Total Inositol Phosphate Formation Research Articles

Abstract 16606: Cardiac Fibroblast-Restricted Enhancement of G q Signaling Prolongs Calcium Transients and Increases Spontaneous Activity in Biomimetic Cardiac Microtissues

Introduction: Enhanced G q signaling in response to hemodynamic stress and injury is well known to induce remodeling in both cardiac myocytes (CMs) and fibroblasts (CFs). However, the effect of CF activation and fibrosis on CMs and integrated myocardial function cannot be easily discerned from concomitant effects on CMs. Hypothesis: Enhanced G q signaling in CFs influences CM function in a 3D microtissue model previously developed in our lab, in which neonatal rat ventricular CMs and CFs are highly interspersed as seen in the myocardium. Methods: CFs infected in suspension (2 hrs, 10 MOI) with empty adenovirus (Ad-Ctr) or Ad encoding wild-type (WT) or constitutively active (Q209L or QL) Gα q were co-seeded with uninfected CMs (CM:CF 1:1) into non-adhesive hydrogels at ~1200 cells per recess (400/800 μm wide/deep). After 2-4 days, we assessed tissue size by microscopy, G q and extracellular matrix proteins proteins by Western Blot analysis, and Ca 2+ transients ([Ca 2+ ] i ) with Rhod 2/AM. Results: Overexpression of Gα q in CFs was associated with a 3-fold (WT) and 7-fold (QL) increase in total inositolphosphate formation, indicating enhanced basal phospholipase β activity. After 4 days, CM:CF QL tissues were larger in size by 30-40% and expressed more laminin and fibronectin. After 3 days, CM:CF CTR paced at 1 Hz had [Ca 2+ ] i duration of 218±3 ms, decay of 141±2 ms, and rise time of 35±4 ms (n=55-69). In CM:CF QL , duration and decay were prolonged by 160 ms and 21 ms, resp., and rise time was decreased by 5 ms (n=10-27). In CM:CF WT with less G q signaling, [Ca 2+ ] i duration and decay were also prolonged but only by 30 ms and 7 ms, resp. (n=46-51). These effects were similar but less pronounced after 2 days and/or at 2 Hz. Spontaneous [Ca 2+ ] i activity was seen in 50% of CM:CF QL , 11% of CM:CF WT , but only 2% of CM:CF CTR microtissues. The gap junction inhibitor carbenoxolone (100 μM) decreased this activity in CM:CF QL by 80% and in CM:CF WT by 15%. Conclusion: We show that enhanced CF-restricted G q signaling in biomimetic cardiac microtissues increases tissue size, prolongs [Ca 2+ ] i duration and decay, and increases spontaneous Ca 2+ activity. Our findings suggest that depending on the CF activation state, CFs can greatly modulate CM calcium handling and potentially influence arrhythmogenesis.

Involvement of the Adenylyl Cyclase/Protein Kinase A Signaling Pathway in the Stimulatory Effect of PACAP on Frog Adrenocortical Cells

We have previously shown that PACAP stimulates in vitro the secretion of corticosteroids by frog adrenal explants and that PACAP increases cAMP formation and cytosolic calcium concentration ('Ca2+'i) in adrenocortical cells. The aim of the present study was to investigate the involvement of cAMP and 'Ca2+'i in the stimulatory effect of PACAP on steroid production. Incubation of adrenal explants with PACAP resulted in a significant increase in total inositol phosphate formation. Administration of the protein kinase A inhibitor, H89, markedly reduced the stimulatory effect of PACAP on corticosterone and aldosterone secretion by perifused adrenal slices. In contrast, chelation of intracellular or extracellular calcium, or incubation with calcium channel blockers, had no effect on PACAP-evoked steroid secretion. Incubation of the cells with BAPTA or thapsigargin totally suppressed the stimulatory effect of PACAP on 'Ca2+'i. In contrast, suppression of extracellular calcium with EGTA or blockage of voltage-dependent Ca2+ channels did not impair PACAP-induced Ca2+ response. These data indicate that, in frog adrenocortical cells, the stimulatory effect of PACAP on steroid secretion is mediated through activation of the cAMP/PKA pathway. Concurrently, PACAP causes calcium mobilization from IP(3)-dependent intracellular stores through activation of a phospholipase C, while the calcium response is not involved in the stimulatory effect of PACAP on corticosteroid secretion.

Selective Loss of Fine Tuning of Gq/11 Signaling by RGS2 Protein Exacerbates Cardiomyocyte Hypertrophy

Alterations in cardiac G protein-mediated signaling, most prominently G(q/11) signaling, are centrally involved in hypertrophy and heart failure development. Several RGS proteins that can act as negative regulators of G protein signaling are expressed in the heart, but their functional roles are still poorly understood. RGS expression changes have been described in hypertrophic and failing hearts. In this study, we report a marked decrease in RGS2 (but not other major cardiac RGS proteins (RGS3-RGS5)) that occurs prior to hypertrophy development in different models with enhanced G(q/11) signaling (transgenic expression of activated Galpha(q)(*) and pressure overload due to aortic constriction). To assess functional consequences of selective down-regulation of endogenous RGS2, we identified targeting sequences for effective RGS2 RNA interference and used lipid-based transfection to achieve uptake of fluorescently labeled RGS2 small interfering RNA in >90% of neonatal and adult ventricular myocytes. Endogenous RGS2 expression was dose-dependently suppressed (up to 90%) with no major change in RGS3-RGS5. RGS2 knockdown increased phenylephrine- and endothelin-1-induced phospholipase Cbeta stimulation in both cell types and exacerbated the hypertrophic effect (increase in cell size and radiolabeled protein) in neonatal myocytes, with no major change in G(q/11)-mediated ERK1/2, p38, or JNK activation. Taken together, this study demonstrates that endogenous RGS2 exerts functionally important inhibitory restraint on G(q/11)-mediated phospholipase Cbeta activation and hypertrophy in ventricular myocytes. Our findings point toward a potential pathophysiological role of loss of fine tuning due to selective RGS2 down-regulation in G(q/11)-mediated remodeling. Furthermore, this study shows the feasibility of effective RNA interference in cardiomyocytes using lipid-based small interfering RNA transfection.

Expression, Characterization, and Purification of C-terminally Hexahistidine-tagged Thromboxane A2 Receptors

Thromboxane A2 (TxA2) receptors belong to the class of G-protein-coupled receptors. Knowledge of the relationship of structure to function for TxA2 receptors is limited because of their low levels of expression, lengthy purification procedures and poor recoveries. A C-terminal hexahistidine-tag (C-His) was ligated to the alpha-isoform of TxA2 receptors and expressed in COS-7 and Chinese hamster ovary cells. The C-His-TxA2 receptors bound the radioligands 125I-7-[(1R,2S,3S,5R)-6, 6-dimethyl-3-(4-benzenesulfonylamino)bicyclo[3.1. 1]hept-2-yl]-5(Z)-heptenoic acid, an antagonist, and 125I-[1S-1alpha, 2beta(5Z),3alpha(1E,3S*), 4alpha]-7-[3[(3-hydroxy-4-(4'-phenoxy)-1butenyl)-7-oxabicycl o-[2.2. 1]heptan-2-yl]-5-heptanoic acid, an agonist, with affinities not significantly different from those of the wild type (wt)-TxA2 receptors. LipofectAMINE transfection of the cDNAs resulted in high levels of expression (Bmax = 95 +/- 6 pmol/mg) of the C-His-TxA2 receptors. In competition binding studies the IC50 values of five different ligands were not significantly different between C-His-TxA2 and wt-TxA2 receptors. Agonist-induced stimulation of cAMP and total inositol phosphate formation were not significantly different between the two receptors. Purification on a Ni2+-NTA column resulted in a rapid (within 4 h) purification with a 36 +/- 2% recovery and a 30 +/- 6-fold purification (n = 5). The partially purified receptors were resolved on SDS-polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane, dissolved in acetone/trifluoroacetic acid/hexafluoroisopropanol/sinapinic acid, and successfully subjected to matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis. The results suggest that the combination of a high level of expression of C-His-TxA2 receptors and a rapid purification procedure followed by SDS- polyacrylamide gel electrophoresis may provide a useful approach for mass-spectrometry based structure-function and other studies of TxA2 receptors.

Neurotrophic factors alter gene expression in mouse hippocampal neurons

The possible age-related involvement of two different signal transduction pathways in the rat CNS was investigated. In the phosphytidyl inostiol (PI) response, higher phospholipase-C (PL-C) activity and drastically higher (almost 2.5-fold) inositol (1,4,5)trisphosphate (Ins(1,4,5) P3) concentration in the corpus striatum (caudate-putamen) of extremely old (approximately 40 months) female Wistar rats in comparison to young adult (approximately 3.5 months) rats were observed. In the adenosine 3′:5′-cyclic monophosphate (cAMP) cascade, a significantly higher endogenus cAMP level and a significant decline of the adenylate cyclase (AC, ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1.) activity were observed in striatal tissue from young rats in comparison with aged rats. Binding saturation experiments with [3H]SCH 23390 at the dopamine (DA) D1 receptor (D1) and [3H]spiperone at the DA D2 receptor (D2) revealed no change in the affinity (Kd) but a significant decrease in the density (Bmax) of D1 (−31%, p<0.005) and of D2 (−22%, p<0.05), respectively, in the aged versus young striata. DA seems to slightly inhibit total inositol phosphate formation and this effect was antagonized by (−)-sulpiride. A significant decrease (p<0.05) in the AC activity stimulated by 10 μM DA in the senescent compared to the young animals was monitored. Apparently, the age-related decline of the AC activity was independent of changes of GS and Gi activity.

Identification of Prostanoid Receptors in Rabbit Non-pigmented Ciliary Epithelial Cells

Preliminary ligand binding studies demonstrated that the membrane preparations of the rabbit non-pigmented ciliary epithelial cell line have 3H-prostaglandin E 2binding sites. The binding sites were specific for 3H-prostaglandin E 2as demonstrated by competition with unlabeled prostaglandin E 2. The IC 50of prostaglandin E 2for the inhibition of 3H-prostaglandin E 2binding was 435n m. The stimulation of adenylyl cyclase and phospholipase C by prostanoid receptor agonists, in rabbit non-pigmented ciliary epithelial cells resulted in the formation of either cyclic AMP or inositol phosphates. Prostaglandin E 2and 16-16-dimethyl prostaglandin E 2(both are EP 1, EP 2, EP 3and EP 4receptor agonists), 11-deoxy prostaglandin E 1(EP 2, EP 3and EP 4receptor agonist), butaprost (EP 2receptor agonist), and prostaglandin D 2(DP receptor agonist) stimulated the formation of cyclic AMP in a dose-dependent manner. Maximal stimulation occurred between 1.25 and 2.5μ mfor prostaglandin E 2and 16,16-dimethyl prostaglandin E 2and between 10 and 20μ mfor 11-deoxy prostaglandin E 1and prostaglandin D 2. Prostaglandin E 2and 16,16-dimethyl prostaglandin E 2were more potent (EC 50of 0.25μ mand 0.42μ mrespectively) than 11-deoxy prostaglandin E 1, butaprost or prostaglandin D 2. The formation of cyclic AMP by prostaglandin D 2was inhibited by BW868C, a highly selective DP receptor antagonist. 17-phenyl trinor prostaglandin E 2, prostaglandin F 2αand U46619, the EP 1, FP and TP receptor agonists, respectively stimulated phospholipase C (as measured by the formation of total inositol phosphates) in a dose-dependent manner. The agonists 11-deoxy prostaglandin E 1and butaprost coupled to adenylyl cyclase via guanine nucleotide binding protein, G s, did not increase the turnover of inositol phosphates. The results of the present study suggest that rabbit non-pigmented ciliary epithelial cells express EP 1, EP 2, DP, FP and TP receptors.

Regulation of endothelin-induced Ca2+ mobilization in smooth muscle cells by protein kinase C

We have investigated the role of protein kinase C (PKC) in regulating vascular smooth muscle cell responses to endothelin (ET). During the initial phase of the response, ET stimulated rapid formation of diacylglycerol due to rapid and transient activation of phosphatidyl inositol-specific phospholipase C and to rapid and prolonged activation of phospholipase D. Concurrently, ET stimulated translocation of PKC activity that reached a peak at 1 min and remained elevated for at least 20 min. Activation of PKC produced early inhibitory effects. Treatment of cells with phorbol 12-myristate 13-acetate (PMA) 5 min before stimulation with ET inhibited total inositol phosphate formation by > 50%. Because each inositol phosphate isomer was equally affected, the target appears to be either phospholipase C or some upstream component of the receptor coupling mechanism. Activation of PKC was important for sustained response to ET. Treatment of cells with staurosporine significantly reduced sustained elevation of cytosolic free Ca2+ concentration ([Ca2+]i) normally seen with ET. We had previously shown that sustained elevation of [Ca2+]i initiated by ET was due to continued activity of L-type Ca2+ channels. Our current data suggest that PKC is important in this response. For example, staurosporine inhibited both ET-induced 45Ca2+ and Mn2+ entry occurring 10 min after stimulation of influx mechanisms by the agonist. Similarly, pretreatment of cells for 18 h with phorbol dibutyrate depleted the cells of PKC and blocked the sustained activity of Ca2+ entry mechanisms stimulated by ET. Finally, PMA initiated a slowly developing, sustained elevation of [Ca2+]i.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of prostaglandin F2 alpha on Ca2+ influx in osteoblast-like cells: function of tyrosine kinase.

We previously reported that pertussis toxin-sensitive GTP-binding protein is involved in prostaglandin F2 alpha (PGF2 alpha)-induced phosphoinositide (PI) hydrolysis in osteoblast-like MC3T3-E1 cells [Miwa et al. (1990): Biochem Biophys Res Commun 171:1229-1235]. In the present study, we investigated the mechanism of PGF2 alpha-induced Ca2+ influx in MC3T3-E1 cells. PGF2 alpha-induced formation of total inositol phosphates (IPs) was markedly reduced by the depletion of extracellular Ca2+ with EGTA. On the other hand, the depletion of extracellular Ca2+ had little effect on PGF2 alpha-induced inositol 1,4,5-trisphosphate formation. PGF2 alpha stimulated 45Ca2+ influx dose dependently, attaining a maximum effect at 10 nM. Dose of PGF2 alpha above 10 nM caused less than maximal stimulation. Genistein, an inhibitor of protein tyrosine kinase, which by itself had little effect on 45Ca2+ influx, significantly suppressed the PGF2 alpha-induced 45Ca2+ influx in a dose-dependent manner in the range between 1 micrograms/ml and 0.1 mg/ml. Sodium orthovanadate, an inhibitor of protein tyrosine phosphatases, enhanced the PGF2 alpha-induced 45Ca2+ influx. Genistein also suppressed the PGF2 alpha-induced total IPs formation dose dependently in the range between 1 micrograms/ml and 0.1 mg/ml. However, it had little effect on the PGF2 alpha-induced inositol 1,4,5-trisphosphate formation. The pretreatment with pertussis toxin had little effect on the PGF2 alpha-induced 45Ca2+ influx. These results strongly suggest that PGF2 alpha stimulates Ca2+ mobilization from extracellular space and PI hydrolysis via independent pathways in osteoblast-like cells, and the PGF2 alpha-induced Ca2+ influx is regulated by protein tyrosine kinase, resulting in the promotion of PI hydrolysis.

Signal transduction in aging

The possible age-related involvement of two different signal transduction pathways in the rat CNS was investigated. In the phosphytidyl inostiol (PI) response, higher phospholipase-C (PL-C) activity and drastically higher (almost 2.5-fold) inositol (1,4,5)trisphosphate (Ins(1,4,5) P(3)) concentration in the corpus striatum (caudate-putamen) of extremely old (approximately 40 months) female Wistar rats in comparison to young adult (approximately 3.5 months) rats were observed. In the adenosine 3':5'-cyclic monophosphate (cAMP) cascade, a significantly higher endogenus cAMP level and a significant decline of the adenylate cyclase (AC, ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1.) activity were observed in striatal tissue from young rats in comparison with aged rats. Binding saturation experiments with [(3)H]SCH 23390 at the dopamine (DA) D(1) receptor (D(1)) and [(3)H]spiperone at the DA D(2) receptor (D(2)) revealed no change in the affinity (K(d)) but a significant decrease in the density (B(max)) of D(1) (-31%, p<0.005) and of D(2) (-22%, p<0.05), respectively, in the aged versus young striata. DA seems to slightly inhibit total inositol phosphate formation and this effect was antagonized by (-)-sulpiride. A significant decrease (p<0.05) in the AC activity stimulated by 10 muM DA in the senescent compared to the young animals was monitored. Apparently, the age-related decline of the AC activity was independent of changes of G(S) and G(i) activity.

Alterations of inositol phosphate turnover in striatum of aged rats

Age-related inositol phosphate turnover in the rat central nervous system was investigated. Higher phospholipase-C activity and drastically higher (almost 2.5-fold) inositol 1,4,5-trisphosphate concentration in the corpus striatum (caudate-putamen) of extremely old (approximately 40 months) female Wistar rats in comparison to the young adult (approximately 3.5 months) rats were observed. Dopamine seems to slightly inhibit total inositol phosphate formation and this effect was antagonized by (−)-sulpiride.

Interaction of parathyroid hormone-related peptide-responsive dual signal transduction systems in osteoblastic osteosarcoma cells: Role in PTHrP-induced homologous desensitization

In osteoblastic UMR-106 cells, 10(-7) M human (h) PTH-related peptide (PTHrP)-(1-34) significantly induced the formation of total inositol phosphates to the same degree as 10(-7) M hPTH-(1-34), confirming that in addition to cAMP-dependent protein kinase (PKA), PTHrP possesses another signal transduction system, calcium/protein kinase C (Ca/PKC). Experiments were therefore performed to characterize the cross talk of these dual-signal transduction systems and its participation in the PTHrP-induced homologous desensitization of cAMP and cytosolic calcium (Cai) response in osteoblasts. Preincubation with 10(-7) M hPTHrP-(1-34) caused homologous desensitization, resulting in a remarkable decrease in cAMP accumulation in response to further exposure to PTHrP. This effect was significant after 2 h pretreament and reached a maximum at 6 h. Pretreatment with the PKC-activating phorbol ester phorbol 12-myristate-13-acetate (PMA, 10(-6) M) for 30 minutes and 6 h caused a significant increase and decrease in cAMP responsiveness to PTHrP, respectively. Pretreatment with calcium ionophores (A23187 or ionomycin, 10(-6) M), not for 30 minutes but for 6 h, caused a significant decrease in cAMP responsiveness to PTHrP. H-7 (an inhibitor of PKC, 50 microM) significantly blocked not only PMA- but also PTHrP-induced desensitization of the cAMP response. PTHrP caused the complete homologous desensitization of an increase in Cai within 30 minutes. Pretreatment with dibutyryl-cAMP (10(-4) M) for 30 minutes caused significant inhibition of the PTHrP-induced increase in Cai, and pretreatment with Sp-cAMPS (10(-4) M), a direct activator of PKA, for 30 minutes completely blocked the PTHrP-induced increase in Cai.(ABSTRACT TRUNCATED AT 250 WORDS)

Differences in inositol phosphate production in blood vessels of normotensive and spontaneously hypertensive rats

1. Total inositol phosphate formation was measured in labelled femoral and iliac arteries and veins of 14 week-old spontaneously hypertensive rats (SHR) and age-matched Wistar Kyoto (WKY) controls, either unstimulated or in the presence of noradrenaline. 2. Basal levels of [3H]-inositol phosphates and [3H]-phosphatidylinositol were significantly enhanced in SHR femoral artery, but not in the other 3 vessels, compared with WKY. 3. Noradrenaline stimulated phosphoinositide hydrolysis in all four vessels of SHR and WKY. Pretreatment with prazosin (10(-7)-10(-6) M) but not with yohimbine (10(-7) M), inhibited the noradrenaline-induced inositol phosphate formation indicating an alpha 1-adrenoceptor-mediated response. 4. In the femoral artery of SHR compared to WKY, [3H]-inositol phosphate accumulation induced by noradrenaline (10(-7)-10(-5) M) was significantly reduced when expressed relative to basal values although the response to higher concentrations (10(-4)-10(-3) M) was not altered. In contrast, a significant reduction of inositol phosphates was seen only with 10(-7) M noradrenaline when absolute values were compared. In the other three vessels, no difference in noradrenaline-induced [3H]-inositol phosphate formation was observed between strains. 5. These data suggest that phosphoinositide hydrolysis-mediated by alpha 1-adrenoceptors may be reduced in some but not all blood vessels of adult SHR.

Potential Mechanisms Involved in the Negative Coupling Between Serotonin 5‐HT1A Receptors and Carbachol‐Stimulated Phosphoinositide Turnover in the Rat Hippocampus

Serotonin 5-HT1A receptors have been reported to be negatively coupled to muscarinic receptor-stimulated phosphoinositide turnover in the rat hippocampus. In the present study, we have investigated further the pharmacological specificity of this negative control and attempted to elucidate the mechanism whereby 5-HT1A receptor activation inhibits the carbachol-stimulated phosphoinositide response in immature or adult rat hippocampal slices. Various 5-HT1A receptor agonists were found to inhibit carbachol (10 microM)-stimulated formation of total inositol phosphates in immature rat hippocampal slices with the following rank order of potency (IC50 values in nM): 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) (11) greater than ipsapirone (20) greater than gepirone (120) greater than RU 24969 (140) greater than buspirone (560) greater than 1-(m-trifluoromethylphenyl)piperazine (1,500) greater than methysergide (5,644); selective 5-HT1B, 5-HT2, and 5-HT3 receptor agonists were inactive. The potency of the 5-HT1A receptor agonists investigated as inhibitors of the carbachol response was well correlated (r = 0.92) with their potency as inhibitors of the forskolin-stimulated adenylate cyclase in guinea pig hippocampal membranes. 8-OH-DPAT (10 microM) fully inhibited the carbachol-stimulated formation of inositol di-, tris-, and tetrakisphosphate but only partially antagonized (-40%) inositol monophosphate production. The effect of 8-OH-DPAT on carbachol-stimulated phosphoinositide turnover was not prevented by addition of tetrodotoxin (1 microM), by prior destruction of serotonergic afferents, by experimental manipulations causing an increase in cyclic AMP levels (addition of 10 microM forskolin), or by changes in membrane potential (increase in K+ concentration or addition of tetraethylammonium). Prior intrahippocampal injection of pertussis toxin also failed to alter the ability of 8-OH-DPAT to inhibit the carbachol response. Carbachol-stimulated phosphoinositide turnover in immature rat hippocampal slices was inhibited by the protein kinase C activators phorbol 12-myristate 13-acetate (10 microM) and arachidonic acid (100 microM). Moreover, the inhibitory effect of 8-OH-DPAT on the carbachol response was blocked by 10 microM quinacrine (a phospholipase A2 inhibitor) but not by BW 755C (100 microM), a cyclooxygenase and lipoxygenase inhibitor. These results collectively suggest that 5-HT1A receptor activation inhibits carbachol-stimulated phosphoinositide turnover by stimulating a phospholipase A2 coupled to 5-HT1A receptors, leading to arachidonic acid release. Arachidonic acid could in turn activate a gamma-protein kinase C with as a consequence an inhibition of carbachol-stimulated phosphoinositide turnover. This inhibition may be the consequence of a phospholipase C phosphorylation and/or a direct effect on the muscarinic receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

Pasteurella multocida toxin, a potent mitogen, increases inositol 1,4,5-trisphosphate and mobilizes Ca2+ in Swiss 3T3 cells.

Pasteurella multocida toxin, both native and recombinant, is an extremely potent mitogen for Swiss 3T3 cells and acts to enhance the formation of total inositol phosphates (Rozengurt, E., Higgins, T., Changer, N., Lax, A.J., and Staddon, J.M. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 123-127). P. multocida toxin also stimulates diacylglycerol production and activates protein kinase C (Staddon, J.M., Chanter, N., Lax, A.J., Higgins, T.E., and Rozengurt, E. (1990) J. Biol. Chem. 265, 11841-11848). Here we analyze, by [3H]inositol labeling and high performance liquid chromatography, the inositol phosphates in recombinant P. multocida toxin-treated cells. Recombinant P. multocida toxin stimulated increases in [3H]inositol 1,4,5-trisphosphate ([3H]Ins(1,4,5)P3) and its metabolic products, including Ins(1,3,4,5)P4, Ins(1,3,4)P3, Ins(1,4)P2, Ins(4/5)P, and Ins(1/3)P. The profile of the increase in the cellular content of these distinct inositol phosphates was very similar to that elicited by bombesin. Furthermore, recombinant P. multocida toxin, like bombesin, mobilizes an intracellular pool of Ca2+. Recombinant P. multocida toxin pretreatment greatly reduces the Ca2(+)-mobilizing action of bombesin, consistent with Ca2+ mobilization from a common pool by the two agents. The enhancement of inositol phosphates and mobilization of Ca2+ by recombinant P. multocida toxin were blocked by the lysosomotrophic agents methylamine, ammonium chloride, and chloroquine and occurred after a dose-dependent lag period. The stimulation of inositol phosphate production by recombinant P. multocida toxin persisted after removal of extracellular toxin, in contrast to the reversibility of the action of bombesin. Recombinant P. multocida toxin, unlike bombesin and guanosine 5'-O-(gamma-thiotriphosphate), did not cause the release of inositol phosphates in permeabilized cells. These data demonstrate that recombinant P. multocida toxin, acting intracellularly, stimulates the phospholipase C-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate.

Inositol phosphate formation in arterial smooth muscle from rabbits with perinephritis hypertension

Total inositol phosphate formation was measured in the aorta and femoral artery from rabbits at 1, 2 and 6 weeks after kidney wrapping, at which times the mean arterial pressures were 88 +/- 4, 96 +/- 3 and 126 +/- 7 mmHg against a control pressure of 74 +/- 3 mmHg. Noradrenaline-stimulated (10(-7) to 10(-4) mol/l) inositol phosphate formation was increased in the aorta and femoral artery from hypertensive rabbits at 2 weeks (aorta noradrenaline 10(-6) mol/l sham, 105 +/- 14%; hypertensive, 164 +/- 20% of control). Noradrenaline-stimulated inositol phosphate formation was unchanged at 1 and 6 weeks in the aorta. Endothelin-stimulated inositol phosphate formation was unchanged at 2 weeks. Basal inositol phosphate formation was not significantly different in normotensive and hypertensive animals. In perinephritis hypertension there is an alteration in phosphatidylinositol metabolism in arterial smooth muscle. This occurs at the time when the blood pressure is rising rapidly. This alteration may affect a specific phosphatidylinositol pool that is linked to the alpha-adrenoceptor but not to the endothelin receptor.

Glutamate‐Stimulated, Guanine Nucleotide‐Mediated Phosphoinositide Turnover in Astrocytes Is Inhibited by Cyclic AMP

The potential for cross-talk between the adenyl cyclase and phosphoinositide (PPI) lipid second messenger system was investigated in astrocytes cultured from neonatal rat brain. Glutamate-stimulated PPI turnover, measured by the formation of total inositol phosphates from myo-[3H]inositol-labeled lipids, was inhibited in a concentration-dependent manner by the elevation of intracellular cyclic AMP levels produced either by stimulation of the isoproterenol receptor linked to adenyl cyclase or by its direct activation by forskolin. N6,2'-O-Dibutyryl cyclic AMP, an analogue that can also activate cyclic AMP-dependent kinase, inhibited glutamate-stimulated PPI turnover in a concentration-dependent manner as well, a result suggesting that cyclic AMP-dependent kinase is involved in mediating the inhibition. Inclusion of an inhibitor of cyclic AMP-dependent kinase, 1-(5-isoquinolinesulfonyl)-2 methylpiperazine dihydrochloride or N-(2-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride, blocked the cyclic AMP-mediated inhibition in a concentration-dependent manner, a finding further supporting this hypothesis. The site of inhibition of the phosphoinositol lipid pathway by cyclic AMP was probed using a digitonin-permeabilized cell system. Guanosine 5'-O-(3-thiotriphosphate), a nonhydrolyzable analogue of GTP, stimulated PPI turnover and potentiated glutamate-stimulated PPI turnover, and guanosine 5'-O-(3-thiodiphosphate) inhibited glutamate-stimulated PPI turnover in these cells, results providing evidence that glutamate receptors are coupled to phospholipase C by a guanine nucleotide binding protein in astrocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential potentiation of mitogen-stimulated phosphoinositide hydrolysis in protein kinase C-depleted Swiss 3T3 cells

In Swiss 3T3 cells, depletion of protein kinase C (PKC) by prolonged incubation with phorbol esters potentiates the formation of total inositol phosphates in response to bombesin or vasopressin [Blakeley, Corps & Brown (1989) Biochem. J. 258, 177-185]. The characteristics of the accumulation of inositol phosphates in control and PKC-depleted cells stimulated by bombesin, vasopressin or prostaglandin F2 alpha (PGF2 alpha) have now been compared. The potentiation of the PGF2 alpha response was greater than that of the vasopressin response which was, in turn, greater than that of the bombesin response. The time courses of the responses to all three agonists were biphasic, and both phases of the response were amplified in the PKC-depleted cells. These results provide further evidence for the involvement of a PKC-mediated negative-feedback loop regulating phosphoinositide hydrolysis in response to several 3T3 cell mitogens. The differential potentiation of the response to these agonists suggests that PKC might act at multiple sites within the signal transduction pathway.

Pharmacological modulation of oscillations in the blowfly salivary gland

lation counting and expressed as d.p.m./culture well. (ii) Alternatively, the various inositol phosphates within the sample were separated by h.p.1.c. on a Partisil Sax column eluted with a 0-3 M-ammonium formate/O.l M-formic acid gradient at a flow rate of 1.25 ml/min. Fractions (0.5 min) were collected and radioactivity in them determined as above expressed as d.p.m./fraction. Treatment of 3T3 cultures with FGF stimulates total inositol phosphate formation in cells. Total [3HH]inotisol phosphates in control 3T3 cultures was 12.5 1 f 36 d.p.m./culture well; in cultures exposed to basic FGF (50 ng/ml) for 2 h it was 2 126 f 77 d.p.m./culture well. Table 1 shows different inositol phosphates produced during this response analysed by h.p.1.c. By 10 s of stimulation, inositol trisphosphate levels are raised, as are levels of inositol tetrakisand pentakisphosphate. After 10 min, the inositol trisand tetrakis-phosphate levels have fallen to control levels while the raised inositol pentakisphosphate levels are maintained. By 30 min of stimulation, although still higher than controls, the inositol pentakisphosphate levels are falling towards control values. This observation of a rapid and transient inositol trisphosphate signal accompanied by a much more persistent inositol pentakisphosphate signal suggests a multiple signalling pathway coupled by phospholipase C and activated by FGF. What mechanisms the two intracellular signals are targeting remain to be established, but it might be that the more highly phosphorylated inositol derivatives are involved in mediating the long-term regulation of cell growth. The responses to FGF in vitro can be divided into very early events, which include ionic fluxes, protein kinase activation and jntracellular pH changes, and later events such as gene transcription and protein synthesis (Gospodarowicz, 1087). It is interesting to speculate that the transient and persistent inositol phosphate signals observed here may be linked t o early and late responses to FGF. Overall these data establish that FGF does stimulate phosphoinositide hydrolysis, at least in Balb/c 3T3 fibroblast cells. However, the relative importance of this signalling pathway for mitogenesis has still to be defined.

Regulation of inositol trisphosphate accumulation by muscarinic cholinergic and H1-histamine receptors on human astrocytoma cells. Differential induction of desensitization by agonists.

Although activation of muscarinic cholinergic receptors on 1321N1 human astrocytoma cells results in a linear accumulation of inositol phosphates for up to 60 min in the presence of LiCl [Masters, Quinn & Brown (1985) Mol. Pharmacol. 27, 325-332], activation of H1-histamine receptors resulted in an increase in total inositol phosphate formation that was maintained for less than 5 min. The effects of stimulation of these two receptors on accumulation of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], inositol 1,3,4-trisphosphate [Ins(1,3,4)P3] and inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] were also examined. Incubation of 1321N1 cells with carbachol resulted in a rapid accumulation of all three inositol phosphates, reaching a maximum within 30 s; this elevated value was maintained for up to 60 min. The rate of disappearance of Ins(1,3,4)P3 from carbachol-treated cells after the addition of atropine paralleled or exceeded the rate of disappearance of Ins(1,4,5)P3. Although the initial rates of accumulation of Ins(1,4,5)P3, Ins(1,3,4)P3 and Ins(1,3,4,5)P4 in the presence of histamine were similar to that observed with carbachol, the amounts of these inositol phosphates had returned to control values within 5 min after the addition of histamine. The results indicate that, although the acute effects of muscarinic receptor and H1-histamine receptor stimulation on phosphoinositide hydrolysis are very similar, the histamine receptor is desensitized rapidly, whereas the muscarinic receptor is not. This effect on histamine-receptor function is apparently homologous, since preincubation of 1321N1 cells with histamine did not decrease the subsequent response to carbachol.