Neurokinin3-receptors are linked to inositol phospholipid hydrolysis in the guinea-pig ileum longitudinal muscle-myenteric plexus preparation.

1. Tachykinin-stimulated inositol phospholipid hydrolysis was examined in slices of longitudinal muscle from guinea-pig ileum. 2. Substance P, neurokinin A and neurokinin B induced a concentration-dependent accumulation of total [3H]-inositol phosphates in the presence of 12 mM lithium with similar maximal responses and EC50 values. 3. The selective NK1-receptor agonist, substance P methyl ester, and the selective NK3-receptor agonist succ-[Asp6, MePhe8]-SP(6-11) (senktide) also stimulated [3H]-inositol phosphate formation with maximum responses of 50.69 +/- 0.96 and 45.64 +/- 1.17% relative to 10 microM substance P, respectively. Substance P methyl ester was approximately equipotent with substance P, whereas senktide was approximately 100 times more potent. 4. When added together, maximally effective concentrations of substance P methyl ester and senktide gave responses that were fully additive. In contrast, responses to substance P and neurokinin B were not additive. 5. The stimulation of [3H]-inositol phosphate formation by substance P, neurokinin B and senktide was not affected by atropine (2 microM) or tetrodotoxin (TTX, 0.3 microM). 6. The contractile effect of senktide was inhibited completely by TTX and partially blocked by atropine. Contractions induced by substance P methyl ester were not changed in the presence of TTX or atropine. 7. [D-Pro4, D-Trp7,9,10]-SP(4-11) competitively antagonized the action of substance P methyl ester on inositol phospholipid hydrolysis and contraction, but had no significant effect on senktide-induced inositol phospholipid breakdown or contraction. 8. These results suggest that NK3-receptors in the guinea-pig ileum are coupled to inositol phospholipid hydrolysis.     

Differential effects of elevated calcium ion concentrations on inositol phospholipid responses in mouse and rat cerebral cortical slices

Inositol phospholipid turnover in cerebral cortical slices from mouse and rat was assessed using a [3H]inositol pre-labelling technique followed by anion exchange chromatography to isolate [3H]inositol phosphates ([3H]InsP chi). In both mouse and rat cerebral cortical slices, elevating the CaCl2 concentration of the Krebs medium from 1.3 to 4 mM did not significantly enhance the accumulation of [3H]InsP chi in the absence of any stimulus, or in the presence of glutamate (3 mM), depolarizing concentrations of KCl (25 mM), 5-hydroxytryptamine (0.3 mM), the calcium ionophore A23187 (33 microM) or carbachol (1 mM). However, the accumulations of [3H]InsP chi induced by histamine (1 mM) or noradrenaline (0.1 mM) were significantly increased by between 95 and 178% in cerebral cortical slices from both species by the elevation of extracellular calcium. Analysis of the individual inositol phosphates revealed that elevated ambient calcium enhanced the histamine-generated accumulations of [3H]InsP2, [3H]InsP3 and [3H]InsP4 by up to two-fold, while only the [3H]InsP3 response to carbachol was significantly increased. Under the same conditions, histamine, but not carbachol, selectively increased the accumulation of [3H]PtdInsP2 by up to 50%. The [3H]InsP chi responses to histamine and noradrenaline in combination with the calcium ionophore A23187 were greater-than-additive, inferring an enhancement of the receptor response by raised intracellular calcium. However, the combination of A23187 with glutamate or KCl resulted in significantly less-than-additive [3H]InsP chi responses. The [3H]InsP chi response to carbachol or 5-hydroxytryptamine was not significantly altered in the presence of A23187. Taken together, these results indicate heterogeneity between the mechanisms of inositol phospholipid turnover induced by these various stimuli in mammalian cerebral cortical slices.     

Inositol phospholipid hydrolysis in human brain; adenosine inhibition of the response to histamine.

1. Inositol phospholipid hydrolysis was examined in human cerebral cortex slices by a [3H]-inositol prelabelling assay. 2. Enhancement of [3H]-inositol phosphates accumulation was observed in the presence of carbachol, noradrenaline, histamine, 5-hydroxytryptamine (5-HT) and depolarizing concentrations of KCl. 3. Despite having no effect alone, adenosine (and its analogue 2-chloroadenosine) selectively inhibited the direct response to histamine. 4. The inhibition due to adenosine was antagonized by theophylline, but not by 8-cyclopropyltheophylline.     

G-protein involvement in muscarinic receptor-stimulation of inositol phosphates in longitudinal smooth muscle from the small intestine of the guinea-pig.

1. Aluminium fluoride (AlF), pertussis toxin (PTX) and cholera toxin (ChTX) have been used to examine the involvement of G-proteins during muscarinic acetylcholine receptor (AChR) stimulation of inositol phospholipid hydrolysis in fragments of longitudinal smooth muscle from the small intestine of the guinea-pig. 2. Carbachol (CCh) induced time- and concentration-dependent increases in [3H]-inositol monophosphates, [3H]-inositol (1,4) bisphosphate, [3H]-inositol (1,3,4) trisphosphate, [3H]-inositol (1,4,5) trisphosphate ([3H]-Ins (1,4,5)P3) and [3H]-inositol tetrakisphosphates measured by h.p.l.c. These increases were inhibited > 95% in the presence of the muscarinic AChR antagonist atropine (0.5 microM). 3. AlF transiently increased the basal levels of [3H]-Ins (1,4,5)P3 but increases in the levels of the other [3H]-inositol phosphates occurred more slowly. CCh-induced increases in the levels of all the [3H]-inositol phosphates were strongly inhibited in the presence of AlF. 4. PTX had no effect on basal levels of any of the [3H]-inositol phosphates but reduced the effects of CCh on these; ChTX had no effects on either basal or CCh-stimulated levels. 5. It was concluded that muscarinic AChR-stimulated increases in the levels of [3H]-inositol phosphates occur via both a PTX-sensitive G-protein and a PTX-insensitive mechanism. The actions of AlF may suggest the involvement of an inhibitory G-protein in the regulation of muscarinic AChR-stimulated inositol phospholipid turnover.     

Pharmacological characterization of tachykinin-stimulated inositol phospholipid hydrolysis in peripheral tissues.

1. Tachykinin-stimulated inositol phospholipid hydrolysis was examined in slices of rat parotid gland, hamster urinary bladder and guinea-pig ileum longitudinal muscle. 2. In the presence of lithium, substance P and other naturally-occurring and synthetic tachykinins induced large, dose-dependent increases in [3H]-inositol monophosphate accumulation. 3. In slices of rat parotid gland, [pGlu6,L-Pro9]SP(6-11) was considerably more potent in stimulating inositol phospholipid hydrolysis than [pGlu6,D-Pro9]SP(6-11). 4. In contrast, in slices of hamster urinary bladder, [pGlu6,D-Pro9]SP(6-11) exhibited greater potency in evoking inositol phospholipid breakdown than [pGlu6,L-Pro9]SP(6-11). 5. The differential selectivity of these C-terminal fragments of substance P suggests that they may be useful tools for distinguishing between NK1 and NK2 receptors. 6. L-659,837 and L-659,874 antagonized eledoisin-stimulated inositol phospholipid hydrolysis in slices of hamster urinary bladder. Neither compound significantly reduced substance-P evoked inositol phospholipid breakdown in slices of rat parotid gland, or senktide-induced inositol phospholipid hydrolysis in slices of guinea-pig ileum. 7. L-659,837 and L-659,874 had no effect on the atropine-sensitive, carbachol-stimulated inositol phospholipid hydrolysis in slices of rat parotid gland. 8. These data further support the notion that L-659,837 and L-659,874 are potent and selective NK2 receptor antagonists.     

Inhibition of muscarinic receptor-induced inositol phospholipid hydrolysis by caffeine, beta-adrenoceptors and protein kinase C in intestinal smooth muscle.

1. The effects of caffeine, isoprenaline, dibutyryl cyclic AMP, isobutylmethylxanthine (IBMX), 12-O-tetradecanoylphorbol-13-acetate (TPA) or 1-oleoyl-2-acetylglycerol (OAG), (protein kinase C (PKC) activators), 2-methoxy verapamil (D600), thapsigargin and ryanodine on muscarinic acetylcholine receptor (AChR)-stimulated inositol phospholipid hydrolysis were studied in smooth muscle fragments from the longitudinal layer of the small intestine of the guinea-pig. 2. Incubation of the fragments with the muscarinic agonist, carbachol (CCh) (100 microM) resulted in rapid increases in the levels of all the inositol phosphate isomers with maximal increases in the [3H]-inositol (1,4,5) trisphosphate ([3H]-Ins(1,4,5)P3) isomer occurring 10 s following incubation. 3. The beta-adrenoceptor agonist, isoprenaline (10 microM) and dibutyryl cyclic AMP (10 microM), a membrane permeant analogue of cyclic AMP both reduced the CCh stimulation, but not the basal levels of [3H]-inositol phosphates. This inhibition by dibutyryl cyclic AMP was enhanced in the presence of the phosphodiesterase inhibitor, IBMX. CCh inhibited the isoprenaline-induced increases in the levels of cyclic AMP and this was via a pertussi toxin (PTX)-sensitive G-protein mechanism. 4. TPA (1 microM) and OAG (100 microM) a 1,2-diacylglycerol (DAG) analogue both reduced the CCh-induced increases in [3H]-inositol phosphates levels but neither affected basal values nor the basal levels of cyclic AMP. 5. D600 (10 microM), which blocks voltage-dependent Ca2+ channels, also reduced the CCh-stimulated levels of [3H]-inositol phosphates suggesting that some of the agonist-induced increases are due to a potentiating effect of Ca2+ entering the cell. 6. Caffeine (0.5-30 mM) significantly inhibited both the basal and CCh-induced increases in all the [3H]-inositol phosphate isomers. Its inhibitory action was not due to increases in cyclic AMP since caffeine had no effect on the levels of cyclic AMP at concentrations up to 30 mM. 7. Incubation with thapsigargin (1 microM) and ryanodine (10 microM) had no effect on either basal or CCh-induced inositol phospholipid hydrolysis or cyclic AMP levels. 8. The results indicate a reciprocal inhibition by beta-adrenoceptors and muscarinic AChRs of their effects on cyclic AMP and inositol phosphate levels respectively. Ca2+ entering the cell (but not the action of ryanodine or thapsigargin) potentiates while caffeine inhibits muscarinic AChR-induced rises in inositol phosphate levels. Diacylglycerols may exert a negative feedback inhibition on inositol phosphate production.     

Studies on the adenosine-receptor mediating the augmentation of histamine-induced inositol phospholipid hydrolysis in guinea-pig cerebral cortex.

Incubation (45 min) of slices of guinea-pig cerebral cortex with adenosine alone had no significant effect on the accumulation of [3H]-inositol phosphates but enhanced the response to histamine H1-receptor stimulation in a concentration-dependent manner. The effect of adenosine on agonist-stimulated inositol phospholipid hydrolysis appeared to be selective for histamine H1-receptor stimulation since it did not augment the phosphoinositide responses to carbachol, noradrenaline, 5-hydroxytryptamine or elevated KCl. The accumulation of [3H]-inositol phosphates induced by histamine increased linearly between 5 and 45 min incubation with agonist. However, following the simultaneous addition of histamine and adenosine, there was a marked delay in the appearance of the augmentation produced by adenosine. The augmentation of [3H]-inositol phosphate accumulation was mimicked by a number of adenosine analogues. The rank order of potency was; cyclopentyladenosine greater than R-phenyl-isopropyladenosine 5'-N-ethylcarboxamidoadenosine greater than 2-chloroadenosine. This is consistent with the order expected for an adenosine A1-receptor effect but the EC50 values were in the micro- rather than nanomolar range. The response to 2-chloroadenosine was antagonized by the xanthine adenosine-antagonists, cyclopropyltheophylline, 8-phenyltheophylline, 3-isobutyl-1-methylxanthine and theophylline, and the non-xanthine alloxazine.     

gamma-Aminobutyric acid inhibition of histamine-induced inositol phosphate formation in guinea-pig cerebellum: comparison with guinea-pig and rat cerebral cortex.

1. gamma-Aminobutyric acid (GABA), 2 mM, inhibited basal accumulation of [3H]-inositol monophosphate ([3H]-IP1) in lithium-treated slices of guinea-pig cerebellum preincubated with [3H]-inositol. In contrast, 2 mM GABA stimulated the accumulation of [3H]-IP1 in rat cerebral cortical slices over a 60 min incubation period, but had no significant effect in slices of guinea-pig cerebral cortex. The estimated IC50 for the inhibitory action of GABA in guinea-pig cerebellar slices was 0.52 +/- 0.12 mM. 2. GABA inhibited histamine-induced [3H]-IP1 accumulation in guinea-pig cerebellar slices in a non-competitive manner. The best-fit value for the maximum level of inhibition was 74 +/- 6%. The estimated IC50 for GABA was 0.77 +/- 0.15 mM and was not significantly different from the IC50 for inhibition of the basal accumulation of [3H]-IP1. The response to histamine in guinea-pig and rat cerebral cortical slices was also inhibited by 2 mM GABA. 3. In guinea-pig cerebellar slices 2 mM GABA potentiated histamine-induced [3H]-inositol bisphosphate ([3H]-IP2) accumulation, whereas in both guinea-pig and rat cerebral cortex the effect was inhibition. 4. Isoguvacine and muscimol, GABAA-selective agonists, and (-)-baclofen, GABA(B)-selective, had no significant effect on basal or histamine-stimulated accumulation of [3H]-IPs in guinea-pig cerebellar slices. (-)-Baclofen had only a weak inhibitory effect on [3H]-IP1 accumulation in guinea-pig-cerebral cortex (16 +/- 6% inhibition with 10 microM (-)-baclofen), whereas in rat cerebral cortex (-)-baclofen mimicked the inhibitory effect of GABA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the effects of lithium on phosphatidylinositol (PI) cycle activity in human muscarinic m1 receptor-transfected CHO cells.

1. The effects of lithium on [3H]-inositol and [3H]-cytidine incorporation into [3H]-inositol monophosphates ([3H]-InsP1) and [3H]-cytidine monophosphorylphosphatidate ([3H]-CMP-PA), respectively, and inositol 1,4,5-trisphosphate (InsP3) and inositol 1,3,4,5-tetrakisphosphate (InsP4) mass were studied in carbachol-stimulated human m1 muscarinic receptor-transfected Chinese hamster ovary cells (m1 CHO cells). 2. Lithium alone (10 mM) had no appreciable effects on any of the four parameters measured; it was only in carbachol-stimulated cells that the effects of lithium became apparent. 3. In the presence of carbachol (1 mM), lithium (10 mM) caused a relatively rapid (within 5 min) accumulation of [3H]-InsP1 and [3H]-CMP-PA which continued up to about 20-30 min, after which accumulation slowed down. On the other hand, the elevation in InsP3 and InsP4 levels produced by carbachol was not altered by lithium in the short-term and only at later times (> 20-30 min) was the response attenuated, with InsP3 and InsP4 levels approaching basal. 4. The effects of lithium on carbachol-stimulated [3H]-InsP1 and [3H]-CMP-PA accumulation and the attenuation of the carbachol-induced elevation of InsP3 and InsP4 were all dose-dependent, with EC50s in the region of 1 mM. 5. The lithium-induced effects on [3H]-CMP-PA and InsP3 and InsP4 in carbachol-stimulated cells could be reversed, in a dose-dependent manner, by preincubation with exogenous myo-inositol (EC50 = 2-3 mM) but not by the inactive analogue scyllo-inositol, indicating that these effects occur as a consequence of depletion of inositol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of monovalent ions upon G proteins coupling muscarinic receptors to phosphoinositide hydrolysis in the rat cerebral cortex

It has been suggested that K+, Li+ and Fl- affect the function of G proteins coupled to signal transducing enzymes. Lithium, at concentrations which were found to reduce forskolin-stimulated adenylate cyclase activity, was without effect on either membrane [3H]phosphatidylinositol-4,5-bisphosphate ([3H]PIP2) hydrolysis measured in the absence or presence of 5'-guanylyl-imidodiphosphate (Gpp(NH)p), or (at greater than or equal to 2.3 mM Li+) upon the stimulation of rat cerebral cortical inositol phospholipid breakdown by either carbachol, noradrenaline or NaF measured at either 6 or 18 mM K+. The increase in assay [K+] greatly enhanced the inositol phospholipid response to carbachol but not to NaF. The inhibitory effect of carbachol upon forskolin-stimulated adenylate cyclase was not affected by raising the [K+] from 6 to 18 mM. At 6 mM K+ (both in the absence and presence of 15 microM AlCl3), the effects of carbachol and NaF upon inositol phospholipid breakdown were essentially additive, whereas at 18 mM K+, the breakdown response to carbachol (antagonised by pirenzepine with a pA2 value of 7.6) was similar in the absence and presence of NaF. It is concluded that in the rat cerebral cortex: (a) Li+ does not affect the function of either the phosphoinositide-specific phospholipase C enzyme itself or the Gp coupled to this enzyme; (b) the difference between the additivity between NaF and carbachol seen at different assay [K+] may reflect the K(+)-dependent changes in the tetrodotoxin-resistant and tetrodotoxin-sensitive pathways of carbachol stimulation of inositol phospholipid breakdown reported by Gurwitz and Sokolovsky (1987, Biochemistry 26, 633); and (c) the effect of K+ on muscarinic receptor-coupled inositol phospholipid breakdown is not found for muscarinic receptors inhibitorily coupled to adenylate cyclase. Evidence is also presented to suggest that NaF affects the dephosphorylation of the formed [3H]inositol polyphosphates.     

Inhibition of histamine-stimulated inositol phospholipid hydrolysis by agents which increase cyclic AMP levels in bovine tracheal smooth muscle.

1. The effect on histamine-stimulated [3H]-inositol phosphate accumulation of a range of agents which increase the accumulation, or mimic the actions, of cyclic AMP has been investigated in bovine tracheal smooth muscle. 2. Salbutamol (1 microM), forskolin (1 microM) and vasoactive intestinal peptide (VIP, 1 microM) inhibited the inositol phosphate response to 0.1 mM histamine and increased the accumulation of [3H]-cyclic AMP in [3H]-adenine-labelled slices of bovine tracheal smooth muscle. The effect on inositol phospholipid hydrolysis was mimicked by the membrane permeant analogues of cyclic AMP, dibutrylcyclic AMP (1 mM) and 8-bromo-cyclic AMP (1 mM). 3. In contrast to salbutamol, which was equally effective at producing the two effects, forskolin produced large increases in [3H]-cyclic AMP accumulation (EC50 = 1.2 microM) at much higher concentrations than those required for inhibition of histamine-stimulated [3H]-inositol phosphate accumulation (EC50 = 0.09 microM). However, significant increases in [3H]-cyclic AMP accumulation, of similar magnitude to those obtained with salbutamol and VIP, were observed over the concentration range appropriate for inhibition of the inositol phosphate response to histamine. 4. In the presence of histamine (0.1 mM), isobutylmethylxanthine (IBMX, 1 mM) and rolipram (0.1 mM) both significantly (P less than 0.05) elevated tissue [3H]-cyclic AMP levels. IBMX, rolipram and (to a lesser extent) SKF 94120 significantly (P less than 0.05) reduced histamine-stimulated [3H]-inositol phosphate accumulation by 81%, 68% and 20%, respectively. M&B 22948 was without a significant effect on either [3H]-cyclic AMP or histamine-induced [3H]-inositol phosphate accumulation. 5. Both rolipram and forskolin reduced the increase in incorporation of [3H]-inositol into membrane phospholipids which followed stimulation with histamine. However, a significant inhibition of [3H]-inositol phosphate accumulation could be demonstrated under conditions in which there was no change in the level of [3H]-inositol incorporation.     

Stimulation of alpha 1-adrenoceptors in rat kidney mediates increased inositol phospholipid hydrolysis.

The molecular events which follow activation of alpha 1-adrenoceptors in rat kidney were investigated by measuring inositol phospholipid hydrolysis. Slices were labelled with [3H]-inositol (0.25 microM) and the accumulation of [3H]-inositol phosphates ([3H]-IP's) was measured after stimulation with alpha-adrenoceptor agonists. Phospholipid labelling was both time- and Ca2+-dependent. In kidney, Ca2+ (1 mM) increased the incorporation of [3H]-inositol by 49% and in cerebral cortex reduced it by 46%. Following addition of noradrenaline (NA, 1 mM), accumulation of [3H]-IP's increased linearly for at least 60 min. In Ca2+-free buffers a 2.1 fold increase in [3H]-IP accumulation was observed and further increases in stimulated and control levels were produced in the presence of Ca2+ (2.5 mM). These responses were attenuated by the inclusion of indomethacin (10 microM) and abolished in the presence of EGTA (0.5 mM). Responses to (-)-NA were more than 4 fold higher in the renal cortex than in the medulla. Separation of the IP's which accumulate after alpha-adrenoceptor agonists showed that after 60 min stimulation the major products were glycerophosphoinositol and inositol-phosphate with smaller amounts of inositol-bisphosphate and inositol-trisphosphate. The most effective agonists tested for stimulation of accumulation of [3H]-IP's were (-)-NA greater than phenylephrine greater than methoxamine, (+)-NA. Clonidine and (-)-isoprenaline were ineffective at concentrations up to 100 microM. The order of effectiveness of alpha-adrenoceptor antagonists was prazosin greater than BE2254 greater than phentolamine greater than idazoxan greater than rauwolscine. The results indicate that alpha 1-adrenoceptors in rat kidney are linked to phosphoinositide hydrolysis and that this response is localized mainly to the renal cortex.     

Effect of gastric secretagogues on the formation of inositol phosphates in isolated gastric cells of the rat.

The effects of compounds affecting gastric acid secretion were studied on the formation of inositol phosphates after prelabelling with [3H]-inositol in enriched gastric parietal cells of the rat, prepared by isopycnic centrifugation with Percoll. In cell preparations with 60 to 70% parietal cells, carbachol (10(-6)-10(-2) M) enhanced the accumulation of [3H]-inositol monophosphate ([3H]-IP1), [3H]-inositol bisphosphate ([3H]-IP2) and [3H]-inositol trisphosphate ([3H]-IP3) in a concentration-dependent manner, an effect which was antagonized by 10(-8) M atropine. Li+ (0.5-30 mM) enhanced the basal and carbachol-induced accumulation of all three [3H]-inositol phosphates, the formation of [3H]-IP1 being more sensitive to Li+ than those of [3H]-IP2 and [3H]-IP3. The concentration of Ca2+ in the incubation medium did not affect the relative stimulation of the accumulation of [3H]-inositol phosphates by carbachol, although the basal formation was higher in the presence of Ca2+ in the medium. In the absence of added Ca2+, the incorporation of [3H]-inositol into phospholipids was increased--an effect which was further enhanced by the addition of EGTA to the medium. Gastrin and pentagastrin (10(-8)-10(-5) M) enhanced the formation of [3H]-inositol phosphates, although they were clearly less effective than carbachol. Histamine (10(-6)-10(-3) M) had no effect of its own, but slightly attenuated the effect of carbachol. Cholecystokinin octapeptide (10(-9)-10(-6) M) slightly increased the formation of [3H]-inositol phosphates. Indomethacin (10(-4) M) had no consistent effect on the basal and carbachol-induced accumulation of [3H]-inositol phosphates, nor did prostaglandin E2 (10(-5) M) modify it. Adrenaline (10(-3) M), 5-hydroxytryptamine (10(-3) M), forskolin (10(-5) M), vasopressin (10(-5) M), angiotensin II (10(-5) M) and bombesin (10(-9)-10(-6) M) were all without effect. We suggest that the hydrolysis of inositol phospholipids may be involved in the signal transduction mechanism by which the activation of the muscarinic and gastrin receptors on the parietal cells leads to Ca2+ mobilization and the stimulation of hydrogen ion secretion.     

Aluminum impairs glucose utilization and cholinergic activity in rat brain in vitro

The effects of AlCl3 on the production of 14CO2 from [U-14C]glucose and high affinity choline transport in rat brain synaptosomes, and on carbachol-stimulated hydrolysis of phosphoinositides in cortical slices were studied. In buffer containing either high K+ (50 mM) or low K+ (4.9 mM), 1 mM AlCl3 significantly depressed the synaptosomal production of 14CO2 from [U-14C]glucose to 54% and 44% of control rates, respectively. At a concentration of 0.1 mM, AlCl3 depressed the evolution of 14CO2 from [U-14C]glucose from synaptosomes incubated in the high K+ buffer, but did not significantly change 14CO2 production from synaptosomes in the low K+ buffer. Aluminum chloride also inhibited high affinity choline transport in synaptosomes prepared from rat cortex and from hippocampus with an IC50 of approximately 0.5 mM. In brain slices the carbachol-stimulated hydrolysis of phosphoinositides was inhibited by AlCl3 in a dose-dependent manner. One millimolar, 0.5 mM and 0.1 mM AlCl3 inhibited the carbachol-stimulated release of inositol phosphates by 75%, 44% and 33%, respectively. These same concentrations of AlCl3 inhibited the incorporation of [3H]inositol into phospholipids. This inhibitory effect was not dose-dependent as all 3 concentrations of AlCl3 inhibited phospholipid labelling to the same extent (27-37%). These results are discussed in relation to the in vivo neurotoxicity of aluminum.     

Modulatory effects of NMDA on phosphoinositide responses evoked by the metabotropic glutamate receptor agonist 1S,3R-ACPD in neonatal rat cerebral cortex.

1. The effect of NMDA-receptor stimulation on phosphoinositide signalling in response to the metabotropic glutamate receptor agonist 1-aminocyclopentane-1S,3R-dicarboxylic acid (1S,3R-ACPD) has been examined in neonatal rat cerebral cortex slices. 2. Total [3H]-inositol phosphate ([3H]-InsPx) accumulation, in the presence of 5 mM LiCl, in [3H]-inositol pre-labelled slices was concentration-dependently increased by 1S,3R-ACPD (EC50 16.6 microM) and, at a maximally effective concentration, 1S,3R-ACPD (300 microM) increased [3H]-InsPx accumulation by 12.8 fold over basal values. 3. [3H]-InsPx accumulation stimulated by 1S,1R-ACPD was enhanced by low concentrations of NMDA (3-30 microM), but not by higher concentrations (> 30 microM). [3H]-InsPx accumulations stimulated by 1S,3R-ACPD in the absence or presence of 10 microM NMDA were linear with time, at least over the 15 min period examined; however, in the presence of 100 microM NMDA the initial enhancement of 1S,3R-ACPD-stimulated phosphoinositide hydrolysis progressively decreased with time. 4. In the presence of a maximal enhancing concentration of NMDA (10 microM), the response to 1S,3R-ACPD (300 microM) was increased 1.9 fold and the EC50 for agonist-stimulated [3H]-InsPx accumulation decreased about 4 fold. The enhanced response to the metabotropic agonist was concentration-dependently inhibited by competitive and uncompetitive antagonists of NMDA-receptor activation. 5. 1S,3R-ACPD also stimulated inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) mass accumulation with an initial peak response (5-6 fold over basal) at 15 s decaying to a smaller (2 fold), but persistent elevated accumulation (1-10 min).(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of inositol phospholipid synthesis and norepinephrine-stimulated hydrolysis in rat brain slices by excitatory amino acids

Inhibition by excitatory amino acid agonists of norepinephrine (NE)-stimulated phosphoinositide hydrolysis was studied in rat brain slices. Inhibition was not observed in cortical slices prelabeled with [3H]inositol but was observed when slices were incubated simultaneously with [3H]inositol, glutamate, and NE. Therefore, we hypothesized that glutamate inhibits the synthesis of inositol phospholipids available to the alpha 1-adrenergic receptor, thereby reducing NE-stimulated phosphoinositide hydrolysis. To test this hypothesis, the distribution of [3H]inositol in cortical slices was measured after 5, 10, 20, 40 and 60 min of incubation, with some slices being exposed to 200 microM NE, 1 mM glutamate, 1 mM N-methyl-D-aspartate (NMDA), 1 mM kainate, 1 mM quisqualate, or to NE in the presence of each of the excitatory amino acid agonists. Glutamate had little effect on the slice content of free [3H]inositol, but it severely reduced the synthesis of [3H]inositol phospholipids, in the presence or absence of NE. Glutamate also abolished NE-induced production of [3H]inositol monophosphate, [3H]inositol bisphosphate and [3H]inositol trisphosphate. Quisqualate mimicked the effects of glutamate, whereas NMDA and kainate caused less inhibition of the synthesis of [3H]inositol phospholipids and did not inhibit the response to NE. Glutamate produced similar inhibitory effects in slices from hippocampus and striatum. To test if the inhibitory effect of glutamate was the result of irreversible cell damage, cortical slices were incubated with 1 mM glutamate for 60 min prior to exposure to [3H]inositol and NE. Preincubation with glutamate did not reduce the synthesis of [3H]inositol phospholipids or inhibit NE-stimulated [3H]inositol monophosphate production. These results indicate that glutamate impairs the synthesis of inositol phospholipids. Each of the excitatory amino acid agonists, quisqualate, NMDA and kainate, inhibited [3H]inositol phospholipid synthesis, but only quisqualate affected [3H]inositol phospholipids available to the alpha 1-adrenergic receptor.     

Comparative effects of BRL 38227, nitrendipine and isoprenaline on carbachol- and histamine-stimulated phosphoinositide metabolism in airway smooth muscle.

1. The ability of BRL 38227 and nitrendipine to affect muscarinic agonist and histamine-stimulated [3H]-inositol phosphate accumulation in slices of bovine tracheal smooth muscle has been studied and compared with the established inhibitory effects of isoprenaline on this pathway. 2. Pre-addition of BRL 38227 (5 microM), nitrendipine (1 microM) or isoprenaline (10 microM) significantly inhibited the subsequent inositol phosphate response to histamine at all concentrations studied (10- 1000 microM). BRL 38227 and nitrendipine also significantly inhibited the [3H]-inositol phosphate response to low (1 microM), but not high (100 microM) concentrations of carbachol. Isoprenaline had no effect at any concentration of carbachol studied. 3. Nitrendipine (IC50 = 95 nM) and BRL 38227 (IC50 = 322 nM) caused concentration-related inhibitions of the inositol phosphate response to histamine (100 microM). Similar maximal inhibitions were caused by each agent (55-58%). Inhibitory effect of BRL 38227 was reduced in potency (IC50 = 5.5 microM), but not magnitude, in the presence of glibenclamide (0.5 microM). 4. Time-course studies comparing the effects of BRL 38227 addition 15 min before, and 10 min after histamine challenge showed that for pre-addition a distinct (less than 2 min) lag occurred following histamine addition before the inhibitory effect of BRL 38227 was manifest. In contrast, when BRL 38227 was added 10 min after histamine, an inhibitory effect was immediately apparent. 5. Further evidence for an initial, 'protected' phase of inositol phosphate accumulation was provided by the finding that BRL 38227 pre-addition had no effect on the early (0-300 s) time-course of inositol 1,4,5-trisphosphate mass accumulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of acute and chronic lithium treatment on pilocarpine-stimulated phosphoinositide hydrolysis in mouse brain in vivo.

1. Measurements were made of the in vivo formation of inositol phosphates in the brains of C57/B1/601a mice treated acutely or chronically with lithium chloride (LiCl). 2. A single injection of LiCl (10 mEquiv kg-1, s.c.) 18 h before death increased the accumulation of [3H]-inositol phosphates ([3H]-Ins P's) in the brains of mice injected i.c.v. with [3H]-myo-inositol 24 h previously. 3. Pilocarpine (200 mg kg-1, i.p.) injected 15 min before death further enhanced the formation of [3H]-Ins P's in the brains of LiCl-treated, but not saline-treated, mice. The enhancement due to pilocarpine was abolished by injection of atropine sulphate (10 mg kg-1, i.p.) 10 min earlier. 4. Chronic (14 days) LiCl feeding produced an accumulation of [3H]-Ins P's significantly less than that due to a single injection of LiCl, but the response to pilocarpine was markedly greater in mice chronically fed with LiCl when compared with mice acutely injected with LiCl. 5. Mass measurements of endogenous inositol 1,4,5 triphosphate revealed increases due to pilocarpine and chronic LiCl feeding alone. A combination of the two treatments produced levels greater than either alone. 6. These results demonstrate that LiCl treatment enhances both basal and pilocarpine-stimulated inositol phospholipid hydrolysis in vivo and this might be relevant to its therapeutic effects.     

Relationship between stimulated inositol lipid hydrolysis and contractility in guinea-pig visceral longitudinal smooth muscle

Carbamylcholine caused a marked, concentration-dependent stimulation of [3H]Ins P, [3H] InsP2 and to a lesser extent [3H]InsP3 production in guinea-pig longitudinal smooth muscle prelabelled with myo-[3H]inositol. Accumulation of these three inositol phosphates showed differential sensitivity to LiCl. Muscle contraction was apparent at lower concentrations of carbamylcholine. Both responses were mediated via muscarinic-type receptors. An association of inositol phosphate production and contractility was also observed in response to substance P, histamine and noradrenaline, the latter via an alpha-adrenergic mechanism. The Ca2+-channel agonist CGP 28392 failed to stimulate inositol phosphate production despite inducing a contractile response. Carbamylcholine -induced inositol phosphate production persisted in the presence of D600 or Mn2+ despite loss of contractile activity. However, both responses showed a similar, marked dependence on the presence of Ca2+ in the extracellular medium. Mn2+ could restore basal and stimulated inositol phosphate production in low Ca2+ solutions but could not substitute for Ca2+ in restoring contractility. The results suggest that stimulated inositol lipid hydrolysis in longitudinal smooth muscle does not result from Ca2+ entry into the tissue, although the response does depend on the concentration of divalent cations in the extracellular medium. This dependency may be related to the maintenance of membrane potential and possibly phospholipid conformation.     

Inhibition of receptor-coupled phosphoinositide hydrolysis by sulfur-containing amino acids in rat brain slices

Sulfur-containing amino acids were found to inhibit norepinephrine-stimulated [3H]phosphoinositide hydrolysis in rat cortical slices. Of the amino acids tested, L-cysteine was the most potent, inhibiting the response by 42 and 85% at concentrations of 50 and 500 microM respectively. L-Cystine and L-serine-O-sulfate also inhibited the response to norepinephrine, but to a lesser degree than did L-cysteine. L-Homocysteic acid slightly potentiated phosphoinositide hydrolysis at a concentration of 100 microM, but caused inhibition at 500 microM. L-Cysteine sulfinate produced effects intermediate to those of L-cysteine and L-homocysteic acid, having no effect on the response to norepinephrine at 50 microM, but causing 84% inhibition at 500 microM. The D-isomers of cysteine and homocysteic acid were much less potent than were the L-isomers. Examination of the time course of the inhibition of norepinephrine-stimulated [3H]phosphoinositide hydrolysis by L-cysteine showed that it was inhibited almost completely after 15, 30, 45 and 60 min of incubation. L-Cysteine and L-homocysteic acid caused similarly strong inhibitions of the production of [3H]inositol monophosphate, [3H]inositol bisphosphate and [3H]inositol trisphosphate. The hydrolysis of [3H]phosphoinositides stimulated by norepinephrine in slices from rat hippocampus and striatum were inhibited by L-cysteine to an extent similar to that occurring in cortical slices. These results demonstrate that several sulfur-containing amino acids, some of which have been proposed to be endogenous excitatory amino acid neurotransmitters, effectively modulate the response to norepinephrine of the phosphoinositide second messenger system in rat brain.