Effect of typical inducers of microsomal drug-metabolizing enzymes on phospholipid metabolism in rat liver.

The effect of administration of phenobarbital (PB), polychlorinated biphenyls(PCBs) or 3-methyl-cholanthrene (3-MC) on the metabolism of phospholipids in rat liver was studied by the i.p. injection of [³²P]orthophosphate or [Me-¹⁴C]choline chloride. The inducers were given to animals for 2 successive days PB had no significant effect on the incorporation rate of ³²Pi into liver microsomal phospholipid classes 48 hr after the first administration. The rate of incorporation of [¹⁴C]choline into phosphatidylcholine (PC) in both subcellular components of the liver and blood plasma decreased slightly at this experimental period. In addition, the ratio of [¹⁴C] sp. act. of phosphorylcholinc to that of microsomal phosphulipid was considerably higher on PB-trcated rats as compared with the ratio in control rats. These and previous findings strongly suggest that proliferation of liver endoplasmic reticulum (ER) membranes induced by PB would be accompanied by the stimulation of phospholipid synthesis at the early process of induction and subsequently followed by the decrease in turnover rate of microsomal phospholipids. The administration of PCBs, on the other hand, caused strong inhibition of both ³²Pi and ¹⁴C incorporation into PC in liver subcellular fractions. The secretion of PC from liver cells to blood plasma was also strongly depressed. In addition, phospholipid catabolizing activity was found to be depressed in the liver. These results indicate that hypertrophy of ER membranes in the liver after PCBs administration could be due to a depression of both secretion of lipoprotein from liver cells to plasma and catabolic activity toward membranous phospholipids in liver cells. The decrease in ³²Pi incorporation into liver microsomal PC was also observed in rats treated with 3-MC. There occurred a considerable accumulation of ¹⁴C activity in phosphorylcholine in the liver of rats treated with either PCBs or 3-MC, suggesting strongly that these drugs caused an inhibition of PC synthesis at the site of CDP-choline formation, namely the inhibition of the reaction catalyzed by cholinephosphate cytidylyltransferase.     

Effect of polychlorinated biphenyls (PCBs) administration on phospholipid biosynthesis in rat liver

The effect of PCBs or phenobarbital on the biosynthesis of phospholipids in hepatic endoplasmic reticulum of rats was studied by the intraperitoneal injection of [³²P]orthophosphate, [Me?¹⁴ C]choline or [2?³H]glycerol. Significant increases in liver microsomal phospholipid content after the administration of either PCBs or phenobarbital indicated the actual proliferation of endoplasmic reticulum membranes. The rate of both [³²P] and [¹⁴C] incorporations into microsomal choline-containing phospholipids, such as phosphatidylcholine, sphingomyelin and lysophosphatidylcholine, was reduced to one fifth by PCBs administration compared with control animals. The incorporation of [³²P]orthophosphate into phosphatidylethanolamine or other phospholipid classes was less or not affected, respectively, by PCBs administration. The specific inhibitory effect of PCBs on the incorporation into cholinecontaining phospholipids was not observed when [2?³-H]glycerol was used as a precursor. Phenobarbital administration, however, increased significantly the rate of [³²P] incorporation into liver phospholipids, especially phosphatidylcholine. It is suggested that the increase in microsomal phospholipid content by PCBs administration is not due to the stimulation of synthesis but to the inhibition of the catabolism of membrane phospholipids and that the increase in content caused by phenobarbital is due at least in part, to the stimulation of synthesis. The possible site(s) of PCBs-induced inhibition of phospholipid biosynthesis in rat liver is discussed.     

Angiotensin II stimulation of phospholipase D in rat renal mesangial cells is mediated by the AT1 receptor subtype

Angiotensin II stimulated a biphasic 1,2-diacylglycerol formation in [³H]arachidonic acid-labelled mesangial cells. In contrast, in cells labelled with [³H]myristic acid, a tracer that preferentially marks phosphatidylcholine, angiotensin II induced a delayed monophasic production of 1,2-diacylglycerol. This delayed peak of 1,2-diacylglycerol generation was associated with a concomitant increase in choline formation, suggesting that stimulation of mesangial cells with angiotensin II causes a phospholipase D-mediated phosphatidylcholine hydrolysis. This conclusion is supported by the observation that angiotensin II stimulated the accumulation of phosphatidylethanol, when ethanol was added to mesangial cells. The production of choline and phosphatidylethanol stimulated by angiotensin II was completely blocked by the angiotensin II AT₁ receptor-selective antagonist DuP 753 with an IC₅₀ value of 8 nM, but not by the angiotensin II AT₂ receptor selective ligand CGP 42112A. Furthermore, angiotensin(1–7) and angiotensin(1–6) had only weak effects on choline generation. These data clearly indicate that angiotensin II AT₁ receptors trigger phospholipase D-mediated phosphatidylcholine hydrolysis in rat mesangial cells.     

Mechanism of carbachol-stimulated diacylglycerol formation in rat parotid acinar cells

We studied the relationship between phosphoinositide hydrolysis, phosphatidylcholine hydrolysis, and sn-1,2-diacylgglycerol (DAG) formation in response to carbachol stimulation in rat parotid acinar cells. Previously, we demonstrated that DAG formation stimulated with 1 μM carbachol was biphasic: the first peak occurred at 5 min and the second one at 20 min. It was also demonstrated that the second peak was regulated in part by a calmodulin/protein kinase C-dependent mechanism. Based on the kinetic analysis of DAG formation and [³²P]phosphoinositide breakdown, the first peak of carbachol (1 μM)-stimulated DAG accumulation was found to be related to the breakdown of [³²P]phosphatidylinositol 4-monophosphate ([³²P]PIP) and [³²P]phosphatidylinositol 4,5-biphosphate ([³²P]PIP₂). The second peak was found to be related to [³²P]PIP₂ breakdown. Carbachol stimulated the release of [³H]phosphocholine into the medium, indicating that the predominant pathway for phosphatidylcholine hydrolysis was via phospholipase C. Moreover, carbachol stimulated the release of [³H]choline metabolites in a time- and dose-dependent manner. This agonist slightly stimulated the release of [³H]ethanolamine metabolites. A calmodulin/protein kinase C-dependent mechanism was also studied and was found to be involved in carbachol-stimulated phosphatidylcholine hydrolysis; W-7, a calmodulin inhibitor and staurosporine, a protein kinase C inhibitor, inhibited the carbachol (1-μM)-induced release of [³H]choline metabolites at 20 min in a dose-dependent manner, but did not have inhibitory effects at 5 min. These results suggest that the first peak of DAG accumulation induced by carbachol is predominantly associated with the breakdown of [³²P]PIP and [³²P]PIP₂ and that the second peak is predominantly associated with [³²P]PIP₂ breakdown and phosphatidylcholine hydrolysis.     

Action of sulfhydryl reagents on cholinergic mechanisms in synaptosomes.

The mechanism by which certain sulfhydryl blocking reagents affect the release of radioactive acetylcholine ([³H]ACh) from synaptosomal preparations of rat cerebral cortex was studied. N-Ethylmaleimide (NEM) or p-hydroxymercuribenzene sulfonate (pHMBS), at a concentration of 0.5 mM, produced a marked release of [³H]choline and [³H]ACh. The release induced by these reagents was independent of [Ca²⁺]₀ and was observable within 30 sec of incubation. Pretreatment for 2 min with pHMBS inhibited high affinity uptake of choline. Using a fluorescence probe, these blocking reagents were shown to cause a rapid increase of synaptosomal fluorescence, indicating that these reagents had depolarized the preparation. Although the incorporation of [³H]NEM into the synaptosomal cytoplasm and particulate fractions was very rapid, an impermeant-NEM derivative (glutathione-maleimide) also release [³H]ACh from the P₂ fraction, suggesting that the release of ACh by sulfhydryl blocking reagents is at least partly due to a membrane effect.     

Effect of 4-chlorobiphenyl on substrate transport and phospholipid metabolism in mouse L5178Y lymphoma cells

The toxicity of 4-chlorobiphenyl, a constituent of Aroclor 1221, was studied in mouse L5178Y cells, in vitro. 4-Chlorobiphenyl had a varied effect on the uptake of small precursor molecules. Uptake of [³H]l-leucine, [³H]l-serine, [³H]uridine and [³H]thymidine was inhibited, while that of [³H]inositol was stimulated. There was no significant effect on either [¹⁴C]ethanolamine or [¹⁴C]choline uptake. However 4-chlorobiphenyl significantly inhibited incorporation of [¹⁴C]ethanolamine into phosphatidylethanolamine and caused a 2- to 3-fold stimulation in the incorporation of [¹⁴C]choline into phosphatidylcholine. This effect on phosphatidylcholine metabolism depended on the adsorption and continued presence of 4-chlorobiphenyl on the cell plasma membrane. The stimulation of [¹⁴C]choline incorporation was reversed when treated cells were placed in fresh growth medium under conditions where 95 per cent of the 4-chlorobiphenyl was desorbed from the cell surface. The effect of 4-chlorobiphenyl on substrate uptake and phospholipid metabolism appears to depend upon the interaction of the agent with the cell membrane surface.     

Evaluation by reverse phase HPLC of [3H]acetylcholine release evoked from the myenteric plexus of the rat

Summary Myenteric plexus-longitudinal muscle strips isolated from the small intestine of rats were incubated with [³H]choline to measure the synthesis and the release of [³H]acetylcholine. To separate different radioactive compounds (acetylcholine, choline, phosphorylcholine) from both the tissue and the overflow a new method, the reverse phase HPLC, was used.The radiochromatogram following the injection of a [³H]choline-standard and a [¹⁴C]acetylcholine-standard onto the HPLC showed a clear separation of both isotopes with a recovery rate of roughly 100%. Incubation of the muscle strips with [³H]choline caused the synthesis of [³H]acetylcholine (30,000 dpm/preparation) that increased 2-fold, when the electrical field stimulation during labelling was increased from 0.2 Hz to 1 Hz. Electrical field stimulation (3 Hz, 2 min) caused an increase in tritium efflux that was abolished by the removal of extracellular calcium or by the addition of tetrodotoxin. Analysis by reverse phase HPLC of the overflow showed that the stimulated increase in tritium overflow was balanced by the enhanced release of [³H]acetylcholine, whereas the overflow of [³H]choline was not affected by the electrical field stimulation. Oxotremorine (1 mol/l) suppressed the release of [³H]acetylcholine by 60%. Scopolamine (0.1 mol/l) prevented this inhibition and, given alone, enhanced the release of [³H]acetylcholine by 43%. The release of [³H]acetylcholine evoked at 0.2, 2 or 20 Hz did not consistently decline at increasing frequencies.The present experiments show the synthesis and the calcium-dependent release of [³H]acetylcholine from the myenteric plexus-longitudinal muscle preparation of rats correspondingly to the same in-vitro preparation isolated from guinea-pigs. Muscarinic autoinhibition operates also in the small intestine of rats. However, some differences (frequency-dependency of [³H]acetylcholine release, spontaneous neuronal activity) are evident between both species. Reverse phase HPLC is a useful method to separate radioactive choline and acetylcholine with a high recovery rate.Send offprint requests to I. Wessler at the above address     

Decreased Biosynthesis of Lung Surfactant Constituent Phosphatidylcholine Due to Inhibition of Choline Transporter by Gefitinib in Lung Alveolar Cells

Purpose We investigated whether gefitinib, an anticancer agent, inhibits phosphatidylcholine (PC) biosynthesis and choline uptake by alveolar epithelial type II cells. Materials and Methods Uptake of choline and PC biosynthesis were examined in vitro, using human alveolar epithelia-derived cell line A549 and rat alveolar type (AT) II cells as models. Results Gefitinib reduced the incorporation of [³H]choline into PC in A549 and rat ATII cells. The uptake of [³H]choline by A549 and rat ATII cells was concentration-dependent, and the Km values were 15.0 and 10–100 μM, respectively. The uptake of [³H]choline by A549 and rat ATII cells was weakly Na⁺-dependent, and inhibited by hemicholinium-3. RT-PCR revealed expression of choline transporter-like protein (CTL)1 and organic cation transporter (OCT)3 mRNAs in both cells. The choline uptake by A549 and rat ATII cells was strongly inhibited by gefitinib with the IC₅₀ value of 6.77 μM and 10.5 μM, respectively. Conclusions Our results demonstrate that gefitinib reduces PC biosynthesis via inhibition of cellular choline uptake by A549 and rat ATII cells, which is mainly mediated by CTL1, resulting in abnormality of lung surfactant that can be one of mechanisms of the interstitial lung disease associated with gefitinib.     

An in vitro study of hemicholinium-3 on phospholipid metabolism of Krebs II ascites cells

With [Me-¹⁴C]choline as marker and after separation of choline and phosphocholine by ion-exchange column chromatography or thin layer chromatography on alumina, it is shown that 40 μM hemicholinium-3 (HC-3) inhibits the cytosolic choline-kinase of rat liver and Krebs cells. This inhibition is competitive (Km different, Vm similar) in the first case and mixed in the second (Km and Vm different). Despite this general inhibition of the phosphocholine formation, the synthesis of phosphatidylcholine (PC) by post-nuclear supernatants of rat liver and Krebs cells is different when tested with HC-3. It is unaffected in rat liver; however, HC-3 induces a PC deficiency in Krebs cells which is time-course dependent between 15 and 120min and proportional to the drug concentrations in the interval 5–40 μM. Incorporation of AT-[γ³²P] or [2-¹⁴C]ethanolamine into phospholipids shows that the sequential methylation pathway is not detectable in Krebs cells. These results are discussed in relation to those established concerning HC-3 action on phospholipid metabolism in other tissues.     

Kinetics of [3H]choline and [3H]acetylcholine metabolism in several regions of rat brain following intracerebroventricular injection of [3H]choline: Effects of haloperidol

Intracerebroventricular (icv.) injection of [³H]choline in conscious rats produced a rapid, efficient labeling of brain choline and acetylcholine (ACh) stores without altering steady-state levels of endogenous ACh. The kinetics of [³H]choline and [³H]ACh metabolism were measured in seven brain regions for up to 10 min following icv. administration of [³H]choline. The initial rate of formation of [³H]ACh varied in different brain areas, being greatest in the striatum and least in the hypothalamus. In contrast, the rate of [³H]choline metabolism was similar in all regions of the brain. Pretreatment of rats with haloperidol resulted in an increase in the apparent synthesis rate of ACh only in the striatum and rostral hypothalamus, pointing to possible dopaminergic-cholinergic interaction in these regions.     

Effect of morphine in vivo on uptake of [3H]choline and release of [3H]acetylcholine from rat striatal synaptosomes

The effect of morphine on the rat striatal cholinergic system was investigated in vitro by measuring the rates of [³H]choline uptake and [³H]acetylcholine release in striatal synaptosomes after in vivo injections of morphine sulfate. Morphine caused a 50 per cent increase in the V_max of [³H]choline uptake. Although a concomitant increase was also measured in the amount of [³H] acetylcholine released, it could be explained by the previous increase in uptake. It is suggested that morphine had an overall stimulatory effect on the striatal cholinergic system which may be a transynaptic phenomenon rather than a direct effect on the cholinergic cell.     

Effects of dl-propranolol on the synthesis of glycerolipids by rabbit iris muscle

Propranolol (0.03?0.3 mM), an amphiphilic cationic drug which is used therapeutically as a β-blocker, was found to alter significantly the incorporation of [¹⁴C]glucose, [¹⁴C]glycerol, [¹⁴C]acetate, ³²Pi, [³H]cytidine, [³H]inositol, [¹⁴C]choline, [¹⁴C]ethanolamine and [¹⁴C]serine into phospholipids of the iris muscle. Furthermore, it was found to exert a stimulatory effect on the [¹⁴C]serine incorporation into phosphatidylserine of the muscle and microsomes. In contrast, sotalol, another β-blocker-but lacking the hydrophobicity of propranolol-exerted no effect on lipid metabolism. Whereas norepinephrine stimulated only the turnover of the phosphate moiety of phosphatidic acid and phosphatidylinositol, in general propranolol caused the following changes: (a) it stimulated by 2- to 6-fold the labelling of phosphatidic acid and phosphatidylinositol from [¹⁴C]glucose, [¹⁴C]glycerol, [¹⁴C]acetate, ³²Pi and [³H]inositol, (b) it increased by 5- and 38-fold the incorporation of ³²Pi and [³H]cytidine, respectively into CDP-diglyceride, (c) it inhibited appreciably the incorporation of [¹⁴C]glucose, [¹⁴C]glycerol, [¹⁴C]acetate and ³²Pi into phosphatidylcholine and phosphatidylethanoalmine. However, while it inhibited significantly the [¹⁴C]choline incorporation into the former, it stimulated by 60 per cent the ethanolamine incorporation into the latter phospholipid. These results indicate that propranolol probably redirects phospholipid synthesis de novo, by inhibiting phosphatidate phosphohydrolase, such that the increase obtained in the biosynthesis of phosphatidylinositol is accompanied by a corresponding decrease in the synthesis of phosphatidylcholine and phosphatidylethanolamine.Propranolol also caused a 250 per cent increase in the [¹⁴C]serine incorporation into phosphatidylserine of the iris muscle and 28 per cent increase in that of microsomes. The drug appears to stimulate the Ca²⁺ -uptake by muscle and microsomes, which in turn could act to stimulate the Ca²⁺-catalyzed base-exchange reaction.In addition the metabolic pathways involved in the biosynthesis of the major phospholipids of the iris, a smooth muscle, are reported for the first time. These pathways were found to be essentially similar to those reported for other tissues.     

Actylcholine output from the cerebral cortex,choline uptake and muscarinic receptors in morphine-dependent,freely-moving rats

Spontaneous acetylcholine (ACh) output from the cerebral cortex, choline high affinity uptake and [³H]-QNB binding to muscarinic receptors in the cerebral cortex and caudate nucleus in freely moving rats made morphine-dependent by morphine pellet subcutaneous implantation were investigated before and during naloxone-induced withdrawal syndrome. The frequency and intensity of the withdrawal signs were also assessed.No significant change in ACh output was found in tolerant rats when compared with that of placebopellet implanted rats. During naloxone-induced withdrawal syndrome a 60% increase in ACh output occurred.In rats made dependent after a large septal lesion or treated for ten days with calcium gluconate (10 mg/kg i.m.) no increase in ACh output was found during the withdrawal syndrome. The intensity of some of its signs was also reduced.During the withdrawal syndrome a marked increase in choline high affinity uptake in the cerebral cortex and caudate nucleus was detected.The affinity of muscarinic receptors (K_D) for [³H]-QNB was significantly increased in the cerebral cortex and caudate nucleus of morphine-dependent rats before naloxone administration. It returned to normal during the withdrawal syndrome. In the caudate nucleus the number of binding sites (B_max) was decreased before and after the withdrawal syndrome.These findings emphasize the role of cholinergic mechanisms in opiate addiction.     

Effect of phenobarbital on the incorporation of 3H or 14C labeled precursors into fatty acids

The incorporation of various ³H or ¹⁴C labeled precursors into hepatic fatty acids was studied in control and phenobarbital-treated rats. In vitro, phenobarbital had no effect on fatty acid synthesis from the tritiated precursors ³H₂O, 1-³H glucose, 2-³H lactate, 2,3-³H succinate and 2-³H acetyl CoA or from the ¹⁴C labeled precursors 1-¹⁴C acetate and 1,3-¹⁴C malonyl CoA in liver supernatant or supernatant + microsomes preparations. In vivo, phenobarbital stimulated the incorporation of 1-¹⁴C acetate, ³H₂O, 2-³H lactate and 2,3-³H succinate but had no stimulatory effect on the incorporation of 1-³H glucose. The activities of lactic dehydrogenase, glucose 6-phosphate dehydrogenase and succinate cytochrome c reductase were not modified by administration of phenobarbital but that of NADPH-cytochrome c reductase was increased. These results indicate that the NADH and NADPH pools are not quite in equilibrium, and that the endoplasmic reticulum probably competes with the cytoplasm for NADPH utilization and thus may play a part in the regulation of fatty acid synthesis. The increased incorporation of ¹⁴C precursors observed in vivo in phenobarbital-treated rats was not due to stimulation of the synthesis of the key enzymes of fatty acid synthesis but could be related to an activation of these enzymes which, in vivo, are probably fixed on the endoplasmic reticulum.     

Studies on accumulation and metabolic fate of (N-Me3h)choline in human term placenta fragments.

Fragments from human term placenta accumulated [N-Me³H]choline against a concentration gradient. Uptake was linearly related to incubation time and temperature. Analysis of the kinetics of choline accumulation revealed the concurrent existence of a diffusional component occuring both at low temperature as well as high (50 mM) choline concentration and a carrier-mediated transport which had characteristics predicted by the Michaelis-Menten equation showing a K_m = 3.46 × 10^?4 M and a V_max of 75 nmoles/ml intracellular water × min^?1. Net concentration ratios, corrected for diffusion and extracellular water, were larger than 1.0 within 5 min and about 4.0 after 15 min. Hemicholinium-3 was a competitive inhibitor of choline uptake with a K_i of 0.45 mM. [³H]Choline accumulation was decreased by conditions known to lower intracellular ATP levels. Thus, 2,4-dimitrophenol (1 mM), sodium cyanide (5 mM) and anaerobic incubation reduced [³H]choline accumulation 36, 54 and 33 per cent respectively. Ouabain (0.1 mM) also decreased the concentration ratios by 50 per cent. Modification of the ionic environment led to an increase of 36 per cent in the amount of tritium in intracellular water when Na⁺ was reduced to one half of the usual 145 mM or 150 per cent when it was completely omitted and replaced by an osmotically equivalent amount of sucrose. Li⁺ was without effect, while high K⁺ (> 25mM), Rb⁺ and Cs⁺ (145 mM) depressed [³H]choline accumulation. The metabolic fate of [³H]choline was studied. Following a 5-min incubation with 5 μM [³H]choline 95 per cent of the radioactivity was acid-soluble and 5 per cent remained in the acid-insoluble fraction. After 30 min the distribution was 88 and 12 per cent, respectively. Paper high voltage electrophoretic analysis of the acid-soluble material showed that after 5 min 55 per cent of the ³H had a mobility equal to authentic choline, 35 per cent equal to acetylcholine, 6 per cent to phosphorylcholine and 1 per cent to betaine. After 20 min it was 25 per cent in choline, 60 per cent in acetylcholine, 10 per cent in phosphorylcholine and 2 per cent in betaine. A chloroform-methanol extract from the acid-insoluble residue revealed a linear increase of ³H-content suggesting incorporation of [³H]choline into phospholipids.     

Binding characteristics of [3H] guanfacine to rat brain α-adrenoceptors: Comparison with [3H]clonidine

The tritium-labeled α-adrenoceptor agonist and antihypertensive drug guanfacine, N-amidino-2-(2,6-dichlorophenyl)-acetamide (sp. act. 24.2 $\text{Ci}\text{mmole}$) was employed for a direct identification and characterization of α-adrenoceptors in rat brain membranes. Its usefulness as a radioligand was studied in comparison with [³H]clonidine (sp. act. 26.7 $\text{Ci}\text{mmole}$). The nonspecific binding of [³H]guanfacine to rat cerebral membranes was considerably more pronounced than that observed for [³H]clonidine. The specific binding of [³H] guanfacine (0.1–20 nM) and [³H]clonidine (0.1–20 nM) as defined as the excess over blanks containing (?)-norepinephrine (10μM) was saturable. Scatchard analyses of these binding data indicated single populations of binding sites for both ligands. K_D values of 3.9 ([³H]guanfacine) and 3.7 nM ([³H]clonidine) were calculated. Maximal number of specific binding sites amounted to 220 and 195 $\text{fmole}\text{mg}$ protein for [³H]guanfacine and [³H]clonidine, respectively. In case unlabeled guanfacine (1 μM) was used to characterize the specific binding of [³H] guanfacine, K_d value and maximal number of binding sites were about twice as high as determined in the presence of excess (?)-norepinephrine. The rate of association of both radioligands was rapid. Binding reached equilibrium by about 10–15 min of incubation. Half-maximal binding was attained at approximately 1–2 min. The rates of dissociation were biphasic. A rapid and a slow component were identified. The specific binding sites of [³H] guanfacine in rat brain possess the general characteristics of α₂-adrenoceptors. Selective antagonists of α₂-adrenoceptors, like yohimbine and rauwolscine strongly interfered with this binding. However, preferential blocking agents of α₁-adrenoceptors, such as prazosin and corynanthine, were weak competitors. The relative potency of agonists and antagonists in displacing [³H]guanfacine was identical to their effectiveness in competing for [³H]clonidine specific binding sites. It is concluded that [³H]guanfacine labels the same α₂-adrenoceptor population in rat brain as [³H]clonidine. However, [³H]guanfacine seems not as suitable as [³H]clonidine for routine use in the direct identification of α₂-adrenoceptors in view of its relatively high nonspecific binding.     

In vitro and in vivo effects of lead,methyl mercury and mercury on inositol 1,4,5-trisphosphate and 1,3,4,5-tetrakisphosphate receptor bindings in rat brain

In vitro and in vivo effects of mercury (Hg), methyl mercury (MM) and lead (Pb) on [³H]inositol 1,4,5-trisphosphate (IP₃) and [³H]linositol 1,3,4,5-tetrakisphosphate (IP₄) receptor binding in the Sprague-Dawley rat brain cerebellar membranes were studied. In vitro studies indicate that binding of [³H]IP₃ and [³H]IP₄ to cerebellar membranes was inhibited by Hg while they were stimulated by MM or Pb in a concentration-ependent manner. MM was more potent (EC₅₀ 3,4 μM) than Pb (EC₅₀ 18.2 μM) in stimulating the [³H]IP₃ receptor binding activity whereas Pb (IC₅₀ 30 μM) was more potent than MM (IC₅₀ 133 μM) in stimulating the [³H]IP₄, receptor binding. When the rats were treated (i.p) with Hg (5 mg/kg body wt.) or MM (5 mg/kg body wt.) or Pb (25 mg/kg body wt.) for 3 or 24 h, no significant alterations in[³H]IP₃ receptor binding were observed in cerebellum and cerebral cortex. But the above treatment of Pb or MM for 3 or 24 h to rats resulted in an increase of [³H]IP₄ receptor binding in the membranes of cerebral cortex. However, the rats treated with Hg (1 mg/kg body wt./day) or Pb (25 mg/kg body wt./day) for 7 days did not show any alteration in binding of [³H]IP₃ to its receptors in cerebellar membranes but an increase in this receptor binding was noticed with the treatment of MM (2.5 mg/kg body wt./day) for 7 days. The cerebellum and cerebral cortex of rats with the above treatment of MM or Pb for 7 days exhibited an increase in [³H]IP₄ receptor binding. These in vitro and in vivo data suggest that alterations in inositol polyphosphate receptor binding by metals could result in alterations in intracellular calcium levels which may influence neuronal activity.     

Effects of ceruletide on the dopamine receptor-adenylate cyclase system in striatum and frontal cortex of rats chronically treated with haloperidol

Chronic treatment of rats with haloperidol decanoate (30 mg/kg and 100 mg/kg IM every 4 weeks for 52 weeks) increased [³H] SCH 23390 binding in striatal membranes by 25% and 50% and in frontal cortical membranes by 56% and 125% in 30 and 100 mg/kg haloperidol treatment groups, respectively. These increases in [³H] SCH 23390 binding to the membranes were restored to control levels after ceruletide treatment (100 µg/kg IP twice a day for 5 days). [³H] Spiperone binding to the rat striatal and cortical membranes also increased after chronic haloperidol treatment (by 66% and 99% in striatal membranes and by 27% and 62% in cortical membranes in the 30 and 100 mg/kg haloperidol treatment groups, respectively). Administration of ceruletide to haloperidol-treated rats reduced the increased [³H] spiperone binding to the cortical membranes toward the control level, but ceruletide was not effective in reducing the haloperidol-induced increase of [³H] spiperone binding to the striatal membranes. Activation of adenylate cyclase by dopamine (1 µM or 100 µM) or Gpp(NH)p (1 µM) was reduced in striatal and cortical membranes from haloperidol-treated rats. Ceruletide restored the lowered level of dopamine-stimulated or Gpp(NH)p-stimulated adenylate cyclase activity in the membranes from haloperidol-treated rats to control levels. These findings indicate that systemically administered ceruletide is capable of reversing alterations in D₁ dopamine receptor/D₁ dopamine receptor coupling to adenylate cyclase in striatum and frontal cortex induced by chronic treatment of rats with haloperidol decanoate.     

Release of [3H]acetylcholine from a modified rat phrenic nerve-hemidiaphragm preparation

Summary Two different preparations of the rat phrenic nerve-hemidiaphragm (whole nerve-muscle preparation, end-plate preparation) were used for studying synthesis and release of radioactive acetylcholine in the absence and presence of cholinesterase inhibitors.When the whole nerve-muscle preparation (110–180 mg) was incubated with [³H]choline, only small amounts of radioactive acetylcholine were synthesized within the tissue. Electrical nerve stimulation of the whole nerve-muscle preparation produced no increase in tritium outflow.Incubation of the end-plate preparation (16–29 mg) which was obtained after removal of most of the muscle mass led to the formation of large amounts of [³H]acetylcholine. Synthesis depended on nerve activity and increased 13-fold during a high loading stimulation (50 Hz), as compared to the synthesis at rest. In a denervated end-plate preparation the formation of [³H]acetylcholine was reduced to 4% of the control preparation. Electrical nerve stimulation of the end-plate preparation produced a release of tritium that could be attributed entirely to the release of [³H]acetylcholine. The stimulated tritium efflux was completely suppressed in a calcium-free medium or in the presence of tetrodotoxin (300 nM). Release could even be detected during a short train of 50 pulses (5 Hz) with a fractional release of about 0.04% of the [³H]acetylcholine tissue content per pulse.It is concluded that the large muscle mass interferes with nerve labelling by a reduction of the [³H]choline supply to the nerve terminals when the whole nerve-muscle preparation is used. Removal of most of the muscle fibres reduces the possibility for [³H]choline to be captured by them and then more radioactive choline can enter the end-plate region. From this end-plate preparation a calcium-dependent release of radioactive transmitter can be measured in the absence of cholinesterase inhibitors.     

Determination of urinary excretion of a methadone metabolite as an indirect measurement of methadone metabolism in the rat

The relationship between the metabolism of methadone in the rat liver and the urinary excretion of methadone metabolites was studied. The concentrations of water-soluble metabolite ([¹⁴C]WSM) of methadone in the liver and urine, expressed as a percentage of total ¹⁴C in the liver or urine, increased with time after [¹⁴C]methadone administration. Acute administration of desipramine (20 mg/kg, ip) I hr before sc administration of [¹⁴C]methadone (5 mg/kg body weight, 3 μCi/mg) decreased the percentage of total ¹⁴C as [¹⁴C]WSM in both liver and urine 3 hr after [¹⁴C]methadone administration. Conversely, phenobarbital pretreatment increased the percentage of total ¹⁴C as [¹⁴C]WSM in liver and urine. The changes in percentage of total ¹⁴C as [¹⁴C]WSM in the urine of rats pretreated with desipramine and phenobarbital were directly proportional to the changes of this percentage in the liver. When the data obtained from rats pretreated with desipramine, phenobarbital, diazepam, and methadone were pooled together, a highly significant correlation between the percentage of total ¹⁴C as [¹⁴C]WSM in the urine and liver was established. The results suggest that determination of percentage of total ¹⁴C as [¹⁴C]WSM in urine reflects the rate of methadone metabolism in the liver and provides an indirect measurement of methadone metabolism in vivo.