Effects of the 5-HT2 receptor antagonist ritanserin on halothane-induced increase of inositol phosphates in porcine malignant hyperthermia

Recent studies have shown a significant increase of inositol phosphates (IPs) in skeletal muscle during episodes of halothane-induced malignant hyperthermia (MH) in pigs. After treatment with dantrolene and disappearance of MH crisis the IP concentrations returned to basal levels. In order to examine if the increase of IPs during halothane-induced MH may be related to an enhanced IP synthesis in response to activation of 5-HT₂ (5-hydroxytryptamine) receptors, the effects of ritanserin, a selective 5-HT₂ receptor antagonist, on IP levels were investigated. Biopsies of skeletal muscle of the hindlimbs were obtained in random order and IPs were determined in homozygous MH-susceptible (MHS) and MH-non-susceptible (MHN) swine in the following order: (1) basal, (2) after treatment with ritanserin (2.0 mg/kg), (3) after halothane challenge (3 vol% for 20 min). Basal concentrations of all IPs were higher in MHS than in MHN swine. Ritanserin did not cause any significant changes of IP levels compared to the basal concentrations in MHS and MHN pigs. In MHS pigs, ritanserin did not prevent a halothane-induced MH-crisis. After halothane challenge, 1,3,4-IP₃, 1,3,4,6-IP₄ and 1,3,4,5-IP₄ levels were increased in MHS (during MH crisis) vs. basal concentrations, whereas no changes were found in MHN pigs. Since the increases of IP levels in MHS pigs during MH crisis found in the present study were comparable to those without pretreatment with ritanserin, shown by recent studies, it may be concluded that ritanserin does not prevent the increase of IPs during a halothane-induced MH. Thus, the present data indicate that increases of IP levels during halothane-induced MH in swine are due to other mechanisms than 5-HT mediated enhancement of IP synthesis.     

Changes in extracellular calcium within the physiological range influence receptor-mediated inositol phosphate responses in brain and tracheal smooth muscle slices

Summary The effect of changes in extracellular calcium concentration ([Ca²⁺]_e) on the incorporation of myo-[2-³H]-inositol into phosphoinositides and agonist-stimulated ³H-inositol phosphates (³H-InsPs) was examined in rat cerebral cortex and bovine tracheal smooth muscle slices. In brain slices, reduction in [Ca²⁺]_e from 2.4 to 1.2 mmol/l resulted in an approximate doubling of the carbachol and noradrenaline-stimulated ³H-InsP response with no effect on the EC₅₀ values. An identical effect of varying [Ca²⁺]_e was observed for carbachol-stimulated ³H-InsP formation in tracheal smooth muscle with a further increase in ³H-InsPs evident at [Ca²⁺]_e 0.6 mmol/l. In this tissue the effect of changes in [Ca ²⁺]_e on the incorporation of myo-[2-³H]-inositol into the total phosphoinositide pool directly paralleled the changes in ³H-InsPs except in conditions of no added calcium when ³H-InsP responses were markedly impaired. Additional studies in brain slices using buffer where the added calcium varied between 0 and 2.4 mmol/l, showed that both the carbachol stimulated formation of separate inositol phosphates during short incubation periods and incorporation of myo-[2-³H]-inositol into PtdInsP and PtdInsP₂ under basal conditions was maximal at [Ca²⁺]_e 0.3 mmol/l. Omitting Ca²⁺]_e from the buffer resulted in maximal labelling of PtdIns but a decrease in PtdInsP and PtdInsP₂ labelling (compared with the level at [Ca²⁺]_e 0.3 mmol/l) and a markedly impaired inositol polyphosphate response. Alterations in [Ca²⁺]_e following ³H-inositol labelling but immediately prior to carbachol stimulation did not influence ³H-inositol polyphosphate responses. It is therefore clear that even relatively small changes in [Ca²⁺]_e markedly influence agonist-stimulated ³H-InsP responses in brain and tracheal smooth muscle slices and that these reflect changes in the labelling of substrate inositol lipids. These findings have important practical implications for studies examining ³H-InsP responses in central and peripheral tissues and the differential effect of very low [Ca²⁺]_e on PtdIns and PtdlnsP/PtdInsP₂ labelling may explain in part the severe decrease in ³H-InsPs seen under these conditions despite apparent maximal total phosphoinositide labelling. Send offprint requests to S. R. Nahorski at the above address     

Effect of desipramine on inositol phosphate formation and inositol phospholipids in rat brain and human platelets.

To examine the mechanism of action of antidepressant drugs, we studied the effect of desipramine (DMI) in vitro on agonist-stimulated inositol phosphate formation and inositol phospholipids in rat brain and human platelets. We observed that DMI inhibited thrombin-stimulated 3H-inositol bisphosphate (IP2) and 3H-inositol trisphosphate (IP3) but not 3H-inositol monophosphate (IP1) formation in human platelets. DMI also inhibited norepinephrine (NE) and serotonin (5-HT) stimulated 3H-IP1 formation in rat cerebral cortex. DMI increased levels of all three 3H-inositol phospholipids, 3H-phosphatidyl inositol (PI), 3H-PI-4-phosphate (PIP), and 3H-PI 4,5-bisphosphate (PIP2), in both platelets and rat cortex. The decreased formation of inositol phosphates and increased levels of [3H]-PI, [3H]-PIP, and [3H]-PIP2 by DMI appears to be due to the inhibition of the enzyme phospholipase C rather than its effects on receptors. It is thus possible that interaction of tricyclic antidepressant drugs with the PI-signaling system may be related to their mechanism of action.     

Studies of carbohydrate metabolism in rat kidney after pharmacological blockade of the pentose phosphate pathway

Summary After application of 6-aminonicotinamide (6-AN), a strong accumulation of 6-phosphogluconate was found in rat kidney. The accumulation of 6-phosphogluconate influences the activity of the phosphoglucose isomerase.The enzymic determination of the substrate concentrations of the carbohydrate metabolism after application of 6-AN revealed that fructose-6-phosphate and lactate as well as phosphoenolpyruvate are significantly increased in the kidney as compared to the controls. The increase in phosphoenolpyruvate might be caused by stimulated gluconeogenesis, as the application of 6-AN increases the release of corticosterone from the adrenal cortex.After adrenalectomy, the accumulation of 6-phosphogluconate is decreased, the ratio glucose-6-phosphate/fructose-6-phosphate is normalized, and also the values for lactate and phosphoenolpyruvate decrease.The effects of adrenaline seem to involve the passage of glucose through the pentose phosphate pathway. There is probably a relationship between the blockade of the oxidative pentose phosphate pathway and the stimulation of gluconeogenesis in the kidney, the mechanism of which is unknown.In contrast to the findings in the brain, no changes could be established in the concentrations of citrate and 2-oxoglutarate.This work was supported by a grant from the Deutsche Forschungsgemeinschaft.We wish to thank Mrs. Ingrid Monden and Miss Kristina Vangehr for their skilful technical assistance in this work.     

Muscarinic cholinoceptor subtypes mediating tracheal smooth muscle contraction and inositol phosphate generation in guinea pig and rat.

The effects of the muscarinic cholinoceptor antagonists atropine (non-selective), pirenzepine (M1-selective), methoctramine (M2-selective) and 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP; M3-selective) were examined on the responsiveness of guinea pig and rat tracheal tissue to acetylcholine and carbachol. Results indicate that smooth muscle contraction in isolated tracheal tissue from both species was mediated primarily by muscarinic M3 cholinoceptors. The effects of atropine, pirenzepine and 4-DAMP were similar against the contractile actions of acetylcholine and carbachol in both species and in epithelium-intact and epithelium-denuded tissue. In contrast, differences in the effects of methoctramine in antagonising contractile responses to acetylcholine and carbachol were observed between the two species and following epithelium removal in the guinea pig. Thus, whilst this study has found that tracheal smooth muscle contraction in the guinea pig and rat is mediated primarily by muscarinic M3 cholinoceptors, anomalies in the functional inositol phosphate generation results obtained with the muscarinic cholinoceptor antagonists highlight species differences in the actions of acetylcholine and carbachol in eliciting smooth muscle contraction suggesting the possible existence of functional non-M3 muscarinic cholinoceptors.     

Effect of bremazocine,a kappa-opioid receptor agonist,on inositol phosphate formation in isolated iris-ciliary bodies

The goal of the current study was to examine the effect of the kappa-opioid agonist, bremazocine (BRE), on inositol phosphate (IP) formation in the rabbit iris-ciliary body (ICB). Concentrations of BRE (10(-7) to 10(-5) M) augmented levels of IP. Incubation of ICBs with BRE (10(-6) M) produced a time-dependent increase in IP levels that peaked at 60 s and declined to basal levels by 5 min. The increase in IP levels produced by BRE (10(-6) M) was inhibited by the kappa-opioid receptor antagonist, nor-binaltorphimine (nor-BNI, 10(-7) to 10(-5) M) and by activation of PKC with PDBu (10(-7) M). These results demonstrate that kappa-opioid receptor activation by BRE in the rabbit ICB is linked to IP production. Thus, opioid agonist-induced increases in IP activity could play a role in BRE-induced increases in atrial natriuretic peptide release and alterations in aqueous humor dynamics.     

Effect of serotonin on cyclic AMP level in rat hypothalamus slices during development.

The effect of serotonin (5-HT) on cyclic AMP levels in rat hypothalamic slices was age-dependent. The sensitivity of the cyclic AMP system to 5 X 10(-5) M 5-HT already existed in the foetus at 21 days of pregnancy. It reached a maximum on the 7th postnatal day and then decreased with age. In adult tissue the response was still present and was antagonized by two serotonergic antagonists (methiothepin and metergoline). When compared to the progressive increase of 5-HT level in the hypothalamus the data suggest a different evolution of serotonergic innervation and of 5-HT receptors.     

Selective dopaminergic mechanism of dopamine and SKF38393 stimulation of inositol phosphate formation in rat brain.

We have previously reported that dopamine and the D1 receptor-selective agonist, SKF38393, stimulate the formation of inositol phosphates in rat brain slices (Undie and Friedman, 1990, J. Pharmacol. Exp. Ther. 253, 987). The present experiments were conducted to determine if actions at alpha-adrenoceptors or at serotonergic sites may contribute to, or interact with, the observed stimulation of phosphoinositide hydrolysis by dopamine receptor agonists. Rat striatal slices prelabeled with [3H]inositol were treated with up to 500 microM dopamine, norepinephrine, serotonin (5-HT), or the dopamine D1 receptor agonist, SKF38393, and accumulated inositol phosphates determined. The action of norepinephrine was dose-dependently blocked by the selective alpha 1-adrenoceptor antagonist, prazosin, but not by SCH23390. The actions of dopamine and SKF38393 were dose-dependently blocked by the dopamine D1 receptor antagonist, SCH23390, but not by prazosin. The effects of 5-HT were blocked by the nonselective 5-HT antagonist, methiotepin, the selective 5-HT2 antagonist, ketanserin, the mixed 5-HT2/5-HT1C antagonist, mianserin, and, with much less potency, by the selective 5-HT1C antagonist, mesulergine. On the contrary, the serotonin receptor antagonists did not block the response to SKF38393, and there was no dose-dependent blockade of the 5-HT response by SCH23390. These observations indicate that the actions of dopamine and SKF38393 in stimulating inositol phosphate formation are selectively mediated through a D1-like dopamine receptor.     

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

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

Effect of hyperosmotic conditions on flavin-containing monooxygenase activity,protein and mRNA expression in rat kidney

Flavin-containing monooxigenases (FMOs) are a polymorphic family of drug and pesticide metabolizing enzymes, found in the smooth endoplasmatic reticulum that catalyze the oxidation of soft nucleophilic heteroatom substances to their respective oxides. Previous studies in euryhaline fishes have indicated induction of FMO expression and activity in vivo under hyperosmotic conditions. In this study we evaluated the effect of hypersaline conditions in rat kidney. Male Sprague–Dawley rats were injected intraperitoneal with 3.5 M NaCl at a doses ranging from 0.3 cm³/100 g to 0.6 cm³/100 g in two separate treatments. Three hours after injection, FMO activities and FMO1 protein was examined in the first experiment, and the expression of FMO1 mRNA was measured in the second experiment from kidneys after treatment with NaCl. A positive significant correlation was found between FMO1 protein expression and plasma osmolarity (p < 0.05, r = 0.6193). Methyl-p-tolyl sulfide oxidase showed a statistically significant increase in FMO activity, and a positive correlation was observed between plasma osmolarity and production of FMO1-derived (R)-methyl-p-tolyl sulfoxide (p < 0.05, r = 0.6736). Expression of FMO1 mRNA was also positively correlated with plasma osmolality (p < 0.05, r = 0.8428). Similar to studies in fish, these results suggest that expression and activities of FMOs may be influenced by hyperosmotic conditions in the kidney of rats.     

Effect of clofibrate on DNA synthesis in rat liver and kidney

A single dose of clofibrate (400 mg/kg), given to rats, increased the incorporation of (^3H)thymidine into liver DNA, in a period of 20–30 h after administration. However, (^3H)thymidine incorporation into hepatic DNA of rats treated repeatedly was identical to that of control animals. After the administration of a single dose of clofibrate a small increase in (^3H)thymidine incorporation also occurred in kidney DNA; repeated doses, however, resulted in a marked suppression of labeling.Dedicated to Professor Dr. med. Herbert Remmer on the occasion of his 65th birthday     

Effect of nucleotides on the cytosolic free calcium activity and inositol phosphate formation in human glomerular epithelial cells.

1. Glomerular epithelial cells (GEC) were cultured from human kidneys and immunologically characterized. 2. The effect of extracellular nucleotides on the cytosolic free calcium activity [Ca2+]i was investigated with the fura-2 microfluorescence method. Extracellular UTP, UDP, UMP, ATP, adenosine 5'-O-(3-thio)-trisphosphate (ATP-gamma-S), inosine-triphosphate (ITP), guanyltriphosphate (GTP), 2-methylthio-ATP, AMP, alpha,beta-methylene-ATP and adenosine led to a rapid, transient, concentration-dependent increase of [Ca2+]i, followed by a plateau above the baseline level. 3. In a calcium-free extracellular solution, the rapid increase of [Ca2+]i was still present, whereas the plateau level was abolished. 4. ATP and UTP (ED50 both: 10(-5) M) stimulated inositol trisphosphate (InsP3) formation in GEC. 5. The order of potency for the purine nucleotides in stimulating InsP3 formation was ATP = ATP-gamma-S greater than ADP greater than 2-methylthio-ATP greater than AMP = a,beta methylene-ATP = adenosine. 6. The increase of InsP3 induced by ATP (10(-5) M) could be inhibited by the P2 receptor blocker suramin (greater than 10(-4) M). Reactive blue 2 exhibited a weak stimulating effect on the InsP3 formation and only a weak inhibitory effect at a concentration of 10(-3) M was observed. 7. Protein kinase C activation by preincubation of GEC with phorbol 12-myristate 13-acetate (PMA, 100 ng ml-1, 15 min) abolished the effect of ATP (10(-5) M) on InsP3 formation. Downregulation of protein kinase C by long term incubation (18 h) with PMA had no significant effect on the phosphoinositol turnover induced by ATP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of dioxane on cytochrome P450 enzymes in liver, kidney, lung and nasal mucosa of rat

The effect of acute and chronic dioxane administration on hepatic, renal, pulmonary and nasal mucosa P450 enzymes and liver toxicity were investigated in male rats. The acute treatment consisted of two doses (2 g/kg) of dioxane given for 2 days by gavage, whereas the chronic treatment consisted of 1.5% of dioxane in drinking water for 10 days. Both the acute and chronic dioxane treatments induced cytochrome P450 2B1/2- and P450 2E1-dependent microsomal monooxygenase activities (pentoxyresorufin O-depentylase and p-nitrophenol hydroxylase) in the liver, whereas in the kidney and nasal mucosa, only the 2E1 marker activities were enhanced. In addition in the liver, an induction of 2-testosterone hydroxylase (associated with the constitutive and hormone-dependent P450 2C11) was also revealed, whereas the hepatic P450 4A-dependent -lauric acid hydroxylase was not enhanced by any dioxane treatment. These inductions were mostly confirmed by western blot analysis of liver, kidney and nasal mucosa microsomes. In the lung, no alteration of P450 activities was observed. To assess the mechanism of 2E1 induction, the hepatic, renal and nasal mucosa 2E1 mRNA levels were also examined. Following two kinds of dioxane administration, in the liver the 2E1 induction was not accompanied by a significant alteration of 2E1 mRNA levels, while both in the kidney and nasal mucosa the 2E1 mRNA increased about 2- to 3-fold, indicating an organ-specific regulation of this P450 isoform. Furthermore, dioxane was unable to alter the plasma alanine aminotransferase activity and hepatic glutathione (GSH) content, examined as an index of toxicity, when it was administered into rats with P450 2B1/2 and 2E1 preinduced by phenobarbital or fasting pretreatment. These results support the lack of or a poor formation of reactive and toxic intermediates during the biotrasformation of this solvent, even when its metabolism was enhanced by P450 inducers. The chronic administration of dioxane was also unable to induce the palmitoyl CoA oxidase, a marker of peroxisome proliferation, excluding this as a way to explain its toxicity. Thus, although the mechanism of dioxane carcinogenicity remains unclear, the present results suggest that the induction of 2E1 following a prolonged administration of dioxane might provide oxygen radical species, and thereby contribute to its organ-specific toxicity.     

The effects of dietary selenium on cadmium binding in rat kidney and liver

In acute studies, approximately 70–90% of cytosolic cadmium in liver and kidney has been shown to be bound to metallothionein, a low-molecular weight protein. In this study, we report on the influence of dietary selenium on the distribution of cadmium in rat kidney and liver. Contrary to the findings of most acute studies, our results indicate that only a relatively small proportion of cadmium (approximately 14% in the kidney and 44% in the liver) is bound to metallothionein when cadmium is administered for 7 weeks in the diet and via osmotic minipumps to selenium-deficient rats. Feeding rats the same diet supplemented with 1.0 ppm selenium results in no detectable cadmium-metallothionein peak in the kidney, and only about 10% of the cytosolic cadmium elutes as cadmium bound to metallothionein in the liver. In animals fed the selenium-supplemented diet, the bulk of the cadmium is recovered in the low-molecular weight fraction. Dietary selenium did not significantly affect the distribution of zinc and copper in the kidney or liver.Supported by NIEHS Center Grant ES-00159 and a grant from the Selenium — Tellurium Development Association     

Effects of the diuretics furosemide,ethacrynic acid,and chlorothiazide on gluconeogenesis from various substrates in rat kidney cortex slices

Summary The effects of wide concentration ranges of furosemide, ethacrynic acid, and chlorothiazide on glucose formation by rat kidney cortex slices were investigated from fructose, pyruvate, lactate, and -ketoglutarate and partly at two different pH.Under control conditions without the drugs, more glucose was synthetized in moles/g dry weight · hour at the lower pH of 7.21 than at pH 7.49 without substrate as well as with the 5 substrates studied.Furosemide stimulated the rate of gluconeogenesis concentration dependent if fructose, pyruvate, or lactate were present as substrates. The percent increase was higher at pH 7.49 but the rates achieved with the maximal effective concentrations were about the same at both pH. The rate of glucose formation was only slightly enhanced from the substrates malate and -ketoglutarate. Ethacrynic acid enhanced the rate of glucose synthesis from fructose, pyruvate, and lactate, too, but had no stimulating effects, if malate or -ketoglutarate were the substrates. It exerted its effects at even lower concentrations than furosemide, but the maximal rates which could be observed were in the same range. Gluconeogenesis was not stimulated by chlorothiazide.Various and different sites of action on enzyme activities in the Embden-Meyerhof pathway are discussed for the two drugs influenced either directly by them or indirectly by changing concentrations of ATP or cyclic AMP.Preliminary reports of the results were presented in part at the joint meeting of the German and the Scandinavian Pharmacological Societies in Copenhagen 1971 and at the meeting of the Gesellschaft für Nephrologie in Aachen 1971.Supported by Deutsche Forschungsgemeinschaft Fu 45/4.     

硼替佐米对大鼠肾移植AMR的保护作用及机制研究

目的研究硼替佐米对肾移植抗体介导排斥反应(AMR)的保护作用及机制。方法采用输血致敏法构建大鼠肾移植AMR模型。在此模型基础上,静脉注射硼替佐米,研究其对移植肾功能和病理损害的保护作用。流式细胞仪检测供体特异性抗体(DSA)的水平,免疫组织化学检测移植肾C4d表达,免疫荧光法检测CD68表达以反映巨噬细胞的浸润。结果硼替佐米可以明显改善肾移植AMR大鼠移植肾功能(P<0.05),减轻移植肾病理损害。硼替佐米显著降低大鼠肾移植AMR模型血清中IgM、IgG 2b和IgG 2c等DSA亚型的水平(P<0.05),同时抑制移植肾组织中C4d的表达和巨噬细胞浸润。结论硼替佐米对肾移植AMR具有明显的保护作用,其机制可能与抑制受体DSA产生及减少移植肾组织中巨噬细胞浸润有关。     

Effects of ageing on muscarinic receptor subtypes and function in rat urinary bladder

We compared the density and function of M₂ and M₃ muscarinic acetylcholine receptor subtypes in the urinary bladder of young adult (3 months) and old (23 months) male Wistar rats. Old rats had a reduced density of muscarinic receptors (96±10 vs. 156±21 fmol/mg protein), but competition experiments with the M₃-selective darifenacin did not indicate alterations in the relative roles of M₂ and M₃ receptors, with the former being more abundant. The amount of immunodetectable -subunits of various G-proteins potentially linked to muscarinic receptor function was unchanged. The potency of carbachol to contract bladder strips was also unaltered; its maximum effects as well as those of a single KCl concentration were unchanged if raw data or those corrected for strip length were analysed, but somewhat reduced when those corrected for strip weight were analysed. Antagonistic effects of atropine, the M₂-selective Ro 320–6206 and the M₃-selective darifenacin were unchanged. Agonistic effects of the M₃-sparing agonist 4-(2-oxo-2,3-dihydro-benzoimidazol-1-yl)-[1,4]bipiperidinyl-1-carboxylic acid ethyl ester were similarly poor in young and old rats. Additional experiments were concomitantly performed in submandibular glands from the same animals. While total muscarinic receptor density in submandibular glands was not significantly affected by age (56±5 vs. 61±4 fmol/mg protein), the relative contribution of M₃ receptors significantly declined from 68±3% to 57±2% based upon darifenacin competition curves. We conclude that aged Wistar rats express fewer muscarinic receptors in their urinary bladder, but there is no change in the relative abundance of M₂ and M₃ receptors; this is accompanied by only minor if any alterations in receptor responsiveness. In contrast, submandibular gland expresses similar receptor numbers in young and old rats, but slightly fewer M₃ receptors in old animals.     

Myocardial adenine nucleotides,hexose phosphates and inorganic phosphate,and the regulation of phosphofructokinase activity during fluoroacetate poisoning in the rat

Myocardial levels of adenine nucleotides, inorganic phosphate (Pi) and hexose phosphates were measured as a function of the time after fluoroacetate (FAc) administration (6 mg/kg i.p.) to the rat. ATP content was progressively depleted, reaching at 6 hr 60 per cent and at 18 hr 45 per cent of the control values. AMP and ADP levels increased during the initial 2 hr and later declined. Intracellular Pi accumulated in great amounts, with a maximum at 4 hr, while serum Pi increased continuously throughout the experimental period. Citrate levels reached a maximum at 6 hr and remained nearly constant thereafter. These results suggest that the energy reserves of the tissue are progressively exhausted during the intoxication, and that the reactivation of the citric acid cycle by the accumulated citrate, postulated by previous authors, does not occur under the present conditions. Fructose diphosphate (FDP) levels were unaltered during the intoxication, while fructose-6-phosphate (F6P) and glucose-6-phosphate only showed transient initial increases. The FDP/F6P ratio, which is indicative of intracellular phosphofructokinase (PFK) activity, was not significantly altered, in spite of the striking changes produced in the levels of PFK effectors. This suggests that the PFK activation usually associated with a depletion of high-energy phosphates and Pi accumulation is blocked in the poisoned tissue. In vitro experiments were performed in which PFK activity was evaluated in the presence of concentrations of substrates and metabolites simulating those found in vivo. It was observed that in the physiological pH range (6·9–7·1) PFK is strikingly activated in the assays corresponding to intoxicated hearts, regardless of the high citrate levels. This suggests that the activation associated with the fall of ATP is quantitatively more important than the citrate inhibition. However, at lower pH values this activation is not produced, irrespective of the levels of ATP, AMP, Pi or citrate. It is suggested that intracellular acidification, possibly associated with the accumulation of citric acid, might be the main factor responsible for the blockade of PFK activation observed in the intoxicated hearts in vivo.     

Potassium bromate content of selected bread samples in Ilorin,Central Nigeria and its effect on some enzymes of rat liver and kidney

Bread samples from five different locations (Gaa-Akanbi, Saw-Mill, Oloje, Fate-Basin and Zango) in Ilorin metropolis, Central Nigeria were analyzed for their potassium bromate content before they were employed as a source of carbohydrate in the formulation of diet for albino rats. A total of sixty (60) albino rats (Rattus norvegicus) were grouped into six (6) of ten (10) rats each. The rats in the first group served as control and they were placed on diet formulated with bromate-free bread. Animals in Groups 2–6 were placed on diet formulated with bread samples obtained from the five different locations in Ilorin metropolis. At the expiration of thirty (30) days feeding period, the animals were sacrificed and blood samples, liver and kidney tissues were collected for the assay of ALP, AST and ALT activities. The results showed a significant reduction (p < 0.05) in the activities of these enzymes in the tissues when compared with the control. However, a significant increase (p < 0.05) was observed in the activities of the selected serum enzymes. Overall, the data indicate adverse effects on the liver and kidney of rats fed on diet containing potassium bromate.     

The distribution and density of receptor subtypes for endothelin-1 in peripheral lung of the rat, guinea-pig and pig.

1. Quantitative autoradiographic studies were conducted to determine the distributions and densities of endothelin-A (ETA) and ETB receptor subtypes in peripheral lung alveolar wall tissue of the rat, guinea-pig and pig, with a view to assessing the potential suitability of these tissues as models for investigations of ET receptor function in human alveolar tissue. 2. High levels of specific [125I]-ET-1 binding were detected in peripheral lung components from all three species tested. In mature porcine alveolar wall tissue, specific binding increased in a time-dependent manner to a plateau, consistent with the previously described pseudo-irreversible binding of this ligand to a finite population of specific binding sites. 3. [125I]-ET-1 was associated specifically with both ETA and ETB binding site subtypes in alveolar wall tissue of foetal pig lung as early as 36 days gestation, raising the possibility of a functional role for ET-1 in lung development. In addition, both ETA and ETB binding site subtypes were detected in alveolar wall tissue and in peripheral airway smooth muscle of mature lung parenchyma from all three species. However, the binding subtype proportions differed in these tissues. For example, in porcine peripheral bronchial smooth muscle, ETA sites apparently predominated, whereas ETB sites constituted the major subtype detected in alveolar wall in this species. These data suggest significant shifts in ET receptor subtype expression at different levels in the respiratory tract. 4. ET binding site subtype proportions in the alveolar wall also differed markedly between species. In rat lung alveoli, ETA and ETB sites were detected in similar proportions (52 +/- 3% and 43 +/- 5% respectively). In contrast, in guinea-pig peripheral lung, ETB binding sites clearly predominated, constituting approximately 80% of total specific binding, with ETA sites accounting for only 12%. Porcine alveolar wall tissue also contained a mixture of these ET receptor subtypes, with ETA and ETB binding comprising 23 +/- 3% and 65 +/- 1% respectively of the total population of specific binding sites detected. These latter proportions are similar to values previously obtained in human peripheral lung tissue, suggesting that porcine lung might be a useful model of the human peripheral lung in subsequent studies of the functions of these pulmonary ET receptor subtypes.