Cell-to-cell variation in store-operated calcium entry in HEK-293 cells and its impact on the interpretation of data from stable clones expressing exogenous calcium channels.

To investigate the effect of cell-to-cell variation in store-operated calcium entry (SOCE) on the evaluation of data from stable cell clones selected following gene transfection, we measured SOCE in 2700 individual HEK-293 cells from the parent population and in 1900 individual cells from a clonal subpopulation of HEK-293 cells. We applied statistical resampling techniques to model conditions where one would compare the average SOCE in n control clones to the average SOCE in n experimental clones (n = 1-200). For an overexpression experiment with n = 1, there is a 27% chance of observing a 100% or higher difference in SOCE between clones, with n = 10 there is a 34% probability of observing a 20% or greater difference in SOCE, and with n = 100, there is less than a 10% chance of seeing a 10% or greater difference in SOCE, based solely on random selection of clones from the parent HEK-293 cell population. To assure that the degree of cell-to-cell variation was predictive of the degree of clone-to-clone variation, we measured SOCE in 270 clones, each arising from a single cell, and found the variation to be very similar to that observed for individual cells.

Functional significance of human trp1 and trp3 in store-operated Ca(2+) entry in HEK-293 cells.

The Drosophila trp (transient receptor potential) gene appears to encode the Drosophila store-operated channel (SOC), and some mammalian trp homologues have been proposed to encode mammalian SOCs. This study provides evidence for the expression of three trp homologues (Mtrp2, Mtrp3, and Mtrp4) in fibroblasts from wild-type and src knockout mice, and four trp homologues (Htrp1, Htrp3, Htrp4, and Htrp6) in human embryonic kidney (HEK-293) cells based on RT-PCR techniques. In HEK-293 cells stably transfected with a 323-bp Htrp3 antisense construct (Htrp3AS), Northern blot analysis revealed that the expression of a 4-kb transcript was dramatically suppressed in comparison to that observed in cells stably transfected with a short Htrp3 sense construct (Htrp3S). Activity of SOCs, monitored as Ba(2+) influx following Ca(2+) store depletion with thapsigargin, was reduced by 32% in Htrp3AS cells in comparison with Htrp3S cells. Transient transfection of a 369-bp Htrp1 antisense construct in cells stably expressing Htrp3AS induced a higher level of inhibition (55%) of store-operated Ca(2+) entry. These data suggest that Htrp1 and Htrp3 may be functional subunits of SOCs.

The role of pp60c-src in the regulation of calcium entry via store-operated calcium channels.

In many cell types, G protein-coupled receptors stimulate a transient Ca2+ release from internal stores followed by a sustained, capacitative Ca2+ entry, which is mediated by store-operated channels (SOCs). Although it is clear that SOCs are activated by depletion of internal Ca2+ stores, the mechanism for this process is not well understood. Previously, we have reported that inhibitors of tyrosine kinase activity block the bradykinin- and thapsigargin-stimulated Ca2+ entry in fibroblasts, suggesting that a tyrosine kinase activity may be involved in relaying the message from the empty internal Ca2+ stores to the plasma membrane Ca2+ channel (Lee, K.-M., Toscas, K., and Villereal, M. L. (1993) J. Biol. Chem. 268, 9945-9948). We also have demonstrated that bradykinin activates the nonreceptor tyrosine kinase c-src (Lee, K.-M., and Villereal, M. L. (1996) Am. J. Physiol. 270, C1430-C1437). We investigated whether c-src plays a role in the regulation of SOCs by monitoring capacitative Ca2+ entry in 3T3-like embryonic fibroblast lines derived from either wild type or src-/src- (Src-) transgenic mice. We report that Ca2+ entry, following store depletion by either bradykinin or thapsigargin, is dramatically lower in Src- fibroblasts than in wild type fibroblasts. The level of capacitative Ca2+ entry in Src- cells is restored to nearly normal levels by transfecting Src- cells with chicken c-src. These data suggest that c-src may play a major role in the regulation of SOCs.

Examination of the role for Ca2+ in regulation and phosphorylation of the Na+/H+ antiporter NHE1 via mitogen and hypertonic stimulation.

It has been suggested that Ca2+ transients, acting through calmodulin-binding proteins, play a role in the activation of the Na+/H+ exchanger isoform NHE1 (Owen and Villereal, 1982a, Biochem. Biophys. Res. Commun., 109:762-768; 1982b, Proc. Natl. Acad. Sci. U.S.A., 79:3537-3541, Ober and Pardee, 1987, J. Cell. Physiol., 132:311-317). This is supported by a recent report that NHE1 is a calmodulin-binding protein and that loss of the high-affinity calmodulin-binding site results in alterations in antiporter function (Bertrand, et al., 1994, J. Biol. Chem., 269:13703-13709). An additional mechanism by which NHE1 is activated by mitogens is thought to be phosphorylation (Sardet, et al., 1990, Science 247:723-726). Although the calmodulin-binding region appears vital to antiporter activation, the role of phosphorylation is unclear. The studies presented here examine a role for Ca2+ in the activation and phosphorylation of NHE1 induced by serum and hypertonicity. It is apparent that the microsomal Ca2+ ATPase inhibitor thapsigargin activates antiporter function in human foreskin fibroblasts (HSWP) as determined by increased intracellular alkalinization examined by image analysis. This effect is Ca(2+)-dependent as the alkalinization is blocked when cells are preincubated with BAPTA, an intracellular Ca2+ chelator. Similarly, the effects of serum-induced intracellular alkalinization are inhibited by BAPTA. In contrast, activation of NHE1 by increased osmolarity was not inhibited by BAPTA. This suggests that serum, and not hypertonicity, increases intracellular pH via a Ca(2+)-dependent process. It was also observed that both thapsigargin and hypertonicity activate NHE1 by a phosphorylation-independent mechanism and that BAPTA did not block the serum-induced increase in phosphorylation of NHE1. These results indicate that Ca2+ plays the predominant role in the serum-induced activation of NHE1, while phosphorylation plays only a minor, if any, role in this process. However, Ca2+ does not appear to be involved in the osmotic regulation of NHE1.

Tyrosine phosphorylation and activation of pp60c-src and pp125FAK in bradykinin-stimulated fibroblasts.

Bradykinin (BK) stimulates protein tyrosine phosphorylation in human foreskin fibroblasts (K.-M. Lee, K. Toscas, and M. L. Villereal, J. Biol. Chem. 268:9945-9948, 1993). The major tyrosine phosphorylation occurs in proteins of a molecular mass of 130 and 70 kDa. In this report, we demonstrate that focal adhesion-associated tyrosine kinase, pp125FAK, is one component of the 130-kDa phosphotyrosine band. The BK-stimulated pp125FAK tyrosine phosphorylation level is well correlated with increased kinase activity, as assessed by in vitro immune complex kinase assays. We have identified paxillin, a protein that is localized in focal adhesions, as a component of the 70-kDa phosphotyrosine band. In addition to identifying the two proteins responsible for the major phosphotyrosine bands, we also report that pp60c-src is tyrosine phosphorylated and activated in response to BK, as analyzed by immunoblotting and in vitro kinase assays, respectively. These findings indicate, for the first time, that the BK receptor is coupled to the important protooncogene c-src and that the src pathway may mediate some of the events downstream from BK binding.

Multiple intracellular Ca2+ pools exist in human foreskin fibroblast cells: the effect of BK on release and filling of the non-cytosolic Ca2+ pools.

Previously, we have used the classical approach to examine intracellular calcium stores in human foreskin fibroblasts (HSWP) cells. In this classical protocol cells are first permeabilized and then allowed to fill their Ca2+ reservoirs with 45Ca2+ in the presence of ATP. In this paper we present an alternative method to examine intracellular calcium pools. In this alternate protocol, whole cells are loaded to isotopic steady-state with 45Ca2+ and then permeabilized using digitonin. Comparison of the Ca2+ content of cells treated with these two methodologies reveals that cells treated with the alternate protocol have a Ca2+ content 3 orders of magnitude higher than those treated with the classical protocol. Using this alternative technique we demonstrate that there are 3 intracellular calcium pools in HSWP cells. These pools are: (i) an IP3-sensitive, thapsigargin-sensitive Ca2+ pool; (ii) an IP3-insensitive, thapsigargin-sensitive Ca2+ pool; and (iii) an ionomycin sensitive Ca2+ pool. The relationship between the Ca2+ pool mobilized by BK treatment and by IP3 treatment is also explored. Microinjection data shown here suggest that IP3 can mobilize all of the intracellular Ca2+ mobilized by BK. However, in the permeabilized system BK pretreatment followed by IP3 treatment can release more Ca2+ than can be release by IP3 treatment alone. We suggest one plausible explanation for this observation: when cells are treated using the alternative permeabilization protocol, communication occurs between an IP3-sensitive and an IP3-insensitive calcium pool. Thus BK pretreatment would empty the IP3-sensitive Ca2+ pool. This pool would subsequently be refilled with Ca2+ from a previously untapped, IP3-insensitive, Ca2+ reservoir and more Ca2+ would be available for subsequent release by IP3 treatment.

Expression and phosphorylation of NHE1 in wild-type and transformed human and rodent fibroblasts.

Activation and phosphorylation of the Na+/H+ exchanger occurs with a diverse group of mitogens such as phorbol myristate acetate (PMA), epidermal growth factor (EGF), thrombin and serum (Sardet et al., 1990, Science, 247:723-726). Some of these growth factors have been shown to differentially activate the Na+/H+ exchanger in various fibroblasts (Etscheid et al., 1990, Am. J. Physiol., 259:C549-C556; Muldoon, L.L., et al., 1987, Am. J. Physiol., 253:C219-C229). However, alterations in the expression and phosphorylation of NHE1 in various fibroblasts has not been examined with respect to a potential mechanism of differential activation of the exchanger. To pursue this question, a novel antibody, anti-XB17, directed to the cytoplasmic tail of NHE1 was characterized and then utilized to examine the expression of NHE1 protein and the level of phosphorylation of the serum stimulated exchanger in human embryonic lung fibroblasts (WI-38), SV40-transformed WI-38, and nontransformed human foreskin fibroblasts (HSWP) cells. The level of mRNA expressed in these cells was also examined. Results indicate that the parental cell lines and other nontransformed fibroblasts express NHE1. Although the transformed cell lines express NHE1 mRNA in approximately similar abundance to the parental lines, they contain decreased quantity of NHE1 exchanger/mg membrane protein as recognized by anti-XB17 Ab. The mechanism that results in the apparent decrease in NHE1 protein levels in the transformed cells is not known. Also, the SV40-transformed cells express an exchanger with a higher apparent molecular weight. The WI-38 cells demonstrate phosphorylation of NHE1 in response to mitogenic stimulation. ALthough the nontransformed HSWP cells have a high level of Na+/H+ exchanger protein, they do not show a significant increase in phosphorylation following serum stimulation, when examined by immunoprecipitation, and analysis on 1-D gels. However, subsequent studies of tryptic phosphopeptides from the immunoprecipitated exchanger reveal that serum-stimulated phosphorylation of one tryptic peptide does occur but may be masked in the first dimension by differential phosphorylation of other tryptic peptides that are more heavily phosphorylated in unstimulated cells.

Inhibition of bradykinin- and thapsigargin-induced Ca2+ entry by tyrosine kinase inhibitors.

We examined the involvement of tyrosine kinase activity in the bradykinin (BK)-mediated signal transduction process. Immunoblots with anti-phosphotyrosine antibodies following BK stimulation of human fibroblasts showed tyrosine phosphorylation of specific proteins that could be inhibited by the tyrosine kinase inhibitors genistein and tyrphostin. Image analysis data from individual cells stimulated by BK in the presence of genistein and tyrphostin showed that these inhibitors block the plateau phase but not the rapid transient phase of the BK-induced calcium response. That the loss of the plateau phase was due to blockage of calcium entry rather than stimulation of calcium pump activity was confirmed by examining the influx of Ba2+ following BK stimulation. The Ca2+ imaging results were confirmed by 45Ca2+ uptake measurements and extended to another tyrosine kinase inhibitor (methyl 2,5-dihydroxycinnamate), which was found to interfere with the fura-2 signal and therefore could not be used in imaging experiments. The fact that three structurally distinct inhibitors of tyrosine kinase activity inhibited BK-stimulated calcium influx, while an inactive analogue of genistein (daidzein) did not, strongly suggests the involvement of tyrosine kinases in the regulation of a BK-induced calcium entry pathway. To our knowledge, this is the first report of tyrosine kinase involvement in the regulation of calcium entry following activation of a receptor that lacks endogenous tyrosine kinase activity and is known to be coupled to phosphatidylinositol turnover. We found that calcium entry in HSWP (human foreskin fibroblast) cells can also be stimulated by emptying the intracellular calcium stores with thapsigargin. Genistein also inhibits the plateau phase of the thapsigargin-induced calcium response while leaving the transient phase intact. This suggests that the Ca2+ influx pathway induced by depletion of intracellular calcium stores with thapsigargin also may be regulated via a tyrosine kinase pathway.

Dihydropyridine-sensitive L-type Ca2+ channels in human foreskin fibroblast cells. Characterization of activation with the growth factor Lys-bradykinin.

We have previously characterized the calcium response of cultured human fibroblasts (HSWP cells) to stimulation by the mitogen Lys-bradykinin (BK). We have reported a biphasic response which includes a rapid rise to a peak that appears to result from mobilization of internal calcium, and a plateau phase, which is due to influx of external calcium (Byron, K., Babnigg, G., Villereal, M. L. (1992) J. Biol. Chem. 267, 108-118). In this paper we examine participation of L-type voltage operated calcium channels in the calcium entry phase of BK-stimulated HSWP cells. We show that there is an increase in 45Ca2+ uptake and an increase in intracellular free calcium concentration ([Ca2+]i) as measured by fura-2, when HSWP cells are stimulated with the L-channel agonist Bay K 8644 under depolarizing conditions. Furthermore, both of these effects are inhibited by low doses of the dihydropyridine antagonist nitrendipine. We also report that BK stimulation of 45Ca2+ uptake can be significantly inhibited by low doses of nitrendipine, while nitrendipine treatment has no effect on the BK-induced rise in [Ca2+]i, as measured by fura-2. These results suggest that under normal conditions the portion of the BK-stimulated Ca2+ influx which is mediated by a nitrendipine-sensitive entry pathway is invisible to the fura-2 technique used to measure [Ca2+]i. This suggest that the nitrendipine-sensitive influx pathway admits calcium preferentially into an intracellular store that is isolated from fura-2. This idea is supported by the observation that in media where calcium has been replaced by 2 mM Ba2+ nitrendipine inhibits most of the BK-stimulated Ba2+ influx.

Calcium signals in growth factor signal transduction.

There is a substantial amount of information which has been obtained concerning the effects of growth factors on [Ca2+]i in proliferating cells. A number of different mitogens are known to induce elevations in [Ca2+]i and some characterization of the Ca2+ response to different classes of mitogens has been obtained. In addition, much is known about whether the Ca2+ response to a particular growth factor occurs as the result of an influx of external Ca2+ or a mobilization of internal Ca2+ stores. In addition, a considerable amount of information is available on the mechanism by which the Ins(1,4,5)P3-sensitive internal Ca2+ store takes up and releases Ca2+. However, there is still a large deficiency in our information concerning other Ca2+ stores in proliferating cells as well as in our knowledge of the mechanisms for regulating Ca2+ entry pathways. Much more data addressing these issues exists for other types of agonist-stimulated cells, and we have discussed much of it in this review article. While the wealth of data in nonproliferating cells provides some indications of what mechanisms might be involved in the growth factor-induced changes in [Ca2+]i, it is clear that much work must be done in proliferating cells to fully understand how external factors such as growth factors control [Ca2+]i. In addition, much work remains to be done in identifying the mechanisms for the internal control of [Ca2+]i as cells move through the cell cycle and in identifying the role that these changes in [Ca2+]i may play throughout the cell cycle.

Bradykinin-induced Ca2+ entry, release, and refilling of intracellular Ca2+ stores. Relationships revealed by image analysis of individual human fibroblasts.

Lys-Bradykinin (BK), a mitogen for human foreskin fibroblasts (HSWP cells) (Owen, N. E., and Villereal, M. L. (1983) Cell 32, 979-985), elicits a rapid, transient elevation of intracellular free Ca2+ concentration ([Ca2+]i) in these cells. We have used image analysis of fura-2-loaded HSWP cells to examine the BK-induced [Ca2+]i changes in individual cells. BK-stimulated Ca2+ entry and release of intracellular Ca2+ stores can be distinguished by stimulating cells in the presence or absence of extracellular Ca2+, or by inhibiting Ca2+ entry with 5 mM NiCl2. BK-sensitive intracellular Ca2+ stores can be depleted by exposure of the cells to BK in Ca(2+)-free medium; refilling of the stores requires extracellular Ca2+. A component of BK-stimulated Ca2+ entry persists after removal of agonist, but inactivates with a t1/2 of approximately 5 min. Although previous studies have attributed the Ca2+ entry which persists after agonist removal to a "capacitative Ca2+ entry" pathway activated by the depletion of the intracellular Ca2+ stores, we find that a large component of this BK-stimulated Ca2+ entry is not due to capacitative Ca2+ entry since (1) ionomycin can deplete the BK-sensitive intracellular Ca2+ stores without appreciably stimulating Ca2+ entry and without inhibiting the BK-stimulated Ca2+ entry and (2) this Ca2+ entry pathway inactivates at a time when the Ca2+ pools are still empty and a capacitance entry pathway should still be open. On the other hand, refilling of the intracellular Ca2+ stores can occur after the noncapacitative Ca2+ entry component has inactivated or when it is inhibited by Ni2+; in these cases refilling occurs without a detectable elevation of [Ca2+]i suggesting that refilling of internal Ca2+ pools might occur by a capacitative route.

Regulation of bradykinin receptor level by cholera toxin, pertussis toxin and forskolin in cultured human fibroblasts.

1. The effect of bacterial toxins on bradykinin-triggered release of arachidonic acid was studied in serum-deprived human foreskin (HSWP) fibroblasts prelabelled with [3H]-arachidonic acid. An 18-h exposure of HSWP cells to cholera toxin, pertussis toxin, or forskolin enhanced the bradykinin-stimulated release of arachidonic acid and metabolites. 2. Prolonged treatment of HSWP cells with these agents also caused a 3 to 4 fold rise in cell surface [3H]-bradykinin binding. The rise was inhibited by concurrent incubation with cycloheximide or actinomycin D. In addition, cholera toxin and foreskolin increased [3H]-bradykinin binding in wildtype PC12 cells, but not in mutant PC12 cells with reduced cyclic AMP-dependent protein kinase type II activity. 3. In conclusion, cholera toxin, pertussis toxin and forskolin enhanced arachidonic acid release in response to bradykinin, and increased the number of bradykinin receptors in HSWP fibroblasts. A cyclic AMP-dependent mechanism appears to mediate the actions of the toxins and forskolin.

Transduction of the bradykinin response in human fibroblasts: prolonged elevation of diacylglycerol level and its correlation with protein kinase C activation.

Stimulation of quiescent human fibroblasts with the peptide mitogen bradykinin (BK) led to a biphasic elevation in cellular 1,2-diacylglycerol (DAG), as estimated by either measurement of total DAG mass or [3H]arachidonate incorporation. A rapid initial transient that peaked 15 s after BK addition was followed by a decline to near basal levels then a second rise to a plateau phase during which DAG levels remained elevated for less than or equal to 45 min. The source of the initial DAG transient appeared to be primarily polyphosphoinositides as these phospholipids were rapidly hydrolyzed after BK addition. This transient correlates well temporally with previous observations of the kinetics of inositol trisphosphate accumulation and intracellular free [Ca2+] observed in the same cells. Cultures preincubated with [3H]myristic acid incorporated label predominantly into the phosphatidylcholine (PC) pool. Subsequent addition of BK under these conditions caused only a relatively slow accumulation of [3H]DAG to a plateau level, without an initial transient. Together with the observation that PC was found to decrease upon BK stimulation, these observations suggest that the late phase of DAG accumulation may involve breakdown of other phospholipids including PC. To investigate the consequences of DAG elevation we examined the phosphorylation of an acidic 80 kDa protein, whose phosphorylation is solely dependent on the activation of protein kinase C (PK-C). The 80 kDa fibroblast protein could be immunoprecipitated by an antibody to bovine brain "myristoylated and alanine-rich C-kinase substrate" (MARCKS) and phosphopeptide maps of brain and fibroblast MARCKS were similar. Stimulation of [32P]-prelabeled fibroblasts with serum, BK, vasopressin, or 12-O-tetradecanoyl phorbol acetate, but not epidermal growth factor or calcium ionophores, resulted in the rapid phosphorylation of MARCKS. With BK or serum this phosphorylation showed an initial transient peak at less than 1 min then rose again to a plateau level that was sustained for less than or equal to 45 min. Removal of BK resulted in a rapid decline in MARCKS phosphorylation. These studies show that the biphasic DAG signal in BK-stimulated human fibroblasts correlates well with the state of activation of PK-C. However, the persistent activation of PK-C does not appear to require continued high levels of Ca2+.

Loss of bradykinin receptors and TPA-stimulated Na+ influx in SV40-transformed WI-38 cells.

Mitogenic stimulation of Na(+)-H+ exchange activity, as defined by the level of 5-(N,N-hexamethylene)amiloride (HMA)-sensitive Na+ influx, was compared in WI-38 and SV40 virus-transformed WI-38 fibroblasts. Serum or bradykinin dramatically stimulated HMA-sensitive Na+ influx in WI-38 cells, whereas in SV40-transformed WI-38 cells, serum, but not bradykinin, produced a large increase in HMA-sensitive Na+ influx. This lack of a bradykinin response was traced to a dramatic reduction in the number of bradykinin receptors, from 470 fmol/mg protein in WI-38 cells to 29 fmol/mg protein in the SV40-transformed WI-38 cells. Transformation of WI-38 cells with SV40 virus also altered the mechanism by which HMA-sensitive Na+ influx is stimulated. In WI-38 cells, 12-O-tetradecanoylphorbol 13-acetate (TPA) dramatically stimulated HMA-sensitive Na+ influx. In SV40-transformed WI-38 cells, TPA alone had no effect on HMA-sensitive influx and inhibited serum-stimulated HMA-sensitive Na+ influx. Down-regulation of protein kinase C activity decreased serum- and TPA-stimulated HMA-sensitive Na+ influx in the WI-38 cells and relieved the TPA inhibition of serum-stimulated HMA-sensitive Na+ influx in the SV40-transformed WI-38 cells.

Structure-activity studies of the nonphorbol tumor promoter palytoxin in Swiss 3T3 cells.

Derivatives of palytoxin have been prepared which are modified on either the hydroxyl terminus or the amino terminus of the molecule. Previously we have shown that palytoxin, a non-12-O-tetradecanoylphorbol-13-acetate-type tumor promoter, can inhibit epidermal growth factor binding in Swiss 3T3 cells through a pathway which is sodium dependent but not calcium or protein kinase C dependent. We used the epidermal growth factor receptor system to determine whether the specific chemical modifications of palytoxin present in these derivatives alter the cellular mechanism of action of the toxin. The dose response and ion dependence of palytoxin, the hydroxyl terminus derivative palytoxin-COOH, and the amino terminus derivatives N-acetylpalytoxin and N-(p-bromobenzoyl)palytoxin were compared with respect to inhibition of epidermal growth factor binding. The potency of palytoxin-COOH was similar to that of palytoxin. By contrast, N-acetylpalytoxin and N-(p-bromobenzoyl)palytoxin were approximately 1/100 as potent as palytoxin in this assay. All three derivatives were at least 100-fold less toxic than palytoxin. Like palytoxin, the activities of palytoxin-COOH, N-acetylpalytoxin and N-(p-bromobenzoyl)palytoxin were dependent upon the presence of extracellular sodium. However, there was a significant difference in the dependence of the derivatives on extracellular calcium. Our results suggest that the hydroxyl terminus is important for determining the calcium dependence of the molecule and the amino terminus is important for determining the biological potency of palytoxin. We conclude that modification of the hydroxyl terminus region is an effective means of reducing the toxicity of palytoxin while retaining the biological effects.

Mitogen-induced [Ca2+]i changes in individual human fibroblasts. Image analysis reveals asynchronous responses which are characteristic for different mitogens.

Temporal changes in intracellular free Ca2+ concentration ([Ca2+]i) in cultured human foreskin fibroblasts were investigated using image analysis techniques to simultaneously monitor Lys-bradykinin (BK)- or thrombin-induced elevations of [Ca2+]i in each individual cell within a microscopic field. Responses to BK are heterogeneous with respect to the shapes of the [Ca2+]i time courses. Furthermore, the onsets of these responses follow a variable lag period such that the individual cell responses occur asynchronously. The asynchrony and heterogeneity of individual cell responses are not related to cell cycle differences since noncycling cells respond in a similar manner. When cells are ranked according to order of an initial response to BK (the first cell to respond is ranked 1, the second to respond is 2, etc.), restimulation of the same cells with BK elicits a similar order of cell responses, and the shape of the [Ca2+]i time course of an individual cell is similar for both responses to BK. If cells that were stimulated with BK are washed and restimulated with thrombin (which produces [Ca2+]i changes similar to those induced by BK), the response order to thrombin does not correspond to the response order following BK stimulation. These data suggest that the asynchrony of [Ca2+]i changes induced by BK or thrombin is characteristic for each mitogen and may be determined by cell-to-cell variation in receptor number.

Sodium as a mediator of non-phorbol tumor promoter action. Down-modulation of the epidermal growth factor receptor by palytoxin.

Previous studies have shown that palytoxin, a non-(12-O-tetradecanoylphorbol-13-acetate)-type tumor promoter, is able to down-modulate the epidermal growth factor (EGF) receptor through a sodium-dependent pathway in Swiss 3T3 cells. A role for sodium is supported by the observation that the sodium proton exchanger monensin and the sodium-conducting ionophore gramicidin mimic palytoxin action by causing a decrease in both high and low affinity EGF binding. However, in addition to causing sodium influx, these agents can induce other cellular effects including changes in membrane polarization, intracellular pH, and macromolecular synthesis. To determine whether any of these factors might be responsible for palytoxin action in our system, we examined the role of each of them in palytoxin-induced inhibition of EGF binding. Although palytoxin depolarizes the membrane, the observation that potassium-induced depolarization of the membrane does not cause a decrease in EGF binding, in conjunction with the fact that monensin hyperpolarizes the membrane, indicates that depolarization of the membrane is not responsible for palytoxin-induced changes in the EGF receptor. An investigation of intra-cellular pH suggests that the palytoxin effects are not mediated by proton flux. In addition, nigericin-mediated changes in intracellular pH do not cause an inhibition of EGF binding. Finally, studies conducted in the presence of cycloheximide indicate that protein synthesis is not required for palytoxin action and that inhibition of EGF receptor biosynthesis does not account for palytoxin-induced loss of EGF-binding sites. These results suggest that sodium may act as a second messenger in the signal transduction mechanism by which palytoxin modulates the EGF receptor.

Coupling of bradykinin receptors to phospholipase C in cultured fibroblasts is mediated by a G-protein.

In cultured foreskin fibroblasts, bradykinin stimulates inositol phosphate generation, arachidonic acid release, and Na+/H+ exchange, with doses of 1-3 nM yielding half-maximal stimulation. Binding of 3H-bradykinin to these cells demonstrates a single receptor site with a Kd of 2.0 nM and a Bmax of 91 fmoles/mg protein. Bradykinin analogs of the B2 type inhibit this binding. GTP synergizes with bradykinin to stimulate phosphatidylinositol turnover in permeabilized fibroblasts and GTP-gamma-S decreases the Bmax of bradykinin binding to fibroblast membranes, indicating that a G-protein couples the receptor to phospholipase C. Pretreatment of fibroblasts with either cholera or pertussis toxin enhances bradykinin stimulation of inositol phosphate accumulation.

Multiple intracellular pathways induce expression of a zinc-finger encoding gene (EGR1): relationship to activation of the Na/H exchanger.

Intracellular pathways that rapidly stimulate the expression of a mitogen-inducible, zinc-finger encoding gene, EGR1 (Sukhatme et al., Cell 53:37-43), have been characterized in two human fibroblasts strains (WI-38 and HSWP). Serum and epidermal growth factor (EGF) were each found to strongly stimulate EGR1 expression in both cell types. Comparably high levels of expression could also be induced by treatment with the phorbol ester TPA. In cells rendered deficient in PK-C, serum and EGF were each still capable of inducing high levels of EGR1 mRNA, demonstrating that additional non-protein kinase C pathways are capable of stimulating EGR1 expression. In both fibroblasts strains, stimulation of EGR1 expression by all these agents exhibited rapid, transient kinetics and could be superinduced if protein synthesis was inhibited through the addition of cycloheximide. Finally, various agents, known to stimulate/inhibit the activation of another early mitogenic response, the activation of Na/H exchange, were analyzed for their effect on EGR1 expression. Interestingly bradykinin, vasopressin, and Ca ionophores, which dramatically stimulate Na/H exchange, were only weak stimulants of EGR1 expression. Conversely, EGF, which stimulates Na/H exchange poorly, strongly activated EGR1 expression. Hence while EGR1 expression could be triggered by multiple intracellular pathways, its expression does not appear to require the prior activation of Na/H exchange.