Stimulation of polyphosphoinositide hydrolysis by thrombin in membranes from human fibroblasts.

One of the earliest actions of thrombin in fibroblasts is stimulation of a phospholipase C (PLC) that hydrolyses phosphatidylinositol 4,5-bisphosphate (PIP2) to inositol 1,4,5-trisphosphate (IP3) and diacylglycerol. In membranes prepared from WI-38 human lung fibroblasts, thrombin activated an inositol-lipid-specific PLC that hydrolysed [32P]PIP2 and [32P]phosphatidylinositol 4-monophosphate (PIP) to [32P]IP3 and [32P]inositol 1,4-bisphosphate (IP2) respectively. Degradation of [32P]phosphatidylinositol was not detected. PLC activation by thrombin was dependent on GTP, and was completely inhibited by a 15-fold excess of the non-hydrolysable GDP analogue guanosine 5'-[beta-thio]diphosphate (GDP[S]). Neither ATP nor cytosol was required. Guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG) also stimulated polyphosphoinositide hydrolysis, and this activation was inhibited by GDP[S]. Stimulation of PLC by either thrombin or p[NH]ppG was dependent on Ca2+. Activation by thrombin required Ca2+ concentrations between 1 and 100 nM, whereas stimulation of PLC activity by GTP required concentrations of Ca2+ above 100 nM. Thus the mitogen thrombin increased the sensitivity of PLC to concentrations of free Ca2+ similar to those found in quiescent fibroblasts. Under identical conditions, another mitogen, platelet-derived growth factor, did not stimulate polyphosphoinositide hydrolysis. It is concluded that an early post-receptor effect of thrombin is the activation of a Ca2+- and GTP-dependent membrane-associated PLC that specifically cleaves PIP2 and PIP. This result suggests that the cell-surface receptor for thrombin is coupled to a polyphosphoinositide-specific PLC by a GTP-binding protein that regulates PLC activity by increasing its sensitivity to Ca2+.     

Ca2+ regulation of phosphatidylinositol turnover in the plasma membrane of tobacco suspension culture cells.

The biochemical properties of the enzymes involved in phosphatidylinositol (PI) turnover in higher plants were investigated using the plasma membrane isolated from tobacco suspension culture cells by aqueous two-phase partitioning. Submicromolar concentrations of Ca2+ inhibited PI kinase and phosphatidylinositol 4-phosphate (PIP) kinase and stimulated phospholipase C. Diacylglycerol (DG) kinase was inhibited by Ca2+, but required a higher concentration than the physiological level. From the above results we postulate the following scheme: signal coupled activation of phospholipase C produces IP3 which induces Ca2+ release from the intracellular Ca2+ compartment, the increased cytoplasmic Ca2+ in turn activates phospholipase C and causes a further increase of the cytoplasmic Ca2+ level. This inhibits PI kinase and PIP kinase and brings about a limited supply of PIP2, the substrate of phospholipase C. Consequently, IP3 production decreases and Ca2+ mobilization ceases. Then cytosolic Ca2+ returns to the stationary level by the Ca2+ pump at the plasma membrane and at the endoplasmic reticulum and Ca2+/H+ antiporter at the plasma membrane and at the tonoplast.     

ATP-induced activation of human B lymphocytes via P2-purinoceptors.

ATP-specific P2-purinoceptors expressed on various cell types have been shown to trigger cell activation via a phospholipase C pathway. In the present study, we provide evidence that P2-purinoceptors are expressed on B lymphocytes but not on T lymphocytes. ATP at concentrations of 10 to 100 microM triggered a dose-dependent increase in inositol 1,4,5-trisphosphate (IP3) levels as well as total inositol phosphate in human B lymphocytes. As expected from the changes in IP3, incubation of B cells with increasing concentrations of ATP lead to a dose-dependent increase in cytosolic free Ca+2 ([Ca+2]i). Extracellular ATP also induced increases in the levels of c-fos and c-myc mRNA. Because no responses were elicited by other nucleotides, the increase in IP3 production, the rise in [Ca+2]i levels, and the enhanced expression of c-fos and c-myc mRNA seem to be mediated by P2-purinoceptors. These responses were exclusive to B lymphocytes, in that ATP had no effect on IP3, [Ca+2]i, or oncogene expression in T cells. The results show that binding of extracellular ATP to P2-purinoceptors on quiescent B cells leads to the activation of genes associated with cell activation. This appears to be mediated via the phospholipase C signal transduction pathway.     

Interplay between ryanodine and IP3 receptors in ATP-stimulated mouse luteinized-granulosa cells

In mouse luteinized-granulosa cells (MGLC), ATP induces an increase in intracellular Ca2+ concentration by stimulating phospholipase C (PLC) associated with purinergic receptors, leading to production of inositol 1,4,5-trisphosphate (IP3) and subsequent release of Ca2+ from intracellular stores. In this study, we examined the cross-talk between the ryanodine receptors (RyR) and IP3 receptors (IP3R) in response to ATP in MGLC. Specifically, the effect of RyR modulators on ATP response was examined. The results showed that ATP-induced intracellular calcium elevation was abolished by inhibitors of the RyR, such as dantrolene (25 microM) and ryanodine (80 microM). When the MGLC were stimulated with activators of RyR, 2 microM ryanodine and 10 mM caffeine, the ATP-elicited response was decreased. These actions were independent of IP3 production stimulated by ATP. Hence, ATP-induced intracellular Ca2+ mobilization involves the coordinated action of both types of calcium release channels (CRCs). Using fluorescent probes, it was shown that IP3R is uniformly distributed throughout the cell; in contrast, RyR is mainly found around the nuclei. It is concluded that the IP3R and the RyR are functionally associated, and both play a role in the pattern of Ca2+ increase observed during purinergic stimulation of MGLC. This coupling may provide a highly efficient amplification mechanism for ATP stimulation of Ca2+ mobilization.     

The receptors for ATP and fMetLeuPhe are independently coupled to phospholipases C and A2 via G-protein(s). Relationship between phospholipase C and A2 activation and exocytosis in HL60 cells and human neutrophils.

The relationship between phospholipase A2 and C activation and secretion was investigated in intact human neutrophils and differentiated HL60 cells. Activation by either ATP or fMetLeuPhe leads to [3H]arachidonic acid release into the external medium from prelabelled cells. This response was inhibited when the cells were pretreated with pertussis toxin. When the [3H]arachidonic acid-labelled cells were stimulated with fMetLeuPhe, ATP or Ca2+ ionophore A23187, and the lipids analysed by t.l.c., the increase in free fatty acid was accompanied by decreases in label from phosphatidylinositol and phosphatidylcholine. Moreover, incorporation of label into triacylglycerol and to a lesser extent phosphatidylethanolamine was evident. Activation of secretion was evident with ATP and fMetLeuPhe but not with A23187. The pharmacological specificity of the ATP receptor in HL60 cells was investigated by measuring secretion of beta-glucuronidase, formation of inositol phosphatases and release of [3H]arachidonic acid. External addition of ATP, UTP, ITP, adenosine 5'-[gamma-thio]triphosphate (ATP[S]), adenosine 5'-[beta gamma-imido]triphosphate (App[NH]p), XTP, CTP, GTP, 8-bromo-ATP and guanosine 5'-[gamma-thio]triphosphate (GTP[S]) to intact HL60 cells stimulated inositol phosphate production, but only the first five nucleotides were effective at stimulating secretion or [3H]arachidonic acid release. In human neutrophils, addition of ATP, ITP, UTP and ATP[S] also stimulated secretion from specific and azurophilic granules, and this was accompanied by increases in cytosolic Ca2+ and in [3H]arachidonic acid release. The addition of phorbol 12-myristate 13-acetate (PMA; 1 nM) prior to the addition of either fMetLeuPhe or ATP led to inhibition of phospholipase C activity. In contrast, this had no effect on phospholipase A2 activation, whilst secretion was potentiated. Phospholipase A2 activation by either agonist was dependent on an intact cell metabolism, as was secretion. It is concluded that (1) activation of phospholipase C does not always lead to activation of phospholipase A2, (2) phospholipase A2 is coupled to the receptor independently of phospholipase C via a pertussis-toxin-sensitive G-protein and (3) for secretion to take place, the receptor has to activate both phospholipases C and A2.     

CCK-JMV-180, an analog of cholecystokinin, releases intracellular calcium from an inositol trisphosphate-independent pool in rat pancreatic acini.

In pancreatic acinar cells cholecystokinin and its analogs, caerulein and CCK-JMV-180, stimulate an increase in intracellular free [Ca2+] by releasing Ca2+ from non-mitochondrial intracellular pools. It is generally believed that the caerulein-induced release of Ca2+ is mediated by phospholipase C-catalyzed production of 1,4,5-inositol triphosphate (IP3). In this study we have investigated the source and mechanism of Ca2+ release induced by CCK-JMV-180 using streptolysin O-permeabilized pancreatic acinar cells. Caerulein-stimulated release of Ca2+ was completely blocked by either neomycin, an inhibitor of phospholipase C, or by heparin, an IP3 receptor antagonist. These observations are compatible with the conclusion that caerulein releases Ca2+ from an IP3-sensitive pool. In contrast to caerulein, however, CCK-JMV-180-stimulated release of Ca2+ was not blocked by either neomycin or by heparin, indicating that CCK-JMV-180 releases Ca2+ by mechanisms which do not involve the generation or action of IP3. CCK-JMV-180 stimulated the release of Ca2+ even after the IP3-sensitive pool had been completely emptied by prior exposure to a supramaximally stimulating concentration of IP3 (40 microM). Prestimulation of permeabilized acini with 20 mM caffeine did not abolish the CCK-JMV-180-induced Ca2+ release. These results indicate that CCK-JMV-180 stimulates release of Ca2+ from a hitherto uncharacterized intracellular storage pool which is insensitive to either IP3 or caffeine.     

Effects of heat on cell calcium and inositol lipid metabolism

Hyperthermia causes a large (three-to fivefold) increase in intracellular free calcium ([Ca2+]i) in HA-1 fibroblasts. Increased [Ca2+]i appears initially to be due to release of Ca2+ from an internal store, probably located in the endoplasmic reticulum. A subsequent influx of Ca2+ from the extracellular medium is then observed. These heat-induced changes in Ca2+ homeostasis are correlated with turnover of the phosphoinositides (PI), a class of phospholipids whose metabolism has been shown to regulate Ca2+ in a wide variety of cells (M. J. Berridge and R. F. Irvine, Nature 312, 315 (1984]. Hyperthermia induces rapid release of inositol 1,4,5-trisphosphate (IP3) within 1 min at 45 degrees C; IP3 release precedes the heat-induced rise in [Ca2+]i. IP3 release, a result of phosphatidylinositol 4,5-bisphosphate hydrolysis by phospholipase C, is the initial step in PI turnover. Later accumulation of phosphatidic acid, another metabolite in the PI pathway, is correlated with the delayed, heat-induced influx of 45Ca2+ from the extracellular environment. The data thus indicate that heat-induced changes in Ca2+ homeostasis are correlated with activation of PI turnover. They indicate that this class of lipids may be closely involved in heat-induced changes in cellular Ca2+ homeostasis. Cell Ca2+ appears to be important in some aspects of the cellular response to heat.     

Muscarinic-agonist and guanine nucleotide stimulation of myo-inositol trisphosphate formation in membranes isolated from bovine iris sphincter smooth muscle: effects of short-term cholinergic desensitization

The effect of short-term cholinergic desensitization on muscarinic acetylcholine receptor (mAChR)-mediated activation of phospholipase C was investigated in membranes isolated from the bovine iris sphincter smooth muscle. Membranes prepared from normal or desensitized muscles, prelabeled with either [3H]myo-inositol or 32P from [gamma-32P]ATP, were incubated with a hydrolysis-resistant analogue of GTP, GTP gamma S, or GTP gamma S plus carbachol (CCh), and the production of [3H]myo-inositol 1,4,5-trisphosphate (IP3) and the breakdown of polyphosphoinositides were assessed. In normal membranes, GTP (greater than or equal to 1 mM), GTP gamma S (greater than 10 microM) and GTP gamma S (1 microM) plus CCh (10 microM), but not GDP or GDP beta S, increased phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis and IP3 production. GTP gamma S increased IP3 accumulation in a time- and dose-dependent manner, and CCh, which had no effect on phospholipase C activity in the absence of GTP gamma S, potentiated the effects of GTP gamma S. The effect of CCh plus GTP gamma S on IP3 production was inhibited by atropine, had an absolute requirement for nM amounts of Ca2+ and was not affected by pertussis toxin. At higher concentrations (greater than 1 microM), Ca2+ alone induced PIP2 hydrolysis. Short-term exposure (less than 60 min) of the muscle to CCh (100 microM) did not affect the total number (Bmax) of mAChRs nor their affinity (KD) for [3H]-N-methylscopolamine. Desensitization did, however, result in: (1) a loss of the CCh-high affinity binding state of the sphincter mAChRs in a manner analogous to that produced by GTP gamma S; (2) a loss of the ability of GTP gamma S to affect CCh binding to the receptors; and (3) an attenuation of the GTP gamma S plus CCh-stimulated PIP2 hydrolysis. In conclusion, the data presented suggest that, in the iris smooth muscle, G-proteins are involved in the coupling of mAChRs to phospholipase C and that short-term cholinergic desensitization results in (1) the uncoupling of the receptor-G-protein complex and (2) the attenuation of mAChR-activation of phospholipase C.     

Effects of adenine nucleotides on inositol 1,4,5-trisphosphate-induced calcium release in vascular smooth muscle cells

Effects of adenine nucleotides on the inositol 1,4,5-trisphosphate (IP3)-induced Ca release (IICR) mechanism were studied in smooth muscle cells of the guinea pig portal vein. A microfluorometry method of fura-2 was used to measure Ca release from saponin-skinned thin muscle strips (width approximately 200 microns, thickness 50-70 microns, length 2-3 mm). About 80% of ionomycin-releasable Ca store was sensitive to IP3, of which approximately 20% was also sensitive to caffeine. The rate of Ca release by 0.1 microM IP3 depended biphasically on ATP concentration in the absence of Mg2+; it was dose-dependently enhanced by ATP up to approximately 0.5 mM, and above this concentration the enhancement became smaller. However, the decline of enhancement of the IICR at the higher ATP concentrations was absent at IP3 concentrations greater than 1 microM. This suggests competitive antagonism between IP3 and ATP. Clear effects of ATP were observed not only at pCa 7 or 8, where the Ca-induced Ca release was not activated, but after a ryanodine treatment to excise the functional compartment that possessed the Ca-induced Ca release mechanism. ATP had no effect on the rate of Ca leakage in the absence of IP3 even at pCa 5.5 after the ryanodine treatment. Therefore, ATP has direct biphasic effects on the IP3-induced Ca release mechanism. The Ca release induced by 0.1 microM IP3 at pCa 7 was potentiated not only by ATP, but by 0.5 mM ADP, AMP, or beta, gamma-methyleneadenosine 5'-triphosphate. 0.5 mM GTP had only a little effect on the IP3-induced Ca release. These results extend the functional similarities between Ca- and IP3-induced Ca release mechanisms in that adenine nucleotides enhance Ca release. Millimolar concentration of ATP, which is present physiologically, will shift the dose-response relation of IP3 toward the higher IP3 concentration and enhance the maximal effect of IP3. Thus, ATP is expected to assist the Ca release by higher concentrations of IP3 while having less effect on the Ca release by low levels of IP3. These effects of ATP may be important in the switching of Ca release from the intracellular Ca store by IP3.     

Extracellular ATP increases free cytosolic calcium in rat parotid acinar cells. Differences from phospholipase C-linked receptor agonists.

The effects of extracellular ATP on intracellular free calcium concentration [( Ca2+]i), phosphatidylinositol (PtdIns) turnover, amylase release and Ca2+-activated membrane currents were examined in isolated rat parotid acinar cells and contrasted with the effects of receptor agonists known to activate phospholipase C. ATP was more effective than muscarinic and alpha-adrenergic agonists and substance P as a stimulus for elevating [Ca2+]i (as measured with quin2). The ATP effect was selectively antagonized by pretreating parotid cells with the impermeant anion-exchange blocker 4,4'-di-isothiocyano-2,2'-stilbenedisulphonate (DIDS), which also inhibited binding of [alpha-32P]ATP to parotid cells. By elevating [Ca2+]i, ATP and the muscarinic agonist carbachol both activated Ca2+-sensitive membrane currents, which were measured by whole-cell and cell-attached patch-clamp recordings. However, there were marked contrasts between the effects of ATP and the receptor agonists linked to phospholipase C, as follows. (1) Although the combination of maximally effective concentrations of carbachol, substance P and phenylephrine had no greater effect on [Ca2+]i than did carbachol alone, there was some additivity between maximal ATP and carbachol effects. (2) Intracellular dialysis with guanosine 5'-[beta-thio]diphosphate did not block activation of ion channels by ATP, but did block channel activation by the muscarinic agonist carbachol. This suggests that a G-protein is involved in the muscarinic response, but not in the response to ATP. (3) Despite its pronounced effect on [Ca2+]i, ATP had little effect on PtdIns turnover in these cells, in contrast with the effects of carbachol and other Ca2+-mobilizing agents. (4) Although ATP was able to stimulate amylase release from parotid acinar cells, the stimulation was only 33 +/- 9% of that obtained with phospholipase C-linked receptor agonists. These differences suggest that ATP increases [Ca2+]i through specific activation of a pathway which is distinct from that shared by the classical phospholipase C-linked receptor agonists.     

Models of IP3 and Ca2+ oscillations: frequency encoding and identification of underlying feedbacks

Hormones that act through the calcium-releasing messenger, inositol 1,4,5-trisphosphate (IP3), cause intracellular calcium oscillations, which have been ascribed to calcium feedbacks on the IP3 receptor. Recent studies have shown that IP3 levels oscillate together with the cytoplasmic calcium concentration. To investigate the functional significance of this phenomenon, we have developed mathematical models of the interaction of both second messengers. The models account for both positive and negative feedbacks of calcium on IP3 metabolism, mediated by calcium activation of phospholipase C and IP3 3-kinase, respectively. The coupled IP3 and calcium oscillations have a greatly expanded frequency range compared to calcium fluctuations obtained with clamped IP3. Therefore the feedbacks can be physiologically important in supporting the efficient frequency encoding of hormone concentration observed in many cell types. This action of the feedbacks depends on the turnover rate of IP3. To shape the oscillations, positive feedback requires fast IP3 turnover, whereas negative feedback requires slow IP3 turnover. The ectopic expression of an IP3 binding protein has been used to decrease the rate of IP3 turnover experimentally, resulting in a dose-dependent slowing and eventual quenching of the Ca2+ oscillations. These results are consistent with a model based on positive feedback of Ca2+ on IP3 production.     

Role of a guanine nucleotide regulatory protein in the activation of phospholipase C by different chemoattractants

It is well established that formyl peptide chemoattractants can activate a phospholipase C in leukocytes via a pertussis toxin (PT)-sensitive guanine nucleotide regulatory (G) protein. Whether this pathway is similarly used by chemoattractant receptors as a class has been unclear. We now report that lipid and peptide chemoattractants in direct comparative studies induced similar amounts of initial (less than or equal to 15 sec) inositol trisphosphate (IP3) release in human polymorphonuclear leukocytes, but the response to lipid chemoattractants was more transient. Production of IP3 by all chemotactic factors was inhibited by treatment of the cells with PT, indicating that chemotactic factor receptors as a class are coupled to phospholipase C via a G protein that is a substrate for ADP ribosylation by PT. The peptide and lipid factors had comparable chemotactic activity, which was also inhibitable by PT. However, transient activation of phospholipase C is apparently an insufficient signal for full cellular activation, since the lipid chemotactic factor leukotriene B4 and platelet-activating factor were poor stimuli for O2- production and lysosomal enzyme secretion compared with N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe). Nonetheless, treatment with PT inhibited O2- production and enzyme secretion in response to all chemoattractants, but as previously noted, did not affect Ca2+ ionophores, lectins, or phorbol myristate acetate. Formyl peptide and lipid chemotactic factors induced similar levels of Ca2+ mobilization when monitored by Quin 2 or chlortetracycline (CTC) fluorescence. Although these responses to fMet-Leu-Phe were blocked by PT, the Quin 2 and initial CTC response to the lipid factors were only partially susceptible. Thus, the lipid factors apparently utilize an additional PT-resistant mechanism for redistributing intracellular Ca2+. This latter process requires extracellular Ca2+ and may be independent of the PT-sensitive G protein.     

Multiple Ca2+ signaling pathways converge on CaM kinase in PC12 cells.

The role of multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) in mediating various Ca2+ signaling pathways was examined in PC12 cells. Conversion of the kinase to a Ca(2+)-independent form was used to monitor which neurotransmitters activate the enzyme in situ. CaM kinase responds to Ca2+ influx elicited by ligand-gated Ca2+ channels for ATP and acetylcholine. It also responds to Ca2+ mobilization of IP3-sensitive stores elicited by phospholipase C-linked receptors for ATP and acetylcholine as well as by caffeine. CaM kinase mediates the actions of many neurotransmitters and Ca2+ signaling pathways.     

Pertussis toxin-sensitive GTP-binding proteins may regulate phospholipase A2 in response to thrombin in rabbit platelets

Incubation of rabbit platelets with thrombin resulted in rapid accumulations of inositol trisphosphate (IP3) in [3H]inositol-labeled platelets, increases of [3H]arachidonic acid [( 3H]AA) release, and [3H]serotonin secretion from the platelets prelabeled with these labeled compounds. The experiments using phospholipase A2 or C inhibitor suggested that not only phospholipase C but also phospholipase A2 activity plays an important role in serotonin secretion. We then studied the regulatory mechanisms of phospholipase A2 activity. Guanosine 5'-(3-O-thio)triphosphate (GTP gamma S), guanyl-5'-(beta,gamma-iminio)triphosphate), or AlF4- caused a significant liberation of AA in digitonin-permeabilized platelets but not in intact platelets. Thrombin-stimulated AA release was not observed in permeabilized platelets, whereas thrombin acted synergistically with GTP or GTP analogs to stimulate AA release. GTP analog-stimulated AA release was inhibited by guanosine 5'-(2-O-thio)diphosphate) and was also inhibited by decreased Mg2+ concentrations. Thrombin-induced, GTP-dependent AA release, but not IP3 formation, was diminished by 100 ng/ml of pertussis toxin, associated with ADP-ribosylation of membrane 41-kDa protein(s). Thrombin-stimulated AA release from intact platelets and GTP gamma S-stimulated release from permeabilized platelets were both markedly dependent on Ca2+. However, Ca2+ addition could not enhance AA release without GTP gamma S even when Ca2+ was increased up to 10(-4) M in permeabilized platelets. The results show that thrombin-stimulated AA release from rabbit platelets is mainly mediated by phospholipase A2 activity, not by phospholipase C activity, and that Ca2+ is an important factor to the activation of phospholipase A2 but is not the sole factor to the regulation. GTP-binding protein(s) is involved in receptor-mediated activation of phospholipase A2.     

High reactivity of aortic fibroblasts to vasoactive agents: endothelins, bradykinin and nucleotides.

Cultured aortic fibroblasts express high affinity Et-1 binding sites that poorly discriminate between Et-1 and Et-3. Both endothelins activate phospholipase C hence indicating the presence of ETB receptors. Fibroblasts respond to bradykinin by large activations of phospholipase C and increases in [Ca2+]i in a manner that was abolished by D-Arg, [Hyp3,Thi5,8,D-Phe7]-bradykinin, thus indicating the presence of B2 kinin receptors. Finally, ATP, UTP and ADP increases [Ca2+]i in aortic fibroblasts via a nucleotide receptor that has a higher affinity for ATP and UTP (3 microM) than for ADP (50 microM) and that is distinct from P2x and P2y purinoceptors.     

Adenine-nucleotide binding sites on the inositol 1,4,5-trisphosphate receptor bind caffeine, but not adenophostin A or cyclic ADP-ribose.

Binding of ATP to the inositol 1,4,5-trisphosphate receptor (IP3R) results in a more pronounced Ca2+ release in the presence of inositol 1,4,5-trisphosphate (IP3). We have expressed the cDNAs encoding two putative adenine-nucleotide binding sites of the neuronal form of IP3R-1 as glutathione S-transferase (GST)-fusion proteins in bacteria. Specific [alpha-32P]ATP binding was observed for the two GST-fusion proteins, representing aa 1710-1850 and aa 1944-2040 of IP3R-1. The ATP-binding sites in both fusion proteins had the same nucleotide specificity as found for the intact IP3R (ATP > ADP > AMP > GTP). Smaller GST-fusion proteins (aa 1745-1792 and aa 2005-2023) displayed a much weaker ATP-binding activity. CoA, which also potentiated IP3-induced Ca2+ release in A7r5 cells, interacted with the ATP-binding sites on the fusion proteins. Such interaction was not observed for 1,N6-etheno CoA and 3'-dephospho-CoA, which are much less effective in potentiating IP3-induced Ca2+ release. Since the adenine-containing compounds adenophostin A, caffeine and cyclic ADP-ribose modulate IP3-induced Ca2+ release, a possible effect of these compounds on the ATP-binding sites was examined. ATP stimulated adenophostin A- and IP3-induced Ca2+ release in A7r5 cells with an EC50 of respectively 21 and 20 microM. Also the threshold concentration of ATP for stimulating the release was similar for the two agonists. Adenophostin A (100 microM) and cyclic ADP-ribose (100 microM) were ineffective in displacing [alpha-32P]ATP from the binding sites of both GST-fusion proteins. Caffeine (50 mM), however, inhibited [alpha-32P]ATP binding to both fusion proteins by more than 50%. These data provide evidence for a direct interaction of caffeine but not of adenophostin A or cyclic ADP-ribose with the adenine-nucleotide binding sites of the IP3R.     

Collagen stimulates [3H]inositol trisphosphate formation in indomethacin-treated human platelets.

The present study investigates the pathway of metabolism of inositol phospholipids in human platelets exposed to collagen. Platelet activation by collagen was preceded by a lag phase usually lasting 10-20 s. Formation of [3H]inositol trisphosphate (IP3) was not observed during this period, but occurred in parallel with the onset of aggregation, release of ATP and phosphorylation of a 20 000 Da and a 40 000 Da protein. Indomethacin treatment partially inhibited all of these responses. Aggregation and ATP release, but not IP3 formation, were further inhibited in indomethacin-treated platelets loaded with the fluorescent Ca2+ indicator, quin2. Under these conditions there was no detectable mobilization of Ca2+. These results demonstrate that activation of platelets by collagen is associated with rapid hydrolysis of polyphosphoinositides by phospholipase C, thereby producing IP3. This observation is discussed in relation to IP3 as a possible Ca2+-mobilizing agent.     

The calcium-mobilizing effect of inositol-1,4,5-triphosphate in rod outer segments and disks]

The effect of inositol-1,4,5-trisphosphate (IP3) on the release of calcium ions from retinal rod discs was studied. It was shown that the release of Ca2+ from discs is an electroneutral process. The intradiscal calcium concentration during the release of the ion from the organelle decreases by 1 mM. It was found that the IP3-dependent release of Ca2+ ions from discs is activated by guanosine triphosphate and beta gamma-transducin. The increase in calcium concentration in the medium also activates the IP3-dependent release of Ca2+ ions from discs, which probably is due to the stimulation of phospholipase C. It is suggested that the functional role of the release of ions in related not to phototransduction but to slow regulatory and adaptation processes in the photoreceptor cell.     

Signal transduction in cells following binding of chemoattractants to membrane receptors

Binding of chemoattractants to specific cell surface receptors on human polymorphonuclear leukocytes (PMNs) initiates a variety of biologic responses, including directed migration (chemotaxis), release of superoxide anions, and lysosomal enzyme secretion. Chemoattractant receptors belong to a large class of receptors which utilize the hydrolysis of polyphosphoinositides to initiate Ca2+ mobilization and cellular activation. Receptor occupancy leads to phospholipase C-mediated hydrolysis of polyphosphoinositol 4,5-bisphosphate (PIP2) yielding inositol 1,4,5-trisphosphate (IP3) and 1,2 sn-diacylglycerol (DAG). These products synergize to initiate cell activation via calcium mobilization (IP3) and protein kinase C activation (DAG). Pertussis toxin, which ADP-ribosylates and inactivates some GTP binding proteins (G proteins), abolishes all chemoattractant-induced responses, including Ca2+ mobilization, IP3 and DAG production, enzyme secretion, superoxide production and chemotaxis. Direct evidence for chemoattractant receptor: G protein coupling was obtained using PMN membrane preparations which contain a Ca2+-sensitive phospholipase C. Hydrolysis of polyphosphoinositides at resting intracellular Ca2+ levels (100 nm) was only observed when the membranes were stimulated with the chemoattractant N-formyl-methyl-leucyl-phenylalanine (fMet-Leu-Phe) in the presence of GTP. Myeloid cells contain two distinct pertussis toxin substrates of similar molecular weight (40 and 41 kD). The 41 kD substrate resembles Gi, whereas a 40 kD substrate is physically associated with a partially purified fMet-Leu-Phe receptor preparation and may therefore represent a novel G protein involved in chemoattractant-stimulated responses. Metabolism of 1,4,5-IP3 to inositol proceeds via two distinct pathways in PMNs: (1) degradation to 1,4-IP2 and 4-IP1 or (2) conversion to 1,3,4,5-IP4, 1,3,4-IP3, 3,4-IP2 and 3-IP1. Initial formation (0-30 s) of 1,4,5-IP3 and DAG occurs at ambient intracellular Ca2+ levels, whereas formation of 1,3,4-IP3 and a second sustained phase of DAG production (30 s-10 min) require elevated cytosolic Ca2+ influx. The later peak of DAG, which is not derived from phosphoinositides, appears to be required for stimulation of respiratory burst activity. Products formed during activation can feed back to attenuate chemoattractant receptor-mediated stimulation of phospholipase C by uncoupling receptor-G protein-phospholipase C interaction.     

Bradykinin stimulation of inositol polyphosphate production in porcine aortic endothelial cells

Bradykinin stimulation of inositol polyphosphate production was followed using [3H]inositol-labeled porcine aortic endothelial cells grown in culture. Bradykinin stimulated a significant increase in inositol trisphosphate (IP3) production within 15 s. This increase reached a maximum value of 5-fold above control at 30 s and returned toward baseline by 90 s. Production of inositol bisphosphate increased with time reaching 4-fold by 60 s. Bradykinin stimulated the production of IP3 and inositol biphosphate in a dose-dependent manner with an EC50 of 9 X 10(-9) M. Labeled pools of phosphatidylinositol-4,5-bisphosphate (PIPP) decreased by 50% within 30 s, corresponding to the rise in IP3, while labeled lysophosphatidylinositol pools increased 3-fold by 60 s. Pertussis toxin, a protein which ribosylates GTP-binding proteins, did not inhibit bradykinin-stimulated inositol polyphosphate production. Incubation of labeled cells in the absence of extracellular Ca2+ also did not affect bradykinin-stimulated inositol polyphosphate production. Further, A23187, a Ca2+ ionophore, failed to stimulate PIPP metabolism. Finally, Ca2+ influx into cell monolayers occurred with a time course which paralleled rather than preceded the increase in IP3 levels. These data suggest that bradykinin stimulates phospholipase C metabolism of PIPP to IP3 by a mechanism which does not contain a pertussis toxin sensitive GTP-binding protein. Also, this receptor-linked phospholipase C activity does not appear to be activated by extracellular Ca2+ influx. The results support the proposal that IP3 production initiates Ca2+ mobilization and suggest that the calcium-dependent step in arachidonate release is distal to IP3 production.