Reassessment of the Ca2+ sensing property of a type I metabotropic glutamate receptor by simultaneous measurement of inositol 1,4,5-trisphosphate and Ca2+ in single cells

Transient transfection of Chinese hamster ovary or baby hamster kidney cells expressing the Group I metabotropic glutamate receptor mGlu1alpha with green fluorescent protein-tagged pleckstrin homology domain of phospholipase Cdelta1 allows real-time detection of inositol 1,4,5-trisphosphate. Loading with Fura-2 enables simultaneous measurement of intracellular Ca(2+) within the same cell. Using this technique we have studied the extracellular calcium sensing property of the mGlu1alpha receptor. Quisqualate, in extracellular medium containing 1.3 mm Ca(2+), increased inositol 1,4,5-trisphosphate in all cells. This followed a typical peak and plateau pattern and was paralleled by concurrent increases in intracellular Ca(2+) concentration. Under nominally Ca(2+)-free conditions similar initial peaks in inositol 1,4,5-trisphosphate and Ca(2+) concentration occurred with little change in either agonist potency or efficacy. However, sustained inositol 1,4,5-trisphosphate production was substantially reduced and the plateau in Ca(2+) concentration absent. Depletion of intracellular Ca(2+) stores using thapsigargin abolished quisqualate-induced increases in intracellular Ca(2+) and markedly reduced inositol 1,4,5-trisphosphate production. These data suggest that the mGlu1alpha receptor is not a calcium-sensing receptor because the initial response to agonist is not sensitive to extracellular Ca(2+) concentration. However, prolonged activation of phospholipase C requires extracellular Ca(2+), while the initial burst of activity is highly dependent on Ca(2+) mobilization from intracellular stores.     

Inducible nitric-oxide synthase attenuates vasopressin-dependent Ca2+ signaling in rat hepatocytes

Increases in both Ca(2+) and nitric oxide levels are vital for a variety of cellular processes; however, the interaction between these two crucial messengers is not fully understood. Here, we demonstrate that expression of inducible nitric-oxide synthase in hepatocytes, in response to inflammatory mediators, dramatically attenuates Ca(2+) signaling by the inositol 1,4,5-trisphosphate-forming hormone, vasopressin. The inhibitory effects of induction were reversed by nitric oxide inhibitors and mimicked by prolonged cyclic GMP elevation. Induction was without effect on Ca(2+) signals in response to AlF(4)(-) or inositol 1,4,5-trisphosphate, indicating that phospholipase C activation and release of Ca(2+) from inositol 1,4,5-trisphosphate-sensitive Ca(2+) stores were not targets for nitric oxide inhibition. Vasopressin receptor levels, however, were dramatically reduced in induced cultures. Our data provide a possible mechanism for hepatocyte dysfunction during chronic inflammation.     

The orexin OX1 receptor activates a novel Ca2+ influx pathway necessary for coupling to phospholipase C

Ca(2+) elevations in Chinese hamster ovary cells stably expressing OX(1) receptors were measured using fluorescent Ca(2+) indicators fura-2 and fluo-3. Stimulation with orexin-A led to pronounced Ca(2+) elevations with an EC(50) around 1 nm. When the extracellular [Ca(2+)] was reduced to a submicromolar concentration, the EC(50) was increased 100-fold. Similarly, the inositol 1,4,5-trisphosphate production in the presence of 1 mm external Ca(2+) was about 2 orders of magnitude more sensitive to orexin-A stimulation than in low extracellular Ca(2+). The shift in the potency was not caused by depletion of intracellular Ca(2+) but by a requirement of extracellular Ca(2+) for production of inositol 1,4,5-trisphosphate. Fura-2 experiments with the "Mn(2+)-quench technique" indicated a direct activation of a cation influx pathway by OX(1) receptor independent of Ca(2+) release or pool depletion. Furthermore, depolarization of the cells to +60 mV, which almost nullifies the driving force for Ca(2+) entry, abolished the Ca(2+) response to low concentrations of orexin-A. The results thus suggest that OX(1) receptor activation leads to two responses, (i) a Ca(2+) influx and (ii) a direct stimulation of phospholipase C, and that these two responses converge at the level of phospholipase C where the former markedly enhances the potency of the latter.     

Inositol 1,4,5-trisphosphate induces aggregation and release of 5-hydroxytryptamine from saponin-permeabilized human platelets

Inositol 1,4,5-trisphosphate induces aggregation and the release of [3H]5-hydroxytryptamine from human platelets rendered permeable with saponin. This action of inositol 1,4,5-trisphosphate is associated with a significant formation of thromboxane B2, activation of phospholipase C, and phosphorylation of 20,000- and 40,000-dalton proteins, which are the substrates for myosin light chain kinase and protein kinase C, respectively. All of these responses are blocked by the cyclooxygenase inhibitors indomethacin and aspirin and the dual cyclooxygenase and lipoxygenase inhibitor 3-amino-1-[m-(trifluoromethyl)phenyl]-2-pyrazoline (BW 755C). These data indicate that platelet activation by inositol 1,4,5-trisphosphate is initiated by the mobilization of Ca2+, which leads to phospholipase A2 activation. The thromboxanes and endoperoxides that are subsequently generated then induce activation via cell surface receptors.     

How do inositol phosphates regulate calcium signaling?

Activation of a variety of cell surface receptors results in the phospholipase C-catalyzed hydrolysis of the minor plasma membrane phospholipid phosphatidylinositol 4,5-bisphosphate, with concomitant formation of inositol 1,4,5-trisphosphate and diacylglycerol. There is strong evidence that inositol 1,4,5-trisphosphate stimulates Ca2+ release from intracellular stores. The Ca2+-releasing actions of inositol 1,4,5-trisphosphate are terminated by its metabolism through two distinct pathways. Inositol 1,4,5-trisphosphate is dephosphorylated by a 5-phosphatase to inositol 1,4-bisphosphate; alternatively, inositol 1,4,5-trisphosphate can also be phosphorylated to inositol 1,3,4,5-tetrakisphosphate by a 3-kinase. Although the mechanism of Ca2+ mobilization is understood, the precise mechanisms involved in Ca2+ entry are not known; the proposal that inositol 1,4,5-trisphosphate secondarily elicits Ca2+ entry by emptying an intracellular Ca2+ pool is considered.     

Evidence for coupling of bradykinin receptors to a guanine-nucleotide binding protein to stimulate arachidonate liberation in the osteoblast-like cell line, MC3T3-E1.

The effect of bradykinin on the activation production of inositol 1,4,5-trisphosphate and prostaglandin E2 (PGE2) was examined in the murine osteoblastic cell line, MC3T3-E1. Bradykinin, at concentrations ranging from 1 to 1000 nM, stimulated the production of inositol 1,4,5-trisphosphate 2.5- to 3-fold within 10 s, and elevated cytosolic-free Ca2+, even in the absence of external Ca2+. This process is mediated through the activation of phospholipase C. Bradykinin at the same concentration also stimulated the production of PGE2 and caused a release of 3H radioactivity from the cells prelabeled with [3H]arachidonic acid, probably via the activation of phospholipase A2. Pretreatment of the cells with pertussis toxin inhibited the stimulation of PGE2 production and 3H radioactivity release, while the elevation in cytosolic Ca2+ and the production of inositol 1,4,5-trisphosphate were not altered by toxin-pretreatment. The addition of an unhydrolyzable analog of GTP, guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) to the beta-escin-permeabilized cells prelabeled with [3H]arachidonic acid enhanced the release of 3H radioactivity. The simultaneous presence of bradykinin with GTP gamma S further activated the 3H radioactivity release in the beta-escin-permeabilized cells. These results provide evidence that receptors for bradykinin in the MC3T3-E1 couple stimulating arachidonate release, probably via the activation of phospholipase A2, through a guanine nucleotide binding protein sensitive to pertussis toxin.     

Quantitative changes in inositol 1,4,5-trisphosphate in chemoattractant-stimulated neutrophils

myo-Inositol 1,4,5-trisphosphate is an intracellular second messenger generated from the hydrolysis of phosphatidylinositol 4,5-bisphosphate by phospholipase C. In the present study, we have used the abilities of inositol 1,4,5-trisphosphate to inhibit inositol 1,4,5-tris[32P]phosphate binding and to stimulate release of sequestered stores of 45Ca2+ to assay the mass of inositol 1,4,5-trisphosphate in extracts derived from [3H]inositol-prelabeled chemoattractant-stimulated neutrophils. These assays are specific for inositol 1,4,5-trisphosphate since the relative capacity of the extracts to compete with inositol 1,4,5-tris[32P]phosphate binding and to release 45Ca2+ correlated well with the [3H]inositol 1,4,5-trisphosphate content of the extract as determined by high pressure liquid chromatography. No correlation of these activities was observed with the content in the extract of either [3H]inositol 1,3,4-trisphosphate or [3H]inositol 1,3,4,5-tetrakisphosphate, whose formation exhibited kinetics distinct from [3H]inositol 1,4,5-trisphosphate. Thus, within 10 s of stimulation with 10 nM formyl-methionyl-leucyl-phenylalanine, the inositol 1,4,5-trisphosphate content of the extract increased from 0.05 to 0.55 pmol/10(6) cells, equivalent to a change in intracellular concentration from 100 nM to 1.1 microM. These studies demonstrate that neutrophils produce sufficient quantities of inositol 1,4,5-trisphosphate to mobilize Ca2+ from intracellular stores.     

Signaling mechanism for receptor-activated canonical transient receptor potential 3 (TRPC3) channels

Canonical transient receptor potential 3 (TRPC3) is a receptor-activated, calcium permeant, non-selective cation channel. TRPC3 has been shown to interact physically with the N-terminal domain of the inositol 1,4,5-trisphosphate receptor, consistent with a "conformational coupling" mechanism for its activation. Here we show that low concentrations of agonists that fail to produce levels of inositol 1,4,5-trisphosphate sufficient to induce Ca(2+) release from intracellular stores substantially activate TRPC3. By several experimental approaches, we demonstrate that neither inositol 1,4,5-trisphosphate nor G proteins are required for TRPC3 activation. However, diacylglycerols were sufficient to activate TRPC3 in a protein kinase C-independent manner. Surface receptor agonists and exogenously applied diacylglycerols were not additive in activating TRPC3. In addition, inhibition of metabolism of diacylglycerol slowed the reversal of receptor-dependent TRPC3 activation. We conclude that receptor-mediated activation of phospholipase C in intact cells activates TRPC3 via diacylglycerol production, independently of G proteins, protein kinase C, or inositol 1,4,5-trisphosphate.     

Regulation of epidermal growth factor-stimulated formation of inositol phosphates in A-431 cells by calcium and protein kinase C

Epidermal growth factor (EGF) treatment of A-431 cells induces a biphasic increase in the levels of inositol phosphates. The growth factor produces an initial, rapid increase in the level of inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) due to hydrolysis of phosphatidyl-inositol-4,5-bisphosphate (Wahl, M., Sweatt, J. D., and Carpenter, G. (1987) Biochem. Biophys. Res. Commun. 142, 688-695). The level of inositol 1,3,4,5-tetrakisphosphate (Ins-1,3,4,5-P4) also rises rapidly in response to treatment with EGF. The initial formation (less than 1 min) of Ins-1,4,5-P3 and Ins-1,3,4,5-P4 does not require Ca2+ present in the culture medium. However, the addition of Ca2+ to the medium at levels of 100 microM or greater potentiates the growth factor-stimulated increases in the levels of all inositol phosphates at later times after EGF addition (1-60 min). The data suggest that EGF-receptor complexes initially stimulate the enzyme phospholipase C in a manner that is independent of an influx of extracellular Ca2+. The presence of Ca2+ in the medium allows prolonged growth factor activation of phospholipase C. Treatment of A-431 cells with Ca2+ ionophores (A23187 and ionomycin) did not mimic the activity of EGF in producing a rapid increase in the formation of the Dowex column fraction containing Ins-1,4,5-P3, Ins-1,3,4,5-P4, and inositol 1,3,4-trisphosphate (InsP3). However, the initial EGF-stimulated formation of inositol phosphates was substantially diminished in cells loaded with the Ca2+ chelator Quin 2/AM. EGF receptor occupancy studies indicated that maximal stimulation of InsP3 accumulation by EGF requires nearly full (75%) occupancy of available EGF binding sites, while half-maximal stimulation requires 25% occupancy. 12-O-Tetradecanoylphorbol-13-acetate (TPA), an exogenous activator of Ca2+/phospholipid-dependent protein kinase (protein kinase C), causes a dramatic, but transient, inhibition of the EGF-stimulated formation of inositol phosphates. Tamoxifen and sphingosine, reported pharmacologic inhibitors of protein kinase C activity, potentiate the capacity of EGF to induce formation of inositol phosphates. Neither TPA nor tamoxifen significantly affects the 125I-EGF binding capacity of A-431 cells; however, TPA appeared to enhance internalization of the ligand. Ligand occupation of the EGF receptor on the A-431 cell appears to initiate a complex signaling mechanism involving production of intracellular messengers for Ca2+ mobilization and activation of protein kinase C.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cell side-specific sensitivities of intracellular Ca2+ stores for inositol 1,4,5-trisphosphate, cyclic ADP-ribose, and nicotinic acid adenine dinucleotide phosphate in permeabilized pancreatic acinar cells from mouse.

In pancreatic acinar cells hormonal stimulation leads to a cytosolic Ca(2+) wave that starts in the apical cell pole and subsequently propagates toward the basal cell side. We used permeabilized pancreatic acinar cells from mouse and the mag-fura-2 technique, which allows direct monitoring of changes in [Ca(2+)] of intracellular stores. We show here that Ca(2+) can be released from stores in all cellular regions by inositol 1,4,5-trisphosphate. Stores at the apical cell pole showed a higher affinity to inositol 1,4,5-trisphosphate (EC(50) = 89 nm) than those at the basolateral side (EC(50) = 256 nm). In contrast, cADP-ribose, a modifier of Ca(2+)-induced Ca(2+) release, and nicotinic acid adenine dinucleotide phosphate (NAADP) were able to release Ca(2+) exclusively from intracellular stores located at the basolateral cell side. Our data agree with observations that upon stimulation Ca(2+) is released initially at the apical cell side and that this is caused by high affinity inositol 1,4,5-trisphosphate receptors. Moreover, our findings allow the conclusion that in Ca(2+) wave propagation from the apical to the basolateral cell side observed in pancreatic acinar cells Ca(2+)-induced Ca(2+) release, modulated by cADP-ribose and/or NAADP, might be involved.     

Cross-linking of FcepsilonRI causes Ca2+ mobilization via a sphingosine kinase pathway in a clathrin-dependent manner

Clathrin-coated pits are now recognized to be involved in cell signaling in addition to receptor down-regulation. Here we tried to identify signaling pathways that might be dependent on clathrin. Our initial data with pharmacological inhibitors of formation of clathrin-coated pits or lipid-rafts indicated that Ca(2+) response evoked by cross-linking of the high affinity receptors for IgE (FcepsilonRI) was dependent on clathrin. To confirm this finding, we created clathrin-knockdown cells by transfecting the mast cell line RBL-2H3 with a shRNA-clathrin heavy chain construct. In these cells, the FcepsilonRI-mediated Ca(2+) response was almost completely abolished, which was accompanied by the inhibition of sphingosine 1-phosphate (S1P) production with no changes in inositol 1,4,5-trisphosphate (IP(3)) production. This suggests that the Ca(2+) signaling pathway via a sphingosine kinase (SK) is dependent on clathrin. Furthermore, antigen-induced tyrosine phosphorylation of p85 and p110 subunits of PI3K was almost completely inhibited in clathrin-knockdown cells. In contrast, antigen-induced tyrosine phosphorylation of phospholipase Cgamma was not affected by clathrin-knockdown and tyrosine phosphorylation of Syk and degranulation were partially inhibited in clathrin-knockdown cells. The present study identifies the SK/Ca(2+) pathway to be dependent on clathrin.     

Requirement for PLCγ2 in IL-3 and GM-CSF-stimulated MEK/ERK phosphorylation in murine and human hematopoietic stem/progenitor cells

Even though the involvement of intracellular Ca(2+) Ca(i)(2+) in hematopoiesis has been previously demonstrated, the relationship between Ca(i)(2+) signaling and cytokine-induced intracellular pathways remains poorly understood. Herein, the molecular mechanisms integrating Ca(2+) signaling with the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway in primary murine and human hematopoietic stem/progenitor cells stimulated by IL-3 and GM-CSF were studied. Our results demonstrated that IL-3 and GM-CSF stimulation induced increased inositol 1,4,5-trisphosphate (IP(3) ) levels and Ca(i)(2+) release in murine and human hematopoietic stem/progenitor cells. In addition, Ca(i)(2+) signaling inhibitors, such as inositol 1,4,5-trisphosphate receptor antagonist (2-APB), PKC inhibitor (GF109203), and CaMKII inhibitor (KN-62), blocked phosphorylation of MEK activated by IL-3 and GM-CSF, suggesting the participation of Ca(2+) -dependent kinases in MEK activation. In addition, we identify phospholipase Cγ2 (PLCγ2) as a PLCγ responsible for the induction of Ca(2+) release by IL-3 and GM-CSF in hematopoietic stem/progenitor cells. Furthermore, the PLCγ inhibitor U73122 significantly reduced the numbers of granulocyte-macrophage colony-forming units after cytokine stimulation. Similar results were obtained in both murine and human hematopoietic stem/progenitor cells. Taken together, these data indicate a role for PLCγ2 and Ca(2+) signaling through the modulation of MEK in both murine and human hematopoietic stem/progenitor cells.     

Bradykinin-induced changes in phosphoinositides, inositol phosphate production and intracellular free calcium in cultured bovine aortic endothelial cells

Acute hydrolysis of phosphoinositides has been demonstrated in bovine aortic endothelial cells (BAEC) treated with bradykinin (BK) (10(-7)M). The first phosphoinositide to decrease was phosphatidylinositol-4,5-bisphosphate (PIP2) indicating this to be the initial substrate of phospholipase action. Other lipid changes associated with the stimulation of BAEC were an increase in diacylglycerol (DAG) and arachidonic acid (AA) with a sustained production of phosphatidic acid (PA). The changes in cell phospholipids were accompanied by the release of inositol phosphates. Inositol-1,4,5-trisphosphate (Ins-1,4,5-P3) was produced within 10 s of stimulation with BK. There was no evidence for the production of inositol-1,3,4-trisphosphate. The release of ionic calcium (Ca2+) intracellularly was demonstrated. The timecourse of the rise in intracellular Ca2+ was consistent with the timecourse of production of IP3. Intracellular Ca2+ rose from 127 +/- 21 nM to 462 +/- 27 nM. The Ca2+ peak was at 7.0 +/- 0.4 s and took 3 min to reach a steady state which remained above the basal level. When extracellular Ca2+ was depleted in the extracellular medium a spike of intracellular Ca2+ release was measured with an immediate return to basal. Entry of extracellular Ca2+ into the cell after ionophore A23187 treatment does not induce inositol phosphate release, indicating that phosphoinositide hydrolysis is likely to be the cause rather than consequence of the elevation in cytosolic Ca2+. These data indicate action of phospholipase C (PLC) on PIP2 after BK stimulation of BAEC with the subsequent production of InsP3 causing the resulting intracellular Ca2+ release.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium signaling in the nucleus

Cytosolic Ca(2+) is a versatile secondary messenger that regulates a wide range of cellular activities. In the past decade, evidence has accumulated that free Ca(2+) within the nucleus also plays an important messenger function. Here we review the mechanisms and effects of Ca(2+) signals within the nucleus. In particular, evidence is reviewed that the nucleus contains the machinery necessary for production of inositol 1,4,5-trisphosphate and for inositol 1,4,5-trisphosphate receptor-mediated Ca(2+) release. The role of Ca(2+) signals within the nucleus is discussed including regulation of such critical cell functions as gene expression, activation of kinases, and permeability of nuclear pores.     

Urokinase activates calcium-dependent potassium channels in U937 cells via calcium release from intracellular stores.

The urokinase receptor (uPAR) is highly expressed in the human promyelocytic cell line U937 and contributes to transmembrane signalling. However, the signalling mechanisms are poorly understood. We used the patch-clamp technique to demonstrate that urokinase (uPA) binds to uPAR and thereby stimulates Ca(2+)-activated K+ channels in U937 cells. uPA transiently increased K+ currents within 30 s. The K+ currents were pertussis toxin-sensitive and were also observed in Ca(2+)-free solution. However, when cells were dialysed with EGTA, uPA did not affect K+ currents. The intracellular Ca2+ response to uPA was independent of extracellular Ca2+, was pertussis toxin-sensitive, and was blocked by both thapsigargin and the phospholipase C inhibitor U-73122. The uPA-induced increase in intracellular Ca2+ was independent of uPA proteolytic activity. Furthermore, uPA initiated a rapid formation of inositol 1,4, 5-trisphosphate [Ins(1,4,5)P3]. The amino-terminal uPA fragment and uPA inactivated with diisopropyl fluorophosphate or with inhibitory antibody, elicited the same Ca2+ signal. On the other hand, Ca2+ signalling required the intact uPAR because the effects were abrogated by PtdIns-specific phospholipase C, which removes the uPAR from the cell surface. The prevention of glycosyl phosphatidylinositol moiety synthesis and interference with uPAR anchoring to the cell surface using mannosamine also abolished Ca2+ signals. Taken together, our findings indicate that uPA binds to uPAR and stimulates the production of Ins(1,4,5)P3 via a G-protein- and phospholipase C-dependent mechanism. Ins(1,4,5)P3 in turn liberates Ca2+ from intracellular stores, which leads to the stimulation of Ca(2+)-activated K+ channels.     

The mechanism for synergism between phospholipase C- and adenylylcyclase-linked hormones in liver. Cyclic AMP-dependent kinase augments inositol trisphosphate-mediated Ca2+ mobilization without increasing the cellular levels of inositol polyphosphates.

The ability of cAMP-dependent hormones to modulate the actions of Ca2(+)-mobilizing hormones was studied in single fura-2-injected guinea pig hepatocytes. In 91% of cells the cAMP-linked hormone, isoproterenol, applied alone, did not alter cytosolic Ca2+ concentration. In 78% of cells which had been pre-exposed to a low concentration of angiotensin II, isoproterenol was able to increase cytosolic Ca2+. Isoproterenol did not, however, increase inositol 1,4,5-trisphosphate or inositol tetrakisphosphate on its own, or in the presence of angiotensin II. Isoproterenol was also able to raise cytosolic Ca2+ concentration in cells microinjected with inositol 2,4,5-trisphosphate or a photoactivatable derivative of inositol 1,4,5-trisphosphate. The elevation of cytosolic Ca2+ concentration induced by isoproterenol in angiotensin II-treated cells and cells injected with caged inositol 1,4,5-trisphosphate was blocked by heparin, implying that the effect was mediated by an inositol 1,4,5-trisphosphate receptor agonist. In permeabilized hepatocytes, inositol 1,4,5-trisphosphate-induced Ca2+ release was enhanced by 8-bromo-cAMP and the catalytic subunit of cAMP-dependent kinase. Cyclic AMP-dependent kinase shifted the dose-response curve for inositol 1,4,5-trisphosphate-mediated Ca2+ release to the left by a factor of 4 and increased the total amount of Ca2+ released by 25%. These results indicate that increased sensitivity of the intracellular Ca2+ releasing organelle to inositol 1,4,5-trisphosphate is responsible for synergism between phospholipase C- and adenylylcyclase-linked hormones in the liver.     

Alkaline pH shifts Ca2+ sparks to Ca2+ waves in smooth muscle cells of pressurized cerebral arteries

The effects of external pH (7.0-8.0) on intracellular Ca(2+) signals (Ca(2+) sparks and Ca(2+) waves) were examined in smooth muscle cells from intact pressurized arteries from rats. Elevating the external pH from 7.4 to 7.5 increased the frequency of local, Ca(2+) transients, or "Ca(2+) sparks," and, at pH 7.6, significantly increased the frequency of Ca(2+) waves. Alkaline pH-induced Ca(2+) waves were inhibited by blocking Ca(2+) release from ryanodine receptors but were not prevented by inhibitors of voltage-dependent Ca(2+) channels, phospholipase C, or inositol 1,4,5-trisphosphate receptors. Activating ryanodine receptors with caffeine (5 mM) at pH 7.4 also induced repetitive Ca(2+) waves. Alkalization from pH 7.4 to pH 7.8-8.0 induced a rapid and large vasoconstriction. Approximately 82% of the alkaline pH-induced vasoconstriction was reversed by inhibitors of voltage-dependent Ca(2+) channels. The remaining constriction was reversed by inhibition of ryanodine receptors. These findings indicate that alkaline pH-induced Ca(2+) waves originate from ryanodine receptors and make a minor, direct contribution to alkaline pH-induced vasoconstriction.     

Cholinergic Stimulation of Inositol Phosphate Formation in Bovine Adrenal Chromaffin Cells: Distinct Nicotinic and Muscarinic Mechanisms

The ability of cholinergic agonists to activate phospholipase C in bovine adrenal chromaffin cells was examined by assaying the production of inositol phosphates in cells prelabeled with [3H]inositol. We found that both nicotinic and muscarinic agonists increased the accumulation of [3H]inositol phosphates (mainly inositol monophosphate) and that the effects mediated by the two types of receptors were independent of each other. The production of inositol phosphates by nicotinic stimulation required extracellular Ca2+ and was maximal at 0.2 mM Ca2+. Increasing extracellular Ca2+ from 0.22 to 2.2 mM increased the sensitivity of inositol phosphates formation to stimulation by submaximal concentrations of 1,1-dimethyl-4-phenyl-piperazinium iodide (DMPP) but did not enhance the response to muscarine. Elevated K+ also stimulated Ca2+-dependent [3H]inositol phosphate production, presumably by a non-receptor-mediated mechanism. The Ca2+ channel antagonists D600 and nifedipine inhibited the effects of DMPP and elevated K+ to a greater extent than that of muscarine. Ca2+ (0.3-10 microM) directly stimulated the release of inositol phosphates from digitonin-permeabilized cells that had been prelabeled with [3H]inositol. Thus, cholinergic stimulation of bovine adrenal chromaffin cells results in the activation of phospholipase C by distinct muscarinic and nicotinic mechanisms. Nicotinic receptor stimulation and elevated K+ probably increased the accumulation of inositol phosphates through Ca2+ influx and a rise in cytosolic Ca2+. Because Ba2+ caused catecholamine secretion but did not enhance the formation of inositol phosphates, phospholipase C activation is not required for exocytosis. However, diglyceride and myo-inositol 1,4,5-trisphosphate produced during cholinergic stimulation of chromaffin cells may modulate secretion and other cellular processes by activating protein kinase C and/or releasing Ca2+ from intracellular stores.     

Receptor-mediated release of inositol 1,4,5-trisphosphate and inositol 1,4-bisphosphate in rat basophilic leukemia RBL-2H3 cells permeabilized with streptolysin O

Antigen-mediated exocytosis in intact rat basophilic leukemia (RBL-2H3) cells is associated with substantial hydrolysis of membrane inositol phospholipids and an elevation in concentration of cytosol Ca2+ ([ Ca2+i]). Paradoxically, these two responses are largely dependent on external Ca2+. We report here that cells labeled with myo-[3H]inositol and permeabilized with streptolysin O do release [3H]inositol 1,4,5-trisphosphate upon stimulation with antigen or guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) at low (less than 100 nM) concentrations of free Ca2+. The response, however, is amplified by increasing free Ca2+ to 1 microM. The subsequent conversion of the trisphosphate to inositol 1,3,4,5-tetrakisphosphate is enhanced also by the increase in free Ca2+. Although [3H]inositol 1,4,5-trisphosphate accumulates in greater amounts than is the case in intact cells, [3H]inositol 1,4-bisphosphate is still the major product in permeabilized cells even when the further metabolism of [3H]inositol 1,4,5-trisphosphate is suppressed (by 77%) by the addition of excess (1000 microM) unlabeled inositol 1,4,5-trisphosphate and the phosphatase inhibitor 2,3-bisphosphoglycerate. It would appear that either the activity of the membrane 5-phosphomonoesterase allows virtually instantaneous dephosphorylation of the inositol 1,4,5-trisphosphate under all conditions tested or both phosphatidylinositol 4-monophosphate and the 4,5-bisphosphate are substrates for the activated phospholipase C. The latter alternative is supported by the finding that permeabilized cells, which respond much more vigorously to high (supraoptimal) concentrations of antigen than do intact RBL-2H3 cells, produce substantial amounts of [3H]inositol 1,4-bisphosphate before any detectable increase in levels of [3H]inositol 1,4,5-trisphosphate.     

Vascular endothelial growth factor signals endothelial cell production of nitric oxide and prostacyclin through flk-1/KDR activation of c-Src.

Vascular endothelial growth factor (VEGF) is a potent endothelial cell-specific mitogen that promotes angiogenesis, vascular hyperpermeability, and vasodilation by autocrine mechanisms involving nitric oxide (NO) and prostacyclin (PGI(2)) production. These experiments used immunoprecipitation and immunoassay procedures to characterize the signaling pathways by which VEGF induces NO and PGI(2) formation in cultured endothelial cells. The data showed that VEGF stimulates complex formation of the flk-1/kinase-insert domain-containing receptor (KDR) VEGF receptor with c-Src and that Src activation is required for VEGF induction of phospholipase C gamma1 activation and inositol 1,4,5-trisphosphate formation. Reporter cell assays showed that VEGF promotes a approximately 50-fold increase in NO formation, which peaks at 5-20 min. This effect is mediated by a signaling cascade initiated by flk-1/KDR activation of c-Src, leading to phospholipase C gamma1 activation, inositol 1,4,5-trisphosphate formation, release of [Ca(2+)](i) and nitric oxide synthase activation. Immunoassays of VEGF-induced 6-keto prostaglandin F(1alpha) formation as an indicator of PGI(2) production revealed a 3-4-fold increase that peaked at 45-60 min. The PGI(2) signaling pathway follows the NO pathway through release of [Ca(2+)](i), but diverges prior to NOS activation and also requires activation of mitogen-activated protein kinase. These results suggest that NO and PGI(2) function in parallel in mediating the effects of VEGF.