Carbachol-stimulated Inositol Phospholipid Research Articles

Investigation into the effects in-vitro of the 5-hydroxytryptamine reuptake inhibitor, alaproclate, on carbachol-stimulated inositol phospholipid breakdown in the rat cerebral cortex.

The effect of alaproclate in carbachol-stimulated inositol phospholipid (PI) breakdown in rat cerebral cortical miniprisms has been investigated. Carbachol-stimulated PI breakdown was greatly enhanced by increasing the assay potassium concentration from 5.88 to 18.2 mM. Alaproclate, on the other hand, did not influence carbachol-stimulated PI breakdown over the concentration range tested (0-100 microM) at either assay [K+]. The elution pattern of the inositol phosphates from the Dowex-1 columns was also unaffected by alaproclate both in the absence and presence of carbachol. Thus, the potentiation by alaproclate of tremor and salivation induced by the muscarinic agonist oxotremorine in-vivo reported previously is not seen when muscarinic function is measured in-vitro using carbachol-stimulated PI breakdown.

Long-lasting microencephaly following exposure to cocaine during the brain growth spurt in the rat

In utero exposure to cocaine has been shown to produce somatic and behavioral effects. As microencephaly is often present in children born from cocaine-addicted mothers, aim of the present study was to develop an animal model for cocaine-induced microencephaly. Rats were treated with cocaine (20, 30 or 50 mg/kg/day, s.c., each dose divided in two equal doses given 3 h apart) from postnatal day 4 through 10. None of the doses had any effect on growth, however, at 50 mg/kg, cocaine caused a significant decrease in brain weight, measured on day 12. The effect of cocaine was similar in male and female rats, and microencephaly was still present in 45-day-old animals. When the same dose of cocaine was given as a single daily injection, long-lasting microencephaly was also present, but it was accompanied by a decrease in body weight and significant toxicity. Ethanol (4 g/kg), used as a positive control, also caused microencephaly without affecting body weight, but, differently from cocaine, its effect was more pronounced in female animals. Blood and brain levels of cocaine and its metabolites norcocaine and benzoylecgonine were measured by HPLC during treatment (postnatal day 8). After administration of the 50 mg/kg dose, concentrations of cocaine were 1.92 μg/g in brain and 0.94 μg/ml in blood. These levels are encountered in cases of cocaine overdoses and have been found in meconium of newborns from crack-addicted mothers. As cocaine and norcocaine have been shown to inhibit in vitro muscarinic receptor-stimulated phosphoinositide metabolism, which may be associated with ethanol's induced microencephaly, this biochemical response was measured in cerebral cortex from cocaine-treated rats. A dose-dependent inhibition of carbachol-stimulated inositol phospholipid metabolism was observed. In summary, these experiments establish a rodent model for cocaine-induced microencephaly, which should be useful to further investigate this aspect of the developmental neurotoxicity of cocaine and its underlying mechanism(s).

Ambient glucose and aldose reductase-induced myo-inositol depletion modulate basal and carbachol-stimulated inositol phospholipid metabolism and diacylglycerol accumulation in human retinal pigment epithelial cells in culture.

Physiological hyperglycemia has been speculated to alter phosphoinositide (PPI; inositol phospholipid) signal transduction in cells prone to diabetic complications by two separate mass-action mechanisms with antiparallel putative effects on diacylglycerol (DAG): (i) sorbitol-induced depletion of myo-inositol leads to diminished PPI synthesis and turnover and DAG release, and (ii) elevated glucose-derived DAG precursors enhance de novo DAG synthesis. Because the first mechanism is mediated by aldose reductase (AR2), which converts glucose to sorbitol, the effects of glucose on basal and stimulated PPI signaling were explored in lines of cultured human retinal pigment epithelial cells differing widely in their basal AR2 gene expression and enzymatic activity. The results suggest that the effects of glucose on PPI signaling vary inversely with the level of AR2 activity and parallel the extent of AR2-induced myo-inositol depletion.

Carbachol induces secretion in a mast cell line (RBL-2H3) transfected with the ml muscarinic receptor gene

The possibility that the ml muscarinic receptor subtype can induce release of intracellular granules and transmitters was studied by transfecting a cultured mast cell line, RBL-2H3 cells, with the ml receptor gene. Comparisons were made between carbachol- and antigen-induced activation of various secretory responses. Like antigen, carbachol stimulated inositol phospholipid hydrolysis and release of arachidonic acid with concomitant dose-dependent secretion of granular contents. Carbachol also stimulated a biphasic increase in intracellular calcium, as measured by single cell fura-2 measurements. Although the kinetics of the carbachol-induced rise in intracellular calcium differed from that induced by antigen, they both utilized the same intracellular pool of calcium, and the second phase of the rise in intracellular calcium was dependent on extracellular calcium in both cases. Thus, the ml muscarinic receptor activates release of granules by a mechanism ostensibly similar to that of antigen.

Antagonism by 8-hydroxy-2 (DI-N-propylamino) tetraline and other serotonin agonists of muscarinic M1-type receptors coupled to inositol phospholipid breakdown in human IMR-32 and SK-N-MC neuroblastoma cells

IMR-32 and SK-N-MC cells were found to contain [ 3H]quinuclidinyl benzilate specific binding sites inhibited by pirenzepine in a manner suggesting the presence of both M1-type and M2-type muscarinic receptor recognition sites. Neither cell had detectable [ 3H]8-OH-DPAT binding sites. Carbachol stimulated the rate of inositol phospholipid breakdown in IMR-32 and SK-N-MC human neuroblastoma cells with an EC 50 value of about 50 μM in both cases. Pirenzepine inhibited the carbachol (100 μM)-stimulated inositol phospholipid breakdown in both cells with Hill slopes of unity and IC 50 values of 15 nM (IMR-32) and 12 nM (SK-N-MC). The 5-HT 1A receptor agonist 8-OH-DPAT competitively inhibited carbachol-stimulated inositol phospholipid breakdown with pA 2 values of 5.78 (IMR-32) and 5.61 (SK-N-MC). These values are consistent with the inhibitory potency of 8-OH-DPAT towards [ 3H]quinuclidinyl benzilate binding in these cells. The 5-HT agonists 5-MeODMT and buspirone at micromolar concentrations inhibited carbachol-stimulated breakdown in IMR-32 cells. The inhibition by 8-OH-DPAT and 5-MeODMT was not affected by preincubation with (−)alprenolol. 5-HT (10–100 μM) was without effect on either basal or carbachol-stimulated breakdown. It is concluded that IMR-32 and SK-N-MC neuroblastoma cells express muscarinic M1-type but not serotoninergic receptors coupled to phosphoinositide-specific phospholipase C. 8-OH-DPAT acts as a weak antagonist at these muscarinic receptors.

Modulation of carbachol-stimulated inositol phospholipid breakdown in rat cerebral cortical miniprisms by excitatory amino acids and by bay K-8644 is dependent upon the assay calcium and potassium concentrations used

The calcium and potassium ion dependencyof the inositol phospholipid breakdown response to stimulatory agents has been investigated in rat cerebral cortical miniprisms. The calcium channel agonist BAY K-8644 (10 μM) potentiated the response to carbachol at 6 mM K + when Ca 2+-free, but not when 2.52 mM Ca 2+ assay buffer was used. In Ca 2+-free buffer, verapamil (10 μM) inhibited the response to carbachol at both 6 and 18 mM K +, but higher concentrations (30–300 μM) were needed when 2.52 mM Ca 2+ was used. At these higher concentrations, however, verapamil inhibited the binding of 2 nM [ 3H]pirenzepine to muscarinic recognition sites. N-Methyl-D-Aspartate (NMDA, 100 μM) significantly reduced the basal phosphoinositide breakdown rate at 18 mM K + at 1.3 mM Ca 2+, but was without effect on the basal rate at other K + and Ca 2+ concentrations. In the presence of NMDA (100 μM) or quisqualate (100 μM), the responses to carbachol were reduced, the degree of reduction showing a complex dependency upon the assay K + and Ca 2+ concentrations used. These results indicate that the inositol phospholipid breakdown response to carbachol in cerebral cortical miniprisms can be modulated in a manner dependent upon the extracellular calcium and potassium concentrations used.

The Na +, K +-ATPase inhibitor ouabain and the Na + ionophore monensin have opposite effects upon carbachol-stimulated inositol phospholipid breakdown in rat cerebral cortical miniprisms

The effects of ouabain and monensin upon basal and carbachol-stimulated inositol phospholipid breakdown in rat cerebral cortical miniprisms have been investigated. Basal inositol phospholipid breakdown was increased by both compounds at both 6 and 18 mM K +. Enhancement of the carbachol reponse at 6 mM, but not at 18 mM K +, was found with high concentrations of ouabain. On the other hand, monensin blocked the response to carbachol. Monensin also inhibited the specific binding of [ 3H]pirenzepine to cerebral cortical membranes, but this was found only at concentrations higher than required to affect the basal and carbachol-stimulated inositol phospholipid breakdown responses. Quabain did not affect [ 3H]pirenzepine binding at any of the concentrations tested (6–600 μM). It is concluded that agents that increase the intracellular sodium ion concentration affect the inositol phospholipid breakdown response to carbachol, but that the modulation can be both to potentiate and to inhibit the response.

Multiple actions of fluoride ions upon the phosphoinositide cycle in the rat brain

The effects of sodium fluoride upon basal and agonist-stimulated inositol phospholipid breakdown have been investigated in rat brain miniprisms. NaF concentration dependently increased basal inositol phospholipid breakdown, with a maximum effect being seen at 20 mM. NaF reduced the inositol phospholipid breakdown responses to stimulation by carbachol, noradrenaline, serotonin and quisqualate, but not to the stimulation produced by raising the assay [K +] from 6 to 18 mM. More detailed study demonstrated NaF to have a ‘levelling’ effect, reducing all InsP/(Lipid + InsP) valuesn 0.15 (i.e. produce by carbachol at raised [K +], noradrenline and by 50 mM K +) to about this Time-course experiments indicated that NaF treatment reduced the rate of carbachol-stimulated inositol phospholipid breakdown up to this InsP/(Lipid + InsP) level and thereafter blocked further breakdown. Inhibitory effects upon carbachol-stimulated inositol phospholipid phosphoinositides from [ 3H]myo-inositol, NaF reduced the total Lipid + InsP labelling by about 20%. NaF in addition inhibits the activity of Ins(1,4)P 2-phosphatase in cerebral cortical homogenates. It is concluded that fluoride ions inhibit agonist-stimulated inositol phospholipid breakdown via actions not only on G-proteins but also on phosphoinositide-specific phospholipase C substrate availability.

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.

Differential enhancement by potassium ions of M1-type and M2-type muscarinic receptor-mediated phosphoinositide breakdown in the rat brain

Raising the assay [K+] from 6 to 18 mM enhances the inositol phospholipid breakdown response to carbachol in rat brain miniprisms. In the frontal cortex, the degree of enhancement by K+ was independent of the carbachol concentration used, whereas in the striatum, a significantly higher degree of enhancement was seen at 1000 than at 50 microM carbachol. The carbachol-stimulated inositol phospholipid breakdown was antagonized by pirenzepine at both [K+] with potencies suggesting involvement of M1-type muscarinic receptors in the frontal cortex and both M1- and M2-type muscarinic receptors in the striatum. It is suggested that the response mediated by the M1-type receptors is enhanced to a greater degree by raised [K+] than that mediated by the M2-type receptors.

Enhancement of carbachol-stimulated inositol phospholipid breakdown in rat brain miniprisms by potassium ions: mechanistic studies

Enhancement of carbachol-stimulated inositol phospholipid breakdown in rat brain miniprisms by potassium ions: mechanistic studies

Enhancement by Potassium of Carbachol‐Stimulated Inositol Phospholipid Breakdown in Rat Cerebral Cortical Miniprisms: Comparison with Other Depolarising Agents

Increasing the [K+] in the assay medium from 5.7 to 17.8 mM produces a large enhancement of the inositol phospholipid breakdown response to the muscarinic agonist carbachol in rat cerebral cortical miniprisms, with minor effects on basal inositol phospholipid breakdown. This effect is also found with Rb+. The enhancement by a raised [K+] is not accompanied by a change in the composition of the labelled polyphosphoinositides. The carbachol-stimulated inositol phospholipid breakdown at 17.8 and 42.7 mM K+ was antagonised by veratrine (5-80 microM), 4-aminopyridine (5 mM), and tetraethylammonium (20 mM). These compounds, however, also inhibited the binding of [3H]quinuclidinyl benzilate to cortical membranes. BRL 34915 (0.2-20 microM) was without significant effect on carbachol-stimulated inositol phospholipid breakdown at either 5.7 or 17.8 mM K+.Mg2+ (10 mM) considerably reduced the carbachol-stimulated inositol phospholipid breakdown at 17.8, but not 42.7, mM K+. Inositol phospholipid breakdown was also stimulated, albeit to a small extent, by L-glutamate (100-3,000 microM) and quisqualate (1-100 microM), with the stimulation being additive to that produced by carbachol at both 5.7 and 17.8 mM K+. N-Methyl-D-aspartate (10-1,000 microM in Mg2+-free medium) had no significant effect on basal inositol phospholipid breakdown and had little or no effect on carbachol-stimulated inositol phospholipid breakdown at either 5.7 or 17.8 mM K+. It is concluded that it may not be correct to ascribe wholly the enhancement by K+ of carbachol-stimulated inositol phospholipid breakdown to the tissue-depolarising actions of this ion and that other actions of K+ may be involved.

Dopamine receptor stimulation does not affect phosphoinositide hydrolysis in slices of rat striatum.

The effect of dopamine receptor stimulation on the accumulation of labelled inositol phosphates in rat striatal slices under basal and stimulated conditions was examined following preincubation with [3H]inositol. Incubation of striatal slices with the selective D-1 agonist SKF 38393 or the selective D-2 agonist LY 171555 for 5 or 30 min did not affect the basal accumulation of labelled inositol mono-, bis-, tris-, and tetrakisphosphate. Resolution by HPLC of inositol trisphosphate into inositol-1,3,4-tris-phosphate and inositol-1,4,5-trisphosphate isomers revealed that under basal conditions dopamine did not influence the accumulation of inositol-1,4,5-trisphosphate. Depolarisation evoked by KCl, or addition of the muscarinic receptor agonist carbachol, produced a marked increase in the accumulation of labelled inositol phosphates in both the presence and absence of lithium. Addition of dopamine did not reduce the ability of KCl or carbachol to increase inositol phospholipid hydrolysis. In the presence of lithium, dopamine (100 microM) enhanced KCl-stimulated inositol phospholipid hydrolysis, but this effect appears to be mediated by alpha 1 adrenoceptors because it was blocked by prazosin. SKF 38393 (10 microM) or LY 171555 (10 microM) also did not affect carbachol-stimulated inositol phospholipid hydrolysis. These data, in contrast to recent reports, suggest that striatal dopamine receptors do not appear to be linked to inositol phospholipid hydrolysis.

Modulation of carbachol-stimulated inositol phospholipid hydrolysis in rat cerebral cortex

Cortical slices from rat brain were used to study carbachol-stimulated inositol phospholipid hydrolysis. Omission of calcium during incubation of slices with [3H]inositol increased its incorporation into receptor-coupled phospholipids. Carbachol-stimulated hydrolysis of [3H]inositol phospholipids in slices was dose-dependent, was affected by the concentrations of calcium and lithium present and resulted in the accumulation of mostly [3H]inositol-1-phosphate. Incubation of slices with N-ethylmaleimide or a phorbol ester reduced the response to carbachol. Membranes prepared from cortical slices labeled with [3H]inositol retained the receptor-stimulated inositol phospholipid hydrolysis reaction. The basal rate of inositol phospholipid hydrolysis was higher than in slices and addition of carbachol further stimulated the process. Addition of GTP stimulated inositol phospholipid hydrolysis, suggesting the presence of a guanine nucleotide-binding protein coupled to phospholipase C. Carbachol and GTP-stimulated inositol phospholipid hydrolysis in membranes was detectable following a 3 min assay period. In contrast to slices, increased levels of inositol bisphosphate and inositol trisphosphate were detected following incubation of membranes with carbachol. These results demonstrate that agonist-responsive receptors are present in cortical membranes, that the receptors may be coupled to phosphatidylinositol 4, 5-bisphosphate, rather than phosphatidylinositol, hydrolysis and that a guanine nucleotide-binding protein may mediate the coupling of receptor activation to inositol phospholipid hydrolysis in brain.