Effects of ethanol on muscarinic receptor-stimulated phosphoinositide metabolism during brain development

The pattern of muscarinic receptor-stimulated inositol metabolism during postnatal development has a striking resemblance with the curve of rat brain growth spurt. Therefore, the enhanced hydrolysis of membrane inositol lipids by cholinergic agonists during this period may have a relevant role in cell proliferation and differentiation. In this study we have investigated whether exposure to EtOH to rat pups during the brain growth spurt, known to cause a permanent microencephaly, would alter muscarinic receptor-stimulated inositol metabolism in cerebral cortex. Female Long-Evans rats were administered 4 g/kg of EtOH, in two doses of 2 g/kg, by gastric intubation from postnatal day 4 to day 10. This treatment did not have any effect on the pups' body weight as compared to an equally handled, sucrose-fed group of animals. Blood EtOH concentration in the pups during exposure ranged between 237 and 283 mg/dl. Muscarinic receptor-stimulated inositol metabolism was measured in cerebral cortex slices of control and EtOH-treated rats at days 4, 7, 10, 12, 20 and 45 of age. Carbachol-induced accumulation of [3H] inositol phosphates was reduced significantly in EtOH-exposed animals on day 7 and 10, but not at other ages. This decrease was not due to an alteration of muscarinic receptor density or affinity. Exposure to EtOH had no effect on phosphoinositide metabolism stimulated by norepinephrine, serotonin or histamine in cerebral cortex, whereas the effect of glutamate was increased. Similar results were observed in the hippocampus. An identical treatment with EtOH in adult rats did not cause alteration in any of the biochemical parameters of the cholinergic system measured.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of cholinergic muscarinic receptor-stimulated phosphoinositide metabolism in brain from immature rats

Hydrolysis of phosphoinositides elicited by stimulation of cholinergic muscarinic receptors has been studied in brain from neonatal (7-day-old) rats in order to determine: 1) whether the neonatal rat could provide a good model system to study this signal-transduction pathway; and 2) whether potential differences with adult nerve tissue would explain the differential, age-related effects of cholinergic agonists. Accumulation of [3H] inositol phosphates in [3H]inositol prelabeled slices from neonatal and adult rats was measured as an index of phosphoinositide metabolism. Full (acetylcholine, methacholine, carbachol) and partial (oxotremorine, bethanechol) agonists had qualitatively similar, albeit quantitatively different, effects in neonatal and adult rats. Atropine and pirenzepine effectively blocked the carbachol-induced response with inhibition constants of 1.2 and 20.7 nM, respectively. In all brain areas, response to all agonists was higher in neonatal than adult rats, and in hippocampus and cerebral cortex the response was higher than in cerebellum or brainstem. The relative intrinsic activity of partial agonists was higher in the latter two areas (0.6-0.7) than in the former two (0.3-0.4). Carbachol-stimulated phosphoinositide metabolism in brain areas correlated well with the binding of [3H]QNB (r2 = 0.627) and, particularly, with [3H]pirenzepine (r2 = 0.911). In cerebral cortex the effect of carbachol was additive to that of norepinephrine and glutamate. The presence of calcium (250-500 microM) was necessary for maximal response to carbachol to be elicited; the EC50 value for Ca2+ was 65.4 microM. Addition of EDTA completely abolished the response. Removal of sodium ions from the incubation medium reduced the response to carbachol by 50%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Carbachol- and norepinephrine-stimulated phosphoinositide metabolism in rat brain: effect of chronic cholinesterase inhibition

Activation of cholinergic muscarinic receptors leads to several biochemical events including an increased turnover of phosphoinositides. In this study we have investigated whether repeated administration of the organophosphorus insecticide disulfoton, known to cause the development of tolerance to this compound, would affect phosphoinositide metabolism in rat brain. Basal and carbachol-stimulated phosphoinositide metabolism were measured in cerebral cortex slices, by measuring the accumulation of inositol phosphates (InsPs) in the presence of lithium. In control animals carbachol caused a 600% increase in InsPs accumulation with an EC50 of 100 microM. Maximal effect occurred with a LiCl concentration of 7.5 mM and required the presence of calcium. Administration of disulfoton for 10 days (2 mg/kg/day by gavage), decreased the number of muscarinic receptors in cortex from 1.1 to 0.7 pmol/mg of protein without changing the affinity of the receptors (both measured by binding of [3H]quinuclidinyl benzilate). Acetylcholinesterase was inhibited by 85%. Basal InsPs accumulation was unchanged in disulfoton-treated rats, whereas carbachol-stimulated InsPs accumulation decreased by 18%. No changes of norepinephrine-stimulated InsPs formation and of alpha-1 adrenoceptors were present in cortices from disulfoton-treated rats. Recovery of muscarinic receptor binding and carbachol-stimulated InsPs accumulation occurred at a similar rate and was completed 2 to 3 weeks after the end of the treatment, whereas acetylcholinesterase activity was still 38% inhibited 3 weeks later. These results support the hypothesis that a functional adaptation of muscarinic receptors is involved in the development of tolerance to organophosphates.     

Pharmacological characterization of the muscarinic receptors mediating phosphoinositide hydrolysis in rat myometrium

Activation of rat uterine myometrial muscarinic receptors with a variety of agonists results in increased phosphatidylinositol metabolism. Activation with carbachol is concentration- and time-dependent and is most apparent by following the accumulation of inositol monophosphate although there are small but significant increases of inositol bisphosphate and inositol trisphosphate. Carbachol stimulation of phospholipid turnover is greatest in the upper third of the uterus. The carbachol-induced increase of inositol monophosphate is antagonized by atropine and by the selective M-3 muscarinic receptor antagonist 4-diphenylacetoxy-N-methylpiperidine methobromide. Pirenzepine, a selective M-1 receptor antagonist is less active, whereas gallamine and 11-2[[(diethylamino)methyl]-1-piperidinyl]acetyl]-5, 11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepine-6-one, selective M-2 receptor antagonists, are minimally effective suggesting that muscarinic M-3 receptors modulate phospholipid turnover in the rat myometrium. Displacement of tritium-quinuclidinyl benzilate binding by muscarinic antagonists also supports the presence of M-3 receptors in the uterus. Incubation with phorbol 12, 13-dibutyrate significantly reduced the accumulation of inositol monophosphate induced by carbachol implying that protein kinase C might modulate the responsiveness of the M-3 receptors in the rat uterus. Our results suggest that the intracellular concentration of calcium required for the contraction of the rat myometrium may be modulated, in part, through M-3 muscarinic receptors coupled to phospholipase C-activated turnover of phosphoinositides.     

Developmental changes in the muscarinic stimulation of canine Purkinje fibers

Acetylcholine (ACh) hyperpolarizes adult canine Purkinje fibers and induces a decrease in their automaticity. In Purkinje fibers from young dogs, there is a biphasic effect on automaticity, which increases at low and decreases at high ACh concentrations. We used standard microelectrode techniques to study these actions of ACh. In fibers from young dogs, 10(-10) to 10(-9) M ACh increased automaticity and 10(-5) M ACh decreased automaticity. The decrease was blocked by the M2 muscarinic blocker AFDX-116, whereas the increase was blocked by the predominant M1 blocker pirenzepine. The M2 agonist oxotremorine never increased automaticity. Rather, it decreased automaticity and hyperpolarized adult and young fibers, the former more than the latter. The hyperpolarization and biphasic effect on automaticity of ACh in fibers from young dogs failed to occur after treatment with pertussis toxin, suggesting that these effects are dependent on a pertussis toxin-sensitive G protein. These electrophysiologic studies suggest that postsynaptic M1 and M2 muscarinic processes modulate the automatic response of Purkinje fibers from young dogs and that the postsynaptic M1 pathway is no longer seen in the adult.     

Comparison of norepinephrine- and veratrine-induced phosphoinositide hydrolysis in rat brain

Stimulation of phosphoinositide hydrolysis by depolarization with veratrine was compared to that produced by stimulation of alpha-1 adrenoceptors by norepinephrine. The phosphoinositides in rat cerebral cortex were labeled with [myo-3H]inositol and the effects of the drugs on the formation of the following inositol phosphates were determined: inositol 1-phosphate (IP); inositol 1,4-bisphosphate (IP2); mixture of inositol 1,4,5-trisphosphate and inositol 1,3,4-trisphosphate (IP3). Termination of the hydrolysis by trichloroacetic acid resulted in lower basal levels and more reproducible results than termination by water lysis or a chloroform-methanol mixture (CHCl3-MeOH). The amounts of IP and IP2 formed by a maximal concentration of veratrine were about one half of that formed by a maximal concentration of norepinephrine although the amount of IP3 formed after stimulation by veratrine was only about 10% of that produced by norepinephrine. The increase in IP was linear with time (30 min) for both norepinephrine and veratrine. Stimulation of IP2 and IP3 formation by veratrine reached a maximum at 5 min whereas that produced by norepinephrine continued to increase for 30 min. Blockade of voltage-dependent calcium channels with manganese produced nearly complete antagonism of the veratrine response while only partially antagonizing the norepinephrine response. Norepinephrine-induced IP2 formation was less sensitive to manganese than was formation of IP or IP3. These data suggest that either veratrine and norepinephrine cause hydrolysis of different pools of phosphoinositide or that the hydrolysis occurs by different mechanisms. The data also suggest that IP and IP2 may be produced directly from phosphatidylinositol and phosphatidylinositol 4-phosphate rather than solely as a metabolite of IP3.     

Irreversible and quaternary muscarinic antagonists discriminate multiple muscarinic receptor binding sites in rat brain

The maximal number of binding sites (Bmax) of [3H]quinuclidinyl benzilate (QNB) binding was greater than the Bmax of N-[3H]methylscopolamine (NMS) binding to homogenates of rat brain. The competition of NMS for [3H]QNB demonstrated that NMS discriminates multiple muscarinic binding sites. Similarly, pirenzepine competition of [3H]QNB binding also revealed multiple muscarinic binding sites. Pirenzepine competition for [3H]NMS also was shallow and demonstrated the presence of binding sites with similar affinities to those labeled by [3H]QNB. These data were consistent with the presence of at least three populations of muscarinic binding sites with similar affinities for [3H]QNB: the M1 and M2 binding sites having high and low affinity for pirenzepine, respectively, but which cannot be discriminated by [3H]NMS, and a third site with high affinity for [3H]QNB which has low affinity for NMS. The classical muscarinic antagonists, atropine and scopolamine, also appear to have slightly different affinities for the putative M1 and M2 binding sites. The use of the irreversible antagonists, N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) and propylbenzilylcholine mustard (PBCM), were used to elucidate the distinct properties of these multiple muscarinic binding sites. Both PBCM and EEDQ irreversibly decreased the Bmax of [3H]QNB and [3H]NMS binding in cortex. PBCM did not appear to discriminate putative M1 and M2 binding sites but selectively alkylated the high affinity NMS and QNB binding sites. In contrast, EEDQ modified the low affinity NMS binding sites such that they still bound [3H]QNB but their affinity for other muscarinic antagonists was reduced.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regional characterization of agonist and depolarization-induced phosphoinositide hydrolysis in rat brain

The ability of various receptor agonists and elevated extracellular potassium to initiate inositol phospholipid hydrolysis in various regions of rat brain was examined by using a direct assay which involves prelabeling slices with [3H]inositol and assaying [3H]inositol phosphates ([3H]IPs) in the presence of lithium. Both carbachol and noradrenaline evoked an increase in [3H]IP accumulation in all cerebral regions, although there were marked topographical differences in maximal responsiveness. These marked differences do not seem to be due to regional differences in coupling as similar apparent affinities of full agonists and the relative intrinsic activities of partial agonists were obtained. Both carbachol and noradrenaline responses were antagonized equipotently in all the brain regions tested by the muscarinic and alpha-1 antagonists atropine and prazosin, respectively. However, the putatively selective muscarinic antagonist pirenzepine did show an (approximately 10-fold) apparent selectivity against the carbachol responses elicited in the forebrain regions from those in the pons-medulla. Evaluation of extracellular potassium to 18 mM resulted in an increased production of [3H]IPs in all brain regions except the cerebellum. Incubation of slices with the cholinesterase inhibitor physostigmine (50 microM) and the dihydropyridine Ca++ channel activator BAY-K-8644 (1 microM) greatly enhanced the responses produced by elevated K+ in the forebrain regions but had markedly weaker effects in the hindbrain regions. The elevated K+ response alone and the enhanced response in the presence of BAY-K-8644 were both antagonized significantly by the dihydropyridine antagonist (+)-PN-205-033 in all brain regions, by 70 to 80 and 70 to 95%, respectively.     

Heterogeneity of binding of muscarinic receptor antagonists in rat brain homogenates

The binding properties of (-)-[3H]quinuclidinyl benzilate and [3H] N-methylscopolamine to muscarinic acetylcholine receptors have been investigated in rat brain homogenates. The binding of both antagonists demonstrated high affinity and saturability. Analysis of the binding data resulted in linear Scatchard plots. However, (-)-[3H]quinuclidinyl benzilate showed a significantly higher maximal binding capacity than that of [3H]N-methylscopolamine. Displacement of both ligands with several muscarinic receptor antagonists resulted in competition curves in accordance with the law of mass-action for quinuclidinyl benzilate, atropine and scopolamine. A similar profile was found for the quaternary ammonium analogs of atropine and scopolamine when [3H]N-methylscopolamine was used to label the receptors. However, when these hydrophilic antagonists were used to displace (-)-[3H] quinuclidinyl benzilate binding, they showed interaction with high- and low-affinity binding sites. On the other hand, the nonclassical muscarinic receptor antagonist, pirenzepine, was able to displace both ligands from two binding sites. The present data are discussed in terms of the relationship of this anomalous heterogenity of binding of these hydrophilic muscarinic receptor antagonists and the proposed M1 and M2 receptor subtypes.     

Developmental changes in the rat atriopeptin hormonal system.

We undertook a study of fetal synthesis, storage, and release of atriopeptin (AP). Plasma levels of both atriopeptin immunoreactivity (APir) and the NH2-terminal fragment of the prohormone immunoreactivity (NTFir) were very high in the fetus (4 and 20 times the maternal plasma, respectively). However, the atrial content of the AP was low, but surprisingly, ventricular content of AP was quite high (relative to the adult) in the fetus and fell postnatally. Atrial AP messenger RNA (mRNA) increased with postnatal age, whereas ventricular mRNA was extremely high in the fetus and fell rapidly after birth. High fetal plasma peptide levels may derive from the mother since infusion of exogenous atriopeptin 24 into the mother resulted in parallel increases in fetal and maternal peptide levels. Fetal plasma APir and NTFir levels partially reflect the markedly reduced total renal metabolic capacity compared with that of the adult. Plasma levels fell progressively after birth; whereas neonatal atrial content rose substantially. Plasma AP and NTF were simultaneously elevated in both the maternal and fetal circulation after vasopressin injection of the mother. The fetus can also respond to exogenous stimuli (vasopressin or indomethacin--presumably via ductal closure) and promptly release substantial amounts of peptide into its circulation. Thus, it appears that the AP hormonal system is functional during fetal life and responds avidly to increases in intracardiac pressure as does the mature animal.     

Pharmacological characterization of serotonin-stimulated phosphoinositide turnover in brain regions of the immature rat

The effects of serotonin (5-HT) and related agonists and antagonists on phosphoinositide turnover have been investigated in several brain regions of the immature rat. In the presence of LiCl, 5-HT caused a marked increase in total [3H]inositol phosphate levels in cortical (maximal effect + 420%, EC50 = 7 microM) and to a lesser extent in hippocampal and striatal slices prepared from the immature (8-day-old) rat; the cortical 5-HT-induced phosphoinositide response was tetrodotoxin resistant. The magnitude of the increase in the cortical phosphoinositide response caused by 5-HT was maximal at 1 day postnatal and progressively declined to reach 6% of this maximal response in the adult. After incubation of immature (8-day-old) rat cortical slices for 2.5 min with 5-HT (in the absence of LiCl), inositol 1-phosphate, inositol 1,4-bisphosphate, inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate levels increased about 2-fold. A variety of 5-HT2 or mixed 5-HT1/5-HT2 agonists stimulated total [3H]inositol phosphate formation in the immature rat cortex and hippocampus with a rank order of potency [alpha(+)-methyl-5-HT greater than quipazine greater than MK 212 greater than 5-HT] which resembles their potencies at the 5-HT2 binding site. In contrast, the 5-HT1A agonist 8-hydroxy-2-(di-n-propylamino)tetralin, the 5-HT1B agonists 1-(m-trifluoromethylphenyl)piperazine and 1-(m-chlorophenyl)-piperazine and the 5-HT3 agonist 2-methyl-5-HT were inactive.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chronic lisuride hydrogen maleate administration enhances muscarinic receptor binding in senescent rat brain

Changes in the regional density of muscarinic-1 (M1) receptors and the effect of lisuride hydrogen maleate on these changes were studied in senescent rat brain by in vitro autoradiography. In young adult controls, M1 receptor binding was most dense in the striatum and hippocampus, followed by the cerebral cortex and amygdala. Binding to M1 receptors was markedly lower in these areas of the senescent brain compared with the young adult brain. These decreases were reversed by intraperitoneal administration of 50 micrograms/kg.day lisuride for 14 days. The present results indicate that the therapeutic efficacy of lisuride depends on normalization of not only monoamine systems but also acetylcholine systems.     

无脑部转移的体部恶性肿瘤患者脑静息葡萄糖代谢改变

目的 探讨无脑部转移的恶性肿瘤患者大脑静息状态下对葡萄糖摄取的变化。 方法 57例无脑部疾患的原发性体部恶性肿瘤患者接受常规全身¹⁸F-FDG PET检查,采用统计参数地形图分析软件(SPM)回顾性分析其脑部¹⁸F-FDG PET影像资料,并与年龄、性别相匹配的57名健康体检者进行比较,获得肿瘤患者大脑静息状态下葡萄糖代谢改变。 结果 恶性肿瘤患者两侧额叶(包括两侧额上回、额中回、额下回)、颞叶(包括两侧颞叶颞中回、颞下回)、部分边缘系统结构(扣带回,眶回)及小脑在静息状态下存在广泛的葡萄糖代谢减低区,额叶代谢减低以左侧为著。代谢升高区主要见于两侧海马、海马旁回、扣带回、基底节、岛叶,两侧小脑亦可见部分葡萄糖代谢升高区。 结论 无脑部转移的体部恶性肿瘤患者存在广泛的葡萄糖代谢异常区,这些脑代谢改变可能与肿瘤患者的复杂精神心理状况有关。     

Energy utilization and changes in some intermediates of glucose metabolism in normal and hypoxic rat brain after decapitation

Energy metabolism was studied in the cerebral cortex of rats during and after hypoxia induced by breathing a gas mixture of 7% O2 in N2 for 2 h. Cortical energy stores (2ATP + ADP + phosphocreatine) remained unchanged after hypoxic treatment. Lactate rose over four-fold. Pyruvate, glucose and glucose 6-phosphate concentrations also increased significantly. Metabolic activity in the cortex expressed as the utilization of high-energy phosphates 5, 10 and 30 s after decapitation was decreased by 30% after hypoxia and remained lowered for 3 h during recovery. This was accompanied by elevated glucose consumption and lactate production, suggesting that the maintenance of the energy balance after hypoxia was partly due to activation of the glycolytic pathway. During the recovery period, these metabolic abnormalities returned towards control values, but, after 6 h of recovery the high-energy phosphate utilization increased transitorily above the control values.     

Aging does not alter brain muscarinic receptor-mediated phosphoinositide hydrolysis and its inhibition by phorbol esters, tetrodotoxin and receptor desensitization

The effects of aging on muscarinic receptor-mediated phosphoinositide (PI) hydrolysis in the brain were investigated in Fischer-344 rats. Oxotremorine-M stimulated this response to the same magnitude in young and old rats in the cerebral cortex, striatum, hippocampus, thalamus, hypothalamus and cerebellum, although maximal stimulation varied among brain regions within each age group. The PI response was also equally potentiated by elevated K+ or suppressed by tetrodotoxin in both age groups. In addition, a phorbol ester attenuated muscarinic receptor-mediated PI hydrolysis in the cerebral cortex to the same extent in young and aged rats. Moreover, preincubation with oxotremorine-M resulted in a similar down-regulation of cell-surface receptors and desensitization of receptor function regardless of age. Therefore, under our experimental conditions, PI hydrolysis in response to activation of brain muscarinic receptors does not appear to be sensitive to aging-related alterations.     

Developmental changes in patterns of brain activity associated with moment-to-moment adjustments in control

The current study investigated age-related changes in patterns of brain activity associated with moment-to-moment adjustments in control through the use of fMRI. Fifty-eight participants ranging continuously in age from 9 to 32years were scanned as they performed a task in which the need for rapid adjustments in control was greater in one condition than another. Despite comparable behavioral performance across ages, moment-to-moment adjustments were associated with stronger engagement of anterior cingulate, anterior insula, lateral prefrontal cortex and the intraparietal sulcus in older than in younger participants. The findings confirm the importance of cingulo-insular and fronto-parietal cortices for moment-to-moment adjustments in control, and suggest continuous increases in the utilization of these networks over development.     

Pirenzepine distinguishes between muscarinic receptor-mediated phosphoinositide breakdown and inhibition of adenylate cyclase

Subtypes of muscarinic cholinergic receptors have been proposed to exist, but the biochemical responses mediated by the putative subtypes are unknown. In the present study, muscarinic receptor-mediated phosphoinositide breakdown and inhibition of adenylate cyclase activity were characterized in rat brain as well as rat parotid and heart. To study whether these responses are mediated by separate subtypes of muscarinic receptors, the potencies of agonists and antagonists were determined in both assays. Antagonist potencies were calculated by Schild analysis. In the brain, the putatively selective muscarinic receptor antagonist, pirenzepine, exhibited Ki values of 21 nM in the assay of phosphoinositide breakdown and 310 nM in the assay of adenylate cyclase activity. Similarly, using radioligand binding techniques, it distinguished two binding sites with Kd values of 12 and 168 nM. The antagonist, atropine, on the other hand, was equipotent in the two biochemical assays and the radioligand binding assay with Ki values of approximately 1 to 2 nM. In peripheral tissues with robust muscarinic receptor-mediated phosphoinositide (parotid) and adenylate cyclase (heart) responses, pirenzepine exhibited a similar selectivity (19-fold) for the phosphoinositide assay that was seen in the brain, but it was 6- to 7-fold less potent in both peripheral tissues than in the central nervous system. In addition, the potencies of pirenzepine in binding and functional studies in each tissue were not as well correlated as in the brain. Atropine and other antagonists were 4- to 9-fold selective for inhibiting oxotremorine-stimulated phosphoinositide breakdown in the peripheral tissues.(ABSTRACT TRUNCATED AT 250 WORDS)     

Developmental changes of the G proteins-muscarinic cholinergic receptor interactions in rat heart

The pertussis toxin-sensitive G proteins (guanine nucleotide binding proteins)-muscarinic receptor interactions in 18-day fetal to adult rat hearts were studied. The collective concentration of these G proteins was measured by pertussis toxin-dependent ADP-ribosylation and decreased from 1.5 pmol of [32P]ADP-ribose/mg of protein in the fetal heart to 0.5 pmol of [32P]ADP-ribose/mg of protein in 21-day postpartum and older animals in a nonlinear pattern. The muscarinic receptor density diminished 2-fold from 400 fmol/mg in 1-day-old neonate to 200 fmol/mg of protein in adult hearts in a linear manner. The receptor affinity for the agonist oxotremorine and the effect of guanine nucleotide on that affinity were monitored in heart preparations of the various ages. The IC50 value for oxotremorine/[3H]quinuclidinylbenzilate competition curves in the absence or presence of guanine nucleotide increased gradually with age. Modeling of the competition curves from 1-day-old neonate and adult preparations suggested the receptor has two oxotremorine affinity states in both age groups but the ability of guanine nucleotide to shift receptors from the higher affinity state increases with age. [3H] Oxotremorine binding to the higher affinity state had KD values of 95 and 170 pM in neonate and adult heart preparations. The concentration of 5'-guanylylimidodiphosphate which caused 50% displacement of [3H]oxotremorine binding was 1.3 and 0.22 microM in neonate and adult tissue. These results suggest that although the quantity of the G proteins and muscarinic receptors diminish with development, the sensitivity of the G protein:muscarinic receptor complex to guanine nucleotide increases.