Rapid Rise In Intracellular Ca2 Research Articles

Mechanosensor transient receptor potential vanilloid member 4 (TRPV4) regulates mouse cholangiocyte secretion and bile formation.

Mechanosensitive signaling has emerged as a mechanism for the regulation of cholangiocyte transport and bile formation. The mechanical effect of fluid-flow, or shear, at the apical membrane of cholangiocytes regulates secretion through a process involving increases in [Ca2+]i and activation of Ca2+-activated Cl- channels. However, the initiating steps translating shear force to increases in intracellular calcium concentration ([Ca2+]i) are unknown. Transient receptor potential vanilloid member 4 (TRPV4), a nonselective cation channel present in the apical membrane of cholangiocytes, has been proposed as a potential mechanosensor. The aim of the present studies was to determine the potential role of TRPV4 in initiating mechanosensitive signaling in response to fluid-flow in cholangiocytes. TRPV4 expression was confirmed in both small and large mouse cholangiocytes. Exposure of cells to either fluid flow or specific TRPV4 pharmacological agonists rapidly increased both [Ca2+]i and membrane cation currents. Both flow- and agonist-stimulated currents displayed identical biophysical properties and were inhibited in the presence of TRPV4 antagonists or in cells after transfection with TRPV4 small interfering RNA. Transfection of mouse cholangiocytes with a TRPV4-enhanced green fluorescent protein construct increased the expression of TRPV4 and the magnitude of flow-stimulated currents. A specific TRPV4 agonist significantly increased the biliary concentration of ATP and bile flow in live mice when administered intravenously and increased ATP release from cholangiocyte monolayers when applied exogenously. The findings are consistent with a model in which activation of cholangiocyte TRPV4 translates shear force into an acute rise in membrane cation permeability, [Ca2+]i, ATP release, and bile flow. Understanding the role of mechanosensitive transport pathways may provide novel insights to modulate bile flow for the treatment of cholestatic liver disorders.NEW & NOTEWORTHY These studies functionally characterize TRPV4 as a mechanosensitive channel in mouse cholangiocytes. By mediating a rapid rise in intracellular Ca2+, necessary for Ca2+-dependent secretion, TRPV4 represents a mechanosensor responsible for translating fluid flow into intracellular signaling and biliary secretion. Furthermore, intravenous infusion of a specific TRPV4 agonist increases bile flow in live mice. Understanding the role of TRPV4 in mechanosensitive transport pathways may provide novel insights to modulate bile flow during cholestasis.

Different Membrane Pathways Mediate Ca2+ Influx in Adrenal Chromaffin Cells Exposed to 150-400 ns Electric Pulses.

Exposing adrenal chromaffin cells to 5 ns electric pulses (nsPEF) causes a rapid rise in intracellular Ca2+ ([Ca2+]i) that is solely the result of Ca2+ influx through voltage-gated Ca2+ channels (VGCCs). This study explored the effect of longer duration nsPEF on [Ca2+]i. Single 150, 200, or 400 ns pulses at 3.1 kV/cm evoked rapid increases in [Ca2+]i, the magnitude of which increased linearly with pulse width and electric field amplitude. Recovery of [Ca2+]i to prestimulus levels was faster for 150 ns exposures. Regardless of pulse width, no rise in [Ca2+]i occurred in the absence of extracellular Ca2+, indicating that the source of Ca2+ was from outside the cell. Ca2+ responses evoked by a 150 ns pulse were inhibited to varying degrees by ω-agatoxin IVA, ω-conotoxin GVIA, nitrendipine or nimodipine, antagonists of P/Q-, N-, and L-type VGCCs, respectively, and by 67% when all four types of VGCCs were blocked simultaneously. The remaining Ca2+ influx insensitive to VGCC inhibitors was attributed to plasma membrane nanoporation, which comprised the E-field sensitive component of the response. Both pathways of Ca2+ entry were inhibited by 200 μM Cd2+. These results demonstrate that, in excitable chromaffin cells, single 150–400 ns pulses increased the permeability of the plasma membrane to Ca2+ in addition to causing Ca2+ influx via VGCCs.

Expression and functional activity of bitter taste receptors in primary renal tubular epithelial cells and M-1 cells.

The kidney is essential in the maintenance of in vivo homeostasis by body fluid and electrolyte conservation and metabolic waste removal. Previously, we reported the expression of a novel G protein family (Tas2rs), which includes bitter taste receptors, in the kidney tubule system, including the nephrons and the collecting duct system. Bitter taste receptors could affect kidney function via Ca2+ intake. Alkaloids such as phenylthiocarbamide stimulate these receptors and cause an increase in Ca2+ intake. In this study, we determined the expression of bitter taste receptors in the immature kidney and small intestine and in primary renal epithelial cells and M-1 (collecting tubule cell line) cells, by using QPCR and immunostaining. We found no expression of bitter taste receptors in the immature kidney and small intestine several days after birth; the relative abundance of Tas2rs transcripts varied depending on the developmental stage. Tas2rs were expressed in primary renal epithelial cells and M-1 cells. The traditional Chinese medicinal plant extracts phellodendrine and coptisine caused a rapid rise in intracellular Ca2+ concentration, which was inhibited by the phospholipase C (PLC) inhibitor U-73122. Thus, phellodendrine and coptisine could change the physiological status of renal cells in vitro by mediation of bitter taste receptors in a PLC-dependent manner. Our results provide new insights on the expression and role of bitter taste receptors in renal development and function.

Kisspeptin induction of somatolactin-α release in goldfish pituitary cells: functional role of cAMP/PKA-, PLC/PKC-, and Ca(2+)/calmodulin-dependent cascades.

Although the importance of kisspeptin in the pituitary is firmly established, the signaling mechanisms for the pituitary actions of kisspeptin are still largely unknown. Somatolactin (SL), a member of the growth hormone (GH)/prolactin (PRL) family, is a pituitary hormone with pleiotropic functions in fish, but its regulation by kisspeptin has not been examined. To investigate the functional role of kisspeptin in SL regulation, expression of two paralogues of goldfish Kiss1 receptors (Kiss1ra and Kiss1rb) were confirmed in immunoidentified SLα but not SLβ cells isolated by RT-PCR coupled with laser capture microdissection. In goldfish pituitary cells prepared from neurointermediate lobe (NIL), synthetic goldfish Kiss decapeptides (gKiss1-10 and gKiss2-10) could increase SLα release. Consistent with the lack of Kiss1r expression in SLβ cells, SLβ release was not altered by kisspeptin stimulation. In parallel experiments, goldfish gKiss1-10 could elevate cyclic adenosine monophosphate (cAMP) production, upregulate protein kinase A (PKA) and protein kinase C (PKC) activities, and trigger a rapid rise in intracellular Ca(2+) levels in goldfish NIL cells. Using a pharmacological approach, cAMP/PKA and phospholipase C (PLC)/PKC pathways and subsequent activation of Ca(2+)/calmodulin (CaM)-dependent cascades were shown to be involved in SLα release induced by gKiss1-10. Apparently, the Ca(2+)-dependent cascades were triggered by extracellular Ca(2+) entry via voltage-sensitive Ca(2+) channels and mobilization of inositol trisphosphate-sensitive intracellular Ca(2+) stores. Our results demonstrate that gKiss1-10 can act directly at the pituitary level to trigger SLα release via a complex network of post-receptor signaling mechanisms.

Activation of TRPV4 channel in pancreatic INS-1E beta cells enhances glucose-stimulated insulin secretion via calcium-dependent mechanisms

Transient receptor potential channel vanilloid type 4 (TRPV4) is a Ca2+- and Mg2+-permeable cation channel that influences oxidative metabolism and insulin sensitivity. The role of TRPV4 in pancreatic beta cells is largely unknown. Here, we characterize the role of TRPV4 in controlling intracellular Ca2+ and insulin secretion in INS-1E beta cells. Osmotic, thermal or pharmacological activation of TRPV4 caused a rapid rise of intracellular Ca2+ and enhanced glucose-stimulated insulin secretion. In the presence of the TRPV channel blocker ruthenium red (RuR) or after suppression of TRPV4 protein production, TRPV4 activators failed to increase [Ca2+]i and insulin secretion in INS-1E cells.

Mg2+ ions reduce microglial and THP-1 cell neurotoxicity by inhibiting Ca2+ entry through purinergic channels

Mg2+ is a known antagonist of some Ca2+ ion channels. It may therefore be able to counteract the toxic consequences of excessive Ca2+ entry into immune-type cells. Here we examined the effects of Mg2+ on inflammation induced by Ca2+ influx into microglia and THP-1 cells following activation of purinergic receptors. Using tissue culture, an inflammatory response was induced by treatment with either the P2X7 purinergic receptor agonist 2′,3′-[benzoyl-4-benzoyl]-ATP (BzATP) or the P2Y2,4 receptor agonist uridine 5′-triphosphate (UTP). Both microglia and THP-1 cells expressed the mRNAs for these receptors. Treatment produced a rapid rise in intracellular Ca2+ which was significantly reduced by Mg2+ or the calcium chelator BAPTA-AM. Purinergic receptor stimulation activated the intracellular inflammatory pathway P38 MAP kinase and NFκB. This caused release of TNFα, IL-6, nitrite ions and other materials that are neurotoxic to SH-SY5Y cells. These effects were all ameliorated by Mg2+. They were also partly ameliorated by the P2X7R antagonists, oxATP and KN-62, the P2YR antagonist MRS2179, and the store operated Ca2+ channel blocker, SK96365. These results indicate that elevated Mg2+ is a broad spectrum inhibitor of Ca2+ entry into microglia or THP-1 cells. Mg2+ administration may be a strategy for reducing the damaging consequences Ca2+ induced neuroinflammation in degenerative neurological disorders such as Alzheimer disease and Parkinson disease.

Thapsigargin Modulates Osteoclastogenesis Through the Regulation of RANKL-Induced Signaling Pathways and Reactive Oxygen Species Production

The mechanism by which TG modulates osteoclast formation and apoptosis is not clear. In this study, we showed a biphasic effect of TG on osteoclast formation and apoptosis through the regulation of ROS production, caspase-3 activity, cytosolic Ca2+, and RANKL-induced activation of NF-kappaB and AP-1 activities. Apoptosis and differentiation are among the consequences of changes in intracellular Ca2+ levels. In this study, we investigated the effects of the endoplasmic reticular Ca2+-ATPase inhibitor, thapsigargin (TG), on osteoclast apoptosis and differentiation. Both RAW264.7 cells and primary spleen cells were used to examine the effect of TG on RANKL-induced osteoclastogenesis. To determine the action of TG on signaling pathways, we used reporter gene assays for NF-kappaB and activator protein-1 (AP-1) activity, Western blotting for phospho-extracellular signal-related kinase (ERK), and fluorescent probes to measure changes in levels of intracellular calcium and reactive oxygen species (ROS). To assess rates of apoptosis, we measured changes in annexin staining, caspase-3 activity, and chromatin and F-actin microfilament structure. At concentrations that caused a rapid rise in intracellular Ca2+, TG increased caspase-3 activity and promoted apoptosis in osteoclast-like cells (OLCs). Low concentrations of TG, which were insufficient to measurably alter intracellular Ca2+, unexpectedly suppressed caspase-3 activity and enhanced RANKL-induced osteoclastogenesis. At these lower concentrations, TG potentiated ROS production and RANKL-induced NF-kappaB activity, but suppressed RANKL-induced AP-1 activity and had little effect on ERK phosphorylation. Our novel findings of a biphasic effect of TG are incompletely explained by our current understanding of TG action, but raise the possibility that low intensity or local changes in subcellular Ca2+ levels may regulate intracellular differentiation signaling. The extent of cross-talk between Ca2+ and RANKL-mediated intracellular signaling pathways might be important in determining whether cells undergo apoptosis or differentiate into OLCs.

Activation of CD38 by Interleukin-8 Signaling Regulates Intracellular Ca2+ Level and Motility of Lymphokine-activated Killer Cells

CD38 is an ADP-ribosyl cyclase, producing a potent Ca(2+) mobilizer cyclic ADP-ribose (cADPR). In this study, we have investigated a role of CD38 and its regulation through interleukin-8 (IL8) signaling in lymphokine-activated killer (LAK) cells. Incubation of LAK cells with IL8 resulted in an increase of cellular cADPR level and a rapid rise of intracellular Ca(2+) concentration ([Ca(2+)](i)), which was sustained for a long period of time (>10 min). Preincubation of an antagonistic cADPR analog, 8-Br-cADPR (8-bromo-cyclic adenosine diphosphate ribose), abolished the sustained Ca(2+) signal only but not the initial Ca(2+) rise. An inositol 1,4,5-trisphosphate (IP(3)) receptor antagonist blocked both Ca(2+) signals. Interestingly, the sustained Ca(2+) rise was not observed in the absence of extracellular Ca(2+). Functional CD38-null (CD38(-)) LAK cells showed the initial rapid increase of [Ca(2+)](i) but not the sustained Ca(2+) rise in response to IL8 treatment. An increase of cellular cADPR level by cGMP analog, 8-pCPT-cGMP (8-(4-chlorophenylthio)-guanosine-3',5'-cyclic monophosphate), but not cAMP analog or phorbol 12-myristate 13-acetate was observed. IL8 treatment resulted in the increase of cGMP level that was inhibited by the IP(3) receptor blocker but not a protein kinase C inhibitor. cGMP-mediated Ca(2+) rise was blocked by 8-Br-cADPR. In addition, IL8-mediated LAK cell migration was inhibited by 8-Br-cADPR and a protein kinase G inhibitor. Consistent with these observations, IL8-induced migration of CD38(-) LAK cells was not observed. However, direct application of cADPR or 8-pCPT-cGMP stimulated migration of CD38(-) cells. These results demonstrate that CD38 is stimulated by sequential activation of IL8 receptor, IP(3)-mediated Ca(2+) rise, and cGMP/protein kinase G and that CD38 plays an essential role in IL8-induced migration of LAK cells.

Ca2+ responses to hypoxia are mediated by IP3-R on Ca2+ store depletion.

Inositol 1,4, 5-triphosphate (IP3) is generated from the hydrolysis of phosphatidylinositol 4, 5-triphosphate (PIP2) which is a component of plasma membrane. IP3 acts as a messenger to link with receptors IP3-Rs which are located on intracellular Ca2+ stores, such as the endoplasmic reticulum (ER) (Belousov et al., 1995; Berridge, 1993). The ER also often shares the same domain of the mitochondria which have a low affinity, high-capacity for Ca2+ uptake mechanism, and Ca2+ concentrations influence mitochondrial metabolism (Duchen, 2000). Thus, IP3-Rs are an ideal candidate for Ca2+- related cellular functions, Rizzuto et al. (1993) have shown that [Ca2+]m increases rapidly and transiently upon stimulation with agonists coupled to IP3- R generation. This leads to high [Ca2+]i close to IP3-R and sensed by mitochondria. Conversely, metabolites generated by energy production may influence IP3-mediated Ca2+ dynamics. Depletion of cellular energy resources leads to the accumulation of cytoplasmic reduced NADH (Veech et al., 1970). Hypoxia decreases mitochondrial respiration by inhibiting the terminal step in the electron transport chains, provoking a rapid rise in intracellular Ca2+ (Biscoe and Duchen, 1990; Kaplin et al., 1996; McCormack et al., 1990). Hypoxia also increases cytoplasmic NADH levels as a result of enhanced glycolysis (Yager et al., 1991). Also, NADH selectively stimulates the release of Ca2+ mediated by IP3-R (Ferris and Snyder, 1992).

An Antagonist of cADP-ribose Inhibits Arrhythmogenic Oscillations of Intracellular Ca2+ In Heart Cells

Oscillations of Ca2+ in heart cells are a major underlying cause of important cardiac arrhythmias, and it is known that Ca2+-induced release of Ca2+ from intracellular stores (the sarcoplasmic reticulum) is fundamental to the generation of such oscillations. There is now evidence that cADP-ribose may be an endogenous regulator of the Ca2+ release channel of the sarcoplasmic reticulum (the ryanodine receptor), raising the possibility that cADP-ribose may influence arrhythmogenic mechanisms in the heart. 8-Amino-cADP-ribose, an antagonist of cADP-ribose, suppressed oscillatory activity associated with overloading of intracellular Ca2+ stores in cardiac myocytes exposed to high doses of the beta-adrenoreceptor agonist isoproterenol or the Na+/K+-ATPase inhibitor ouabain. The oscillations suppressed by 8-amino-cADP-ribose included intracellular Ca2+ waves, spontaneous action potentials, after-depolarizations, and transient inward currents. Another antagonist of cADP-ribose, 8-bromo-cADP-ribose, was also effective in suppressing isoproterenol-induced oscillatory activity. Furthermore, in the presence of ouabain under conditions in which there was no arrhythmogenesis, exogenous cADP-ribose was found to be capable of triggering spontaneous contractile and electrical activity. Because enzymatic machinery for regulating the cytosolic cADP-ribose concentration is present within the cell, we propose that 8-amino-cADP-ribose and 8-bromo-cADP-ribose suppress cytosolic Ca2+ oscillations by antagonism of endogenous cADP-ribose, which sensitizes the Ca2+ release channels of the sarcoplasmic reticulum to Ca2+.

The potassium channel opener (-)-cromakalim prevents glutamate-induced cell death in hippocampal neurons.

(-)-Cromakalim, a typical K+-channel opener, prevents neuronal death induced by either glucose and oxygen privation or by high (100 microM) extracellular glutamate in primary cultures of hippocampus. (-)-Cromakalim has no effect on the earliest events associated with exposure to glutamate. It does not prevent the rapid rise of intracellular Ca2+, the initial swelling of neurons, or the induction of c-fos mRNA transcription. (-)-Cromakalim inhibits all delayed effects associated with the excitotoxic effect of glutamate: (a) (-)-cromakalim inhibits the late and major phase of intracellular Ca2+ increase occurring up to hours after glutamate application; and (b) although (-)-cromakalim cannot prevent the initial cellular swelling induced by glutamate, cells that have been pretreated with (-)-cromakalim return to their original size in a few hours, whereas non-(-)-cromakalim-treated cells remain swollen for more prolonged periods. Many neurons surviving the initial necrotic phase of glutamate-induced cell death undergo progressive DNA cleavage leading to apoptosis. This apoptotic process is prevented completely by (-)-cromakalim. Glibenclamide, a potent blocker of the ATP-sensitive K+ channel, abolishes all the beneficial effects of (-)-cromakalim. These findings strongly suggest that (-)-cromakalim has postsynaptic effects that are closely related to the regulation of Ca2+ homeostasis and cell volume.

Stimulation of Intracellular Ca2+ Levels in Human Neutrophils by Soluble Immune Complexes

Soluble immune complexes bind to unprimed neutrophils and generate intracellular Ca2+ transients but fail to activate the NADPH oxidase. Following priming of the neutrophils with either tumor necrosis factor alpha or granulocyte-macrophage colony-stimulating factor, stimulation of the cells with the soluble immune complexes leads to an enhanced Ca2+ signal and significant secretion of reactive oxidants. The enhanced Ca2+ signal observed in primed neutrophils results from the influx of Ca2+ from the external environment and is partly sensitive to tyrosine kinase inhibitors. This is in contrast to the Ca2+ signal observed in unprimed neutrophils, which arises from the mobilization of intracellular stores. When the surface expression of FcgammaRIIIb on primed neutrophils was decreased either through incubation with Pronase or phosphoinositide-specific phospholipase C, the extra enhanced Ca2+ mobilization seen in primed cells was significantly lowered, while the initial rise in intracellular Ca2+ was unaffected. Depletion of FcgammaRIIIb had no significant effect on the Ca2+ transients in unprimed neutrophils. Cross-linking FcgammaRII, but not FcgammaRIIIb, induced increases in intracellular Ca2+ in unprimed neutrophils, while cross-linking either of these receptors increased Ca2+ levels in primed neutrophils. The FcgammaRII-dependent intracellular Ca2+ rise in primed cells was unaffected by incubation in Ca2+-free medium, whereas the FcgammaRIIIb-dependent transient was significantly decreased when Ca2+ influx was prevented in Ca2+-free medium supplemented with EGTA. Cross-linking either FcgammaRII or FcgammaRIIIb in primed or unprimed cells failed to stimulate substantial levels of inositol 1,4,5-trisphosphate production. These results indicate that following stimulation of primed neutrophils with soluble immune complexes the enhanced Ca2+ mobilization observed is the result of a functional activation of the glycosylphosphatidylinositol-linked FcgammaRIIIb.

P48-Triggered transmembrane signaling transduction of human monocytes: mobilization of calcium ion and activation of protein kinase C (PKC)

P48 is a cytokine which induces monocyte differentiation and the induction of cytotoxic activity. In this study, the signal transduction events involved in the stimulation of monocytes with the membrane form of P48 (mP48) were investigated. Monocyte stimulation with mP48 was found to involve the mobilization of intracellular calcium (Ca2+) and the activation and translocation of PKC from the cytosol to the membrane. Membane P48 induced a rapid rise of intracellular Ca2+ in a dose dependent maner. Similarly, the stimulation of monocytes with P48 was found to involve the activation and translocation of PKC. The translocation of PKC was rapid (within 0-5 min) yet transient with PKC activity returning to control levels by 8 min. The functional role of protein kineses in P48 induced TNF secretion was studied using various kinese inhibitors. The PKC inhibitors, H-7 and sphingosine, were found to inhibit P48 induced TNF secretion with 50% inhibition at 5 μ M. HA1004, which inhibts cyclic nucleotide-dependent kinase (PKA, Ki 1.2 μM), did not inhibit TNF secretion. H-8 (PKA inhibitor) was found to be an effective inhibitor of TNF secretion only at high concentrations(30 μM). The Calmodulin-dependent kinase inhibitor, W7 (Ki 12 μM) was found to be effective at concentration above 5 μM. These findings suggest that P48-triggered TNF secretion, involves transmembrane Ca2+ signaling and the subsequent activation of at least two protein kineses, PKC and CaMK.

7,12-Dimethylbenz[a]anthracene activates protein-tyrosine kinases Fyn and Lck in the HPB-ALL human T-cell line and increases tyrosine phosphorylation of phospholipase C-gamma 1, formation of inositol 1,4,5-trisphosphate, and mobilization of intracellular calcium.

Previous studies have shown that the immunosuppressive and carcinogenic polycyclic aromatic hydrocarbon 7,12-dimethylbenz(a)anthracene (DMBA) impairs Ca(2+)-dependent transmembrane signaling in human and murine lymphocytes. The purpose of the present studies was to analyze potential mechanisms of immunosuppression by DMBA and to examine effects on Ca2+ homeostasis and antigen-receptor signaling in human T cells. DMBA produced a rapid and sustained increase in Ca2+ levels in HPB-ALL cells by release of cytoplasmic Ca2+. DMBA also inhibited anti-CD3/CD4 mobilization of Ca2+ in HPB-ALL cells, with half-maximal inhibition occurring at approximately 4 hr. Thus, the kinetics for initial Ca2+ mobilization and inhibition of the anti-CD3/CD4 response differed. The rapid rise in intracellular Ca2+ induced by DMBA alone was accompanied by a rapid but transient increase in inositol 1,4,5-trisphosphate and tyrosine phosphorylation of phospholipase C-gamma 1. The pattern of tyrosine phosphorylation induced by DMBA in HPB-ALL cells was remarkably similar to that induced by anti-CD3/CD4 activation. Thus, DMBA-induced phosphorylation may mimic antigen-receptor activation in T cells, which may lead to alterations in antigen responsiveness. The mechanism of DMBA-induced tyrosine phosphorylation of phospholipase C-gamma 1 may have been due to an increase in protein-tyrosine kinase activity, since it was found that DMBA produced a > 2-fold increase in the activity of the T-cell receptor-associated Src-family kinases Fyn and Lck. The kinetics of activation of protein-tyrosine kinases demonstrated that Fyn activity was increased within 10 min of exposure to DMBA, whereas maximal Lck activation required 30 min. Thus, it is likely that the Fyn kinase or other protein-tyrosine kinases may be responsible for the early tyrosine phosphorylation of phospholipase C-gamma 1, which results in inositol 1,4,5-trisphosphate release and mobilization of intracellular Ca2+.

Regulation of arachidonic acid release in vascular endothelium. Ca2+-dependent and -independent pathways

Ca2+ metabolism and its relationship to arachidonic acid release were studied in cultured pig aortic endothelial cells. When cells were treated with bradykinin, a rapid rise in intracellular Ca2+ concentration ([Ca2+]i) occurred. Arachidonic acid release from cells prelabelled with [3H]arachidonic acid and subjected to flow-through conditions closely followed the changes in [Ca2+]i. Attenuation of the Ca2+ response by chelating extracellular and intracellular Ca2+ or by desensitization of receptors led to comparable attenuation of arachidonate release. Activation of protein kinase C inhibited Ca2+ mobilization in response to bradykinin and stimulated arachidonic acid release. Inhibition of protein kinase C had no effect on bradykinin-stimulated arachidonic acid release, suggesting that protein kinase C does not mediate the bradykinin response. The role of GTP-binding regulatory proteins (G-proteins) in mediating the bradykinin response was also investigated. Bradykinin-stimulated arachidonic acid release was not diminished by preincubation with pertussis toxin. Treatment with the G-protein activator AlF4- resulted in the release of a large pool of arachidonic acid and the formation of lysophospholipids. Combined treatment with AlF4- and bradykinin resulted in a greater than additive effect on arachidonic acid release. In contrast with bradykinin, AlF(4-)-stimulated arachidonic acid release was not dependent on the presence of extracellular Ca2+ or the mobilization of intracellular Ca2+. These results demonstrate Ca(2+)-dependent (bradykinin) and Ca(2+)-independent (AlF4-) pathways of phospholipase A2 activation.