due to the activation of an amiloride-sensitive Na+/H+ antiport. It is unclear whether growth factor-induced activation of the antiport or the resultant increase in pHi is the trigger for proliferation, an obligatory requirement for proliferation, or simply an associated phenomenon. Interleukin 2 (IL 2) acts as a growth factor for mitogen or antigen-stimulated thymus-derived (T) lymphocytes. In this study, we established that IL 2 produces an increase in pHi and determined whether this increase in pHi plays a role in the proliferative response to IL 2. Monitoring pHi with an intracellularly trapped, pH-sensitive, fluorescent dye, 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein, we demonstrated that IL 2 rapidly (less than 90 s) initiates an increase in pHi in IL 2-sensitive human and murine T cells. Because intracellular alkalinization requires extracellular Na+ and is amiloride-sensitive, it likely occurs through activation of the Na+/H+ antiport. Using partitioning of a weak acid, 5,5-dimethyl-2,4-oxazolidinedione, we confirmed that the IL 2-dependent increase in pHi is sustained for several hours and returns to near base-line levels by 18 h. We also investigated the consequence of preventing Na+/H+ exchange on the proliferative response induced by IL 2. IL 2-driven proliferation occurred in nominally bicarbonate-free medium in the presence of concentrations of amiloride analogs sufficient to inhibit the Na+/H+ antiport and prevent intracellular alkalinization. These data suggest that although the antiport is activated by binding of IL 2 to its receptor, intracellular alkalinization is not essential for IL 2-dependent proliferation. It seems unlikely that either cytoplasmic alkalinization or activation of the Na+/H+ antiport are triggers for T cell proliferation.     

IL-1 activates the Na+/H+ antiport in a murine T cell

One of the early events following growth factor exposure is elevation of intracellular pH, a process mediated by the Na+/H+ antiport. We studied the effects of human rIL-1 alpha (HrIL-1 alpha) on intracellular pH (pHi) and calcium ([Ca2+]i) in a murine T cell line (MD10 cells), which proliferates in response to IL-1 alone. By using the intracellularly trapped fluorescent dyes (2(1),7(1)-bis-2-carboxyethyl)-5(and -6) carboxyfluorescein) and indo-1, we monitored immediate to early changes of pHi and [Ca2+]i in response to HrIL-1 alpha. Exposure to HrIL-1 alpha (120 pM) leads to an early, sustained intracellular alkalinization (delta pH = + 0.09 +/- 0.03) that plateaus within 20 min. Lower concentrations of the monokine (12 pM, 1.2 pM) have a positive but not statistically significant effect on pHi. These effects parallel the degree of MD10 IL-1R saturation predicted by the KD (49 pM) as assessed by 125I-HrIL-1 alpha binding by MD10 cells (Bmax = approximately 1300). Both the MD10 IL-1 receptor KD and the HrIL-1 alpha concentration required to induce early measurable alkaline pH shifts, however, exceed by three orders of magnitude the HrIL-1 alpha ED50 (50 fM) required for MD10 proliferation. The IL-1-induced rise in pHi is both sodium dependent and amiloride sensitive, indicative of activation of the Na+/H+ antiport. Additionally, PMA (100 nM) and IL-2 (2 nM) alkalinize MD10 cells, with the rise in pHi as a result of PMA exceeding the maximal IL-1 effect (delta pH = + 0.13 +/- 0.04). Furthermore, although PMA alkalinizes cells previously exposed to HrIL-1 alpha, the monokine does not alter the pHi of PMA-treated MD10 cells. Importantly, intracellular alkalinization induced by either HrIL-1 alpha or PMA is inhibited by staurosporine (1 mu iM). Finally, HrIL-1 alpha does not change MD10 [Ca2+]i, in either an acute or sustained fashion. These results indicate that IL-1 activates the Na+/H+ antiport in T cells by a mechanism that is unrelated to changes in [Ca2+]i but may involve protein kinase C activation.     

Intracellular alkalinization leads to Ca2+ mobilization from agonist-sensitive pools in bovine aortic endothelial cells

Receptor-stimulated phosphoinositide turnover leads to activation of Na+/H+ exchange and subsequent intracellular alkalinization. To probe the effect of increased intracellular pH (pHi) on Ca2+ homeostasis in cultured bovine aortic endothelial cells (BAEC), we studied the effect of weak bases, ammonium chloride (NH4Cl) and methylamine (agents which increase pHi by direct passive diffusion), on resting and ATP (purinergic receptor agonist)-induced Ca2+ fluxes. Changes in cytosolic free Ca2+ ([Ca2+]i) or pHi were monitored in BAEC monolayers using the fluorescent dyes, fura-2 or 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein, respectively. NH4Cl-induced, dose-dependent (5-20 mM) increases in [Ca2+]i (maximum change = 195 +/- 26 nM) which were temporally similar to the NH4Cl-induced pHi increases. Methylamine (20 mM) induced a more sustained pHi increase and also stimulated a prolonged [Ca2+]i increase. When BAEC were bathed in HCO3- buffer, removal of extracellular CO2/bicarbonate caused pHi to increase and also induced [Ca2+]i to increase transiently. Extracellular Ca2+ removal did not abolish the rapid NH4Cl-induced rise in [Ca2+]i, although the response was blunted and more transient. NH4Cl addition to BAEC cultures resulted in an increase in 45Ca efflux and decrease in total cell 45Ca content. BAEC treatment with ATP (100 microM) to deplete inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ pools completely blocked the NH4Cl (20 mM)-induced rise in [Ca2+]i. Likewise, prior NH4Cl addition partially inhibited ATP-induced increases in [Ca2+]i, as well as slowed the frequency of repetitive [Ca2+]i spikes in single endothelial cells due to agonist. NH4Cl augmented the rate of [Ca2+]i increase that occurs in response to the depletion of agonist-sensitive intracellular Ca2+ pools. However, the internal Ca2+ store remained depleted during the continued presence of NH4Cl, as indicated by a decreased [Ca2+]i response to ATP in Ca2(+)-free medium. Finally, NH4Cl exerted these actions without affecting basal or ATP-stimulated IP3 formation. These observations provide direct evidence that increased pHi leads to Ca2+ mobilization from an agonist-sensitive pool and impairs Ca2+ pool(s) refilling mechanisms without altering cellular IP3 levels.     

Regulation of intracellular pH in human platelets. Effects of thrombin, A23187, and ionomycin and evidence for activation of Na+/H+ exchange and its inhibition by amiloride analogs

Intracellular pH (pHi) of human platelets was measured with the fluorescent dye 2',7'-bis(carboxyethyl)5,6-carboxyfluorescein under various conditions. Stimulation by thrombin at 23 degrees C caused a biphasic change in pHi (initial pHi 7.09); a rapid fall of 0.01-0.04 units (correlated with the rise of [Ca2+]i measured with quin2) followed after 10-15 s by a sustained rise of 0.1-0.15 units pHi. The fall of pHi and [Ca2+]i mobilization was reduced by early (5 s) addition of hirudin, but the later elevated pHi was not reversed by hirudin added after 30 s, although this strips thrombin from receptors and rapidly returns [Ca2+]i to basal levels. In Na+-free medium, or in presence of the Na+/H+ antiport inhibitors, 5-(N,N-dimethyl)amiloride (DMA) or 5-(N-ethyl-N-isopropyl)amiloride (EIPA), thrombin caused a greater fall of pHi (0.22-0.26 units) that was sustained. DMA or EIPA could also reverse the alkalinization response to thrombin. Ca2+ ionophores (ionomycin, A23187) decreased platelet pHi by 0.02-0.15 units, but without an increase of pHi comparable to that following thrombin; DMA and EIPA enhanced the fall of pHi (0.14-0.33 units). Cytoplasmic acidification produced by nigericin (K+/H+ ionophore) was followed by return towards normal that was abolished by Na+/H+ antiport inhibitors. The phorbol diester phorbol 12-myristate 13-acetate had little effect on resting pHi but increased the rate of recovery 2-3-fold after cytoplasmic acidification by nigericin, ionomycin, or sodium propionate. These results indicate that elevation of [Ca2+]i by thrombin enhances H+ production, but the subsequent alkalinization is independent of receptor occupancy or elevated [Ca2+]i and stimulation of the Na+/H+ antiporter by thrombin probably involves some mechanism apart from regulation by H+ and protein kinase C.     

Hypertonicity-induced intracellular pH changes in rat mast cells

In a non-isotonic environment, cells can shrink or swell and return to their normal shape by activating ion transport pathways. Changes in intracellular pH (pHi) after osmotic stress have been identified in several cells. In order to study the mechanisms that regulate cytosolic pH of rat mast cells in a hypertonic medium, we used the pH sensitive dye, BCECF. Under these hypertonic conditions, pHi undergoes an alkalinization following an initial acidification. The alkalinization is mediated by a Na+/H+ exchanger, since it is inhibited by amiloride and lack of extracellular sodium. Under these conditions, the alkalinization is increased with the PKC activators, TPA and OAG, and partially blocked with trifluoperazine, an unspecific protein kinase C (PKC) and Ca2+ calmodulin-dependent protein kinases (Ca2+/CaM K) inhibitor. There is also an anion exchanger, blocked with DIDS but not activated by PKC, that participates in the observed alkalinization. However, Na+/H+ exchanger is the main mechanism involved in the alkalinization of pHi of mast cells in a hyperosmotic environment.     

Leu13-psi(CH2NH)Leu14]bombesin is a specific bombesin receptor antagonist in Swiss 3T3 cells

The pseudopeptide [Leu13-psi(CH2NH)Leu14]bombesin blocks bombesin-stimulated mitogenesis in Swiss 3T3 cells in a competitive and reversible manner, but not that of other mitogens. It inhibits the mobilization of intracellular Ca2+ and activation of protein kinase C by bombesin-like peptides. It acts at receptor level, as shown by inhibition of [125I]GRP binding and reduction in cross-linking of the Mr 75-85,000 receptor-associated protein. Thus [Leu13-psi(CH2NH)Leu14]bombesin is a specific bombesin receptor antagonist in Swiss 3T3 cells which blocks long-term growth promoting effects of bombesin-like peptides.     

Hepatocyte growth factor induces calcium mobilization and inositol phosphate production in rat hepatocytes.

The effects of hepatocyte growth factor (HGF) on intracellular Ca2+ mobilization were studied using fura-2-loaded single rat hepatocytes. Hepatocytes microperfused with different amounts of HGF responded with a rapid concentration-dependent rise in the cytosolic free Ca2+ concentration with a maximum increase of 142% at 80 ng/ml of HGF. The lag period of the Ca2+ response was decreased with increasing HGF concentrations, being 64 +/- 12 s, 42 +/- 6 s, and 14 +/- 2 s, respectively, with 8, 20, and 80 ng/ml of HGF. The detailed pattern of Ca2+ transients, however, was variable. Out of 16 cells tested using 20 ng/ml of HGF, 68% showed sustained oscillatory responses, whereas other cells showed a sustained increase in the cytosolic-free Ca2+ upon exposure to HGF, which was dependent on the presence of extracellular Ca2+. HGF also induced Ca2+ entry across the plasma membrane. Mobilization of Ca2+ by HGF was accompanied by a rapid accumulation of inositol 1,4,5-trisphosphate (Ins 1,4,5-P3). The effects of HGF and epidermal growth factor (EGF) were comparable and partly additive for Ins 1,4,5-P3 production and for the sustained phase of Ca2+ mobilization. Preincubation of cells with 10 microM of genistein to inhibit protein tyrosine kinases abolished the HGF-induced Ca2+ response and also inhibited HGF-induced Ins 1,4,5-P3 production in rat liver cells. These data indicate that early events in the signal transduction pathways mediated by HGF and EGF have in common the requirements for tyrosine kinase activity, Ins 1,4,5-P3 production, and Ca2+ mobilization.     

Intracellular pH changes of cultured bovine aortic endothelial cells in response to ATP addition

The changes of the intracellular pH (pHi) of cultured bovine aortic endothelial cells were fluorometrically monitored using 2',7'-bis(carboxyethyl)carboxyfluorescein (BCECF). A biphasic pHi change was observed by addition of ATP: an initial acidification followed by an alkalinization of about 0.2 pH unit above the resting level of pHi 7.23. The alkalinization was dependent on [Na+]o and [H+]o, and was inhibited by 5-(N,N-hexamethylene)amiloride, indicating that the alkalinization is mediated by the Na+/H+ exchanger. The 50% effective concentration of ATP was about 1.4 microM. ADP similarly induced pHi changes, whereas AMP and adenosine were inactive. The pHi changes induced by ATP were dependent on the extracellular Ca2+, and the addition of calcium ionophore A23187 induced similar pHi changes. The results indicate that ATP activates the Na+/H+ exchanger in cultured bovine aortic endothelial cells and the activation is mediated by the P2-purinergic receptor and is dependent on the extracellular Ca2+.     

Mechanisms of bombesin-induced arachidonate mobilization in Swiss 3T3 fibroblasts

A peptide mitogen bombesin, which activates the phospholipase C-protein kinase C signaling pathway, induces a mepacrine-sensitive, dose-dependent increase in the release of [3H]arachidonic acid and its metabolites ([3H]AA) from prelabeled Swiss 3T3 fibroblasts. The effect is temporally composed of two phases, i.e. an initial transient burst that is essentially independent of extracellular Ca2+, and a following sustained phase that is absolutely dependent on the extracellular Ca2+. The initial transient [3H]AA liberation occurs concomitantly with bombesin-induced 45Ca efflux from prelabeled cells: both responses being substantially attenuated by loading cells with a Ca2+ chelator quin2. However, bombesin-induced intracellular Ca2+ mobilization by itself is not sufficient as a signal for the initial transient [3H]AA liberation, since A23187 potently stimulates 45Ca efflux to an extent comparable to bombesin but fails to induce [3H]AA release in the absence of extracellular Ca2+. The second sustained phase of the bombesin-induced [3H]AA release is abolished by reducing extracellular Ca2+ to 0.03 mM, although bombesin effects on phospholipase C and protein kinase C activation are barely affected by the same procedure. A protein kinase C activator phorbol 12,13-dibutyrate induces an extracellular Ca(2+)-dependent, slowly developing sustained increase in [3H]AA release, and markedly potentiates both phases of bombesin-induced [3H]AA release. Down-regulation of cellular protein kinase C completely abolishes all of the effects of phorbol dibutyrate, and partially inhibits the second but not the first phase of bombesin-induced [3H]AA release. These results indicate that bombesin-induced receptor-mediated activation of phospholipase A2 involves multiple mechanisms, including intracellular Ca2+ mobilization for the first phase, protein kinase C activation plus Ca2+ influx for the second phase, and as yet unknown mechanism(s) independent of intracellular Ca2+ mobilization or protein kinase C for both of the phases.     

The effects of bombesin on polyphosphoinositide and calcium metabolism in Swiss 3T3 cells

Bombesin, a peptide mitogen for a variety of cell types, acts as a typical Ca2+-mobilizing hormone in Swiss 3T3 fibroblasts. At its mitogenic concentrations (1-25 nM), bombesin stimulates polyphosphoinositide turnover, i.e. breakdown of phosphatidylinositol 4,5-bisphosphate and a concomitant increase in inositol phosphates in a time- and dose-dependent manner. In particular, bombesin induces an initial transient increase in inositol 1,4,5-trisphosphate concentration, followed by an increase in the concentration of inositol 1,3,4-trisphosphate. Also, within 30 s of bombesin addition, the mass of 1,2-diacylglycerol nearly doubles and remains at this level for up to 60 min. Intracellular [Ca2+] measurements with a photoprotein, aequorin, demonstrate that bombesin stimulates a transient rise in cytosolic free Ca2+ concentration. A mobilization of Ca2+ from an intracellular pool is observed as a dose-dependent, transient increase in 45Ca2+ efflux from prelabeled cells, both in the presence and absence of extracellular Ca2+. Bombesin also induces a sustained increase in Ca2+ influx rate and stimulates 3-O-methyl-D-glucose transport across the plasma membrane. These composite results indicate that the mitogenic effect of bombesin is mediated through an activation of the Ca2+ messenger system.     

Chronic desensitization to bombesin by progressive down-regulation of bombesin receptors in Swiss 3T3 cells. Distinction from acute desensitization

Prolonged exposure (40 h) of Swiss 3T3 cells to bombesin induced homologous desensitization to bombesin and structurally related peptides including mammalian gastrin releasing peptide (GRP). The ability of bombesin to mobilize intracellular Ca2+, inhibit epidermal growth factor binding, and stimulate DNA synthesis was profoundly and selectively inhibited. In contrast, Ca2+ mobilization by either vasopressin or bradykinin was unaffected, indicating that chronic desensitization is mechanistically distinct from acute desensitization of Ca2+ mobilization. Prolonged (24 or 40 h) pretreatment with bombesin also induced a 78 +/- 5% loss of bombesin receptor binding sites in both intact and plasma membrane preparations of Swiss 3T3 cells without an apparent change in receptor affinity (Kd = 1.9 +/- 0.1 x 10(-9) M and Kd = 1.8 +/- 0.2 x 10(-9) M for control and pretreated cells, respectively). Loss of 125I-GRP binding was slow and progressive with half-maximal loss of binding occurring after 7 h and maximal after approximately 14 h. Cross-linking of 125I-GRP to intact cultures and membrane preparations revealed an identical time-dependent loss of the Mr = 75,000-85,000 cross-linked band, previously identified as the bombesin receptor. Prolonged exposure of the cells to phorbol 12,13-dibutyrate, epidermal growth factor, cholera toxin, or mitogenic combinations of these agents did not alter 125I-GRP binding. Receptor down-regulation and loss of mitogenic responsiveness to bombesin were: (a) induced in a parallel dose-dependent manner by bombesin (ED50 = 1 nM), GRP (ED50 = 2 nM), and neuromedin B (ED50 = 20 nM), but not by the biologically inactive fragment GRP (1-16); (b) inhibited by the specific bombesin antagonist [Leu13-psi(CH2NH)-Leu14] bombesin, and (c) reversed upon removal of bombesin with a similar time course (full recovery after 15 h). On the basis of these observations, we propose that prolonged pretreatment of Swiss 3T3 cells with bombesin induces homologous desensitization to peptides of the bombesin family by down-regulation of cell surface bombesin receptors.     

Early events elicited by bombesin and structurally related peptides in quiescent Swiss 3T3 cells. II. Changes in Na+ and Ca2+ fluxes, Na+/K+ pump activity, and intracellular pH

The amphibian tetradecapeptide, bombesin, and structurally related peptides caused a marked increase in ouabain-sensitive 86Rb+ uptake (a measure of Na+/K+ pump activity) in quiescent Swiss 3T3 cells. This effect occurred within seconds after the addition of the peptide and appeared to be mediated by an increase in Na+ entry into the cells. The effect of bombesin on Na+ entry and Na+/K+ pump activity was concentration dependent with half-maximal stimulation occurring at 0.3-0.4 nM. The structurally related peptides litorin, gastrin-releasing peptide, and neuromedin B also stimulated ouabain-sensitive 86Rb+ uptake; the relative potencies of these peptides in stimulating the Na+/K+ pump were comparable to their potencies in increasing DNA synthesis (Zachary, I., and E. Rozengurt, 1985, Proc. Natl. Acad. Sci. USA., 82:7616-7620). Bombesin increased Na+ influx, at least in part, through an Na+/H+ antiport. The peptide augmented intracellular pH and this effect was abolished in the absence of extracellular Na+. In addition to monovalent ion transport, bombesin and the structurally related peptides rapidly increased the efflux of 45Ca2+ from quiescent Swiss 3T3 cells. This Ca2+ came from an intracellular pool and the efflux was associated with a 50% decrease in total intracellular Ca2+. The peptides also caused a rapid increase in cytosolic free calcium concentration. Prolonged pretreatment of Swiss 3T3 cells with phorbol dibutyrate, which causes a loss of protein kinase C activity (Rodriguez-Pena, A., and E. Rozengurt, 1984, Biochem. Biophys. Res. Commun., 120:1053-1059), greatly decreased the stimulation of 86Rb+ uptake and Na+ entry by bombesin implicating this phosphotransferase system in the mediation of part of these responses to bombesin. Since some activation of monovalent ion transport by bombesin was seen in phorbol dibutyrate-pretreated cells, it is likely that the peptide also stimulates monovalent ion transport by a second mechanism.     

Cyclosporin A inhibits kinase C-independent activation of the Na+/H+ exchanger by PDGF and vanadate

Mitogen-induced activation of Na+/H+ exchange was studied in Swiss 3T3 fibroblasts. Phorbol myristic acetate (PMA) caused amiloride inhibitable cell alkalinization. PDGF and vanadate, but not bombesin or thrombin, caused additional alkalinization when given 10 min after a maximal dose of PMA. Down-regulation of kinase C by 24 hr PMA exposure prevented the alkalinization response to bombesin and thrombin, but not to PDGF or vanadate. Cyclosporin A specifically blocked the additional alkalinization after PDGF or vanadate in cells acutely exposed to PMA and in kinase C down-regulated cells. Thus, there are at least two independent pathways which activate Na+/H+ exchange. PMA, bombesin, and thrombin act via kinase C. PDGF and vanadate cause additional stimulation of the Na+/H+ exchanger by a kinase C-independent pathway, inhibitable by cyclosporin A.     

Extracellular superoxide dismutase in the vascular system of mammals.

NIH 3T3 cells, which express a small number of EGF (epidermal growth factor) receptors, are poorly responsive to EGF. However, when the same cells overexpress the cloned human EGF receptor (EGFR T17 cells), they display EGF-dependent transformation. In EGFR T17 cells (but not in the parental NIH 3T3 cells), EGF is shown here to trigger polyphosphoinositide hydrolysis as well as the generation of the ensuing intracellular signals, the increase in the cytosolic Ca2+ concentration ([Ca2+]i) and pH. EGF induced a large accumulation of inositol 1,4,5-trisphosphate, with a peak at 15-30 s and a slow decline thereafter. Other inositol phosphates (1,3,4-trisphosphate and 1,3,4,5-tetrakisphosphate) increased less rapidly and to a lesser degree. [Ca2+]i increased after a short lag, reached a peak at 25 s and remained elevated for several minutes. By use of incubation media with and without Ca2+, the initial phase of the EGF-induced [Ca2+]i increase was shown to be due largely to Ca2+ release from intracellular stores. In contrast with previous observations in human A431 cells, the concentration-dependence of the EGF-triggered [Ca2+]i increase in EGFR T17 cells paralleled that of [3H]thymidine incorporation. It is concluded that polyphosphoinositide hydrolysis, [Ca2+]i increase and cytoplasmic alkalinization are part of the spectrum of intracellular signals generated by the activation of one single EGF receptor type. These processes might be triggered by the receptor via activation of the intrinsic tyrosine kinase activity. Large stimulation of DNA synthesis and proliferation by EGF in EGFR T17 cells could be due to a synergistic interplay between the two signal pathways initiated by tyrosine phosphorylation and polyphosphoinositide hydrolysis.     

Thrombin-induced changes of intracellular [Ca2+] and pH in human platelets. Cytoplasmic alkalinization is not a prerequisite for calcium mobilization

We have studied the effects of thrombin (0.1 U/ml) on intracellular Ca2+ ([Ca2+]i) and pH (pHi) in human platelets loaded with fluorescent indicators. Thrombin produced a transient decrease of pHi which reached its maximum within 15-25 seconds (s) and was followed by a sustained alkalinization which brought pHi above the resting value. [Ca2+]i increased transiently peaking at 5-10 s. The late alkalinization induced by thrombin was antagonized by ethylisopropylamiloride, an inhibitor of Na+-H+ exchange, and by sphingosine, an inhibitor of protein kinase C, with little effect on the [Ca2+]i transient. The early acidification was not inhibited by these treatments. We conclude tha the thrombin-induced changes of [Ca2+]i and pHi are mediated by different mechanisms. The late alkalinization is due to activation of Na+/H+ exchange mediated by protein kinase C and, contrarily to previous proposals (Siffert, W. and Akkerman, J.W.N. (1987) Nature 325, 456-458), it is not necessary for calcium mobilization from intracellular stores.