Regulation of the cell swelling-activated chloride conductance by cholesterol-rich membrane domains.

AIM: The role of high cholesterol-containing microdomains in the signal transduction cascade leading to the activation of volume-regulated anion channels (VRACs) was studied. METHODS: Osmotic cell swelling-induced efflux of 125I- was determined in human epithelial Intestine 407 cells and in skin fibroblasts obtained from healthy controls or Niemann-Pick type C (NPC) patients. Cellular cholesterol content was modulated by pre-incubation with 2-hydroxypropyl-beta-cyclodextrin in the presence of acceptor lipid vesicles. RESULTS: Osmotic cell swelling of human Intestine 407 cells leads to the rapid activation of a compensatory anion conductance. Treatment of the cells with cyclodextrin enhanced the response to submaximal hypotonic stimulation by approx. twofold, but did not further increase the efflux elicited by a saturating stimulus. In contrast, the volume-sensitive anion efflux was markedly inhibited when cholesterol-loaded cyclodextrin was used. Potentiation of the response by cholesterol depletion was maintained in caveolin-1 deficient Caco-2 colonocytes as well as in sphingomyelinase-treated Intestine 407 cells, indicating that cholesterol-rich microdomains are not crucially involved. However, treatment of the cells with progesterone, an inhibitor of NPC1-dependent endosomal cholesterol trafficking, not only markedly reduced the hypotonicity-provoked anion efflux, but also prevented its potentiation by cyclodextrin. In addition, the volume-sensitive anion efflux from human NPC skin fibroblasts was significantly smaller when compared with control fibroblasts. CONCLUSIONS: The results support a model of regulatory volume decrease involving recruitment of volume-sensitive anion channels from intracellular compartments to the plasma membrane.

Signalling mechanisms involved in volume regulation of intestinal epithelial cells.

Most mammalian cells have developed compensatory mechanisms to respond to the variable osmotic stress caused by changes in the concentrations of intracellular osmo-active substances (e.g. glucose, amino acids, lactate) or by variations in the osmolarity of the surrounding medium. In response to osmotic cell swelling, the Regulatory Volume Decrease (RVD) is triggered and directs a reduction in the tonicity of the cell by the concerted opening of cation and anion selective ion channels. To date, the K(+) and Cl(-) conductances activated upon hypo-osmotic stimulation have been characterised electrophysiologically in many different cell systems. The molecular identity of the channels however, as well as the mechanism(s) involved in their activation have not yet been fully clarified and may differ between cell types. In this review, we will evaluate the different signalling pathways activated by osmotic cell swelling and discuss their putative role(s) in ion channel regulation, in maintaining cellular volume homeostasis, and in auto- and paracrinic signal transduction, with emphasis on intestinal epithelial cells.

Protein N-myristoylation: critical role in apoptosis and salt tolerance.

N-myristoylation is a covalent protein modification that can promote the association of proteins with membranes. De Jonge, Hogema, and Tilly discuss how N-myristoylation may be involved in triggering Fas ligand-induced apoptosis in mammals, and in adapting to conditions of high salt in plants. The pro-apoptotic protein BID is unique in that its proteolytic cleavage product, tBID, is posttranslationally myristoylated. In contrast, the plant accessory protein SOS3 undergoes "classical" cotranslational N-myristoylation. N-myristoylation is essential for the proper functioning of these proteins in regulating the signaling pathways (apoptosis and adaptation to salt stress, respectively) in which they are involved.

Osmotic cell swelling-induced ATP release mediates the activation of extracellular signal-regulated protein kinase (Erk)-1/2 but not the activation of osmo-sensitive anion channels.

Human intestine 407 cells respond to hypo-osmotic stress by the rapid release of ATP into the extracellular medium. A difference in the time course of activation as well as in the sensitivity to cytochalasin B treatment and BAPTA-AM [1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid acetoxymethyl ester] loading suggests that ATP leaves the cell through a pathway distinct from volume-regulated anion channels. To evaluate a putative role for nucleotides as autocrinic/paracrinic factors in osmotic signalling, the effects of extracellular ATP on the regulation of volume-sensitive anion channels as well as on the hypotonicity-induced activation of extracellular signal-regulated protein kinases (Erk-1/2) were investigated. Micromolar concentrations of ATP were unable to elicit an isotope efflux from (125)I(-)-loaded cells by itself, but strongly potentiated the hypotonicity-provoked anion efflux through a Ca(2+)-dependent mechanism. The order of potency of nucleotides (ATP = UTP = ATP[S] > ADP = AMP >> adenosine = cAMP) indicated the involvement of P2Y(2) receptors. In contrast, millimolar concentrations of ATP markedly inhibited both the osmotically induced isotope efflux and whole-cell Cl(-) currents. Inhibition of whole-cell Cl(-) currents, not only by millimolar ATP but also by the purinoceptor antagonists suramin and reactive blue, was observed most prominently at depolarizing holding potentials, suggesting a direct interaction with volume-sensitive Cl(-) channels rather than interaction with purinoceptors. Both ATP and UTP, at submicromolar levels, were found to act as potent activators of Erk-1/2 in intestine 407 cells. Addition of the ATP hydrolase apyrase to the bath greatly reduced the hypotonicity-induced Erk-1/2 activation, but did not affect the swelling-induced isotope efflux or whole-cell Cl(-) currents. Furthermore, pre-treatment with suramin or reactive blue almost completely prevented the hypo-osmotic activation of Erk-1/2. The results indicate that extracellularly released ATP functions as an autocrinic/paracrinic factor that mediates hypotonicity-induced Erk-1/2 activation but does not serve as an activator of volume-sensitive compensatory Cl(-) currents.

Osmotic swelling-induced activation of the extracellular-signal-regulated protein kinases Erk-1 and Erk-2 in intestine 407 cells involves the Ras/Raf-signalling pathway.

Human Intestine 407 cells respond to hypo-osmotic stress with a rapid stimulation of compensatory ionic conductances accompanied by a transient increase in the activity of the extracellular-signal-regulated protein kinases Erk-1 and Erk-2. In this study, we examined the upstream regulators of hypotonicity-induced Erk-1/Erk-2 activation and their possible role in cell-volume regulation. The hypotonicity-provoked Erk-1/Erk-2 activation was greatly reduced in cells pretreated with the specific mitogen-activated/Erk-activating kinase inhibitor PD098059 and was preceded by a transient stimulation of Raf-1. Pretreatment of the cells with PMA, GF109203X, wortmannin or Clostridium botulinum C3 exoenzyme did not appreciably affect the hypotonicity-provoked Erk-1/Erk-2 stimulation, suggesting the osmosensitive signalling pathway to be largely independent of protein kinase C and p21(rho). In contrast, expression of dominant negative RasN17 completely abolished the hypotonicity-induced Erk-1/Erk-2 activation. Stimulation of the swelling-induced ion efflux was independent of activation of these mitogen-activated protein kinases, as revealed by hypotonicity-provoked isotope efflux from 125I-- and 86Rb+-loaded cells after pretreatment with PD098059 and after expression of RasN17. In addition, the epidermal-growth-factor-induced potentiation of the hypotonicity-provoked anionic response did not depend on the increase in Erk-1/Erk-2 activity but, instead, was found to depend on Ca2+ influx. Taken together, these results indicate that hypotonic stress induces Erk-1/Erk-2 activation through the Ras/Raf-signalling pathway, and argue against a direct role for this pathway in cell-volume control.

Membrane targeting of cGMP-dependent protein kinase is required for cystic fibrosis transmembrane conductance regulator Cl- channel activation.

A recently cloned isoform of cGMP-dependent protein kinase (cGK), designated type II, was implicated as the mediator of cGMP-provoked intestinal Cl- secretion based on its localization in the apical membrane of enterocytes and on its capacity to activate cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels. In contrast, the soluble type I cGK was unable to activate CFTR in intact cells, although both cGK I and cGK II could phosphorylate CFTR in vitro. To investigate the molecular basis for the cGK II isotype specificity of CFTR channel gating, we expressed cGK II or cGK I mutants possessing different membrane binding properties by using adenoviral vectors in a CFTR-transfected intestinal cell line, and we examined the ability of cGMP to phosphorylate and activate the Cl- channel. Mutation of the cGK II N-terminal myristoylation site (Gly2 --> Ala) reduced cGK II membrane binding and severely impaired cGK II activation of CFTR. Conversely, a chimeric protein, in which the N-terminal membrane-anchoring domain of cGK II was fused to the N terminus of cGK Ibeta, acquired the ability to associate with the membrane and activate the CFTR Cl- channel. The potency order of cGK constructs for activation of CFTR (cGK II > membrane-bound cGK I chimer >> nonmyristoylated cGK II > cGK Ibeta) correlated with the extent of 32P incorporation into CFTR observed in parallel measurements. These results strongly support the concept that membrane targeting of cGK is a major determinant of CFTR Cl- channel activation in intact cells.

cGMP stimulation of cystic fibrosis transmembrane conductance regulator Cl- channels co-expressed with cGMP-dependent protein kinase type II but not type Ibeta.

In order to investigate the involvement of cGMP-dependent protein kinase (cGK) type II in cGMP-provoked intestinal Cl- secretion, cGMP-dependent activation and phosphorylation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels was analyzed after expression of cGK II or cGK Ibeta in intact cells. An intestinal cell line which stably expresses CFTR (IEC-CF7) but contains no detectable endogenous cGK II was infected with a recombinant adenoviral vector containing the cGK II coding region (Ad-cGK II) resulting in co-expression of active cGK II. In these cells, CFTR was activated by membrane-permeant analogs of cGMP or by the cGMP-elevating hormone atrial natriuretic peptide as measured by 125I- efflux assays and whole-cell patch clamp analysis. In contrast, infection with recombinant adenoviruses expressing cGK Ibeta or luciferase did not convey cGMP sensitivity to CFTR in IEC-CF7 cells. Concordant with the activation of CFTR by only cGK II, infection with Ad-cGK II but not Ad-cGK Ibeta enabled cGMP analogs to increase CFTR phosphorylation in intact cells. These and other data provide evidence that endogenous cGK II is a key mediator of cGMP-provoked activation of CFTR in cells where both proteins are co-localized, e. g. intestinal epithelial cells. Furthermore, they demonstrate that neither the soluble cGK Ibeta nor cAMP-dependent protein kinase are able to substitute for cGK II in this cGMP-regulated function.

p38 mitogen activated protein kinase regulates endothelial VCAM-1 expression at the post-transcriptional level.

The cytokine tumor necrosis factor (TNF) alpha was found to stimulate the p38 mitogen activated protein (MAP) kinase signalling cascade in human umbilical vein endothelial cells. TNFalpha increased the activity of the p38 substrate MAP kinase-activated-protein (MAPKAP) kinase 2 and the subsequent phosphorylation of the small heat shock protein Hsp27 about two to three fold. This stimulation was blocked almost completely by the specific p38 MAP kinase inhibitor SB203580. This inhibitor also suppressed the TNFalpha-induced surface expression of the endothelial adhesion molecule vascular cell adhesion molecule (VCAM)-1. In contrast, inhibition of p38 MAP kinase had no effect on the stimulated surface expression of the intercellular cell adhesion molecule (ICAM)-1. VCAM-1 mRNA accumulation induced by TNFalpha was not affected by SB203580, suggesting that the p38 MAP kinase signalling cascade regulates the endothelial expression of VCAM-1 at the post-transcriptional level.

Hypo-osmotic cell swelling activates the p38 MAP kinase signalling cascade.

Hypo-osmotic swelling of human Intestine 407 cells leads to a significant increase of intracellular MAPKAP-kinase 2 activity and Hsp27 phosphorylation. Pre-treatment of the cells with the p38 MAP kinase inhibitor SB-203580 blocks this activation, indicating that the hypotonicity-induced activation of MAPKAP kinase 2 is, similarly to that described for hyperosmotic treatment, the result of an activated p38 MAP kinase cascade. The activation of MAPKAP kinase 2 proceeds with kinetics similar to that of one of the first physiological responses of hypo-osmotic treatment, the opening of compensatory Cl- channels. However, inhibition of the p38 MAP kinase cascade does not block the osmo-sensitive anion efflux and, vice versa, activation of p38 MAP kinase by cytokines and anisomycin does not increase the efflux. These results indicate that the p38 MAP kinase cascade is not directly involved in Cl- channel activation but instead may play a role in subsequent cellular repair processes.

Activation of the osmo-sensitive chloride conductance involves P21rho and is accompanied by a transient reorganization of the F-actin cytoskeleton.

Hypo-osmotic stimulation of human Intestine 407 cells rapidly activated compensatory CL- and K+ conductances that limited excessive cell swelling and, finally, restored the original cell volume. Osmotic cell swelling was accompanied by a rapid and transient reorganization of the F-actin cytoskeleton, affecting both stress fibers as well as apical ruffles. In addition, an increase in total cellular F-actin was observed. Pretreatment of the cells with recombinant Clostridium botulinum C3 exoenzyme, but not with mutant enzyme (C3-E173Q) devoid of ADP-ribosyltransferase activity, greatly reduced the activation of the osmo-sensitive anion efflux, suggesting a role for the ras-related GTPase p21rho. In contrast, introducing dominant negative N17-p21rac into the cells did not affect the volume-sensitive efflux. Cell swelling-induced reorganization of F-actin coincided with a transient, C3 exoenzyme-sensitive tyrosine phosphorylation of p125 focal adhesion kinase (p125FAK) as well as with an increase in phosphatidylinositol-3-kinase (PtdIns-3-kinase) activity. Pretreatment of the cells with wortmannin, a specific inhibitor of PtdIns-3-kinase, largely inhibited the volume-sensitive ion efflux. Taken together, our results indicate the involvement of a p21rho signaling cascade and actin filaments in the activation of volume-sensitive chloride channels.

Expression and regulation of chloride channels in neonatal rat cardiomyocytes.

Using an 125I- efflux assay, we have studied the expression of various types of chloride channels in isolated neonatal rat cardiomyocytes. Three different classes of anion conductances were distinguished: (1) a Ca(2+)-sensitive Cl- conductance, triggered upon stimulation of the cells with endothelin-1 or Ca(2+)-ionophore; (2) a cAMP/protein kinase A-operated Cl- conductance, activated by addition of forskolin. This anion channel could be identified as the Cystic Fibrosis Transmembrane conductance Regulator (CFTR-CI- channel) by Western blotting as well as by its enhanced activity in cultures pretreated with the tyrosine kinase inhibitor genistein; (3) a distinct class of cell volume-regulated Cl- channels, potentiated in the presence of endothelin-1 or the phosphotyrosine phosphatase inhibitor pervanadate. The potential role of each class of Cl- channels in the generation and/or modulation of action potentials as well as in maintaining cell volume is discussed.

Ca(2+)-mobilizing hormones potentiate hypotonicity-induced activation of ionic conductances in Intestine 407 cells.

Human Intestine 407 cells respond to hyposmotic stimulation by activating the conductive efflux of both Cl- and K+ (regulatory volume decrease) through pathways involving protein tyrosine phosphorylation (Tilly, B. C., N. van den Berghe, L. G. J. Tertoolen, M. J. Edixhoven, and H. R. de Jonge. J. Biol. Chem. 268: 19919-19922, 1993). Stimulation of the cells with hormones linked to the phospholipase C signaling cascade (e.g., bradykinin, histamine, or thrombin) or with the phosphotyrosine phosphatase inhibitor vanadate, potentiated the osmosensitive anion efflux by two- to threefold but did not affect anion efflux under isotonic conditions. No substantial increase in intracellular Ca2+ concentration ([Ca2+]i) was observed on mild hypotonicity-induced cell swelling. In addition, loading the cells with the intracellular Ca2+ chelator 1,2-bis(2-amino-phenoxy)ethane- N,N,N',N',-tetraacetic acid acetoxymethyl ester (BAPTA-AM) caused a partial reduction of the osmoshock-induced 125I- efflux but did not affect its potentiation by vanadate. In contrast, bradykinin transiently elevated [Ca2+]i, and its potentiation of the osmosensitive anion efflux was completely inhibited after BAPTA-AM loading. Both the Ca(2+)-mobilizing hormones as well as osmotic cell swelling rapidly triggered the phosphorylation of several proteins on tyrosine residues. However, the effects of the hormones, but not the effect of hypotonicity, on protein tyrosine phosphorylation was largely abolished in BAPTA-loaded cells. Taken together the results indicate a novel role for Ca(2+)-mobilizing hormones, although elevation of [Ca2+]i, in potentiating volume-sensitive ionic efflux even in cell types lacking the expression of Ca(2+)-activated Cl- channels in their plasma membrane.

Protein tyrosine phosphorylation is involved in osmoregulation of ionic conductances.

Using the human Intestine 407 cell line as a model, we investigated a possible role for tyrosine kinase(s) in regulating the ion efflux pathways induced by hyposmotic stimulation (regulatory volume decrease, RVD). Pretreatment of 125I(-)-and 86Rb(+)-loaded cells with the phosphotyrosine phosphatase inhibitor sodium orthovanadate (200 microM) potentiated isotope efflux triggered by mild hypotonicity (10-20%) but did not further increase the efflux in response to more vigorous osmotic stimulation (30% hypotonicity). The tyrosine kinase inhibitors herbimycin A and genistein largely reduced the osmoshock-induced efflux in both control and vanadate-pretreated cells, while not affecting calcium-activated 86Rb+ efflux. Potentiation of the RVD response by vanadate was confirmed by direct measurements of hypotonicity-induced changes in cell volume. Hypotonic shock alone triggered a rapid and transient increase in tyrosine phosphorylation of several proteins as well as phosphorylation of mitogen-activated protein kinase. Furthermore, the potentiating effects of vanadate on hypotonicity-induced ion efflux and mitogen-activated protein (MAP) kinase phosphorylation were mimicked by epidermal growth factor. Neither vanadate nor epidermal growth factor provoked a RVD-like ionic response under isotonic conditions. These results indicate that tyrosine phosphorylation is an essential step in the RVD response and suggest a novel role of growth factors in the cellular defense against osmotic stress.

Clustered CD3/TCR complexes do not transduce activation signals after bispecific monoclonal antibody-triggered lysis by cytotoxic T lymphocytes via CD3.

Bispecfic mAb (bsmAb) directed against the CD3/TCR complex and a tumor-associated Ag (TAA) induces CTL-mediated lysis of TAA+ target cells. We have investigated whether bsmAb-pretargeted CTL can enter multiple lytic cycles. BsmAb-pretargeted CTL retained bsmAb-targeted lytic capacity for at least 24 h when exposed to medium without TAA+ target cells. Exposure of bsmAb-pretargeted CTL to TAA+ target cells resulted in a rapid loss of bsmAb-targeted cytotoxicity of TAA+ or Fc gamma R+ target cells, although the CTL retained surface bsmAb. Moreover, addition of rabbit anti-mouse lg to these CTL did not induce calcium mobilization. These CTL still showed Ag-specific cytotoxicity and cytolysis of anti-CD3 mAb-expressing hybridoma cells. Readdition of bsmAb to CTL that had lost bsmAb-targeted cytotoxicity instantly restored the bsmAb-targeted lytic activity of the CTL. Hence, as in Ag-specific cytolysis, bsmAb-pretargeted CTL can enter multiple bsmAb-targeted cytolytic cycles. Surprisingly, exposure of bsmAb-pretargeted CTL to Fc gamma R+ cells did not result in loss of bsmAb-targeted cytolysis of TAA+ cells. Fluorescence microscopic analysis revealed that bsmAb-mediated interaction with TAA+ cells, but not with Fc gamma R+ cells, resulted in clustering of bsmAb-pretargeted CD3/TCR complexes on the CTL surface. On the basis of the observed correlation between clustered bsmAb-pretargeted CD3/TCR complexes and loss of bsmAb-targeted cytotoxicity, we hypothesize that clustered CD3/TCR complexes can no longer transduce signals.

Nucleotide-activated chloride channels in lysosomal membranes.

Lysosomal membrane vesicles purified from rat liver contain a basal chloride conductance that was enhanced in the presence of ATP, non-hydrolysable ATP-analogs and, to a lesser extent, GTP. Other nucleotides, including AMP, ADP and cAMP, as well as CTP and UTP were not effective. Following fusion of the vesicles with an artificial phosphatidylethanolamine/phosphatidylserine bilayer, we found that ATP gamma S dramatically increased the incidence of 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS)-sensitive chloride channels with a unitary slope conductance of approx. 40 pS in 300 mM/50 mM KCl buffers and 120 pS in symmetrical 300 mM KCl buffers. Since similar results were obtained with AMP-PNP, the results indicate that lysosomes contain a chloride permeable ion channel that is activated by ATP through allosteric interaction.

Cyclic AMP-dependent protein kinase activation of cystic fibrosis transmembrane conductance regulator chloride channels in planar lipid bilayers.

Membrane vesicles, prepared from mouse NIH-3T3 fibroblasts and Chinese hamster ovary cells expressing high levels of cystic fibrosis transmembrane conductance regulator (CFTR), were fused with Mueller-Rudin planar lipid bilayers. Upon addition of the catalytic subunit of cAMP-dependent protein kinase and ATP, low conductance Cl(-)-selective ion channels were observed in 10 of 16 experiments. The channels had a linear current-voltage relationship and a unitary conductance of approximately 6.5 pS. The channels were more permeable to Cl- than to I- and showed no appreciable time-dependent voltage activation. In contrast, addition of cAMP-dependent protein kinase and ATP to lipid bilayers fused with vesicles prepared from mock transfected (n = 14) cells failed to activate Cl- channels. These data support the conclusion that CFTR is a Cl- channel. They indicate that it can be reconstituted in a planar lipid bilayer and that the biophysical and regulatory properties are very similar to those observed in the native cell membrane. These data also argue against the requirement for loosely associated factors for regulation or function of the channel.

G-proteins mediate intestinal chloride channel activation.

The localization of several GTP-binding regulatory proteins in teh apical membrane of intestinal epithelial cells has prompted us to investigate a possible role for G-proteins as modulators of apical Cl- channels. In membrane vesicles isolated from rat small intestine or human HT29-cl.19A colon carcinoma cells, the entrapment of guanosine 5'-O-(3-thiophosphate (GTP gamma S) led to a large increase in Cl- conductance, as evidenced by an increased 125I- uptake and faster SPQ quenching. The enhancement was observed in the presence, but not in the absence of the K+ ionophore valinomycin, indicating that the increased Cl- permeability is not secondary to the opening of K+ channels. The effect of GTP gamma S was counteracted by guanosine 5'-O-(2-thiophosphate (GDP beta S) and appeared to be independent of cytosolic messengers, including ATP, cAMP, and Ca2+, suggesting that protein phosphorylation and/or phospholipase C activation is not involved. Patch clamp analysis of apical membrane patches of HT29-cl.19A colonocytes revealed a GTP gamma S-activated, inwardly rectifying, anion-selective channel with a unitary conductance of 20 +/- 4 pS. No spontaneous channel openings were observed in the absence of GTP gamma S, while the open time probability (Po) increases dramatically to 0.81 +/- 0.09 upon addition with GTP gamma S. Since the electrophysiological characteristics and regulatory properties of this channel are markedly different from those of the more widely studied cAMP/protein kinase A-operated channel, we propose the existence of a separate Cl(-)-selective ion channel in the apical border of intestinal epithelial cells. Our results suggest an alternative regulatory pathway in transepithelial salt transport and a possible site for anomalous channel regulation as observed in cystic fibrosis patients.

Regulation of phosphoinositide hydrolysis induced by histamine and guanine nucleotides in human HeLa carcinoma cells. Calcium and pH dependence and inhibitory role of protein kinase C.

The regulation of phospholipase C has been investigated in both intact and streptolysin-O permeabilized human HeLa carcinoma cells. Stimulation of phospholipase C by histamine and guanosine-5'-O-thiotriphosphate (GTP[S]) requires the presence of at least 10 nM free Ca2+, but is not significantly further increased by raising [Ca2+]i to greater than 10(-6) M. The pH optimum of the inositol phosphate response is at pH 6.8, while small changes in intracellular pH, as occur during hormonal stimulation (0.2-0.4 unit) attenuate the histamine/GTP[S]-induced stimulation of phospholipase C. Increasing cellular cAMP levels, either through addition of cell permeable cAMP analogues to intact cells or by stimulation with isoproterenol, does not affect histamine responsiveness, arguing against cross-talk between both signalling pathways. In contrast, we found that the response to histamine and/or GTP[S] is largely inhibited after brief pretreatment of the cells with phorbol esters or synthetic diacylglycerol prior to permeabilization, suggesting that protein kinase C exerts feedback inhibition at the level of, or downstream from, the putative GTP-binding protein.

Histamine as a growth factor and chemoattractant for human carcinoma and melanoma cells: action through Ca2(+)-mobilizing H1 receptors.

Histamine receptors are present on the surface of various normal and tumor-derived cell types, where their biological function is incompletely understood. Here we report that histamine not only stimulates cell proliferation under serum-free conditions, but also is chemotactic for human carcinoma (Hela and A431) and melanoma (A875) cells expressing H1 type receptors. Histamine was found to be a potent activator of phospholipase C, leading to polyphosphoinositide hydrolysis and subsequent intracellular Ca2+ mobilization. In addition, histamine also causes the protein kinase C-mediated activation of Na+/H+ exchange, as evidenced by an amiloride-sensitive rise in cytoplasmic pH. All histamine-induced responses, including chemotaxis and DNA synthesis, are completely inhibited by the H1 receptor antagonist pyrilamine, but not by cimetidine, an inhibitor of histamine H2 type receptors. Our results suggest that histamine may have a previously unrecognized role in the migration and proliferation of cells expressing H1 receptors.

Histamine-H1-receptor-mediated phosphoinositide hydrolysis, Ca2+ signalling and membrane-potential oscillations in human HeLa carcinoma cells.

In human HeLa carcinoma cells, histamine causes a dose-dependent formation of inositol phosphates, production of diacylglycerol and a transient rise in intracellular [Ca2+]. These responses are completely blocked by the H1-receptor antagonist pyrilamine. In streptolysin-O-permeabilized cells, formation of inositol phosphates by histamine is strongly potentiated by guanosine 5'-[gamma-thio]triphosphate and inhibited by guanosine 5'-[beta-thio]diphosphate, suggesting the involvement of a GTP-binding protein. Histamine stimulates the rapid but transient formation of Ins(1,4,5)P3, Ins(1,3,4)P3 and InsP4. InsP accumulates in a much more persistent manner, lasting for at least 30 min. Studies with streptolysin-O-permeabilized cells indicate that InsP accumulation results from dephosphorylation of Ins(1,4,5)P3, rather than direct hydrolysis of PtdIns. The rise in intracellular [Ca2+] is biphasic, with a very fast release of Ca2+ from intracellular stores, that parallels the Ins(1,4,5)P3 time course, followed by a more prolonged phase of Ca2+ influx. In individual cells, histamine causes a rapid initial hyperpolarization of the plasma membrane, which can be mimicked by microinjected Ins(1,4,5)P3. Histamine-induced hyperpolarization is followed by long-lasting oscillations in membrane potential, apparently owing to periodic activation of Ca2+-dependent K+ channels. These membrane-potential oscillations can be mimicked by microinjection of guanosine 5'-[gamma-thio]triphosphate, but are not observed after microinjection of Ins(1,4,5)P3. We conclude that H1-receptors in HeLa cells activate a PtdInsP2-specific phospholipase C through participation of a specific G-protein, resulting in long-lasting oscillations of cytoplasmic free Ca2+.