Increase In Phosphoinositide Hydrolysis Research Articles

Comparative Effects of Monomethylsuccinate and Glucose on Insulin Secretion from Perifused Rat Islets

In the absence of any other exogenously added fuel, monomethylsuccinate, the methyl ester of succinic acid, at 10-20 mM stimulates insulin release in a biphasic pattern. In quantitative terms, first-phase release evoked by 20 mM MMSucc was comparable to that observed with 20 mM glucose but second-phase release was only 20% of the glucose-induced response. Secretion to both MMSucc and glucose was virtually abolished by the calcium channel antagonist nitrendipine (0.5 microM). In islets that had phosphoinositide pools labeled with [3H]inositol for 2 h, subsequent stimulation with 20 mM MMSucc results in dramatic and sustained increases in [3H]inositol efflux rates. Inositol phosphate levels are also increased. In contrast to secretion, the increase in phosphoinositide hydrolysis caused by MMSucc was largely resistant to nitrendipine, whereas significant reductions in glucose-induced phosphoinositide hydrolysis were observed in the presence of the calcium channel antagonist. MMSucc (2.75-10 mM) substitutes for glucose in that MMSucc supported the insulinotropic effects of the sulfonylurea tolbutamide (200 microM) and the gut hormone cholecystokinin (200 nM). A prior 15-min exposure to 20 mM MMSucc also sensitized islets to the stimulatory effects of 7.5 mM glucose. Finally, a 2-h exposure to 20 mM MMSucc desensitized the islet, in terms of both phosphoinositide hydrolysis and insulin secretion, to a subsequent exposure to 10 mM glucose. Thus, appropriate concentrations of MMSucc can cause qualitatively many of the effects induced by glucose.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of metabotropic glutamate receptors increases cAMP accumulation in hippocampus by potentiating responses to endogenous adenosine

Metabotropic glutamate receptors (mGluRs) are coupled to effector systems through GTP-binding proteins (G-proteins) and appear to mediate slow synaptic responses in the CNS. Although mGluR-mediated increases in phosphoinositide hydrolysis have been well characterized, other mechanisms for signal transduction employed by mGluRs are poorly understood. We recently reported that the selective mGluR agonist 1-aminocyclopentane-1 S,3R-dicarboxylic acid (1S,3R-ACPD) increases cAMP accumulation in rat hippocampal slices. We have now investigated the mechanisms involved in this response. A number of G-protein-linked receptors that are not directly coupled to adenylate cyclase increase cAMP accumulation by potentiating cAMP responses to other agonists. Furthermore, previous studies suggest that glutamate increases cAMP accumulation by a mechanism that is dependent upon the presence of endogenous adenosine. Therefore, we tested the hypothesis that 1S,3R-ACPD-stimulated increases in cAMP accumulation in rat hippocampal slices are dependent upon the presence of endogenous adenosine and are mediated by an mGluR that potentiates cAMP responses to other agonists. We found that adenosine deaminase abolished 1S,3R-ACPD-stimulated cAMP accumulation whereas the adenosine uptake blocker dipyridamole enhanced this response. Additionally, adenosine receptor antagonists blocked mGluR-mediated increases in cAMP accumulation with potencies that were highly correlated with their potencies at A2 adenosine receptors. Furthermore, we performed a series of studies that suggest that 1S,3R-ACPD activates an mGluR subtype that potentiates responses to agonists of other receptors that are coupled to adenylate cyclase and that 1S,3R-ACPD-stimulated increases in cAMP accumulation in hippocampal slices are mediated by potentiation of the cAMP response to low levels of endogenous adenosine that are continuously present extracellularly.

Serotonin agonists increase transferrin levels via activation of 5-HT1C receptors in choroid plexus epithelium

Choroid plexus epithelial cells are enriched in mRNA for proteins such as the iron carrier transferrin, which acts as a trophic factor in the brain. Choroid plexus epithelial cells also have a high density of 5-HT1C receptors linked to activation of the phosphoinositide (PI) hydrolysis second messenger system. The present studies show that the 5-HT1C/5-HT2 receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) potently increases PI hydrolysis and the levels of transferrin in primary cultures of rat choroid plexus epithelial cells. These effects are blocked by the 5-HT1C/5-HT2 receptor antagonists mesulergine and mianserin, but not by the 5-HT2 receptor-selective antagonist spiperone. Similarly, mesulergine and mianserin, but not spiperone, block the increases in transferrin levels and PI hydrolysis elicited by 5-carboxamidotryptamine (5-CT), a 5-HT1 receptor-selective agonist, and by serotonin. We conclude, therefore, that 5-HT1C receptor activation in the choroid plexus leads to an increase in the production of transferrin. By promoting transferrin synthesis in the choroid plexus, 5-HT may indirectly influence brain development and differentiation.

Biochemical characterization of phosphoinositide hydrolysis stimulated by 5-HT3 receptor agonists.

Serotonin (5-HT) stimulates phosphoinositide (PI) turnover in rat fronto-cingulate cortical slices and is probably mediated through the activation of both 5-HT2 and 5-HT3 receptors. We have extended these findings and have assessed whether the increased stimulation of PI turnover is secondary to 5-HT stimulated arachidonate metabolism or to the release of another neurotransmitter. Incubation of the cortical slices by the two 5-HT3 receptor agonists, 2-methyl-serotonin (2-Me-5-HT) and phenylbiguanide (PBG), significantly decreases serotonin-stimulated phosphoinositide turnover, indicating that activation of 5-HT3, receptors by 2-Me-5-HT and PBG caused the desensitized PI hydrolysis to 5-HT. Indomethacin did not affect the increased PI hydrolysis induced by 5-HT, 2-Me-5-HT and PBG, suggesting that neither cyclooxygenase nor lipoxgenase activity is required for the PI response and that it is independent of arachidonic acid metabolism. The stimulation in PI turnover induced by 5-HT and the 5-HT3 receptor agonists was not potentiated by proteinase inhibitors suggesting that the release of a peptide neurotransmitter is not involved in the PI response. In addition, the effects of 5-HT, 2-Me-5-HT and PBG on PI turnover are additive to the effect of KCl and veratrine. In conclusion, our results indicate that the action of 2-Me-5-HT and PBG on PI turnover is direct.

Effect of muscarinic cholinergic drugs on ischemia-induced decreases in glucose uptake and CA1 field potentials in rat hippocampus slices

To clarify the role of muscarinic acetylcholinc receptors in the hypoxia/ hypoglycemia (ischemia)-induced functional deficit in hippocampal neurons, we examined the effect of cholinergic drugs on ischemia-induced impairments of glucose uptake and CA1 field potentials in hippocampus slices. Muscarinic receptors were subdivided into M 1 (high affinity for pirenzepine) and M 1 (low affinity for pirenzcpine) subtypes. The M 1 receptor subtype is coupled to an increase in phosphoinositide hydrolysis and the M 2 receptor subtype is associated with inhibition of adenylate cyclase. The greater potency of carbachol in stimulating phosphoinositide hydrolysis resulted in exacerbated ischemia-induced deficits. Treatment with the muscarinic receptor antagonists scopolamine and pircnzepine (M 1 receptor-selective antagonist) had a strong dose-dependent protective effect against ischemia-induced deficits. Oxotremorine and McN-A-343, weak stimulators of phosphoinositide hydrolysis and strong inhibitors of adenylate cyclase, had a weak neuroprotective action against ischemia-induced deficits. These results suggest that stimulation of M 1 musarinic receptors coupled with an increase in phosphoinositide hydrolysis may play a facilatory role in ischemia-induced deficits. Stimulation of M 2 muscarinic receptors may play an inhibitory role in ischemia-induced neuronal deficits.

Bradykinin B2 receptor-mediated phosphoinositide hydrolysis in bovine cultured tracheal smooth muscle cells.

1. Bovine tracheal smooth muscle cells were established in culture to study agonist-induced phosphoinositide (PI) hydrolysis in this tissue. 2. Bradykinin (0.1 nM-10 microM) evoked a concentration-dependent increase (log EC50 (M) = -9.4 +/- 0.2; n = 8) in the accumulation of total [3H]-inositol phosphates in cultured tracheal smooth muscle cells whereas the selective B1 receptor agonist des-Arg9-bradykinin (10 microM) was significantly less effective (16% of bradykinin maximal response; relative potency = 0.2 with respect to bradykinin = 100). 3. The bradykinin-induced increase in PI hydrolysis was unaffected by the B1 receptor antagonist des-Arg9[Leu8]-bradykinin (1 nM-1 microM) but showed marked attenuation in the presence of the B2 receptor antagonists D-Arg,[Hyp3,D-Phe7]-bradykinin (10 nM-10 microM) or D-Arg[Hyp3,Thi5,8,D-Phe7]-bradykinin (10 nM-10 microM). The estimated KB values obtained for these two compounds, assuming competitive antagonism, were 40 +/- 14 nM and 8.6 +/- 2.8 nM for D-Arg,[Hyp3,D-Phe7]-bradykinin and D-Arg[Hyp3,Thi5,8,D-Phe7]-bradykinin respectively. 4. We conclude that bradykinin B2 receptors are expressed in cultured bovine tracheal smooth muscle cells and are coupled to PI hydrolysis mechanisms.

Biochemical mechanisms involved in monomethyl succinate-induced insulin secretion.

Esters of succinic acid stimulate insulin secretion from pancreatic beta-cells. Using collagenase-isolated rat islets, the transduction mechanisms involved were investigated. In freshly isolated perifused islets, monomethyl succinate (MMSucc), in the presence of basal (2.75 mM) glucose, stimulated insulin release in a biphasic pattern. This secretory response was dependent on extracellular calcium movement into the beta-cell, since the calcium channel blocker nitrendipine (5 microM) abolished it. The glucokinase inhibitor mannoheptulose (20 mM) had no effect on its secretory action, while the protein kinase-C inhibitor staurosporine (20 nM) reduced secretion to MMSucc. In addition, while ineffective alone, the diacylglycerol kinase inhibitor monooleoylglycerol (25 microM) potentiated MMSucc-induced insulin release. A similarly amplified response occurred in the presence of forskolin (0.25 microM), a compound that elevates islet cAMP levels. The sodium salt of succinic acid (20 mM) had no effect on insulin release in the presence or absence of forskolin. Prior treatment with MMSucc in the presence of 2.75 mM glucose sensitized islets to the usually weak insulin secretory effect of 7.5 mM glucose. Other groups of islets were incubated for 2 h with myo-[2-3H]inositol to label their phosphoinositide pools. These islets were subsequently stimulated, and the kinetics of [3H]inositol efflux and insulin secretion were measured. MMSucc induced a rapid and sustained dose-dependent increase in [3H]inositol efflux rates. In batch-incubated islets, MMSucc increased inositol phosphate levels. Finally, MMSucc (20 mM), in the presence of 8 mM glucose, did not influence the detritiation of [5-3H]glucose, but reduced the oxidation of [U-14C] glucose. These results support the following conclusions. First, MMSucc is a potent activator of islet phosphoinositide hydrolysis. Second, the activation of protein kinase-C appears to contribute to the acute insulin secretory effect of MMSucc. Third, MMSucc-induced increases in phosphoinositide hydrolysis contribute at least in part to its ability to acutely stimulate insulin release and prime the beta-cell to subsequent stimulation. Finally, mitochondrial events associated with the oxidative metabolism of MMSucc may underlie its insulinotropic action.

Glucosamine-induced desensitization of beta-cell responses: possible involvement of impaired information flow in the phosphoinositide cycle.

The influence of glucosamine on beta-cell response characteristics of collagenase-isolated rat islets was determined. Groups of islets were incubated for 2 h with myo-[2-3H]inositol to label their phosphoinositide (PI) pools. Also included in some experiments was glucosamine (0.1-10 mM). Subsequently, these islets were perifused, and their responses to 10 mM glucose, 10 mM alpha-ketoisocaproate (KIC), and 1 microM of the phorbol ester phorbol 12-myristate 13-acetate were assessed. Increases in PI hydrolysis were monitored during the perfusion by measuring fractional efflux rates of [3H]inositol. The accumulation of inositol phosphates after the perifusion was also determined. In other experiments, the use of 10 mM glucose was measured after a 2-h exposure to 5 or 10 mM glucosamine. Finally, the ability of glucosamine itself to augment release and activate PI hydrolysis was assessed. The following observations were made. 1) A prior 2-h exposure to 5-10 mM glucosamine resulted in parallel dose-dependent impairments in 10 mM glucose-induced insulin release and PI hydrolysis. 2) Glucosamine (5-10 mM) also impaired the subsequent response to alpha-ketoisocaproate (KIC). Parallel deficits in KIC-induced PI hydrolysis were noted under conditions where insulin secretion was impaired. 3) Under several conditions where glucosamine impaired glucose-induced secretion, it had no adverse effect on phorbol 12-myristate 13-acetate-induced release. 4) The desensitizing effect of 10 mM glucosamine on 10 mM glucose-induced release and PI hydrolysis developed within 30 min of exposure to it. 5) Glucosamine (5-10 mM) preexposure had no adverse effect on the use of 10 mM glucose by desensitized islets. 6) Short term (5-min) exposure to glucosamine (10 mM) alone stimulated PI hydrolysis, while a 30-min exposure to the same level of the hexosamine depressed it. 7) In the presence of 0.25 microM forskolin, 10 mM glucosamine also had a transient stimulatory effect on insulin release. These findings support the concept that the acute and chronic effects of glucosamine on the beta-cell result at least in part from its ability to influence PI hydrolysis in islets.

Adrenalectomy increases phosphoinositide hydrolysis induced by norepinephrine or excitatory amino acids in rat hippocampal slices

Phosphoinositide hydrolysis induced by norepinephrine, quisqulate, or trans-1-amino-1,3-cylopentanedicarboxylic acid (ACPD), but not by carbachol, was approximately 50% greater in hippocampal slices from adrenalectomized (14 days) rats compared with controls. These changes appeared to be selective for the hippocampus because no effects of adrenalectomy on phosphoinositide hydrolysis were detected in cortical or striatal slices. The enhanced response to norepinephrine in hippocampal slices after adrenalectomy was observed throughout the effective concentration range of norepinephrine, was not influenced by in vitro addition of corticosterone, was not mimicked or altered by incubation with dibutyryl cyclic adenosine 3′,5′-monophosphate (AMP), and did not appear to be due to impaired inhibition of the response to norepinephrine which was elicited by activation of protein kinase C or by inclusion of an inhibitory concentration of quisqualate. These findings indicate that adrenalectomy either removes an inhibitory influence of glucocorticoids on the phosphoinositide system in the hippocampus or that the neurodegeneration of granule cells in the dentate gyrus following adrenalectomy is associated with neurotransmitter-selective increases in phosphoinositide hydrolysis. These data provide further evidence that glucocorticoids modify signal transduction in the brain and extends their known influence to the phosphoinositide second messenger system.

Tyrosine kinase-regulated and inositol phosphate-independent Ca2+ elevation and mobilization in T cells.

Perturbation of the T cell antigen-specific receptor leads to a series of signaling events that includes a rapid increase in phosphoinositide hydrolysis, intracellular Ca2+, and tyrosine phosphorylation. We have examined the function of tyrosine phosphorylation in isolation by introducing the v-src tyrosine kinase into a T cell hybridoma. T cell receptor-mediated increases in phosphoinositide hydrolysis and, in particular the generation of inositol 1,4,5-trisphosphate, were comparable between v-src+ and v-src- cells. Unexpectedly, the v-src+ cells exhibited spontaneously elevated intracellular Ca2+ and exaggerated Ca2+ increases when stimulated via the T cell receptor. The enhanced Ca2+ response was not due to tyrosine phosphorylation of the T cell receptor itself, since the phenotype was evident in T cell receptor zeta chain-/v-src+ cells as well. These results demonstrate that an active protein tyrosine kinase can markedly affect intracellular Ca2+ handling by a process independent of inositol 1,4,5-trisphosphate production and T cell receptor tyrosine phosphorylation and raise the possibility that tyrosine kinases may directly regulate T cell receptor-mediated changes in intracellular Ca2+.

Lack of desensitization of muscarinic receptor-mediated second messenger signals in rat brain upon acute and chronic inhibition of acetylcholinesterase.

We studied the effects of acute and chronic in vivo inhibition of acetylcholinesterase on both the density and function of brain muscarinic cholinergic receptors. Adult male rats were treated either once or multiple times over a period of 10 days with the irreversible acetylcholinesterase inhibitor diisopropylfluorophosphate (DFP). The concentration and affinity of muscarinic receptors in various brain regions were determined using radioligand binding techniques. Acute DFP treatment resulted in a significant reduction in receptor number only in the brain stem, while chronic treatment caused receptor down-regulation in the brain stem, cerebral cortex, and striatum. There was no change in ligand affinity in any of the brain regions. In sharp contrast, muscarinic receptor function was fully preserved, in terms of coupling of the receptors to increased phosphoinositide hydrolysis in the cerebral cortex, hippocampus, and striatum, or inhibition of cyclic AMP formation in the cerebral cortex or striatum. Therefore, there is a marked lack or correlation between DFP-induced muscarinic receptor down-regulation and receptor desensitization.

Adenosine A1-receptor coupling to phosphoinositide metabolism in pregnant guinea pig myometrium.

Adenosine contracts pregnant and nonpregnant guinea pig myometrial smooth muscle (MSM). We have 1) described dissociation of A1-adenosine receptors from adenylate cyclase inhibition in nonpregnant MSM (M. A. Smith, J. L. Silverstein, D. P. Westfall, and I. L. O. Buxton, Cell. Signal. 1: 357-365, 1989); 2) described appearance of such inhibitory coupling in pregnant MSM [W. P. Schiemann, D. P. Westfall, and I. L. O. Buxton, Am. J. Physiol. 261 (Endocrinol. Metab. 24): E141-E150, 1991]; and 3) demonstrated a role for myometrial A1 receptors in the rapid formation of D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] in nonpregnant MSM and the cyclooxygenase dependence of this effect (W. P. Schiemann, K. O. Doggwiller, and I. L. O. Buxton. J. Pharmacol. Exp. Ther. 258: 429-437, 1991). To further characterize adenosine action in pregnant tissue, we explored A1 coupling to increased phosphoinositide hydrolysis in near-term pregnant MSM. The A1-receptor agonist (+)-N6-(2-phenylisopropyl)adenosine stimulates the rapid dose-dependent formation of Ins(1,4,5)P3 and stimulates rapid degradation of uterine inositol monophosphates (InsP) in a manner paralleling increases in inositol polyphosphates. Both A1-mediated responses were blocked by the A1 antagonist 8-(p-sulfophenyl)theophylline, and, unlike the effect observed in nonpregnant MSM, treatment of pregnant MSM with either meclofenamate or indomethacin failed to block A1-mediated increases in Ins(1,4,5)P3. Pretreatment of MSM with either Li+ or pertussis toxin failed to alter either Ins(1,4,5)P3 formation or InsP degradation. Furthermore, assay of inositol phosphomonoesterase (InsPase) activity in the presence or absence of Li+ confirmed the existence of an MSM Li(+)-insensitive InsPase enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of clonidine and other imidazole-receptor binding agents on second messenger systems and calcium influx in bovine adrenal chromaffin cells

Clonidine and related imidazoline compounds bind to α 2-adrenergic as well as to newly described non-adrenergic imidazole/imidazoline receptors in brain and peripheral tissues. The present study was undertaken to identify the signal transduction mechanism coupled to this new class of receptors (imidazole receptors) using bovine adrenal chromaffin cells. Clonidine did not modify the basal or forskolin-stimulated production of cyclic AMP (cAMP), suggesting the absence of functionally active α 2-adrenergic receptors in adrenal chromaffin cells. Clonidine also failed to modify the basal and GTPγS- or carbachol-stimulated increase in phosphoinositide hydrolysis. However, clonidine increased significantly the production of cyclic GMP (cGMP) as well as the uptake of 45Ca 2+. The cGMP response to clonidine was slower (peak at 15 min) and smaller (only about 50% over control) than the response to acetylcholine and was not shared by other agents that bind to imidazole receptors. In contrast, all agents that bind to imidazole receptors increased the influx of 45Ca 2+ into chromaffin cells. It is concluded that (a) α 2-adrenergic and imidazole receptors are functionally distinct and linked to different signal transduction mechanisms; (b) the classical G-protein coupled soluble second messenger systems are not coupled to imidazole receptors; (c) clonidine may increase cGMP by a non-receptor-mediated intracellular action; and (d) imidazole receptors may regulate intracellular calcium levels through an ion regulating system that may be different from calcium channels.

A metabotropic glutamate receptor agonist does not mediate neuronal degeneration in cortical culture

In light of the evidence that calcium plays a critical role in excitotoxic neuronal death, it has been speculated that the metabotropic glutamate receptor may also contribute to excitotoxic damage through the mobilization of Ca 2+ from intracellular stores. In the present study, we examined this possibility by studying the neurotoxicity of trans-1-amino-cyclopentyl-1,3-dicar☐ylate(trans-ACPD), a selective agonist of the metabotropic glutamate receptor. Exposure of cortical neurons to 100 μM trans-ACPD substantially increased phosphoinositide hydrolysis and intraneuronal free calcium in the presence of CPP and CNQX. Despite the presence of functional metabotropic receptors on cultured neurons, however, exposure of cultures to as high as 1 mM trans-ACPD for 24 h failed to produce any morphological or chemical signs of neuronal damage. Furthermore, trans-ACPD did not potentiate submaximal neurotoxicity produced by other non-N-methyl- D-aspartate (NMDA) agonists, kainate and D,L-α-amino-3-hydroxy-5-methyl-4-isoxazole-4-propionic acid (AMPA).

Angiotensin-II receptor subtypes in fetal tissue of the rat: autoradiography, guanine nucleotide sensitivity, and association with phosphoinositide hydrolysis.

Quantitative autoradiography revealed large numbers of angiotensin-II (AT) receptors in the 18-day-old rat embryo. The selective AT-1 antagonist DuP 753 readily competed for AT receptors in liver, lung parenchyma, and choroid plexus, and these receptors are classified as AT-1 receptors. The selective AT-2 displacers CGP 42112 A and/or PD 123177 competed with high affinity with AT bound to most receptors located in skeletal muscle, skin, diaphragm, bronchi, and stomach, and these receptors are classified as AT-2 receptors. The amount of AT-2 receptors in fetal tissue was more than 10-fold higher than that of AT-1 receptors. In skeletal muscle and skin, DuP 753 competed with AT in the presence of 10(-7) M CGP 42112 A, indicating the presence of small numbers of AT-1 receptors. In liver and lung parenchyma, binding to AT-1 receptors was sensitive to guanine nucleotides. AT binding to AT-2 receptors in fetal skin and skeletal muscle was insensitive to guanine nucleotides. AT stimulated phosphoinositide hydrolysis in liver (ED50, 64 nM) and in skin and skeletal muscle (ED50, 62 nM); this was inhibited by DuP 753 (liver IC50, 38 nM; skin and skeletal muscle IC50, 26 nM), but not by PD 123177 in concentrations up to the micromolar range. AT-1 receptors are probably coupled to G-proteins, and their stimulation increases phosphoinositide hydrolysis. AT-2 receptors may not be linked to G-proteins, their stimulation is not associated with phosphoinositide hydrolysis, and the nature of their second messenger system(s) is presently unknown.

Long-term changes in phosphoinositide hydrolysis following colchicine lesions of the nucleus basalis magnocellularis

The effect of bilateral colchicine lesions of the nucleus basalis magnocellularis (NBM) on agonist-stimulated phosphoinositide (PI) hydrolysis was examined in cortical slices 1, 3, or 14 months after surgery. Colchicine lesions resulted in a loss of acetylcholinesterase staining in the cortex which recovered to control levels by 14 months. Choline acetyltransferase activity in the cortex was decreased by 43% one month after lesioning, but returned to control levels by 3 months. In vitro stimulation with carbachol produced a concentration-dependent increase in PI hydrolysis, which was enhanced 3 and 14 months after NBM lesions. Norepinephrine and quisqualate-stimulated PI hydrolysis was also enhanced 14 months after NBM lesions. These results suggest a slow up-regulation of postsynaptic receptor function following presynaptic loss of transmitter.

Nerve growth factor stimulates phosphorylation of phospholipase C-gamma in PC12 cells

PC12 cells contain at least three immunologically distinct phospholipase C (PLC) isozymes, PLC-beta, PLC-gamma, and PLC-delta. Treatment of PC12 cells with nerve growth factor (NGF) leads to an increase in the phosphorylation of PLC-gamma, but not of PLC-beta or PLC-delta. This increase can be seen in as little as 1 minute. The increased phosphorylation occurs on both serine and tyrosine residues, with the major increase being in the former. This result suggests the possibility that the NGF-dependent increase in phosphoinositide hydrolysis in PC12 cells is due to selective phosphorylation of PLC-gamma by serine and tyrosine protein kinases associated with the NGF receptor.

Reduced α1-Adrenergic Receptor-Mediated Inositide Hydrolysis in Cardiac Atria of Senescent Rats

We investigated the effect of age on epinephrine stimulation of phosphoinositide hydrolysis in atrial slices prepared from F-344 female rats. Three age groups were chosen for study: young adults (aged 6 months), mature adults (aged 15 months), and senescent animals (aged 25 months). Tissue slices were labeled with [3H]myoinositol and epinephrine-stimulated hydrolysis measured in the presence of LiCl. Epinephrine caused a dose-dependent increase in phosphoinositide hydrolysis in each age group. This increase was blocked by prazosin, suggesting that alpha 1-adrenergic receptors are involved. In animals aged 6 months, epinephrine caused a maximal increase in hydrolysis of 2.8-fold over basal. The maximal response was reduced at 15 months (2.52-fold increase, p less than 0.05) and at 25 months (2.02-fold increase, p less than 0.01). The potency for epinephrine stimulation of phosphoinositide hydrolysis was unchanged with age. The data indicate that alpha 1-mediated phosphoinositide hydrolysis in atria is reduced with age.

Muscarinic M 1 receptors stimulate phosphoinositide hydrolysis in bovine cerebral arteries

The muscarinic agonist oxotremorine-M produced a concentration-dependent increase in phosphoinositide hydrolysis in bovine pial arteries. The maximal effect was 5.9 ± 0.89 fold over basal levels, and the EC 50 for oxotremorine-M was 8.9×10 −6 M. The phosphoinositide response in arteries with the luminal endothelium removed was similar to the response in intact arteries. The specific muscarinic antagonists pirenzepine, 4-DAMP and methoctramine produced parallel shifts of the concentration-response curve to oxotremorine-M, with the following order of potency (pK B): 4-DAMP (8.59 ± 0.10 > pirenzepine (8.12 ± 0.11) > methoctramine (6.77 ± 0.20). These results indicate that muscarinic stimulation activates phosphoinositide hydrolysis in cerebral arteries, and that the muscarinic receptors mediating this increase are similar to the M 1 subtype.

Adenosine and Its Analogs Stimulate Phosphoinositide Hydrolysis in the Kidney

Renal blood flow, glomerular filtration rate and sodium excretion are known to be affected by adenosine. The present studies were undertaken to investigate the actions of adenosine and its analogs (both agonists and antagonists) on phosphoinositide (PI) hydrolysis in the outer medullary slices. Adenosine was found to cause a dose-dependent stimulation of PI hydrolysis (ED50, 2.8 microM) in renal slices from outer medulla. The adenosine analogs 5'-(N-cyclopropyl)-carboxamidoadenosine (NCCA) and 5'-N-ethylcarboxamidoadenosine (NECA) also stimulated PI hydrolysis in renal medulla. Stimulation of PI hydrolysis was blocked by the adenosine antagonists: aminophylline, 1,3-dipropyl-7-methylxanthine (DMX) and 8-(p-sulfophenyl)-theophylline (8-SPT). Caffeine not only antagonized adenosine-stimulated PI hydrolysis but also increased PI hydrolysis independently. These results indicate that adenosine stimulates PI hydrolysis in renal medulla through a receptor-mediated mechanism.