Stimulation Of Inositol Phosphate Formation Research Articles

Regulation of phosphoinositide hydrolysis by GTP-binding proteins, phorbol esters and botulinum toxin type D.

The muscarinic cholinergic receptor in 1321N1 human astrocytoma cells does not inhibit adenylate cyclase activity but regulates cyclic AMP levels by affecting a cyclic AMP phosphodiesterase (1). Because muscarinic activation of phosphodiesterase is calcium-dependent, Ken Harden and I postulated that muscarinic receptor stimulation in these cells caused phosphoinositide hydrolysis and calcium mobilization. Subsequently, in a series of joint and independent publications, we demonstrated that in 1321N1 cells muscarinic agonists stimulate the formation of inositol phosphates, including the active 1,4,5 isomer of InsP3 (2–6) and mobilize calcium by release from intracellular stores (2,4,7). The muscarinic receptor in these cells had been shown to be regulated by guanine nucleotides (8), and the interaction of the receptor with a putative GTPbinding (G-) protein could be shown to correlate with the capacity of an agonist to stimulate inositol phosphate formation and 4 5Ca++ efflux (4). More direct evidence that a G-protein transduced muscarinic receptor binding into phospholipase C activation came from studies with cell free systems. In these washed membranes GTPγS caused marked stimulation of inositol phosphate production, a response that could be potentiated by the agonist carbachol but not effected by the agonist alone in the absence of added guanine nucleotide (9,10). The studies described in this paper deal primarily with our attempts to manipulate and characterize the G-protein that regulates phospholipase C.

Stimulation of inositol phosphate formation in FRTL-5 rat thyroid cells by catecholamines and its relationship to changes in 45Ca2+ efflux and cyclic AMP accumulation.

Catecholamines specifically stimulated the rapid formation of inositol phosphates, bisphosphates and trisphosphates in a concentration-dependent manner in FRTL-5 thyroid cells. Further analysis by high performance liquid chromatography revealed the presence of two isomers of inositol trisphosphate, 1,4,5- and 1,3,4-trisphosphate, suggesting that the 1,4,5-trisphosphate of inositol is further metabolized to the 1,3,4-trisphosphate isomer. The alpha 1-adrenoreceptor antagonist, prazosin, inhibited the effects of epinephrine, while the alpha 2-adrenoreceptor antagonist, yohimbine, was without effect. Treatment of FRTL-5 cells with pertussis toxin (to inhibit Ni) did not abolish the epinephrine effect on inositol trisphosphate formation. Carbachol, N6-[L-2-phenylisopropyl]-adenosine and forskolin were without effect on phosphoinositide metabolism. Both epinephrine and the calcium ionophore A23187 stimulated 45Ca2+ efflux from 45Ca2+-loaded FRTL-5 cells. The time-course of the epinephrine effect indicates that inositol 1,4,5-trisphosphate formation (t1/2 approximately 1 s) precedes both the efflux of 45Ca2+ (t1/2 approximately 30 s) as well as the reduction of cyclic AMP levels (t1/2 approximately 90 s) in response to epinephrine. These results strongly suggest that inositol 1,4,5-trisphosphate has the appropriate properties to act as a second messenger by which alpha 1-adrenergic hormones, through mobilization of intracellular Ca2+ and activation of cyclic AMP phosphodiesterase, reduce cyclic AMP levels in FRTL-5 cells.

Alpha-adrenoceptor stimulation, but not muscarinic stimulation, increases cyclic AMP accumulation in brain slices due to protein kinase C mediated enhancement of adenosine receptor effects.

In the present study, using rat hippocampal slices, we have further examined the stimulatory effect of alpha 1-adrenoceptors on the accumulation of cyclic AMP, which is known to depend on calcium and adenosine. The addition of noradrenaline (NA) stimulated the accumulation of [3H]inositol phosphates in [3H]inositol-treated slices. This effect was shared by carbachol (10-100 mumol l-1) but not by the adenosine receptor agonist 2-chloroadenosine (100 mumol l-1). The stimulatory effect of the alpha-agonists (phenylephrine or NA + propranolol) on cyclic AMP was shared by a diacylglycerol derivative, sn-1-oleyl-2-acetyl glycerol (OAG), and by the tumour-promoting phorbol esters phorboldibutyrate (PDiBu) and tetradecanoyl phorbol acetate (TPA). PDiBu caused a translocation of protein kinase C from soluble to particulate fractions. The effects of PDiBu and alpha-adrenoceptor stimulation on cyclic AMP were not additive. Surprisingly, carbachol (1-1000 mumol l-1) did not stimulate cyclic AMP accumulation in rat hippocampal slices either in the presence or in the absence of an adenosine receptor agonist. The results are compatible with the opinion that alpha-adrenoceptor stimulating drugs enhance the formation of inositol phosphates and diacylglycerol, which synergistically activate protein kinase C, which in turn augments the stimulation of cyclic AMP formation. Thus, a neurotransmitter whose principal biological effect is to stimulate inositol phosphate formation can influence cyclic AMP formation by virtue of an interaction with the actions of the ubiquitous neuromodulator adenosine. The fact that the effect of the alpha-receptor stimulation was not mimicked by a muscarinic agonist could indicate that other factors besides activation of inositol phospholipid hydrolys are important for this receptor-receptor interaction.

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

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

Thyroid-stimulating hormone stimulates increases in inositol phosphates as well as cyclic AMP in the FRTL-5 rat thyroid cell line.

Studies were conducted to determine whether thyroid-stimulating hormone (TSH; thyrotropin), a hormone known to increase cytosol concentrations of cyclic AMP, also stimulates the formation of inositol phosphates in thyroid cells. TSH and noradrenaline both stimulated [3H]inositol phosphate formation in a concentration-dependent manner in the rat thyroid cell line, FRTL-5 cells, which had been prelabelled with [3H]inositol. The threshold concentration of TSH required to stimulate inositol phosphate formation was more than 20 munits/ml, which is approx. 10(3)-fold greater than that required for cyclic AMP accumulation and growth in these cells. We also demonstrate that membranes prepared from FRTL-5 cells possess a guanine nucleotide-activatable polyphosphoinositide phosphodiesterase, which suggests that activation of inositide metabolism in these cells may be coupled to receptors by the G-protein, Gp. Our findings suggest that two second-messenger systems exist to mediate the action of TSH in the thyroid.

Effects of calcium depletion on norepinephrine- and A23187-induced stimulation of inositol phosphate formation

The role of calcium in the stimulation of phosphoinositide (PIn) hydrolysis by norepinephrine and the calcium ionophore A23187 was investigated in chopped cerebral cortex in which the PIns had been labeled previously with ([ 3 H]myo- inositol . The accumulation of the newly formed [ 3H]inositol phosphate ([ 3H]IPs) was used as an index of PIn hydrolysis. Norepinephrine produced a concentration-related increase in the accumulation of [ 3H]inositol-phosphates ([ 3H]IP), and this effect was only partially antagonized by omission of calcium from the incubation medium. Norepinephrine had relatively little effect on the accumulation of [ 3H]inositol 1,4-bisphosphate [ 3H]IP 2 and inositol 1,4,5-trisphosphate and/ or inositol 1,3,4-trisphosphate ([ 3H]IP 3). A23187 also increased the accumulation of [ 3H]IP but this effect was not antagonized by omission of calcium from the incubation medium. When the tissue had been washed extensively with EGTA, the basal levels of [ 3H]IP, [ 3H]IP 2 and [ 3H]IP 3 were decreased markedly, and the effects of both norepinephrine and A23187 were antagonized. Addition of calcium back to the depleted tissue led to an increase in the basal level of [ 3H]IPs as well as restoration of the stimulation produced by norepinephrine and A23187. The calcium threshold for the PIn effect was 0.1 μM. Additional calcium did not affect markedly the stimulation of accumulation of [ 3H]IP produced by norepinephrine and A23187. The results suggest that there is an absolute calcium requirement for PIn phosphodiesterase which is attained at 0.1 μM Ca 2+. A23187 can stimulate the accumulation of [ 3H]IP perhaps by providing access of another form of the enzyme to artificially high concentrations (millimolar range) of calcium.

Serotonergic and cholinergic stimulation of inositol phosphate formation in the rabbit retina. Evidence for the presence of serotonin and muscarinic receptors

A direct assay which involves prelabelling with [ 3H]inositol has been performed to characterize receptor-mediated breakdown of inositol phospholipids in the rabbit retina. In the presence of 10 mM lithium, the receptor agonists, carbachol and serotonin (5-HT), evoked an increase in the accumulation of tritiated inositol phosphates in a dose-dependent manner. A variety of 5-HT and other antagonists were used to show that at least part of the 5-HT-induced response is mediated by 5-HT 2 receptor sites, adding weight to the theory that 5-HT plays a neurotransmitter role in the mammalian retina. The very high rate of carbachol-stimulated inositol phosphate accumulation observed in retinas from young animals (10 days) decreases significantly during development of the rabbit to the adult stage. In contrast 5-HT only induces significant stimulation of inositol phosphate accumulation in retinas of animals matured to at least 17 days. Separation of total accumulated inositol phosphates (after stimulation with both carbachol and 5-HT) showed that the vast majority of tritium label was associated with the monophosphate fraction.

α1Adrenoceptor subtypes linked to different mechanisms for increasing intracellular Ca2+ in smooth muscle

Receptor-mediated increases in intracellular Ca2+ levels can be caused by release from intracellular organelles and/or influx from the extracellular fluid. Noradrenaline (NA) released from sympathetic nerves acts on alpha 1-adrenoceptors to increase cytosolic Ca2+ and promote smooth muscle contraction. In many cells activation of alpha 1-adrenoceptors causes formation of inositol 1,4,5-trisphosphate which promotes Ca2+ release from intracellular stores. The mechanism by which receptor activation opens cell surface Ca2+ channels is not known, although in some cases it may be secondary to formation of inositol phosphates or release of stored intracellular Ca2+ (ref. 3). However, alpha 1-adrenoceptors have recently been shown to have different pharmacological properties in different tissues, and it has been proposed that different alpha 1-adrenoceptor subtypes may control mobilization of intracellular Ca2+ and gating of extracellular Ca2+ influx. We here report evidence for two subtypes of alpha 1-adrenoceptors which cause contractile responses through different molecular mechanisms. One subtype stimulates inositol phosphate (InsP) formation and causes contractions which are independent of extracellular Ca2+, and the other does not stimulate inositol phosphate formation and causes contractions which require the influx of extracellular Ca2+ through dihydropyridine-sensitive channels. These results suggest that neurotransmitters and hormones may control Ca2+ release from intracellular stores and influx through voltage-gated membrane channels through distinct receptor subtypes.

Rapid formation of inositol 1,4,5-trisphosphate in rat pancreatic acini stimulated by carbamylcholine

Cholinergic stimulation of inositol phosphate formation was studied in isolated rat pancreatic acini, prelabelled with myo-[2- 3H]inositol. (1) Carbamylcholine increased incorporation of radioactivity into Ins(1,4,5) P 3 and Ins P 4 within 5 s. Increases in [ 3 H]Ins(1,3,4)P 3 were delayed with marked stimulation occurring between 10 s and 1 min. (2) Inositol polyphosphate formation was less sensitive to carbamylcholine concentration than was stimulation of amylase release. At a low (0.3 μM) carbamylcholine concentration, no increase in inositol polyphosphate formation was detected, whereas stimulation of amylase release, which was not dependent on extracellular calcium, was observed. (3) Ins(1,4,5) P 3 was shown to release actively accumulated 45Ca 2+ from isolated rough endoplasmic reticulum membranes to a similar extent as that released from rough endoplasmic reticulum following cholinergic stimulation of pancreatic acini (Richardson, A.E. et al. (1984) Biochem. Soc. Trans. 12, 1066–1067). (4) The data is consistent with Ins(1,4,5) P 3 being produced rapidly enough to release sufficient calcium from the rough endoplasmic reticulum to cause an observed increases in cytoplasmic free Ca 2+.

Cholinergic stimulation of inositol phosphate production in cultured anterior pituitary cells.

The effects of acetylcholine and of the muscarinic receptor agonist carbachol on inositol phosphate production were studied in cultured rat anterior pituitary cells. In the presence of the cholinesterase inhibitor physostigmine, acetylcholine significantly (p less than 0.05-p less than 0.01) stimulated inositol phosphate formation in a concentration-related fashion: carbachol, but not oxotremorine, produced similar effects. The increase in the amount of inositol phosphates (primarily inositol trisphosphate and inositol bisphosphate) was very rapid, an effect potently antagonized by the muscarinic receptor antagonist atropine. This agent significantly attenuated the stimulatory effect of carbachol on growth hormone (GH) release. These results indicate that the effects exerted by acetylcholine on anterior pituitary function (i.e. GH release) may be mediated, at least in part, by receptor-activated polyphosphoinositide hydrolysis. In addition, acetylcholine and carbachol's relation with other intracellular pathways and with hormone release is discussed.

V 1a vasopressin-induced accumulation of inositol trisphosphate in cultured rat aortic myocytes; modulation by protein kinase C

Arginine vasopressin stimulated the accumulation of labeled inositol phosphate in cultured rat aortic myocytes prelabeled with tritiated myo-inositol. This accumulation was prevented by pretreating the myocytes with the phorbol ester PMA. The time-course and concentration-effect curves were similar for inositol phosphate formation in myocytes and contractile effect on isolated aorta. Vasopressin agonists also stimulated inositol phosphate formation, whereas vasopressin-induced response could be inhibited by V 1a-specific antagonists. These results suggest that stimulation of inositol phosphate formation in myocytes is due to V 1a receptor activation and could be modulated by protein-kinase-C-mediated mechanisms.

Phospholipase A2 and phospholipase C are activated by distinct GTP-binding proteins in response to alpha 1-adrenergic stimulation in FRTL5 thyroid cells.

In FRTL5 rat thyroid cells, norepinephrine, by interacting with alpha 1-adrenergic receptors, stimulates inositol phosphate formation, through activation of phospholipase C, and arachidonic acid release. Recent studies have shown that GTP-binding proteins couple several types of receptors to phospholipase C activation. The present study was undertaken to determine whether GTP-binding proteins couple alpha 1-adrenergic receptors to stimulation of phospholipase C activity and arachidonic acid release. When introduced into permeabilized FRTL5 cells, guanosine 5'-[gamma-thio]triphosphate (GTP[gamma-S]), which activates many GTP-binding proteins, stimulated inositol phosphate formation and arachidonic acid release. Neomycin inhibited GTP[gamma-S]-stimulated inositol phosphate formation but was without effect on GTP[gamma-S]-stimulated arachidonic acid release, suggesting that separate GTP-binding proteins mediate each process. In addition, pertussis toxin inhibited norepinephrine-stimulated arachidonic acid release but not norepinephrine-stimulated inositol phosphate formation. Norepinephrine-stimulated arachidonic acid release but not inositol phosphate formation was also inhibited by decreased extracellular calcium and by TMB-8, suggesting a role for a phospholipase A2. To confirm that arachidonic acid was released by a phospholipase A2, FRTL5 membranes were incubated with 1-acyl-2-[3H]arachidonoyl-sn-glycero-3-phosphocholine. GTP[gamma-S] slightly stimulated arachidonic acid release, whereas norepinephrine acted synergistically with GTP[gamma-S] to stimulate arachidonic acid release. The results show that phospholipase C and phospholipase A2 are activated by alpha 1-adrenergic agonists. Both phospholipases are coupled to the receptor by GTP-binding proteins. That coupled to phospholipase A2 is pertussis toxin-sensitive, whereas that coupled to phospholipase C is pertussis toxin-insensitive.

Guanine nucleotide-dependent pertussis-toxin-insensitive stimulation of inositol phosphate formation by carbachol in a membrane preparation from human astrocytoma cells.

The efficacy of muscarinic-receptor agonists for stimulation of inositol phosphate formation and Ca2+ mobilization in intact 1321N1 human astrocytoma cells is correlated with their capacity for formation of a GTP-sensitive high-affinity binding complex in membranes from these cells [Evans, Hepler, Masters, Brown & Harden (1985) Biochem. J. 232, 751-757]. These observations prompted the proposal that a guanine nucleotide regulatory protein serves to couple muscarinic receptors to the phospholipase C involved in phosphoinositide hydrolysis in 1321N1 cells. Inositol phosphate (InsP) formation was measured in a cell-free preparation from 1321N1 cells to provide direct support for this idea. The formation of InsP3, InsP2 and InsP1 was increased in a concentration-dependent manner (K0.5 approximately 5 microM) by guanosine 5'-[gamma-thio]triphosphate (GTP[S]) in washed membranes prepared from myo-[3H]inositol-prelabelled 1321N1 cells. Both GTP[S] and guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG) stimulated InsP formation by 2-3-fold over control; GTP, GDP and GMP were much less efficacious. Millimolar concentrations of NaF also stimulated the formation of inositol phosphates in membrane preparations from 1321N1 cells. In the presence of 10 microM-GTP[S], the muscarinic cholinergic-receptor agonist carbachol stimulated (K0.5 approximately 10 microM) the formation of InsP above that achieved with GTP[S] alone. The effect of carbachol was completely blocked by atropine. The order of potency of nucleotides for stimulation of InsP formation in the presence of 500 microM-carbachol was GTP[S] greater than p[NH]ppG greater than GTP = GDP. Pertussis toxin, at concentrations that fully ADP-ribosylate and functionally inactivate Gi (the inhibitory guanine nucleotide regulatory protein), had no effect on InsP formation in the presence of GTP[S] or GTP[S] plus carbachol. These data are consistent with the idea that a guanine nucleotide regulatory protein that is not Gi is involved in receptor-mediated stimulation of InsP formation in 1321N1 human astrocytoma cells.

Thyroliberin action in pituitary cells is not inhibited by pertussis toxin.

The effects of pertussis toxin on the responses of rat pituitary-tumour (GH) cells to thyrotropin-releasing hormone (thyroliberin, TRH) were examined. Treatment of cells with pertussis toxin did not alter the affinity or concentration of TRH receptors, or the sensitivity of the TRH receptor to inhibition by guanine nucleotides. TRH caused an increase in low-Km GTPase activity in membrane-containing fractions from both control and pertussis-toxin-treated cells. TRH stimulation of inositol phosphate formation was insensitive to pertussis toxin. TRH caused a biphasic increase in the concentrations of cytosolic free Ca2+ as monitored by intracellularly trapped Quin 2, and this increase was the same in control and toxin-treated cultures. The toxin did not alter the increase in prolactin and growth-hormone (somatotropin) release stimulated by TRH or shift the TRH dose-response curve, and it did not affect the TRH-induced rise in prolactin synthesis measured over 24 h. However, pertussis toxin did block the ability of somatostatin and muscarinic agonists to inhibit prolactin and growth-hormone secretion stimulated by vasoactive intestinal peptide when analysed under the same conditions as those in which the TRH system was unaffected. These data indicate that the guanine nucleotide effects on TRH binding and activity are not mediated by Ni, but possibly by another member of the family of guanine-nucleotide-dependent regulatory proteins.

Guanine nucleotide-dependent pertussis-toxin-insensitive stimulation of inositol phosphate formation by carbachol in a membrane preparation from human astrocytoma cells

Guanine nucleotide-dependent pertussis-toxin-insensitive stimulation of inositol phosphate formation by carbachol in a membrane preparation from human astrocytoma cells

Glutamate stimulates inositol phosphate formation in striatal neurones.

The major excitatory amino acids, glutamate (Glu) and aspartate (Asp), are thought to act at three receptor subtypes in the mammalian central nervous system (CNS). These are termed quisqualate (QA), N-methyl-D-aspartate (NMDA) and kainate (KA) receptors according to the specific agonist properties of these compounds revealed by electrophysiological studies. Although Glu has been shown to stimulate cyclic GMP formation in brain slices, direct regulation of second messenger systems (cyclic AMP, Ca2+ or inositol phosphates) subsequent to activation of excitatory amino-acid receptors, has not been extensively studied. Here we demonstrate that in striatal neurones, excitatory amino acids, but not inhibitory or non-neuroactive amino acids, induce a three- to fourfold increase in inositol mono-, di- and triphosphate (IP, IP, IP) formation with the relative potency QA greater than Glu greater than NMDA, KA. The Glu-evoked formation of inositol phosphates appears to result principally from actions at QA as well as NMDA receptors on striatal neurones. Our results suggest that excitatory amino acids stimulate inositol phosphate formation directly, rather than indirectly by the evoked release and subsequent actions of adenosine or acetylcholine.

5-Hydroxytryptamine stimulates inositol phosphate production in a cell-free system from blowfly salivary glands. Evidence for a role of GTP in coupling receptor activation to phosphoinositide breakdown.

Phosphoinositide breakdown has been linked to the receptor mechanism involved in the elevation of cytosolic Ca2+. In a cell-free system prepared from [3H] inositol-labeled blowfly salivary glands, 5-hydroxytryptamine stimulated the rapid production of inositol phosphates. Within 30 s of hormone addition, there was a 100% increase in inositol trisphosphate formation, a 70% increase in inositol bisphosphate formation, and a 90% increase in inositol monophosphate formation as compared to control homogenates incubated for the same length of time. 5-Hydroxytryptamine did not stimulate inositol or glycerol phosphoinositol formation. Half-maximal activation of inositol phosphate production was obtained with 0.33 microM 5-hydroxytryptamine. Ethylene glycol bis(beta-aminoethyl ether)-N',N',N',N'-tetraacetic acid, (EGTA) (0.3 mM) inhibited the basal formation of inositol phosphates and decreased the net accumulation of inositol bisphosphate and inositol trisphosphate due to hormone as compared to homogenates incubated in the absence of added Ca2+. EGTA, however, had little effect on the per cent stimulation of inositol phosphate production due to hormone. In homogenates, ATP, GTP or guanyl-5'-yl imidodiphosphate (Gpp(NH)p) was required for a hormone effect. Gpp(NH)p, unlike ATP or GTP, increased the basal formation of inositol phosphates. In membranes, GTP, Gpp(NH)p, or guanosine 5'-(3-O-thio)trisphosphate (GTP gamma S) sustained a hormone effect whereas ATP was ineffective. GTP did not affect production while Gpp(NH)p and GTP gamma S increased inositol phosphate production. Half-maximal effects of Gpp(NH)p and GTP gamma S on hormone-stimulated inositol phosphate formation occurred at 10 microM and 100 nM, respectively. In the presence of 1 microM GTP gamma S, 5-methyltryptamine stimulated inositol phosphate formation within 2 s in membranes. These results indicate that in a cell-free system, GTP is involved in mediating the effects of Ca2+-mobilizing hormones on phosphoinositide breakdown.