Pituitary thyrotropin-releasing hormone receptors: local anesthetic effects on binding and responses

Pharmacological agents are widely used to probe the mechanism of action of TRH. A number of these drugs behave as local anesthetics at high concentrations. The effect of local anesthetics on the binding of [3H]Me-TRH to specific receptors was studied using the GH4C1 line of rat pituitary tumor cells. [3H]Me-TRH binding was inhibited by classical local anesthetics with the order of potency (IC50 values): dibucaine (0.37 mM) greater than tetracaine (1.2 mM) greater than lidocaine (3.3 mM) greater than procaine and benzocaine (greater than 10 mM). IC50 values for other drugs with local anesthetic properties that inhibited [3H]Me-TRH were: 100 microM trifluoperazine, 100 microM imipramine, 170 microM chlorpromazine, 300 microM verapamil, and 700 microM propranolol. Inhibition by tetracaine and verapamil increased as the pH was raised from 6 to 8.5, indicating that the free base form of the amine drugs was the inhibitory species, and the local anesthetic effect was greater at 37 C than at 24 C or 0 C. [3H]Me-TRH binding to receptors in isolated membranes was inhibited to the same extent as binding to receptors on intact cells. Local anesthetics were 3- to 20-fold less potent at inhibiting [3H]Me-TRH to digitonin-solubilized receptors than binding to intact cells. In contrast, the potency of chlordiazepoxide, a putative TRH antagonist, to inhibit [3H]Me-TRH binding was equal using cells and solubilized receptors (IC50 = 10 microM). Local anesthetics inhibited TRH-stimulated PRL release and also inhibited basal PRL secretion and secretion stimulated by two nonhormonal secretagogues, (Bu)2cAMP and a phorbol ester.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of agonist-stimulated incorporation of myo-[3H]inositol into inositol phospholipids and [3H]inositol phosphate formation in tracheal smooth muscle.

The effects of the muscarinic agonist carbachol, histamine and bradykinin on incorporation of [3H]inositol into the phosphoinositides and the formation of [3H]InsPs were examined in bovine tracheal smooth-muscle (BTSM) slices labelled with [3H]inositol. These agonists result in substantial and dose-related increases in the incorporation of [3H]inositol into the phospholipids. Carbachol and histamine stimulated the incorporation of [3H]inositol into the phospholipids to the same degree, despite histamine being only 35% as effective as carbachol on [3H]InsP accumulation. Histamine and carbachol, at maximal concentrations, were non-additive with respect to both the stimulated incorporation of [3H]inositol and [3H]InsP formation. For carbachol this effect on incorporation was found to occur to a similar extent in PtdInsP and PtdInsP2 as well as PtdIns. The initial effect of carbachol on [3H]inositol incorporation was rapid (maximal by 10 min); however, with prolonged stimulation large secondary declines in PtdInsP and PtdInsP2 labelling were observed, with depletion of the much larger PtdIns pool only evident in the presence of Li+. Lowering buffer [Ca2+] increased the incorporation of [3H]inositol under basal conditions, but did not attenuate the subsequent agonist-stimulated incorporation effect. The large changes in specific radioactivity of the phosphoinositides, and consequently the [3H]InsP products, after carbachol stimulation resulted in the apparent failure of atropine to reverse the [3H]InsP response completely. Labelling muscle slices with [3H]inositol in the presence of carbachol or labelling for longer periods (greater than 6 h) prevented subsequent carbachol-stimulated effects on incorporation without significantly altering the dose-response relationship for carbachol-stimulated [3H]InsP formation and resulted in steady-state labelling conditions confirmed by the ability of atropine to reverse fully the [3H]InsP response to carbachol. This study demonstrates the profound effects of a number of agonists on [3H]inositol incorporation into the phospho- and polyphosphoinositides in BTSM with important consequent changes in the specific radioactivity of these lipids and the resulting [3H]InsP products. In addition, a selective depletion of PtdInsP and PtdInsP2 over PtdIns has been demonstrated with prolonged muscarinic-receptor stimulation.     

The effects of fatty acids on phosphoinositide synthesis and myo-inositol accumulation in exocrine pancreas

The effects of arachidonic acid (20:4) on phosphoinositide turnover were examined in rat pancreatic acinar cells prelabeled with myo-[3H]inositol. Arachidonic acid (50 microM) increased the accumulation of myo-[3H]inositol, but not that of [3H]inositol monophosphate, [3H]inositol bisphosphate, or [3H]inositol trisphosphate. By contrast, 10 microM carbamoylcholine increased the accumulation of all four compounds. A combination of arachidonic acid plus carbamoylcholine caused a selective and marked accumulation of myo-[3H]inositol, which was abolished by 10 mM LiCl. Arachidonic acid (10-100 microM) produced a concentration-dependent inhibition of myo-[3H]inositol incorporation into phosphoinositides and markedly depressed carbamoylcholine-induced increases in myo-[3H]inositol incorporation into inositol phospholipids. Several other unsaturated and saturated fatty acids failed to elicit a synergistic response with carbamoylcholine in stimulating myo-[3H]inositol accumulation and did not retard the incorporation of myo-[3H]inositol into phosphoinositides. The fact that eicosapentaenoic acid (20:5), but not arachidic acid (20:0), mimicked the depressant effect of arachidonate on phosphoinositide labeling suggests that the degree of unsaturation of the fatty acid, rather than chain length, is important for inhibition of phosphoinositide synthesis. The arachidonate-induced decrease in myo-[3H]inositol incorporation was accompanied by a reduction in the steady state level of [32P]phosphatidylinositol 4,5-bisphosphate. The mass of arachidonic acid liberated in response to carbamoylcholine was measured by gas chromatography-mass spectrometry, and the time course of stimulated arachidonate accumulation paralleled that of inositol phosphate accumulation and amylase release. These observations suggest that in exocrine pancreas, endogenous arachidonic acid serves as a negative feedback regulator of phosphoinositide turnover.     

Inositol metabolism in WRK-1 cells. Relationship of hormone-sensitive to -insensitive pools of phosphoinositides

Previous studies have indicated the existence of two separate pools of phosphoinositides in WRK-1 cells; one is labile and hormone-sensitive with respect to turnover, while the other is stable. Hormonal stimulation results in a rapid increase in 32Pi incorporation into the sensitive pool, while in the absence of hormone, incorporation of 32Pi into this pool is slow. Results are quite different when [3H]inositol is the precursor utilized. Incorporation of [3H]inositol into hormone-sensitive phosphoinositides is not stimulated in the presence of hormone, suggesting entry of this exogenous precursor into the cycle by a route other than the resynthetic phase of the cycle. Furthermore, failure of hormone to induce loss of [3H]phosphoinositide in pulse-chase experiments in the absence of lithium suggests reutilization of the [3H]inositol moiety generated by phosphodiesteratic cleavage of hormone-sensitive phosphoinositide. Time course studies indicate that the relative rates of incorporation of [3H]inositol into sensitive and insensitive phosphoinositide remain constant from 2 to 24 h. Several factors are capable of increasing [3H]inositol incorporation into hormone-insensitive phosphoinositide including vasopressin, calcium ionophores, and manganese. On the other hand, vasopressin treatment appears to decrease incorporation of [3H]inositol into the hormone-sensitive pool, probably by shifting the equilibrium between phosphoinositides and inositol phosphates, since the decrease in radioactivity observed in the phosphoinositides is equaled by the increase observed in that in the inositol phosphates.     

Inositol incorporation into phosphoinositides in retinal horizontal cells of Xenopus laevis: enhancement by acetylcholine, inhibition by glycine

The absorption of light by photoreceptor cells leads to an increased incorporation of [2-3H]inositol into phosphoinositides of horizontal cells in the retina of Xenopus laevis in vitro. We have identified several retinal neurotransmitters that are involved in regulating this response. Incubation with glycine, the neurotransmitter of an interplexiform cell that has direct synaptic input onto horizontal cells, abolishes the light effect. This inhibition is reversed by preincubation with strychnine. Acetylcholine added to the culture medium enhances the incorporation of [2-3H]inositol into phosphoinositides in horizontal cells when retinas are incubated in the dark. This effect is inhibited by preincubation with atropine. However, atropine alone does not inhibit the light-enhanced incorporation of [2-3H]inositol into phosphoinositides in the retina. gamma-Aminobutyric acid, the neurotransmitter of retinal horizontal cells in X. laevis, as well as dopamine and norepinephrine, have no effect on the incorporation of [2-3H]inositol into phosphoinositides. These studies demonstrate that the light-enhanced incorporation of [2-3H]inositol into phosphoinositides of retinal horizontal cells is regulated by specific neurotransmitters, and that there are probably several synaptic inputs into horizontal cells which control this process.     

Induction of Apoptotic Cell Death in a Neuroblastoma Cell Line by Dibucaine

Dibucaine, a local anesthetic known to interact with cell membranes, induced apoptosis in SK-N-MC human neuroblastoma cells in a dose-dependent manner. Apoptosis was demonstrated by direct visualization of morphological nuclear changes using a DAPI staining technique and confirmed by the production of characteristic ladder patterns of DNA fragmentation on gel electrophoresis. At concentrations which induced apoptosis, dibucaine significantly altered membrane fluidity, indicating that fluidity may be a major target for the cytotoxic action of dibucaine. Also, dibucaine increased intracellular calcium levels more effectively in calcium-containing Krebs–Ringer buffer than in calcium-free Krebs–Ringer buffer. Removal of extracellular calcium or addition of antioxidants or protein synthesis inhibitor effectively blocked dibucaine-induced apoptosis. These results suggest that membrane damage, intracellular calcium levels, and oxygen free radicals may be involved in the apoptosis induced by dibucaine.     

Guanine nucleotides stimulate production of inositol trisphosphate in rat cortical membranes.

The guanine nucleotides guanosine 5'[beta, gamma-imido]triphosphate (Gpp[NH]p), guanosine 5'-[gamma-thio]-triphosphate (GTP gamma S), GMP, GDP and GTP stimulated the hydrolysis of inositol phospholipids by a phosphodiesterase in rat cerebral cortical membranes. Addition of 100 microM-Gpp[NH]p to prelabelled membranes caused a rapid accumulation of [3H )inositol phosphates (less than 30 s) for up to 2 min. GTP gamma S and Gpp [NH]p caused a concentration-dependent stimulation of phosphoinositide phosphodiesterase with a maximal stimulation of 2.5-3-fold over control at concentrations of 100 microM. GMP was as effective as the nonhydrolysable analogues, but much less potent (EC50 380 microM). GTP and GDP caused a 50% stimulation of the phospholipase C at 100 microM and at higher concentrations were inhibitory. The adenine nucleotides App[NH]p and ATP also caused small stimulatory effects (64% and 29%). The guanine nucleotide stimulation of inositide hydrolysis in cortical membranes was selective for inositol phospholipids over choline-containing phospholipids. Gpp[NH]p stimulated the production of inositol trisphosphate and inositol bisphosphate as well as inositol monophosphate, indicating that phosphoinositides are substrates for the phosphodiesterase. EGTA (33 microM) did not prevent the guanine nucleotide stimulation of inositide hydrolysis. Calcium addition by itself caused inositide phosphodiesterase activation from 3 to 100 microM which was additive with the Gpp[NH]p stimulation. These data suggest that guanine nucleotides may play a regulatory role in the modulation of the activity of phosphoinositide phosphodiesterase in rat cortical membranes.     

Effects of local anesthetics on guanyl nucleotide modulation of the catecholamine-sensitive adenylate cyclase system and on beta-adrenergic receptors

Tetracaine and other local anesthetics exert multiple actions on the catecholamine-sensitive adenylate cyclase system of frog erythrocyte membranes. Tetracaine (0.2--20 mM) reduces the responsiveness of adenylate cyclase to (a) guanyl-5'-yl-imidodiphosphate and (b) isoproterenol in the presence of GTP or guanyl-5'-yl-imidodiphosphate. Local anesthetics did not affect (a) basal enzyme activity, and (b) enzyme responsiveness to NaF. Tetracaine inhibited stimulation of adenylate cyclase by guanyl-5'-yl-imidodiphosphate over the whole range of nucleotide concentrations. By contrast, inhibition by tetracaine of isoproterenol activity in the presence of GTP was significant only if GTP concentrations exceeded 10(-7) M. Tetracaine also competitively inhibited binding of both the antagonist [3H]dihydroalprenolol and the agonist [3H]hydroxybenzylisoproterenol to beta-adrenergic receptors. However, it was twice as potent in inhibiting [3H]hydroxybenzylisoproterenol as [3H]dihydroalprenolol binding. The greater potency for inhibition of agonist binding was due to the ability of the anesthetics to promote dissociation of the high-affinity nucleotide sensitive state of the beta-adrenergic receptor induced by agonists. Other local anesthetics mimicked the effects of tetracaine on adenylatecyclase and in dissociating high-affinity agonist-receptor complexes. The other of potency for both processes was dibucaine greater than tetracaine greater than bupivacaine greater than lidocaine which agrees with their relative potencies as local anesthetics. By contrast, a different order of potency was observed for competitive inhibition of [3H]dihydroalprenolol binding: dibucaine greater than tetracaine greater than greater than lidocaine greater than bupivacaine.     

Calcium-Dependent Nerve Growth Factor-Stimulated Hydrolysis of Phosphoinositides in PC12 Cells

The effect of nerve growth factor (NGF) on the hydrolysis of phosphoinositides in PC12 cells was examined. Addition of NGF to PC12 cells prelabeled with [3H]-inositol resulted in an increase in the formation of labeled inositol trisphosphate ([3H]IP3), inositol bisphosphate ([3H]IP2), and inositol monophosphate ([3H]IP), an observation indicating that NGF stimulated hydrolysis of the polyphosphoinositides. The increase in these inositol phosphates was detected as early as 15 s after addition of NGF. In the presence of LiCl, the accumulation of [3H]IP was linear for at least 20 min. The NGF-stimulated accumulation of [3H]IP was dose-dependent with a Kact of 0.17 nM and was dependent on the presence of extracellular calcium. In a calcium-free buffer containing EGTA, the NGF-dependent increase in accumulation of [3H]IP was not seen, and the basal level of [3H]IP accumulation was lower than that observed in the presence of extracellular calcium. Lanthanum inhibited both the basal and NGF-stimulated accumulation of [3H]IP, whereas the calcium ionophore A23187, in the absence of NGF, stimulated an accumulation of [3H]IP. The maximal accumulation of [3H]IP in the presence of A23187 was the same as that observed in the presence of NGF. Incubation of the cells with both A23187 and NGF resulted in an accumulation of [3H]IP that was not significantly different from the effect of either agent alone. These results suggest that NGF rapidly stimulates the hydrolysis of phosphoinositides in PC12 cells and that this NGF-stimulated hydrolysis of phosphoinositides occurs by a calcium-dependent mechanism.     

The effect of ischaemia-reperfusion on [3H]inositol phosphates and Ins(1,4,5)P3 levels in cardiac atria and ventricles — a comparative study

In this study incorporation of [³H]inositol into inositol phosphates and phosphoinositides as well as tissue Ins(1,4,5)P₃ levels of the atria and ventricles of isolated, perfused rat hearts were compared. Although the incorporation of [³H]inositol into the phosphoinositides of atria and ventricles was similar, significantly higher (2–3 fold) incorporation rates into inositol phosphates were observed in atrial tissue. Using a D-myo-[³H]Ins(1,4,5)P₃ assay system, the Ins(1,4,5)P₃ levels observed in atria from perfused rat hearts were also significantly higher than those obtained under the same experimental circumstances in the ventricles.Since previous studies on whole hearts showed inhibition of the phosphatidylinositol (PI) pathway during ischaemia with an immediate significant stimulation upon reperfusion [12, 20], the effects of ischaemia and 1 min postischaemic reperfusion were also examined separately in atria and ventricles. The results showed that 20 min of global ischaemia significantly depressed Ins(1,4,5)P₃ levels as well as incorporation of [3H]inositol into ventricular InSP₂ and InSP₃. Reperfusion caused an immediate (within 1 min) increase in Ins(1,4,5)P₃ levels and also [³H]inositol incorporation into all three cytosolic inositol phosphates in the ventricles. However, the effect of ischaemia and reperfusion on Ins(1,4,5)P₃ levels as well as the incorporation of [³H]inositol into the inositol phosphates were less prominent in the atria. It therefore appears that the differential responses of the atria and the ventricles to an oxygen deficiency [41] are also reflected in the differences in PI metabolism during ischaemia-reperfusion.     

Long-term atropine treatment lowers the efficacy of carbachol to stimulate phosphatidylinositol breakdown in the cerebral cortex and hippocampus of rats.

The effect of long-term treatment with atropine, a muscarinic antagonist, known to cause up-regulation of receptor numbers, was examined on the muscarinic-receptor-mediated stimulation of phosphoinositide breakdown in the rat cerebral cortex and hippocampus. Although the numbers of both M1 muscarinic receptors, as measured by [3H]pirenzepine binding, and M1 and M2 receptors increased in both brain regions, the maximal breakdown of myo-[3H]inositol-labelled phosphoinositides was unaltered in the presence of carbachol at a saturating concentration (10(-2) M). In fact the efficacy of carbachol was decreased in slices from atropine-treated cerebral cortex [EC50 (concentration producing half-maximal effect) = 93 microM] as compared with the saline-treated control (EC50 = 23 microM)(P less than 0.005). Similarly the EC50 value (23 microM) in hippocampal slices from saline-treated rats increased in atropine-treated rats to 126 microM (P less than 0.005). This lowered efficacy of muscarinic stimulation could not be explained in terms of residual atropine in the tissue from treated rats. The noradrenaline- or serotonin (5-hydroxytryptamine)-stimulated breakdown or the K+ potentiation of the muscarinic-receptor-stimulated breakdown of [3H]phosphoinositides was not affected by the atropine treatment. Chromatography of the released [3H]inositol phosphates shows that atropine treatment did not cause any qualitative change in the pattern of [3H]inositol phosphates released by carbachol stimulation.     

The role of alpha 1-adrenergic stimulation in inositol phosphate metabolism during post-ischaemic reperfusion.

The aim of this study was to elucidate the mechanism of enhanced inositol phosphate metabolism during reperfusion. Inositol phosphate stores were prelabelled by perfusing isolated rat hearts for 1 h with [3H]inositol (1.5 microCi/ml). LiCl (10 mM) and prazosin (0.3 microM) were subsequently added 15 min before (i) 20 min control perfusion; (ii) 20 min normothermic ischaemic cardiac arrest (NICA); (iii) 20 min NICA followed by 1 min reperfusion. The ventricles were freeze-clamped before determination of isotopical incorporation of [3H]inositol into the inositol phosphates (Dowex anion exchange chromatography) and InsP3 levels (Amersham InsP3 assay system). In addition, noradrenaline release into the perfusate was also assessed (HPLC and electrochemical detection). The results showed: (i) increased noradrenaline release into the perfusate immediately after the onset of reperfusion; (ii) significant depression of [3H]inositol incorporation into inositol phosphates and InsP3 levels after 20 min NICA; (iii) reperfusion caused an immediate significant increase in isotopical incorporation of [3H]inositol into inositol phosphates as well as InsP3 levels; (iv) the alpha 1-adrenergic blocker, prazosin (0.3 microM), completely inhibited the reperfusion-induced increase in inositol phosphate metabolism. These observations suggested that increased alpha 1-adrenergic receptor stimulation by noradrenaline might be responsible for the stimulation of ventricular inositol phosphate metabolism during postischaemic reperfusion.     

Stimulation of Phosphoinositide Hydrolysis by Serotonin in C6 Glioma Cells

5-Hydroxytryptamine (serotonin or 5-HT) stimulated the incorporation of 32Pi into phosphatidylinositol (PI) but not into polyphosphoinositides in C6 glioma cells with an EC50 of 1.2 X 10(-7) M. The phosphoinositide response was blocked by the 5-HT2 antagonists ketanserin and spiperone but inhibited only partly by methysergide and mianserin. Atropine, prazosin, and yohimbine did not block the response, whereas fluphenazine and haloperidol did so partially but also inhibited basal incorporation by approximately 30%. The 5-HT1A agonist 8-hydroxy-2(di-n-propylamino)tetralin did not cause stimulation. Incubation with 5-HT (1 microM) for 1 h increased the incorporation of [2-3H]myoinositol into all phosphoinositides but not into inositol phosphates (IPs). Li+ alone at 10 mM increased labeling in inositol bisphosphate (IP2) and trisphosphate (IP3), whereas labeling in IP and phosphoinositides remained unaltered. Addition of 5-HT had no effect on this increase. Mn2+ at 1 mM enhanced labeling in PI, PI-4-phosphate, lyso-PI, glycerophosphoinositol, and IP, but the presence of 5-HT again did not cause further stimulation. 5-HT also stimulated the release of IPs in cells prelabeled with [2-3H]myo-inositol, incubated with LiCl (10 mM) and inositol (10 mM), and then exposed to 5-HT (1 microM). Radioactivity in IP2 and IP3 was very low, was stimulated approximately 50% as early as 30 s, and remained elevated for at least 20 min. Radioactivity in IP was at least 10 times as high as in IP3 but was increased only from 3 min on with a peak at 20 min, when the elevation was approximately 40 times that in IP3.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stimulation of phosphoinositide hydrolysis by oxytocin and the mechanism by which oxytocin controls prostaglandin synthesis in the ovine endometrium.

Slices of caruncular endometrium from steroid-treated ovariectomized sheep were incubated with myo-[2-3H]inositol to label tissue phosphatidylinositol. Effects of oxytocin were determined on the rate of incorporation of radioactivity into phosphatidylinositol and on the hydrolysis of phosphoinositides to inositol phosphates and diacylglycerol. Incorporation of radioactivity into phosphatidylinositol was linear during 2 h incubations; 10(-7) M (100 nM)-oxytocin caused a 2.8-fold increase in the rate of incorporation. In the presence of Li+, addition of 10(-7) M-oxytocin to slices in which phosphatidylinositol was pre-labelled caused mean increase of 40-fold in the incorporation of radioactivity into inositol mono-, bis- and tris-phosphates. Inositol 1,3,4-trisphosphate was quantitatively the major trisphosphate formed. The action of oxytocin on phosphoinositide hydrolysis was dose- and time-dependent, occurring at concentrations within the range observed in plasma during episodes of secretion in vivo, and with a time course comparable with that of the action of oxytocin on uterine prostaglandin production. The effect of oxytocin on incorporation of radioactivity into inositol phosphates was not affected by inhibitors of prostaglandin synthesis. Diacylglycerol 1- and 2-lipases in caruncular endometrium converted up to 72% of added 2-[3H]arachidonyldiacylglycerol into [3H]arachidonic acid during 30 min incubations at pH 7.0. Caruncular endometrium contained 1.49 mumol of phosphatidylinositol/g, representing approx. 0.2 mumol/g of phosphatidylinositol arachidonic acid. It is proposed that the stimulation of endometrial prostaglandin synthesis by oxytocin is accounted for by increased hydrolysis of phosphoinositides to diacylglycerol and inositol phosphates with subsequent release of arachidonic acid from diacylglycerol.     

Inhibitory effect of local anesthetics on Na+/H+ antiporter in brush border membrane-reconstituted vesicles

The effects of three local anesthetics, lidocaine, dibucaine, and tetracaine, on Na+/H+ antiporter activity were examined in brush border membrane-reconstituted vesicles. Lidocaine at 10 microM inhibited H+ efflux in the presence of an inward Na+ gradient, suggesting that this anesthetic specifically inhibits the Na+/H+ antiporter. On the other hand, dibucaine and tetracaine decreased H+ efflux even in the absence of a Na+ gradient.     

GABAB Receptor-Mediated Inhibition of Histamine H1-Receptor-Induced Inositol Phosphate Formation in Slices of Rat Cerebral Cortex

Histamine-stimulated accumulation of [3H]inositol monophosphate ([3H]IP1) in lithium-treated slices of rat cerebral cortex was inhibited by gamma-aminobutyric acid (GABA) (IC50 0.30 +/- 0.03 mM). The maximum level of inhibition was 69 +/- 2%. GABA alone caused a small stimulation of basal accumulation of [3H]IP1. The inhibitory action of GABA on the response to histamine was mimicked by the GABAB agonist (-)-baclofen, IC50 0.69 +/- 0.04 microM, which was 430-fold more potent as an inhibitor than the (+)-isomer. (-)-Baclofen also inhibited histamine-induced formation of [3H]inositol bisphosphate ([3H]IP2) and [3H] inositol trisphosphate ([3H]IP3). Inhibition curves for GABA and for (-)-and and (+)-baclofen had Hill coefficients greater than unity. (-)-Baclofen, at concentrations that caused inhibition of histamine-induced [3H]IP1 accumulation, did not alter the basal level of [3H]IP1 or the incorporation of [3H]inositol into total inositol phospholipids. Isoguvacine, a GABAA agonist, had no effect on either the histamine-stimulated or basal accumulation of [3H]IP1. GABA had no effect on carbachol-stimulated [3H]IP1 formation.     

Phosphoinositide synthesis and Ca2+ gating in blowfly salivary glands exposed to 5-hydroxytryptamine.

Blowfly salivary glands, previously exposed to 10 microM-5-hydroxytryptamine for 30 min, demonstrated a rapid compensatory resynthesis of [3H]inositol-labelled phosphatidylinositol 4,5-bisphosphate when allowed to recover in medium containing 3-5 microM-inositol. Phosphatidylinositol 4,5-bisphosphate comprised 70% of the total [3H]-phosphoinositide, and there was a corresponding decrease in the formation of [3H]-phosphatidylinositol. Subsequent addition of 5-hydroxytryptamine produced an equivalent breakdown of the newly synthesized phosphoinositides but little 45Ca2+ gating. Increasing the inositol concentration in the medium to 300 microM produced a 14-fold stimulation of phosphatidylinositol synthesis but only a 5-fold increase in phosphatidylinositol 4,5-bisphosphate synthesis. Increasing the inositol concentration in the medium from 3 microM to 300 microM resulted in a progressively greater recovery of the 45Ca2+-gating response. At 300 microM-inositol there was an 85% recovery of 45Ca2+-gating response. These results indicate that conversion of phosphatidylinositol into phosphatidylinositol 4,5-bisphosphate occurs in blowfly salivary glands and is secondary to an initial breakdown of the phosphoinositides. Recovery of Ca2+ gating is dependent on the restoration of both phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate to appropriate concentrations.     

Vasoactive intestinal polypeptide and muscarine mobilize intracellular Ca2+ through breakdown of phosphoinositides to induce catecholamine secretion. Role of IP3 in exocytosis

We have recently shown that vasoactive intestinal polypeptide (VIP) is as potent as acetylcholine in inducing the secretion of catecholamines from the rat adrenal medulla. In the present study we have investigated the molecular mechanism involved in the exocytotic secretion of catecholamines by VIP and the effects of VIP on Ca45 uptake and phosphoinositide breakdown and compared them with those of the classical cholinergic agonists. We now show that omission of Ca2+ from the perfusion medium had almost no effect on VIP-induced secretion; however, addition of 1 mM EGTA to calcium-free medium abolished the secretion. Stimulation with VIP did not result in a net increase in Ca45 uptake and it was not modified by a protein kinase C activator, phorbol ester. All these effects of VIP were comparable to those of muscarine. VIP (0.3 to 10 microM) and muscarine (30 to 100 microM) produced time-and concentration-dependent increase (up to 700%) in the production of [3H]inositol phosphates. The production of [3H]inositol phosphates by VIP and muscarine occurred in calcium-free and EGTA medium. The effect of VIP on [3H]IP, [3H]IP2, and [3H]IP3 production was reduced by (1 to 30 microM) VIP antagonist (an analogue of growth hormone-releasing factor, Ac-Tyr1hGRF) and 1 to 20 microM naloxone. Although nicotine produced a brisk secretory response, there was no change in [3H]inositol phosphates. We conclude that inositol 1,4,5-trisphosphate generated upon activation of VIP and muscarine receptors is linked to exocytotic secretion of adrenal medullary hormones through release of internal Ca2+ ions.     

In Vivo Electrical Stimulation of the Sympathetic Nerve of the Eye Increases Inositol Phosphate Production and Prostaglandin Release in the Rabbit Iris Muscle

The effects of in vivo electrical stimulation of the sympathetic nerve of the eye on phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis in rabbit iris and release of arachidonate and prostaglandin (PG) E2 into aqueous humor were investigated. myo-[3H]Inositol or [1-14C]arachidonate was injected intracamerally into each eye 3 h before electrical stimulation of one of the sympathetic trunks. Tissue phosphoinositides were determined by TLC, and 3H-labeled inositol phosphates were analyzed by either ion-exchange chromatography or HPLC. The aqueous humor was analyzed for 14C-labeled arachidonate and PGE2 by radiochromatography and for unlabeled PGE2 by radioimmunoassay. The results obtained from this study can be summarized as follows: (a) The rates of in vivo incorporation of myo-[3H]inositol into phosphoinositides and accumulation of 3H-labeled inositol phosphates in the iris muscle increased with time and then leveled off between 3 and 5 h. (b) Distribution of 3H radioactivity in inositol phosphates, as determined by HPLC, showed that of the total radioactivity in inositol phosphates, 53.6% was recovered in myo-inositol 1-phosphate, 36% in myo-inositol bisphosphate, 0.95% in myo-inositol 1,3,4-trisphosphate (1,3,4-IP3), and 2.6% in 1,4,5-IP3. (c) Electrical stimulation of the sympathetic nerve resulted in a significant loss of 3H radioactivity from PIP2 and a concomitant increase of that in IP3, an observation indicating that PIP2 is the physiological substrate for alpha 1-adrenergic receptors in this tissue. (d) Release of IP3 and liberation of arachidonate for PGE2 synthesis are dependent on the duration of stimulation and the intensity (voltage) of stimulus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enhanced arterial contractility to noradrenaline in diabetic rats is associated with increased phosphoinositide metabolism.

The purpose of this study was to investigate whether the increased contractile responsiveness of aortae from male rats with 12-14 week streptozotocin-induced diabetes to noradrenaline is associated with alterations in phosphoinositide metabolism. The contractile response to noradrenaline (10 microM) in both the presence and absence of extracellular calcium was significantly enhanced in aortae from diabetic rats. No significant differences were found between control and diabetic arteries in the basal incorporation of 32P and [3H]myo-inositol into phosphoinositides, or in the basal accumulation of [32P]phosphatidic acid and [3H]inositol phosphates. However, noradrenaline (10 microM) caused significantly greater breakdown of [32P]phosphatidylinositol 4,5-bisphosphate and formation of [32P]phosphatidic acid and [3H]inositol phosphates in diabetic aortae than in control preparations. The production of [3H]inositol phosphates induced by noradrenaline was selectively reduced by the alpha 1-adrenoceptor antagonist, prazosin, in both control and diabetic tissues. These results indicate that phosphoinositide metabolism in response to noradrenaline via stimulation of alpha 1-adrenoceptors is enhanced in aortae from chronic streptozotocin-diabetic rats. The increase in inositol 1,4,5-trisphosphate and 1,2-diacylglycerol production that presumably results could be responsible, at least in part, for the enhanced contractile response of aortae from diabetic rats to noradrenaline.