RANTES stimulates Ca2+ mobilization and inositol trisphosphate (IP3) formation in cells transfected with G protein-coupled receptor 75.

BACKGROUND AND PURPOSE: RANTES is an inflammatory chemokine with a critical role in T-lymphocyte activation and proliferation. Its effects are mediated through G protein-coupled heptahelical receptors (GPCRs). We show for the first time that RANTES activates the orphan G protein-coupled receptor 75 (GPR75). EXPERIMENTAL APPROACH: To identify a ligand for GPR75 we have used three different and independent methods, namely luciferase assay, bioluminescence assay and IP3 accumulation assay. KEY RESULTS: Treatment of cells expressing GPR75 with subnanomolar concentrations of RANTES led to stimulation of the luciferase activity in a reporter-gene assay, an increase in inositol trisphosphate, and intracellular Ca2+. The latter effect was blocked by the phospholipase-C inhibitor (PLC) U73122 indicating that Gq proteins mediate GPR75 signaling. RANTES enhanced the phosphorylation of AKT and mitogen-activated protein kinase (MAPK) in GPR75-transfected cells and this effect was blocked by the PLC inhibitor U73122 and the phosphatidylinositol 3-kinase (PI3K) inhibitor, wortmannin. The hippocampal cell line HT22, which expresses GPR75 endogenously, but not the other known RANTES receptors, was used to study the effects of RANTES and GPR75 on neuronal survival. Treatment of HT22 cells with RANTES significantly reduced the neurotoxicity of amyloid-beta peptides, by activating PLC and PI3K. CONCLUSIONS AND IMPLICATIONS: This demonstrate clearly and undoubtedly the ability of RANTES to act on GPR75. Defects in the RANTES/GPR75-signaling pathway may contribute to neuroinflammatory and neurodegenerative processes as observed in Alzheimer's disease.

An oxidized metabolite of linoleic acid increases intracellular calcium in rat adrenal glomerulosa cells.

EKODE, an epoxy-keto derivative of linoleic acid, was previously shown to stimulate aldosterone secretion in rat adrenal glomerulosa cells. In the present study, we investigated the effect of exogenous EKODE on cytosolic [Ca(2+)] increase and aimed to elucidate the mechanism involved in this process. Through the use of the fluorescent Ca(2+)-sensitive dye Fluo-4, EKODE was shown to rapidly increase intracellular [Ca(2+)] ([Ca(2+)](i)) along a bell-shaped dose-response relationship with a maximum peak at 5 microM. Experiments performed in the presence or absence of Ca(2+) revealed that this increase in [Ca(2+)](i) originated exclusively from intracellular pools. EKODE-induced [Ca(2+)](i) increase was blunted by prior application of angiotensin II, Xestospongin C, and cyclopiazonic acid, indicating that inositol trisphosphate (InsP(3))-sensitive Ca(2+) stores can be mobilized by EKODE despite the absence of InsP(3) production. Accordingly, EKODE response was not sensitive to the phospholipase C inhibitor U-73122. EKODE mobilized a Ca(2+) store included in the thapsigargin (TG)-sensitive stores, although the interaction between EKODE and TG appears complex, since EKODE added at the plateau response of TG induced a rapid drop in [Ca(2+)](i). 9-oxo-octadecadienoic acid, another oxidized derivative of linoleic acid, also increases [Ca(2+)](i), with a dose-response curve similar to EKODE. However, arachidonic and linoleic acids at 10 microM failed to increase [Ca(2+)](i) but did reduce the amplitude of the response to EKODE. It is concluded that EKODE mobilizes Ca(2+) from an InsP(3)-sensitive store and that this [Ca(2+)](i) increase is responsible for aldosterone secretion by glomerulosa cells. Similar bell-shaped dose-response curves for aldosterone and [Ca(2+)](i) increases reinforce this hypothesis.

Ketamine suppresses platelet aggregation possibly by suppressed inositol triphosphate formation and subsequent suppression of cytosolic calcium increase.

BACKGROUND: Ketamine has been shown to suppress platelet aggregation, but its mechanisms of action have not been defined. The purpose of the current study is to clarify the effects of ketamine on human platelet aggregation and to elucidate the underlying mechanisms of its action. METHODS: Platelet aggregation was measured using an eight-channel aggregometer, and cytosolic free calcium concentration was measured in Fura-2/AM-loaded platelets using a fluorometer. Inositol 1,4,5-triphosphate (IP3) was measured with use of a commercially available IP3 assay kit. To estimate thromboxane A2 (TXA2) receptor binding affinity and expression, Scatchard analysis was performed using [3H]S145, a specific TXA2 receptor antagonist. TXA2 agonist binding assay was also performed. The membrane-bound guanosine 5'-triphosphatase activity was determined using [gamma-32P]guanosine triphosphate by liquid scintillation analyzer. RESULTS: Ketamine (500 microm) suppressed aggregation induced by adenosine diphosphate (0.5 microm), epinephrine (1 microm), (+)-9,11-epithia-11,12-methano-TXA2 (STA2) (0.5 microm), and thrombin (0.02 U/ml) to 39.1 +/- 30.9, 46.3 +/- 4.3, -2.0 +/- 16.8, and 86.6 +/- 1.4% of zero-control, respectively. Ketamine (250 microm-1 mm) also suppressed thrombin- and STA2-induced cytosolic free calcium concentration increase dose dependently. Although ketamine (2 mm) had no effect on TXA2 receptor expression and its binding affinity, it (1 mm) suppressed intracellular peak IP3 concentrations induced by thrombin and STA2 from 6.60 +/- 1.82 and 4.39 +/- 2.41 to 2.41 +/- 0.98 and 1.90 +/- 0.86 pmol/109 platelets, respectively, and it suppressed guanosine triphosphate hydrolysis induced by thrombin (0.02 units/ml) and STA2 (0.5 microm) to 50.3 +/- 3.2 and 67.5 +/- 5.5% versus zero-control, respectively. CONCLUSION: Ketamine inhibits human platelet aggregation possibly by suppressed IP3 formation and subsequent suppression of cytosolic free calcium concentration. The site of action of ketamine is neither TXA2 nor thrombin binding sites but possibly receptor-coupled mechanisms, including G-protein.

Subcellular interactions between parallel fibre and climbing fibre signals in Purkinje cells predict sensitivity of classical conditioning to interstimulus interval.

Classical conditioning of the nictitating membrane response requires a specific temporal interval between conditioned stimulus and unconditioned stimulus, and produces an increase in Protein Kinase C (PKC) activation in Purkinje cells. To evaluate whether biochemical interactions within the Purkinje cell may explain the temporal sensitivity, a model of PKC activation by Ca2+, diacylglycerol (DAG), and arachidonic acid (AA) is developed. Ca2+ elevation is due to CF stimulation and IP3 induced Ca2+ release (IICR). DAG and IP3 result from PF stimulation, while AA results from phospholipase A2 (PLA2). Simulations predict increased PKC activation when PF stimulation precedes CF stimulation by 0.1 to 3 s. The sensitivity of IICR to the temporal relation between PF and CF stimulation, together with the buffering system of Purkinje cells, significantly contribute to the temporal sensitivity.

Inverse agonist activity of selected ligands of platelet-activating factor receptor.

The receptor for platelet-activating factor (PAFR) is a member of the G protein-coupled receptor (GPCR) family. According to the allosteric ternary complex model, GPCRs exist in an equilibrium between different conformations. Agonist binding promotes and stabilizes the receptor in an active conformation. On the other hand, ligands that stabilize the inactive conformation are known as inverse agonists. Due to the association of platelet-activating factor (PAF) with diverse physiological and pathological processes, considerable efforts have been invested in the development of antagonists to PAFR. A large number of these molecules has been shown to specifically interact with PAFR but, surprisingly, little is known about their impact on the conformation of the receptor and its activity. By using a constitutively active mutant (L231R) of the human PAFR and by transiently coexpressing the wild-type (WT) receptor with the G(alpha)q subunit of the trimeric G protein, we were able to address this issue with ligands of diverse structures such as phospholipids, benzodiazepines, furans, and others. We demonstrated that some of these molecules are potent inverse agonists. For example, when cells (WT PAFR + G(alpha)q) were exposed to WEB2086, SM10661, or alprazolam, the basal inositol phosphate production was reduced by 53 +/- 6, 44 +/- 3, and 54 +/- 4%, respectively. The decrease in basal inositol phosphate production by WEB2086 was significantly inhibited by a more neutral antagonist BN52021, confirming the specificity of the reaction. We demonstrate here that WEB2086 and other known ligands previously considered as antagonists can act as inverse agonists on the human PAF receptor.

Effects of kappa-opioid receptor stimulation in the heart and the involvement of protein kinase C.

1. The role of protein kinase C (PKC) in mediating the action of kappa-receptor stimulation on intracellular Ca2+ and cyclic AMP production was determined by studying the effects of trans-(+/-)-3,4-dichloro-N-methyl-N-(2-[1-pyrrolidinyl] cyclohexyl) benzeneacetamide methanesulphonate (U50,488H), a selective kappa-receptor agonist, and phorbol 12-myristate 13-acetate (PMA), a PKC agonist, on the electrically-induced [Ca2+]i transient and forskolin-stimulated cyclic AMP accumulation in the presence and absence of a PKC antagonist, staurosporine or chelerythrine, in the single rat ventricular myocyte. 2. U50,488H at 2.5-40 microM decreased both the electrically-induced [Ca2+]i transient and forskolin-stimulated cyclic AMP accumulation dose-dependently, effects which PMA mimicked. The effects of the kappa-agonist, that were blocked by a selective kappa-antagonist, nor-binaltorphimine, were significantly antagonized by the PKC antagonists, staurosporine and/or chelerythrine. The results indicate that PKC mediates the actions of kappa-receptor stimulation. 3. To determine whether the action of PKC was at the sarcoplasmic reticulum (SR) or not, the [Ca2+]i transient induced by caffeine, that depletes the SR of Ca2+, was used as an indicator of Ca2+ content in the SR. The caffeine-induced [Ca2+]i transient was significantly reduced by U50,488H at 20 microM. This effect of U50,488H on caffeine-induced [Ca2+]i transient was significantly attenuated by 1 microM chelerythrine, indicating that the action of PKC involves mobilization of Ca2+ from the SR. When the increase in IP3 production in response to K-receptor stimulation with U50,488H in the ventricular myocyte was determined, the effect of U50,488H was the same in the presence and absence of staurosporine, suggesting that the effect of PKC activation subsequent to kappa-receptor stimulation does not involve IP3. The observations suggest that PKC may act directly at the SR. 4. In conclusion, the present study has provided evidence for the first time that PKC may be involved in the action of kappa-receptor stimulation on Ca2+ in the SR and cyclic AMP production, both of which play an essential role in Ca2+ homeostasis in the heart.

Effects of the peroxisome proliferator ciprofibrate and prostaglandin F2 alpha combination treatment on second messengers in cultured rat hepatocytes.

Peroxisome proliferators induce hepatic peroxisome proliferation and hepatic tumors in rodents. These chemicals increase the expression of the peroxisomal beta-oxidation pathway and the cytochrome P-450 4A family, which metabolizes lipids, including eicosanoids. Peroxisome proliferators transiently induce increased cell proliferation in vivo. However, peroxisome proliferators are weakly mitogenic and are not co-mitogenic with epidermal growth factor (EGF) in cultured hepatocytes. Earlier study found that the peroxisome proliferator ciprofibrate is comitogenic with eicosanoids. In order to study possible mechanisms of the comitogenicity of peroxisome proliferator ciprofibrate and eicosanoids, we hypothesized that the co-mitogenicity may result from synergistic or additive increases of second messengers in mitogenic signal pathways. We therefore examined the effect of the peroxisome proliferator ciprofibrate, prostaglandin F2 alpha (PGF2 alpha) and the combination of ciprofibrate and PGF2 alpha with or without growth factors on the protein kinase C (PKC) activity, and inositol-1, 4, 5-triphosphate (IP3) and intracellular calcium ([Ca2+]i) concentrations in cultured rat hepatocytes. The combination of ciprofibrate and PGF2 alpha significantly increased particulate PKC activity. The combination of ciprofibrate and PGF2 alpha also significantly increased EGF, transforming growth factor-alpha (TGF-alpha) and hepatic growth factor (HGF)-induced particulate PKC activity. The combination of ciprofibrate and PGF2 alpha greatly increased [Ca2+]i. However, the increases of PKC activity and [Ca2+]i by ciprofibrate and PGF2 alpha alone were much smaller. Neither ciprofibrate or PGF2 alpha alone nor the combination of ciprofibrate and PGF2 alpha significantly increased the formation of IP3. The combination of ciprofibrate and PGF2 alpha, however, blocked the inhibitory effect of TGF-beta on particulate PKC activity and formation of IP3 induced by EGF. These results show that co-mitogenicity of the peroxisome proliferator ciprofibrate and eicosanoids may result from the increase in particulate PKC activity and intracellular calcium concentration but not from the formation of IP3.

Effects of the anti-allergics astemizole and norastemizole on Fc epsilon RI receptor-mediated signal transduction processes.

The non-sedating anti-allergic drug astemizole, apart from its potential to antagonise H1 receptors, inhibits the release of inflammation mediators from mast cells. To study the mechanism of this inhibition, we investigated the effects of astemizole and one of its active metabolites, norastemizole, on different phases of Fc epsilon RI (the high affinity receptor for the immunoglobulin IgE) receptor-activated signal transduction in rat basophilic leukemia cells (RBL-2H3), leading to exocytosis. Cells were stimulated either through antigen, or thapsigargin, or synergistic combinations of Fc epsilon RI receptor activation with either adenosine A3 receptors or integrins, activated by fibronectin adherence. The effects of the drugs on mediator release, inositol 1,4,5-trisphosphate formation, tyrosine phosphorylation of cellular proteins and Ca2+ fluxes were investigated. Inositol 1,4,5-trisphosphate levels are not affected. Astemizole increased tyrosine phosphorylation in resting cells, especially a 96-kDa protein band. Particularly tyrosine phosphorylation related to post Ca2+ processes is changed after cell triggering in the presence of astemizole. Both drugs inhibit the influx of 45Ca2+, with similar dose response curves as for the inhibition of exocytosis. Astemizole but not norastemizole, when used in resting cells, released Ca2+ from intracellular stores. Astemizole (> 15 microM) also induced exocytosis in resting cells. It did not induce additional changes in its inhibiting effect when cells were triggered with synergistic combinations of Fc epsilon RI receptor activation with either adenosine A3 receptors or integrins. Effects on haemolysis of erythrocytes and differential scanning calorimetry in liposomes showed clear differences in membrane perturbation between astemizole and norastemizole. The observed differences, and the role of membrane perturbation in the action on Ca2+ fluxes, are discussed.

Acute or chronic lithium does not affect agonist-stimulated inositol trisphosphate formation in rat brain in vivo.

Rats were given lithium either acutely by s.c. injection (4 m eq kg-1) or chronically by including 0.2% LiCl in their diet for 3 weeks. Microdialysis probes were inserted into the cortex or hippocampus, using re-usable guides, and perfused with artificial CSF. Fractions were collected beginning 18 h after the end of treatment and were analysed for inositol trisphosphate (IP3). Neither acute nor chronic treatment affected basal levels of IP3 or stimulation of IP3 formation by either carbachol or noradrenaline in the hippocampus. Similarly, neither basal nor carbachol-stimulated IP3 levels in rat cortex were affected by acute Li administration. It would appear that the reductions in these parameters previously reported by other workers using brain slices were due to inositol depletion occurring at the stage of brain slice preparation. The inositol depletion hypothesis for the mechanism of action of lithium does not therefore appear to be supported by in vivo evidence.

Intracellular calcium signalling and vascular reactivity in Bartter's syndrome.

We investigated patients affected by Bartter's syndrome in the attempt to localize the intracellular defect mediating the reduced intracellular Ca2+ mobilization that may be responsible for the decreased vascular reactivity characteristic of Bartter's syndrome. Using the formylmethionyl-leucyl-phenylalanine (fMLP) receptor system, which causes, intracellular calcium release, we investigated fMLP-stimulated intracellular inositol 1,4,5-trisphosphate (IP3) production as well as the number and affinity of fMLP receptors in neutrophils from Bartter's syndrome patients and healthy controls. Scatchard plot analysis of radioactive fMLP binding to neutrophils indicated that there were no differences in either cell receptor number and affinity for ligand between healthy controls (n = 5) and patients with Bartter's syndrome (n = 5): 6,151 +/- 1,431 vs. 7,112 +/- 2,566 receptors/cell; K(D): 0.446 +/- 0.14 vs. 0.454 +/- 0.09 pM of fMLP. 5- and 10-second fMLP-stimulated intracellular IP3 production was instead reduced in patients affected by Bartter's syndrome: 2.479 +/- 1.07 vs. 4.073 +/- 1.04 nmol/10(7) cells at 5 s (n = 8; p < 0.01); 1.673 +/- 0.741 vs. 3.766 +/- 1.348 nmol/10(7) cells at 10 s (n = 8; p < 0.005). The results of this study indicate that the anomaly of intracellular calcium mobilization in patients with Bartter's syndrome arises from a defect at the postreceptor level. The anomalous calcium signalling that takes place in Bartter's syndrome may provide a mechanism for the hyporesponsiveness to pressor stimuli characteristic of these patients.

The role of intracellular calcium and protein kinase C in endothelin-stimulated proliferation of rat type I astrocytes.

The increased expression of immunoreactive endothelin-1 (ET-1) in reactive astrocytes and its mitogenic effects on astrocytes and glioma cell lines, have implicated endothelins in the development of reactive gliosis. In this study, an increase in DNA synthesis in rat type I astrocytes was observed after cultures were transiently exposed to ET-1 for 15 min, suggesting that early signal transduction events are essential and sufficient for the propagation of the ET-1-induced mitogenic signal. Prompt increases in inositol triphosphate (IP3) formation and [Ca2+]i were observed upon the addition of ET-1 to these cells. The ET-1-evoked increase in [Ca2+]i consisted of an initial peak which was preserved in Ca(2+)-free medium, and a sustained phase which was abolished in Ca(2+)-free medium and partly attenuated by nifedipine. ET-1 also increased the activity of membrane-associated protein kinase C (PKC) and induced the in vivo phosphorylation of the 85 kD MARCKS protein, an endogenous PKC-specific substrate. The ET-1-evoked increases in DNA synthesis, IP3, [Ca2+]i, membrane PKC, and 85 kD MARCKS protein phosphorylation in rat cortical astrocytes were prevented by either the selective endothelin ETA receptor antagonist, BQ-123, or the phospholipase C (PLC)-specific inhibitor, U-73122. However, the inhibition of PKC activity did not affect ET-1-induced DNA synthesis in rat cortical astrocytes. These results suggest that ET-1-induced IP3 and/or [CA2+]i responses, but not the activation of PKC, are essential for the growth-factor like actions of ET-1 in rat cortical astrocytes.

Calcium antagonistic actions of tetrandrine depend on cell types.

We examined the effects of tetrandrine (TET) on Ca2+ mobilization in various types of cells using inositol trisphosphate-generating drugs and compared it with those using the microsomal Ca(2+)-ATPase inhibitor thapsigargin (TG) which is a tool for analyzing Ca2+ store-regulated Ca2+ entry (capacitative Ca2+ entry). In rat pheochromocytoma PC12 cells, 100 microM TET abolished high K+ (30 mM)-induced sustained increase in [Ca2+]i and partially inhibited bradykinin (1 microM)-induced or TG (100 nM)-induced Ca2+ entry. In NIH/3T3 fibroblasts, 100 microM TET abolished Ca2+ entry induced by bombesin (1 microM) or TG (100 nM). In rat glioma C6 cells, the addition of 100 microM TET reduced the sustained elevation of [Ca2+]i induced by endothelin 1 (10 nM) or TG (100 nM) declining to the resting level. In rat parotid acinar cells, 100 microM TET abolished a sustained increase in [Ca2+]i induced by carbachol (100 microM) or TG (100 nM). In human leukemia T-cell line Jurkat, 100 microM TET did not inhibit Ca2+ entry evoked by the anti-CD3 antibody OKT3 (10 micrograms/ml) or TG (100 nM). The present results suggest that the action of TET on Ca2+ entry is dependent on cell types.

Molecular and functional characterization of recombinant human metabotropic glutamate receptor subtype 5.

We have isolated and characterized overlapping cDNAs that encode two isoforms of the human metabotropic glutamate receptor subtype 5 (hmGluR5). The deduced amino acid sequences of human and rat mGluR5a are 94.5% identical. However, a region in the putative cytoplasmic domain (SER926-ALA1121) displays significant sequence divergence. Genomic analysis of this region showed that the sequence divergence results from species-specific differences in the genomic sequences, not from alternative splicing. The distribution of mGluR5 mRNA in human brain was most strongly detected throughout the hippocampus, with moderate levels in the caudate-putamen, cerebral cortex, thalamus, and deep cerebellar nuclei, and at low levels in the cerebellar cortex. Activation of both hmGluR5a and hmGluR5b transiently expressed in Xenopus oocytes and HEK293 cells was coupled to inositol phosphate (InsP) formation and elevation of the intracellular free calcium ([Ca2+]i). The agonist rank order of potency for activating recombinant hmGluR5a receptors in either system was quisqualate > L-glutamate > 1S,3R-ACPD. Both the quisqualate stimulated InsP and [Ca2+]i were inhibited by (+)-MCPG. Recombinant human mGluR5a was also stably expressed in mouse fibroblast Ltk- cells, in which the efficacy and potency of quisqualate were unchanged for more than 30 cell passages.

Ontogenic increase in PGE2 and PGF2 alpha receptor density in brain microvessels of pigs.

1. The hypothesis that the relative vasoconstrictor ineffectiveness of prostaglandin E2 (PGE2) and PGF2 alpha on cerebral vessels of newborn pigs might be due to fewer receptors for these prostanoids was tested by comparing receptors for PGE2 (EP) and PGF2 alpha (FP) in cerebral microvessels from newborn and adult pigs. 2. Specific binding of [3H]-PGE2 and [3H]-PGF2 alpha to membranes prepared from brain microvessels showed that EP and FP receptor density (Bmax) in tissues from newborn animals was less than 50% of that determined in tissues from adults. By contrast, estimates of affinity (KD) were unchanged. 3. Specifically bound [3H]-PGE2 to brain microvessels from both the newborn and adult was displaced by AH 6809 (EP1-selective antagonist) by 80-90%, and only by approximately 30-35% by both 11-deoxy PGE1 (EP2/EP3 agonist) and M&B 28,767 (EP3 agonist); butaprost (EP2 agonist) was completely ineffective. 4. PGE2, 17-phenyl trinor PGE2 (EP1 agonist), PGF2 alpha and fenprostalene (PGF2 alpha analogue) caused significantly less increase in inositol 1,4,5-triphosphate (IP3) in brain microvessels from the newborn than in those from adult pigs. The stimulation of IP3 by PGE2 and 17-phenyl trinor PGE2 was almost completely inhibited by the EP1 antagonist, AH 6809. 5. PGE2, 11-deoxy PGE1 and M&B 28,767 produced small reduction of adenosine 3':5'-cyclic monophosphate (cyclic AMP) production in adult vessels but no effect in newborn tissues. 6. The lower density of EP and FP receptors in microvessels of newborn pigs compared to adults may explain the reduced ability of PGE2 and PGF2 alpha to stimulate production of IP3 in tissues from newborn animals. This in turn, may provide an explanation for previous observations demonstrating that these prostanoids elicit contraction of adult cerebral microvessels, but exert minimal effects on these vessels in newborn animals.

Characterization of P2-purinoceptor mediated cyclic AMP formation in mouse C2C12 myotubes.

1. The formation of adenosine 3':5'-cyclic monophosphate (cyclic AMP) and inositol(1,4,5)trisphosphate (Ins(1,4,5)P3), induced by ATP and other nucleotides was investigated in mouse C2C12 myotubes. 2. ATP (100 microM) and ATP gamma S (100 microM) caused a sustained increase in cyclic AMP content of the cells, reaching a maximum after 10 min. The cyclic AMP content reached a maximum in the presence of 100 microM ATP, followed by a decline at higher ATP concentrations. ATP-induced cyclic AMP formation was inhibited by the P2-purinoceptor antagonist, suramin. 3. Myotubes hydrolysed ATP to ADP at a rate of 9.7 +/- 1.0 nmol mg-1 protein min-1. However, further hydrolysis of ADP to AMP and adenosine was negligible. 4. The cyclic AMP formation induced by ADP (10 microM-1 mM) showed similar characteristics to that induced by ATP, but a less pronounced decline was observed than with ATP. ADP-induced cyclic AMP formation was blocked by suramin, while cyclic AMP formation elicited by adenosine (10 microM-1 mM) was insensitive to suramin. 5. The ATP analogue, alpha,beta-methylene-ATP also induced a suramin-sensitive cyclic AMP formation, while 2-methylthio-ATP and the pyrimidine, UTP, did not affect cyclic AMP levels. 6. Stimulation of the myotubes with ATP or UTP (10 microM-1 mM) caused a concentration-dependent increase in the Ins(1,4,5)P3 content of the cells. ADP (100 microM-1 mM) was less effective. Adenosine did not affect Ins(1,4,5)P3 levels. 7. Incubation of the cells with UTP (30 microM- 1 mM) inhibited the ATP- and ADP-induced cyclic AMP formation, suggesting that stimulation of the 'nucleotide' type P2-receptor inhibits P2-purinoceptor mediated cyclic AMP formation in C2C12 myotubes. In contrast, UTP (30 microM-I mM) enhanced adenosine-induced cyclic AMP formation.8. Adenosine-sensitive P1-purinoceptors activating cyclic AMP formation were found in C2C12 myotubes.Further, a novel P2-purinoceptor is postulated, sensitive to ATP, ADP and ATPgammaS, which also activates the formation of cyclic AMP in C2C12 myotubes.

Receptor-evoked Ca2+ mobilization in pancreatic acinar cells: evidence for a regulatory role of protein kinase C by a mechanism involving the transition of high-affinity receptors to a low-affinity state.

In order to establish a regulatory role for phosphoproteins in the process of receptor-stimulated Ca2+ mobilization, isolated pancreatic acinar cells, loaded with fura-2, were stimulated with cholecystokinin-octapeptide (CCK8) in the presence of either staurosporine, a general inhibitor of protein kinase activity, or 12-O-tetradecanoylphorbol 13-acetate (TPA), an activator of protein kinase C. Staurosporine alone did not affect the average free cytosolic Ca2+ concentration ([Ca2+]i,av) in a suspension of acinar cells. However, in the presence of 1.0 microM staurosporine the stimulatory effect of submaximal concentrations of CCK8 was significantly enhanced. The potentiating effect of the inhibitor was paralleled by the increased production of inositol 1,4,5-trisphosphate. In addition, staurosporine evoked a transient increase in [Ca2+]i,av in cells prestimulated with a submaximal concentration of CCK8. The data obtained with staurosporine indicate that CCK8-stimulated phosphorylations exert a negative feedback role in the process of receptor-mediated Ca2+ mobilization. The involvement of protein kinase C was investigated by studying the effects of TPA on CCK8-induced Ca2+ mobilization. The phorbol ester induced a rightward shift of the dose/response curve for the CCK8-evoked increase in [Ca2+]i,av, which, in contrast to the unlimited shift obtained with the receptor antagonist D-lorglumide, reached a maximum of approximately one order of a magnitude at 10 nM TPA. The inhibitory effect of TPA was completely overcome by CCK8 at concentrations at or beyond 10 nM. This observation has led to the hypothesis that protein kinase C, directly or indirectly, converts the CCK receptor from a high-affinity state to a low-affinity state. Substantial evidence in favour of this hypothesis was provided by the observation that the increase in [Ca2+]i,av evoked by the CCK8 analogue JMV-180, which acts as an agonist at the high-affinity receptor, was completely blocked by TPA pretreatment. TPA also evoked a rightward shift of the dose/response curve for the carbachol-induced increase in [Ca2+]i,av, indicating that the protein-kinase-C-mediated transition of the affinity state of receptors is a more general phenomenon. In the presence of submaximal CCK8 concentrations, TPA dose-dependently decreased the poststimulatory elevated [Ca2+]i,av to the prestimulatory level, indicating that protein kinase C also inhibits the process of sustained Ca2+ mobilization. The effects of TPA were counteracted by staurosporine, suggesting that the effects of the inhibitor itself were indeed due to inhibition of the receptor-mediated activation of protein kinase C.(ABSTRACT TRUNCATED AT 400 WORDS)

Desensitization of acetylcholine induced inositol 1,4,5-trisphosphate formation in neuroblastoma SH-SY5Y cells following repetitive acetylcholine stimulations.

In this study, the desensitization of acetylcholine-induced inositol 1,4,5-trisphosphate [I(1,4,5)P3] formation, upon short-time prestimulations, was investigated in cultures of human neuroblastoma SH-SY5Y cells. Four repeated stimulations for 10 seconds with 10 microM acetylcholine were necessary to induce a desensitization of the I(1,4,5)P3 formation. The desensitization was observed 4 hours after the initiation of repetitive stimulations. The same effect was obtained by a single prestimulation with 1 mM acetylcholine. Preincubation of the cells with phorbol 12-myristate 13-acetate (PMA) markedly down-regulated the acetylcholine-induced I(1,4,5)P3 formation. However, the protein kinase C (PKC) inhibitors H7 and staurosporine did not influence the desensitization induced by four repeated stimulations with 20 microM acetylcholine. These results indicate that the signal transduction can be desensitized following repeated stimulations with sub-maximal concentrations of receptor agonist and although activation of PKC can induce the same down-regulation, PKC is most likely not involved in the desensitization induced by repetitive acetylcholine-stimulations.

Effects of verapamil on the cardiac alpha 1-adrenoceptor signalling system in diabetic rats.

We evaluated the effects of chronic verapamil treatment on the cardiac alpha 1-adrenoceptor signalling system in streptozocin-induced diabetic rats. The decrease in maximum cell surface [3H]bunazosin binding (Bmax) in isolated cardiac myocytes from the diabetic group (-46%, P < 0.01) was completely reversed by a 4-week course of verapamil, while Bmax in the verapamil-treated control group was unchanged. Similarly, the reduction in ventricular inositol 1,4,5-trisphosphate (IP3) production after stimulation with 10 microM noradrenaline (NA) seen in diabetes (-30%, P < 0.01) was completely normalized by verapamil, while the response in the verapamil-treated control group was unaffected. These results indicate that verapamil can induce complete recovery of the impaired cardiac alpha 1-adrenoceptor signalling system in the diabetic heart without affecting glucose metabolism.