Calcium channel involvement in potassium depolarization-induced phosphoinositide hydrolysis in rat cortical slices

The stimulation of production of inositol phosphates in rat cortical slices by KCl depolarization and the effects of calcium channel active drugs were investigated. Elevation of K⁺ in the medium up to 48 mM KCl caused a linear concentration-dependent increase in [³H]inositol phosphate accumulation. The KCl stimulated response was not significantly inhibited in the presence of muscarinic or ₁-adrenergic antagonists. KCl stimulated the production of inositol trisphosphate at 60 min but not 10 min. Addition of peptidase inhibitors did not significantly affect KCl-stimulated PI hydrolysis. The KCl-stimulated response was still observed in the absence of extracellular calcium, although the net accumulation of inositol phosphates was greater in the presence of 0.1 or 0.5 mM calcium. KCl (48 mM) inhibited [³H]inositol uptake into phospholipids of cortical slices. The dihydropyridine calcium channel agonist BAY K 8644 stimulated PI hydrolysis in cortical slices in a concentration dependent manner in the presence of 19 mM KCl. The BAY K 8644-stimulated PI response was partially inhibited by 1M atropine but not by 1M prazosin. Calcium channel blockers nitrendipine, verapamil, flunarizine, and nifedipine slightly inhibited the PI response stimulated by 19 mM KCl in the presence or absence of BAY K 8644. The effects of the calcium channel antagonists were attenuated in the presence of 1 M atropine. The peptide calcium channel blocker -conotoxin did not affect KCl-stimulated PI hydrolysis. These results suggest that endogenous muscarinic or adrenergic neurotransmitters are not involved in KCl-stimulated PI hydrolysis in cortical slices. Although extracellular calcium is necessary for optimal KCl-stimulated PI hydrolysis, it is not required for the expression of the KCl-evoked response suggesting that depolarization is the primary trigger for this stimulant.     

S-adenosyl-l-methionine inhibits phosphoinositide metabolism in the rat brain synaptosomal suspensions

S-adenosyl-l-methionine (AdoMet) has been reported to affect events linked to noradrenergic neurotransmission. In the present work, we studied the effect of AdoMet on norepinephrine (NE)-stimulated inositol phosphate production in³H-inositol-labelled crude synaptosomal suspensions of rat brain. AdoMet (50–1000 M) decreased both the synthesis of labelled polyphosphoinositide (30–50%) and the release of inositol mono- and bisphosphate (40–50%). The AdoMet effect was not dependent on NE concentration (10–1000 M), suggesting that the inhibition of inositol phosphate release was not the result of a modification of the norepinephrine binding to its receptor sites. S-adenosyl-L-homocysteine (AdoHcy) (1 mM) an inhibitor of methyltransferase activities, partially inhibited (70%) the AdoMet (0.1 mM) effect, indicating that the methylation processes cannot explain all the effects observed. We conclude that, in addition to previously reported effects of AdoMet on NE transport, AdoMet may reduce NE-linked intracellular signalling.     

Differential responsiveness of metabotropic glutamate receptors coupled to phosphoinositide hydrolysis to agonists in various brain areas of the adult rat

The effects of metabotropic glutamate receptor (mGluR) agonists on inositol phosphates (IP) accumulation were investigated in slices of the cerebral cortex, hippocampus, striatum and cerebellum of adult Sprague-Dawley rats. EC₅₀ values for 1S, 3R-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD) did not differ significantly between various brain areas (range 10⁻⁵ M), quisqualate was the most potent in all the brain areas (range 10⁻⁷−10⁻⁶ M), except the cerebellum (10⁻⁵ M), ibotenate was the most potent in the striatum (range 10⁻⁶ M) and the least potent in the cerebral cortex and hippocampus (range 10⁻⁴ M). The efficacy in the four brain areas showed the following trend of ranking order for ACPD and quisqualate: hippocampus > striatum > cerebral cortex > cerebellum, and for ibotenate: hippocampus > cerebral cortex > striatum > cerebellum, although the observed differences reached the level of statistical significance only in the case of ACPD (hippocampus and striatum vs cerebellum) and ibotenate (hippocampus vs cerebellum). Co-incubation of the agonists at maximally effective concentrations in any pairwise combination resulted in no substantial additivity of IP accumulation. D,L-1-amino-3-phosphonopropionic acid (AP3) and D,L-2-amino-4-phosphonobutyric acid (AP4) at 0.5 mM concentration antagonized ACPD-induced IP accumulation by about 70 and 45%, respectively, without differences between brain areas. On the other hand, the antagonistic effects ofl-serine-o-phosphate (SOP) at 1 mM concentration were the highest in the hippocampus (75%) and the lowest in the cerebellum (25%). The comparative data indicate considerable regional receptor heterogeneity, in terms of different ratios of response to the agonists (but not antagonists, except SOP). There is a robust responsiveness of mGluRs not only in the hippocampus and cerebral cortex, but also in the striatum which exhibits the highest affinity to both quisqualate and ibotenate.     

Differential modulation of carbachol and trans-ACPD-stimulated phosphoinositide turnover following traumatic brain injury

In the fluid percussion model of traumatic brain injury (TBI), we examined muscarinic and metabotropic glutamate receptor-stimulated polyphosphoinositide (PPI) turnover in rat hippocampus. Moderate injury was obtained by displacement and deformation of the brain within the closed cranial cavity using a fluid percussion device. Carbachol and (±)-1-Aminocyclopentane-trans-1,3.-dicarboxylic acid (trans-ACPD)-stimulated PPI hydrolysis was assayed in hippocampus from injured and sham-injured controls at both 1 hour and 15 days following injury. At 1 hour after TBI, the response to carbachol was enhanced in injured rats by up to 200% but the response to trans-ACPD was diminished by as much as 28%. By contrast, at 15 days after TBI, the response to carbachol was enhanced by 25% and the response to trans-ACPD was enhanced by 73%. The ionotropic glutamate agonists N-methyl-D-aspartate (NMDA), and -amino-3 hydroxy-5-methyl-4-isoxazolepropionate (AMPA), did not increase PPI hydrolysis in either sham or injured rats and injury did not alter basal hydrolysis. Thus, hippocampal muscarinic and metabotropic receptors linked to phospholipase C are differentially altered by TBI.Abbreviations used TBI traumatic brain injury - EAA excitatory amino acids - PPI polyphosphoinositides - IP inositol phosphates - NMDA N-methyl-D-aspartate - AMPA -amino-3-hydroxy-5-methylisoxazole-4-propionate - trans-ACPD (±)-1-Aminocyclopentanetrans-1,3-dicarboxylic acid - LTP long term potentiation     

Characterization of ouabain-induced phosphoinositide hydrolysis in brain slices of the neonatal rat

The effect of the Na/K-ATPase inhibitor ouabain on phosphoinositide (Ptdlns) hydrolysis was studied in rat brain cortical slices. Ouabain induced a dose-dependent accumulation of inositol phosphates (InsPs) which was much higher in neonatal rats (1570±40% of basal) than in adult animals (287±18% of basal). For this reason, all experiments were conducted with 7 day-old rats. Strophantidin caused a similar stimulation of Ptdlns hydrolysis, although it was less potent than ouabain. The order of potency for ouabain-stimulated InsPs accumulation in brain areas was hippocampus>cortex>brainstem>cerebellum. The effect of ouabain was not blocked by antagonists for the muscarinic, alpha₁-adrenergic and glutamate receptors. Also ineffective were the K⁺ channel blockers 4-aminopyridine and tetraethylammonium, the sodium channel blocker tetrodotoxin, and the calcium channel blocker verapamil, whereas the Na/Ca exchanger blocker amiloride partially antagonized the effect of ouabain. The accumulation of InsPs induced by ouabain was additive to that of carbachol and norepinephrine, as well as to that induced by high K⁺ and veratrine, but not to that of glutamate. Removal of Na⁺ ions from the incubation buffer completely prevented the accumulation of InsPs induced by ouabain. The effect of ouabain was also dependent upon extracellular calcium and was under negative feedback control of protein kinase C. Despite the higher effect of ouabain on Ptdlns hydrolysis of immature rats, the density of [³H]ouabain binding sites, as well as the activity of Na/K-ATPase were higher in adult animals. Furthermore, a poor correlation was found between ouabain-stimulated Ptdlns hydrolysis and [³H]ouabain binding in brain regions. These results suggest an involvement of Na⁺ pump in the hydrolysis of Ptdlns, possibly related to an effect on Na⁺ and Ca²⁺ homeostasis. The immature rat appear to be an useful model for studying the relationship between Na/K-ATPase and inositol metabolism.     

Characterization of adenosine receptor-mediated generation of cyclic AMP in slices of rat cerebral cortex with chronic epileptic activity

Cyclic AMP accumulations elicited by adenosine analogues 2-chloroadenosine (2-CADO),R-N ⁶-phenylisopropyladenosine (R-PIA), andN ⁶-cyclohexyladenosine (CHA) were investigated in cortical slices of chronic iron-induced epileptic rats. Cyclic AMP accumulation was elicited 9-to 18-fold by 2-CADO and it was elicited 5-to 7-fold by eitherR-PIA or CHA; 2-CADO was more potent thanR-PIA or CHA in eliciting cyclic AMP accumulation. The adenosine analogues elicited cyclic AMP accumulation in a dose-dependent manner, and the elicitation was inhibited by the adenosine antagonist 8-phenyltheophylline. The 2-CADO-elicited accumulation of cyclic AMP was greatly increased in the cortical region on the primary epileptic side, while theR-PIA-or CHA-elicited accumulation did not change in any cortical region. The deviation detected only in the 2-CADO-elicited accumulation of cyclic AMP may be due to the difference in relative potency for adenosine receptors of the adenosine analogues. The results suggest that adenosine receptormediated generation of cyclic AMP is altered in the primary region of iron-induced epileptic cortex, in which heterogeneous alterations in different adenosine receptor subtypes may occur in the epileptic process.     

用基因芯片研究高苯丙氨酸诱导神经元基因表达谱的改变

为深入了解苯丙酮尿症患者高苯丙氨酸损伤神经元的可能机制,我们将体外培养3d的胚鼠神经元随机分为两组：高苯丙氨酸组和对照组。高苯丙氨酸组加入0．9mmol/L苯丙氨酸诱导12h,对照组加入等体积的培养液,抽提RNA,与大鼠神经生物学芯片U34杂交,检测胚鼠原代神经元高苯丙氨酸作用下表达谱的改变,并用实时荧光定量聚合酶链式反应方法验证基因芯片的结果。发现芯片上1323条总探针组合数中有167条基因表达增加(12．6％),表达下调的已知基因数为7条,占总探针组合数的0.5％。按基因功能,表达上升的基因可分为信号转导相关基因、神经元相关基因、细胞骨架基因、代谢相关基因、离子通道基因、转录相关基因、细胞因子基因、凋亡相关基因等。研究结果证实了以前的报道,如Na^ 、K^ -ATP酶、凋亡、氧化应激、NMDA受体、Ca^2 参与了高苯丙氨酸对神经元的损伤过程。结果还显示,在高苯丙氨酸环境下,CaMKⅡ、Ras、P38、钙通道基因表达上升,部分与囊泡形成、神经递质释放有关的基因表达增强,与神经递质谷氨酸有关的受体和转运体基因表达上升。我们推测高苯丙氨酸可能激活轴NMDR Ca^2 -CaMKⅡ-Ras-P38,引起神经元突起异常;高苯丙氨酸环境下,神经递质释放可能异常：兴奋性神经递质谷氨酸可能参与了高苯丙氨酸引起的神经元损伤。高苯丙氨酸对神经元的损伤是多因素作用。     

Modulation of adenylyl cyclase activity in young and adult rat brain cortex. Identification of suramin as a direct inhibitor of adenylyl cyclase

Adenylyl cyclase (AC) in brain cortex from young (12-day-old) rats exhibits markedly higher activity than in adult (90-day-old) animals. In order to find some possibly different regulatory features of AC in these two age groups, here we modulated AC activity by dithiothreitol (DTT), Fe(2+), ascorbic acid and suramin. We did not detect any substantial difference between the effects of all these tested agents on AC activity in cerebrocortical membranes from young and adult rats, and the enzyme activity was always about two-fold higher in the former preparations. Nevertheless, several interesting findings have come out of these investigations. Whereas forskolin- and Mn(2+)-stimulated AC activity was significantly enhanced by the addition of DTT, increased concentrations of Fe(2+) ions or ascorbic acid substantially suppressed the enzyme activity. Lipid peroxidation induced by suitable combinations of DTT/Fe(2+) or by ascorbic acid did not influence AC activity. We have also observed that PKC- or protein tyrosine kinase-mediated phosphorylation apparently does not play any significant role in different activity of AC determined in cerebrocortical preparations from young and adult rats. Our experiments analysing the presumed modulatory role of suramin revealed that this pharmacologically important drug may act as a direct inhibitor of AC. The enzyme activity was diminished to the same extent by suramin in membranes from both tested age groups. Our present data show that AC is regulated similarly in brain cortex from both young and adult rats, but its overall activity is much lower in adulthood.     

Activation of phospholiphase C by different effectors in rat placental cells

The present communication documents the accumulation of inositol phosphates in rat placental cells by fluoride as well as by vanadate. These findings suggest the existence of the phosphoinositide pathway and its modulation by a G-protein. A concomitant action of fluoride on phosphoinositide breakdown was also observed. As is often the case in intact cells from different organs, protein kinase C exerts a feeback regulatory control on this signalling system. Gonadotropin-releasing hormone (GnRH) also stimulated the accumulation of inositol phosphates in cultured cells but no effect could be detected in freshly isolated cells. Therefore, the phosphoinositide pathway seems to be involved in the mechanism of action of GnRH in rat placental cells.     

Chronic methamphetamine administration reduces histamine-stimulated phosphoinositide hydrolysis in mouse frontal cortex

In the present study, it was hypothesized that in vivo pretreatment with repeated methamphetamine would alter the agonist-stimulated phosphoinositide hydrolysis in mouse frontal cortical slices. Male ICR mice that received the methamphetamine injection (1.0mg/kg, intraperitoneally) once a day for five consecutive days showed behavioral sensitization to the same dose of methamphetamine 5 days after the last injection of the initial chronic treatment regimen (test day 10). On test day 10, the reduction of histamine (0.1-1.0mM)-stimulated phosphoinositide hydrolysis in the mouse frontal cortex was observed. The reduction was specific to histamine, but not to norepinephrine (10 microM-0.1mM) or L-glutamate (0.1-0.5mM). The reduction occurred without any change in the expression level of histamine H(1) receptor mRNA. The reduction recovered 25 days after the last injection of the initial chronic treatment regimen (test day 30). The direct application to the slices of a pharmacologically effective concentration of methamphetamine in vitro (10 microM) did not alter the histamine signal transduction. The present results suggest that the reduction is probably one of neuroadaptations in the frontal cortex contributing to behavioral sensitization.     

Characterization of a glucuronyltransferase: neolactotetraosylceramide glucuronyltransferase from rat brain

The properties of a rat brain glucuronyltransferase, which is presumed to be associated with the biosynthesis of the HNK-1 epitope on sulfoglucuronyl glycolipids, are described. The enzyme required divalent cations for reaction, with maximal activity at 10mm Mn²⁺, and exhibited a dual optimum at pH 4–5 and pH 6 depending upon the buffer used, with the highest activity at pH 4.5 in MES buffer. This enzyme strictly recognized the Gal1-4GlcNAc terminal structure, and was highly specific for neolacto (type 2) glycolipids as acceptor. The enzyme was localized specifically in the brain, and was barely detected in other issues, including the thymus, spleen, liver, kidney, lung, and sciatic nerve fibres. Phosphatidylinositol and phosphatidylserine increased the enzymatic reaction 4.4- and 2.3-fold, respectively, whereas phosphatidylcholine slightly decreased the rate.Abbreviations GlcA glucuronic acid - Lc-PA₁₄ lactotetraose-phenyl-C₁₄H₂₉ - nLc-PA₁₄ neolactotetraose-phenyl-C₁₄H₂₉ - nLcOse₄-Cer neolactotetraosylceramide - NP-40 Nonidet P-40 - PC phosphatidylcholine - PE phosphatidylethanolamine - PI phosphatidylinositol - PS phosphatidylserine - SGGL sulfoglucuronyl glycolipid     

Phosphoinositide hydrolysis increase by angiotensin-(1--7) in neonatal rat brain

Angiotensin (Ang)-(1–7) is an endogenous peptide hormone of the renin–angiotensin system which exerts diverse biological actions, some of them counterregulate Ang II effects. In the present study potential effect of Ang-(1–7) on phosphoinositide (PI) turnover was evaluated in neonatal rat brain. Cerebral cortex prisms of seven-day-old rats were preloaded with [³H]myoinositol, incubated with additions during 30 min and later [³H]inositol-phosphates (IPs) accumulation quantified. It was observed that PI hydrolysis enhanced 30% to 60% in the presence of 0.01 nM to 100 nM Ang-(1–7). Neither 10 nM [D-Ala⁷]Ang-(1–7), an Ang-(1–7) specific antagonist, nor 10 nM losartan, an angiotensin II type 1 (AT₁) receptor antagonist, blocked the effect of 0.1 nM Ang-(1–7) on PI metabolism. The effect of 0.1 nM Ang-(1–7) on PI hydrolysis was not reduced but it was even significantly increased in the simultaneous presence of [D-Ala⁷]Ang-(1–7) or losartan. PI turnover enhancement achieved with 0.1 nM Ang-(1–7) decreased roughly 30% in the presence of 10 nM PD 123319, an angiotensin II type 2 (AT₂) receptor antagonist. The antagonists alone also enhanced PI turnover. Present findings showing an increase in PI turnover by Ang-(1–7) represent a novel action for this peptide and suggest that it exerts a function in this signaling system in neonatal rat brain, an effect involving, at least partially, angiotensin AT₂ receptors.     

Sodium-dependent transport of [3H](1D)chiro-inositol by Tetrahymena

The transport characteristics of (1D)chiro-inositol by the ciliate Tetrahymena were examined in competition studies employing [3H](1D)chiro-inositol. (1D)chiro-Inositol transport was competed by unlabeled (1D)chiro-inositol, myo-inositol, scyllo-inositol, and D-glucose in a concentration-dependent manner. Conversely, (1D)chiro-inositol competed for [3H]myo- and [3H]scyllo-inositol transport. Lineweaver-Burke analysis of the competition data indicated a Km of 10.3 mM and a Bmax of 4.7 nmol/min/mg for (1D)chiro-inositol. Transport of (1D)chiro-inositol was inhibited by cytochalasin B, an inhibitor of facilitated glucose transporters, and phlorizin, an inhibitor of sodium-dependent transporters. Removal of sodium from the radiolabeling buffer also inhibited uptake. The presence of 0.64 mM calcium or magnesium ions exerted negligible effects on transport, although potassium was inhibitory. [3H](1D)chiro-Inositol was shown to be incorporated into Tetrahymena phosphoinositides.     

Characterization of [3H]Vesamicol binding in rat brain preparations

The binding of (1)-[³H]vesamicol was characterized in several subcellular fractions and brain regions of the rat. Binding to a lysed P₂ fraction from the rat cerebral cortex reached equilibrium within 4 min at 37°C and was reversible (dissociation half-time 4.9 min). At least two binding affinities were found in P₂ fractions from the cerebral cortex (K_d:21 nM and 980 nM), striatum (K_d:28 nM and 690 nM), and cerebellum (K_d:22 nM and 833 nM). High affinity B_max values were highest in striatum (1.17 pmol/mg protein), followed by cerebellum (0.67 pmol/mg protein), and cerebral cortex (0.38 pmol/mg protein). Low affinity B_max values were highest in cerebellum (5.2 pmol/mg protein), with similar values for cerebral cortex (3.7 pmol/mg protein) and striatum (3.8 pmol/mg protein). High affinity but not low affinity binding in each brain region was stereospecific. Another inhibitor of vesicular ACh-transport also displaced 1-vesamicol binding potently (IC₅₀:17 nM) and efficaciously (over 90%). Both high affinity and low affinity B_max values for [³H]vesamicol-binding were highest in a partially purified synaptic vesicle fraction, followed by puriffied synaptosomes, crude membranes and P₂ fractions. Specific binding was not observed in a mitochondria-enriched fraction. Crude membrane preparations of primary, neuron-enriched whole brain cultures also exhibited high (64 nM) and low affinity (1062 nM) [³H]vesamicol binding. Isoosmotic replaement of 0.18 M KCl in the binding-buffer with NaCl had no effect on binding. These results suggest that at least some high affinity [³H]vesamicol binding in rat brain preparations may be associated with synaptic vesicles, some of which may not be cholinergic in origin.     

Alcohol dehydrogenase activity in rat kidney cortex stimulated by oestradiol

Sex differences in alcohol dehydrogenase activity, determined by the influence of oestrogen hormones, were found to exist in the rat kidney. Oestradiol, but neither testosterone nor progesterone, was shown to be a powerful stimulator of kidney alcohol dehydrogenase activity in rats, maximally 6- to 8-times over control values. The Michaelis-Menten constant for acetaldehyde of both non-stimulated and oestradiol-stimulated kidney alcohol dehydrogenases was found to be similar, 6.7 · 10^?5 M and 7.8 · 10^?5 M, respectively. Actinomycin D was shown to have an additive effect (superinduction) on the oestradiol-induced increase in kidney enzyme activity. The findings suggest the possibility of the higher contribution of kidneys in ethanol metabolism in states with an elevated level of oestradiol, such as chronic ethanol intake and ethanol hepatic disease.     

Phorbol ester-induced phosphoinositide hydrolysis in rat aorta: role of cyclooxygenase products

This study investigates whether phorbol esters increase phosphoinositide hydrolysis in intact vascular smooth muscle, and the mechanism underlying the hydrolysis. Phorbol myristate acetate induced time- and concentration-dependent increases in phosphoinositide hydrolysis, as demonstrated by elevated inositol monophosphate levels, in deendothelialized rat aorta. The phorbol ester-elevated inositol monophosphate levels were abolished by indomethacin, a cyclooxygenase inhibitor, but were only partially decreased by SQ29548, a thromboxane A2/prostaglandin H2 receptor antagonist. SQ29548 also only partially decreased elevated inositol monophosphate levels due to prostaglandin E2, prostaglandin F2alpha, prostaglandin I2 and carbacyclin, a stable prostaglandin I2 analog. SQ29548 abolished elevated inositol monophosphate levels due to U46619, a stable thromboxane A2/prostaglandin H2 receptor agonist. These studies demonstrate that phorbol esters increase phosphoinositide hydrolysis in intact vascular smooth muscle, and that the increase is due, at lease in part, to endogenously released prostaglandins other than prostaglandin H2.     

Day-night cycle of lipidic composition in rat cerebral cortex

A study of the lipidic pattern of the cerebral cortex of the normal adult rat during the daynight cycle was carried out. The changes observed were the following: phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and phosphatidylserine plus phosphatidic acid showed a peak at 16:00 hr possibly due to a general increase in phospholipid biosynthesis. During the nocturanl period the variations of phosphatidylcholine and phosphatidylethanolamine were not clearly observe, they might be due to an increase in the interconversion or exchange reaction, since the ratio phosphatidylcholine/phosphatidylethanolamine showed a significative change at 04:00 hr. This occurred because small but opposite changes in both phospholipids were observed, suggesting an increase in the methylation reactions of phospholipids. Cardiolipin showed a significant peak at 04:00 hr. Plasmalogens exhibited significative changes, an important diminution at 16:00 hr and a prominent peak at 24:00 hr. Cholesterol levels were high during the light period and low in the dark one. Cerebrosides and gangliosides showed no day-night variations. The changes observed indicate a phenomenon of biological rhythmicity synchronized by the photoperiod, suggesting that these fluctuations could act as physiological modulators of the properties and functions of the nerve cell membrane.     

Pertussis toxin-sensitive G proteins mediate the inhibition of basal phosphoinositide metabolism caused by adenosine A1 receptors in rat hippocampal slices

The adenosine A₁ receptor selective agonist, N ⁶-cyclopentyladenosine (CPA, 300 nM) inhibited basal accumulation of [³H]inositol phosphates ([³H]InsPs), but not the total levels of membrane [³H]-phosphoinositides, in rat hippocampal slices. This action of CPA was not significantly modified when synaptic transmission was blocked with tetrodotoxin (TTX, 200 nM) but was prevented in slices pre-incubated with pertussis toxin (PTX, 5 g/mL) for 12-16 hr. Neither PTX nor TTX, when applied in the absence of CPA, influenced basal [³H]InsPs accumulation. It is concluded that the inhibition of the basal phosphatidylinositol metabolism by adenosine A₁ receptor activation is independent of neurotransmission and involves a PTX-sensitive G protein, probably of the G_i/G_o family.     

Radio-label and mass determinations of inositol 1,3,4,5-tetrakisphosphate formation in rat cerebral cortical slices: Differential effects ofmyo-inositol

To investigate the effects of increasing concentrations ofmyo-inositol (inositol) on receptor stimulated [³H]inositol polyphosphate formation in the absence of lithium, slices of rat cerebral cortex were incubated with various concentrations of [³H]inositol (1 to 30 M). Carbachol stimulated formation of [³H]inositol trisphosphate (InsP₃) and [³H]inositol 1,3,4,5-tetrakisphosphate {Ins(1,3,4,5)P₄} increased several fold when the inositol concentration was increased reaching a plateau at approximately 12 M inositol. Time course studies revealed that in the presence of low concentrations of inositol (1 M), [³H]InsP₃ and [³H]Ins(1,3,4,5)P₄ formation in response to carbachol stimulation increased slowly over a 10 to 20 min time period, whereas in the presence of 4 and 12 M inositol, carbachol stimulated [³H]InsP₃ and [³H]Ins(1,3,4,5)P₄ formation was rapid and essentially complete within 3 to 5 min after carbachol addition. Although the carbachol dose response in 12 M inositol had a much greater maximal efficacy, there was no change in potency. Similar to the effects of carbachol on [³H]Ins(1,3,4,5)P₄ formation from prelabeled phosphoinositides, muscarinic receptor stimulation increased Ins(1,3,4,5)P₄ mass formation by seven fold. Furthermore, Li⁺ (8 mM) completely inhibited carbachol stimulated increases in Ins(1,3,4,5)P₄ mass formation. In contrast to the effects of increasing inositol on carbachol stimulated formation of radiolabeled inositol phosphates, increasing inositol had no effect upon mass formation of Ins(1,3,4,5)P₄. These results show that when measuring inositol polyphosphate formation by the radiolabeling technique in the absence of Li⁺, increasing the inositol concentration greatly increases the stimulated component of [³H]InsP₃ and [³H]Ins(1,3,4,5)P₄ formation. However, this inositol induced increase in agonist stimulated Ins(1,3,4,5)P₄ formation is not reflected as an increase in mass formation.     

Age-related changes in receptor-mediated phosphoinositide hydrolysis in various regions of rat brain.

The effects of age on cholinergic markers and receptor-stimulated phosphoinositide hydrolysis was examined in the frontal cortex and striatum of male Fischer-344 rats. Choline acetyltransferase activity was decreased 27% in the striatum of aged (24 month) rats compared to young (3 month) controls. Muscarinic receptor density as measured by [3H]-quinuclidinyl benzilate binding showed a similar 26% decrease in the striatum of aged rats. Phosphoinositide hydrolysis was measured by the release of inositol phosphate (IP) from tissue slices prelabeled with [3H]myoinositol in response to carbachol, norepinephrine, and quisqualate. In the cortex, stimulated IP release was significantly greater in slices from aged rats compared to young rats for all three agonists. In contrast, stimulated IP release was significantly decreased in striatal slices from aged rats compared to young for all three agonists. These data indicate a differential effect of age on agonist-stimulated phosphoinositide hydrolysis in the cortex and striatum. The decreased responsiveness in the latter area may result from the age-related loss of postsynaptic receptors.