Canadian Journal of Physiology and Pharmacology

Published by NRC Research Press
Online ISSN: 1205-7541
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Article
The chronic toxicity of sodium chloride was studied in young male albino rats given 2.57–6.14 g/kg in water by stomach tube once daily for 100 days or until half the animals had died, whichever occurred first. The LD50(0.1 L) or daily dose which killed 50% of the animals after administration for 100 days, i.e. 1/10 the animal's normal lifespan (0.1 L), was 2.69 ± 0.12 g/kg. It is suggested that the LD50(0.1 L) expressed as a percentage of the acute LD50 would provide a useful index of chronic toxicity; this is termed the C/A LD50(0.1 L) index, C meaning chronic and A acute. The value of this index was 72 for sodium chloride, 62 for benzylpenicillin, and 13 for atropine. Rats which survived doses of the order of the LD50(0.1 L) had no change in food intake but lost some body weight as daily dose increased. They had a dose-dependent polydipsia and polyuria. In the initial month of drug administration the rats developed a slight fever, proteinuria, and alkalinuria, and in the terminal month a slight hypothermia and aciduria, all of which were statistically significant but not dose-dependent. When deaths occurred within the first week or two, they were similar clinically and pathologically to those seen in studies on the acute oral toxicity of sodium chloride. Other deaths followed a period of hypothermic cachexia and were due to bronchopneumonia, associated with hepatitis, nephritis, arteriolitis, and occasionally encephalopathy, and accompanied by degeneration of the thymus, adrenals, and testes. Animals which survived for 100 days had developed a hypertrophied gastrointestinal mucosa but most other organs had lost weight, and there was some arteriolitis, myocarditis, pulmonary edema, and nephritis.
 
Article
The ability of a mild irritant to reduce ethanol-induced damage to the rat gastric mucosa was investigated using an ex vivo gastric chamber preparation. Exposure to 0.25 M hydrochloric acid (HCl) did not cause significant damage to the surface epithelium, but did reduce both the lesion area and the extent of superficial epithelial damage caused by subsequent exposure to 40% ethanol (EtOH). "Adaptive cytoprotection" was also demonstrated by the reduction of ethanol-induced changes in transmural potential difference and net K+ efflux, and by rapid recovery of these physiological parameters following the removal of ethanol from the chamber. Pretreatment of rats with indomethacin at a dose that has been shown to significantly inhibit gastric cyclooxygenase activity did not significantly affect the ability of 0.25 M HCl to reduce the effects of ethanol on lesion area, epithelial damage, potential difference, and net K+ efflux.
 
Article
We examined whether there was a minimal change in fetal arterial Po2 necessary to elicit alterations in plasma adrenocorticotropic hormone, arginine vasopressin, or cortisol or to affect the incidence of breathing movements or eye movements in fetal sheep at 106-117 days of gestation. Fetal sheep were exposed to two levels of hypoxemia, mild (4.1 mmHg Po2 drop) (1 mmHg = 133.32 Pa) and moderate (8.4 mmHg Po2 drop), for 1 h without acidemia. Hypoxemia was induced by altering the inspired percent oxygen of the mother. No significant hormonal and biophysical changes were observed in mild hypoxemia. In moderate hypoxemia, there were significant increases of fetal adrenocorticotropic hormone and arginine vasopressin and decreased incidence of fetal breathing movements. However, there were no significant changes in cortisol or eye movements. We conclude that a fetal arterial Po2 drop of between 4.1 and 8.4 mmHg is necessary to elicit responses to hypoxemia in fetal sheep at 106-117 days of gestation in adrenocorticotropic hormone, arginine vasopressin, and fetal breathing movements, but this degree of hypoxemia does not cause changes in cortisol or fetal eye movements.
 
Article
The current proposed mechanism of action of nitrovasodilator drugs involves biotransformation to nitric oxide, which is postulated to be the active vasodilator substance. Our objective was to determine whether nitric oxide was formed from two prototype nitrovasodilator drugs, glyceryl trinitrate (GTN) and sodium nitroprusside (SNP), after incubation with bovine pulmonary vein (BPV) preparations. GTN or SNP was incubated in an argon atmosphere with phosphate buffer, BPV homogenate, or the 10,000 x g supernatant fraction of the homogenate. Nitric oxide formation, as determined by a chemiluminescence-headspace gas method, was measurable following the incubation of SNP with BPV homogenate and 10,000 x g supernatant. There was no detectable formation of nitric oxide from the incubation of GTN with the two BPV preparations, although GTN was biotransformed to glyceryl dinitrate, as determined by gas-liquid chromatography. There was decreased recovery of nitric oxide during the incubation of authentic nitric oxide with the two BPV preparations as compared with buffer. In conclusion, formation of nitric oxide was measured for the interaction of SNP, but not GTN, with BPV preparations. However, the data do not exclude the possible formation of nitric oxide from GTN, as nitric oxide was shown to be sequestered or transformed by the BPV preparations.
 
Body mass (kg), at different times during the experi- ment, of rabbits fed the casein-containing diet. 
Effect of D-003 (50 mg/kg during 30 days) on the heparinsensitive binding of human [ 125 I]-LDL to liver homogenates. The results, expressed as nanograms [ 125 I]-LDL bound per milligram of protein, represent heparin-sensitive binding calculated by subtracting the radioactivity of the filters incubated with heparin (heparin-resistant binding) from the radioactivity of the filter incubated in the absence of heparin (total binding). The nonspecific (heparin-resistant) binding was about 50% of the total binding, and the c.v. of the assay was 12%. Bars show means ± SD for homogenates of liver of five rabbits measured in triplicate. **, significantly different from control, P < 0.01 (MannWhitney U test). 
Article
D-003 is a mixture of very long chain saturated fatty acids (VLCSFA) purified from sugar cane wax with cholesterol-lowering effects proven in animal models and healthy volunteers. D-003 inhibits cholesterol biosynthesis through the regulation of HMG-CoA reductase activity. Rabbits fed diets enriched with casein develop endogenous hypercholesterolemia (EH), making them a very useful model for determining the mechanism of action of drugs affecting lipids. We examined whether D-003 prevented EH. Rabbits were fed a casein diet for 4 weeks, administered simultaneously with D-003 (5, 50, and 100 mg.kg-1.day-1). As expected, nontreated rabbits became hypercholesterolemic; however, as early as 15 days following administration, the treated group (50 and 100 mg.kg-1.day-1) had significantly decreased total cholesterol and low-density lipoprotein cholesterol (LDL-C). Triglycerides were not affected; however, at study completion, HDL-C levels significantly increased at all the doses assayed. D-003 inhibited de novo synthesis of cholesterol, since the incorporation of 3H2O into sterols in the liver and proximal small bowel was significantly depressed. Also, D-003 significantly raised the rate of removal of [125I]-LDL from serum and significantly elevated [125I]-LDL binding activity to liver homogenates. Taken together, these results show that the efficacy of D-003 in reducing casein-derived hypercholesterolemia could involve, at least partially, an inhibition of hepatic cholesterol biosynthesis, which may elicit a decreased cholesterol concentration in hepatocytes, preventing the loss of hepatic LDL receptors induced by casein administration. However, since casein-induced hypercholesterolemia is also a consequence of a stimulation of cholesterol absorption in the lumen and an increase of the output of cholesterol associated with LDL, the effect of D-003 on cholesterol absorption and LDL synthesis by the liver should be investigated.
 
Article
The effects of bosentan (Ro 47-0203), an endothelin A and B receptor antagonist, on responses to endothelin-1, sarafotoxin 6c, angiotensin II, and arginine vasopressin were investigated in the hind-limb vascular bed of the cat. Under constant-flow conditions, intraarterial injections of endothelin-1 and sarafotoxin 6c induced biphasic changes in hind-limb perfusion pressure characterized by an initial decrease followed by a secondary increase in perfusion pressure. The vasodilator and vasoconstrictor components of the biphasic responses to endothelin-1 and sarafotoxin 6c were reduced by bosentan, and the endothelin receptor antagonist reduced baseline systemic arterial and hind-limb perfusion pressures. Bosentan decreased vasoconstrictor responses to lower doses of angiotensin II, whereas responses to higher doses of angiotensin II and responses to vasopressin, U46619, BAY K8644, norepinephrine, acetylcholine, bradykinin, levcromakalim, PGE1, adrenomedullin, and calcitonin gene-related peptide were not altered. Vasoconstrictor responses to ET-1 were not altered by the angiotensin AT1 receptor antagonist DuP 532 or the AT2 receptor antagonist PD123,319. The results of the present study show that bosentan attenuates vasodilator and vasoconstrictor responses to endothelin-1 and sarafotoxin 6c and vasoconstrictor responses to lower doses of angiotensin II in the hind-limb vascular bed of the cat. These results suggest that endothelin may be involved in mediating responses to lower doses of angiotensin II and in the maintenance of baseline tone in the systemic vascular bed of the cat.
 
Effect of BTM-0512 on tube formation of endothelial cells impaired by high glucose (30 mmol/L, 48 h). Glucose-induced tube formation impairment of endothelial cells was improved by BTM-0512 in a concentration-dependent manner. (A) Representatives of tube formation detected by Matrigel assay (magnification 200×). (B) Summarized data of tube formation. Values are means ± SEM (n = 5). † †, P < 0.01 vs. control; *, P < 0.05; **, P < 0.01: vs. D-glucose (30 mmol/L, 48 h). 
Effect of BTM-0512 on expression of sirtuin 1 (SIRT1) and vascular endothelial growth factor (VEGF) mRNA, reactive oxygen species (ROS) production, and tumor necrosis factor-a (TNF-a) release. Glucose (30 mmol/L, 24 h) decreased the expression of SIRT1 (A) and VEGF (C), increased ROS production (B), and increased TNF-a release (D). These effects of glucose were reversed by BTM-0512 in a concentration-dependent manner. BTM-0512 (3 µmol/L) alone increased mRNA expression of SIRT1 (A), but had no effect on the baseline of intracellular ROS production, VEGF expression, and TNF-a release (B, C, and D, respectively). Values are means ± SEM (n = 4). †, P < 0.05; † †, P < 0.01: vs. control; *, P < 0.05; **, P < 0.01: vs. D-glucose (30 mmol/L, 24 h). a.u., arbitrary units. 
Article
Hyperglycemia impairs the function of endothelial cells. Sirtuin 1 (SIRT1) is involved in regulating the function of endothelial cells. Resveratrol, a polyphenol found in many plant species, exerts protective effects on endothelial cells through activation of SIRT1. The aims of this work were to explore whether BTM-0512, a novel derivative of resveratrol, is able to exert beneficial effects on high glucose-induced dysfunction of endothelial cells through regulation of SIRT1. We found that high glucose significantly impaired the function of endothelial cells as shown by reduced tube formation, cell migration, and cell adhesion concomitantly with downregulation of mRNA expression of SIRT1 and vascular endothelial growth factor as well as increased tumor necrosis factor-α release and reactive oxygen species production. These effects of high glucose were inhibited by pretreatment with BTM-0512. The beneficial effects of BTM-0512 on high glucose-induced cell dysfunction were abolished by splitomicin, a specific inhibitor of SIRT1. The regulatory effects of BTM-0512 on high glucose-induced changes in vascular endothelial growth factor mRNA expression and tumor necrosis factor-α release were also abolished by splitomicin. The results suggest that BTM-0512 exerts beneficial effects on high glucose-induced endothelial cell dysfunction through regulation of the SIRT1 - reactive oxygen species - vascular endothelial growth factor - tumor necrosis factor-α pathway.
 
Article
A recent study showed that resveratrol, a polyphenol found in many plant species, exerts dual effects on gastric mucosal injury. By using the model of ethanol-induced gastric mucosal injury in the present study, we explored the effect of trans-3,5,4'-trimethoxystilbene (BTM-0512), a novel analog of resveratrol, on gastric mucosal injury and the possible underlying mechanisms. Gastric mucosal injury in the rat was induced by oral administration of acidified ethanol. The gastric tissues were collected for determination of the gastric ulcer index, asymmetric dimethylarginine (ADMA) and nitric oxide (NO) contents, the activity of dimethylarginine dimethylaminohydrolase (DDAH) and superoxide anion (O2(-)) or hydroxyl radical (OH*) formation. The results showed that acute administration of ethanol significantly increased the gastric ulcer index concomitantly with the decrease in DDAH activity and NO content as well as the increase in ADMA content, effects that were reversed by pretreatment with BTM-0512 (100 mg/kg) or L-arginine (300 mg/kg). Administration of BTM-0512 did not show a significant effect on O2(-) or OH. formation. The results suggest that BTM-0512 could protect the gastric mucosa against ethanol-induced injury, which is mainly related to an increase in DDAH activity and subsequent decrease in ADMA content.
 
Article
MK-0591 (3-[1-(4-chlorobenzyl)-3-(t-butylthio)-5-(quinolin-2-yl-methoxy)- indol-2-yl]-2,2-dimethyl propanoic acid, previously L-686,708) is a potent inhibitor of leukotriene (LT) biosynthesis in intact human and elicited rat polymorphonuclear leukocytes (PMNLs) (IC50 values 3.1 and 6.1 nM, respectively) and in human, squirrel monkey, and rat whole blood (IC50 values 510, 69, and 9 nM, respectively). MK-0591 had no effect on rat 5-lipoxygenase. MK-0591 has a high affinity for 5-lipoxygenase activating protein (FLAP) as evidenced by an IC50 value of 1.6 nM in a FLAP binding assay and inhibition of the photoaffinity labelling of FLAP by two different photoaffinity ligands. Inhibition of activation of 5-lipoxygenase was shown through inhibition of the translocation of the enzyme from the cytosol to the membrane in human PMNLs. MK-0591 was a potent inhibitor of LT biosynthesis in vivo, first, following ex vivo challenge of blood obtained from treated rats and squirrel monkeys, second, in a rat pleurisy model, and, third, as monitored by inhibition of the urinary excretion of LTE4 in antigen-challenged allergic sheep. Inhibition of antigen-induced bronchoconstriction by MK-0591 was observed in inbred rats pretreated with methysergide, Ascaris-challenged squirrel monkeys, and Ascaris-challenged sheep (early and late phase response). These results indicate that MK-0591 is a potent inhibitor of LT biosynthesis both in vitro and in vivo indicating that the compound will be suitable for assessing the role of leukotrienes in pathological situations.
 
Article
We have investigated the inhibitory activity of compound MK-0591 (3-[1-(4-chlorobenzyl)-3-(t-butylthio)-5-(quinolin-2-yl-methoxy)-i ndol-2- yl]-2,2-dimethyl propanoic acid) on 5-lipoxygenase (5-LO) product synthesis in various human phagocytes stimulated with either the ionophore A23187, opsonized zymosan (OPZ), platelet-activating factor (PAF), or formyl-methionyl-leucyl-phenylalanine (fMLP). The lipoxygenase products were analyzed by reversed-phase HPLC. MK-0591 inhibited the formation of 5-hydroxyeicosatetraenoic acid, leukotriene (LT) B4, its omega-oxidation products, and 6-trans-isomers with IC50 values of 2.8-4.8 nM in A23187-stimulated neutrophils. In these conditions, arachidonic acid at a concentration of 10 microM had no effect on MK-0591 inhibitory activity. In neutrophils stimulated with OPZ, the synthesis of LTB4, its omega-oxidation products, and 6-trans-isomers was inhibited with IC50 values of 9.5-11.0 nM. MK-0591 inhibited 5-LO product synthesis in A23187-stimulated blood monocytes, eosinophils, and alveolar macrophages with IC50 values of 0.3-0.9, 3.7-5.3, and 8.5-17.3 nM, respectively. In neutrophils primed with granulocyte--macrophage colony-stimulating factor and stimulated with PAF, lipoxygenase product synthesis was inhibited with IC50 values of 7.7-8.7 nM. At the concentration of 1 microM, MK-0591 had no inhibitory effect on 15-lipoxygenase activity in human polymorphonuclear leukocytes, nor on human platelet 12-lipoxygenase and cyclooxygenase. In conclusion, MK-0591 is a very potent and specific inhibitor of 5-LO product synthesis in various types of human phagocytes.
 
Article
An extensive amount of research has focused on the development of new pharmacological agents to treat schizophrenia. Varying from person to person, schizophrenia is a heterogeneous disease with symptoms of positive, negative, and cognitive deficits. PRX-07034, a 5-hydroxytryptamine6 (5-HT6) receptor antagonist has been evaluated for its potential in treating obesity and cognitive deficits. This study evaluated PRX-07034 (0.1, 0.3, and 1.0 mg/kg body mass, by intraperitoneal (i.p.) injection), in combination with a low dose of prazosin (0.3 mg/kg, i.p.), for its antipsychotic potential. The research utilized a stereotypy assay, an open field test, an object recognition task, and prepulse inhibition. Dizocilpine, a non-competitive N-methyl-d-aspartate (NMDA) antagonist, was also administered in the above-mentioned assays as a psychomimetic. The combination of PRX-07034 and prazosin alleviated stereotypy and hyperlocomotor activity while enhancing memory in an object recognition task, and reversed sensory-gating deficits induced by dizocilpine. Examination of the medial prefrontal cortex revealed that a combination of PRX-07034 and prazosin reduced the dizocilpine-mediated increase of 5-HT. These results suggest that the combination of a 5-HT6 antagonist with low doses of prazosin could have therapeutic potential in the treatment of schizophrenia.
 
Article
The effects of AHN 086 and its reversibly acting structural analogue Ro 5-4864 were studied in the spontaneously beating guinea-pig atria and field-stimulated guinea-pig ileal longitudinal smooth muscle in the presence and absence of dihydropyridine calcium channel modulators. The treatment of guinea-pig atria with AHN 086 followed by extensive washing did not alter contraction. However, AHN 086 (0.5 microM) potentiated (88%) the positive inotropic responses by BAY K 8644, an effect that was not reversed by extensive washing of the tissue. Higher concentrations of AHN 086 (greater than 2 microM) irreversibly inhibited the intropic, but not the chronotropic responses to BAY K 8644, nifedipine, and isoproterenol. Ro 5-4864 (10 microM) produced a reversible enhancement of the inotropic responses and block of the chronotropic responses to BAY K 8644. In guinea-pig ileal longitudinal smooth muscle, both AHN 086 and Ro 5-4864 reversibly inhibited field-stimulated contractions. Neither Ro 5-4864 nor AHN 086 affected the ability of nifedipine to inhibit field-stimulated contractions of ileal longitudinal smooth muscle. Treatment of intact atrial with 5 microM AHN 086 followed by extensive washing resulted in a significant inhibition (30-50%) of [3H]Ro 5-4864 binding to peripheral benzodiazepine receptors and of [3H]nitrendipine binding to voltage-operated calcium channels, but did not affect [3H]dihydroalprenolol binding to beta-adrenergic receptors on atrial membranes. The same treatment applied to intact ileal longitudinal smooth muscle affected neither [3H] (-)-quinuclidinyl benzilate binding to muscarine receptors nor [3H]nitrendipine binding, but did result in a significant inhibition (30-50%) of [3H]Ro 5-4864 binding to ileal longitudinal smooth muscle membranes.(ABSTRACT TRUNCATED AT 250 WORDS)
 
Article
Administration of a single intraperitoneal dose of 1,1-dichloroethylene (125 mg/kg, 1,1-DCE) to mice resulted in bronchiolar injury with selective necrosis of Clara cells. Degenerative changes were manifest in Clara cells as early as 1 h following 1,1-DCE exposure, and were characterized by marked swelling of mitochondria and aggregation of chromatin against the nuclear membrane. Cell death was apparent at 2 h; by 8 h, areas of the bronchiolar epithelium were devoid of lining cells, and at 24 h, the majority of Clara cells were exfoliated. The residual epithelium consisted of flattened cells which formed a thin lining for the airway. Necrosis of Clara cells early in the course of 1,1-DCE exposure coincided with peak covalent binding of [14C]1,1-DCE and significant depression of components of the pulmonary mixed-function oxidase system; cytochrome P-450 and aryl hydrocarbon hydroxylase activity were markedly reduced but not depleted. Liver damage involving centrilobular hepatocytes was observed at 24 h in 30% of treated animals, and coincided with significant inhibition of aryl hydrocarbon hydroxylase activity; cytochrome P-450 content, however, remained unchanged. While changes in the liver evoked by 1,1-DCE were less striking, the results in lung demonstrate positive temporal correlations between structural damage, peak covalent binding and disturbances of monooxygenase enzymes.
 
Article
1,1-Dichloroethylene (1,1-DCE) causes lung and liver necrosis in mice. Covalent binding of [14C]1,1-DCE to isolated lung and liver microsomes from CD-1 mice required NADPH and was strongly inhibited by carbon monoxide. Lung and liver microsomes isolated from animals treated with phenobarbital demonstrated no changes in covalent binding of [14C]1,1-DCE compared with those from vehicle-treated animals. While 3-methylcholanthrene caused no alterations in binding to lung microsomes, the same pretreatment resulted in significantly increased levels of binding to liver microsomes. Piperonyl butoxide caused significant decreases in covalent binding to lung and liver microsomes; SKF 525-A significantly inhibited binding to liver microsomes but had no effect on lung microsomes. The incubation of liver microsomes with inhibitors required more NADPH than those performed with lung microsomes. The results demonstrate that reactive metabolites of 1,1-DCE can be formed by lung and liver microsomes, and suggest the involvement of cytochrome P-450 isozymes in the lung and liver injury induced by the halocarbon. However, metabolic activation by lung and liver microsomes may additionally involve non P-450 dependent mechanisms as evidenced by relatively high levels of nonspecific binding of 1,1-DCE.
 
Incidence of aberrant crypt foci (ACF) in the control and test rat groups. (A) Topographical view of normal crypts (40×). (B) Topographical view of normal crypts from rats supplemented with multivitamins and minerals alone (40×). (C) Topographical view of ACF (arrows) with multiple crypts in the colon from rats treated with 1,2-dimethylhydrazine (DMH, 40×), but without supplements. (D) Topographical view of ACF (arrow) with 2 and 3 crypts in the colon from rats treated with DMH, but with multivitamin and mineral supplementation during weeks 1 – 15 (initiation, 40×). (E) Topographical view of ACF (arrow) with a crypt in the colon from rats treated with DMH, but with multivitamin and mineral supplementation during weeks 16 – 32 (post-initiation, 40×). (F) Topographical view of ACF (arrow) with no crypt in the colon from rats treated with DMH, but multivitamin and mineral supplementation during weeks 1 – 32 (entire experimental period, 40×). 
Histopathological alterations in control and test rats after 32 weeks of treatment. (A) Control groups (groups 1 and 2): shows colon tissue with normal mucosal glands with regular lining of cells (40×). (B) Colon tissue from rats treated with 1,2-dimethylhydrazine (DMH) alone (Group 3): colon shows severe epithelial dysplasia with complete loss of polarity, and randomly placed nuclei (40×). (C) Colon tissue from rats treated with DMH, but with multivitamin and mineral supplementation during weeks 1 – 15 (Group 4, initiation): few areas with mucosal thickening and less severe inflammatory cell infiltration (40×). (D) Colon tissue from rats treated with DMH, but with multivitamin and mineral supplementation during weeks 16 – 32 (Group 5, post-initiation): surface and upper crypt epithelium are normal with scattered inflammatory cell infiltration (40×). (E) Colon tissue from rats treated with DMH, but with multivitamin and mineral supplementation during weeks 1 – 32 (Group 6, entire experimetal period): normal mucosa with well-oriented crypts (40×). 
Effect of multivitamins on intestinal and colonic lipid peroxidation of control and test rats after 32 weeks of treatment.
Article
This study was performed to determine the chemopreventive and antioxidant status of multivitamin and mineral (0.01% in drinking water, ad libitum) supplements in 1,2-dimethylhydrazine (DMH)-induced experimental colon carcinogenesis. Experimental colon carcinogenesis was induced in male albino Wistar rats by injecting DMH (20 mg·(kg body mass)(-1)) once weekly for 15 consecutive weeks, and administering a multivitamin supplement in 3 regimes (initiation, post-initiation, and entire experimental period) for 32 weeks. We studied lipid peroxidation products (thiobarbituric acid reactive substances, lipid hydroperoxides, conjugated dienes) in the circulation and in the tissues, antioxidant status (superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, and non-enzymatic antioxidant-reduced glutathione) of the tissues, aberrant crypt foci (ACF), and histopathological alterations. DMH-induced rats had an increase in lipid peroxidation products and a lower antioxidant status compared with control animals. Multivitamin and mineral supplementation during the initiation, post-initiation, and the entire study period significantly decreased the levels of lipid peroxidation products in circulation and colonic tissues, significantly elevated the activities of the antioxidant enzymes and reduced glutathione to near normalcy in DMH-induced rats. The incidence of ACF was reduced by [corrected] 84.1% in rats supplemented with multivitamin and minerals for the entire study and prevented the colonic tissue from histopathological alterations induced by DMH.
 
Article
This study was designed to determine the effect of a newly synthesized proton pump inhibitor, 2-dimethylamino-4,5-dihydrothiazolo[4,5:3,4]pyridol[1,2-a]be nzimidazole (YJA20379-2), on gastric H(+)-K(+) ATPase activity, acid secretion, and experimental gastroduodenal lesions or ulcers in rats. YJA20379-2 inhibited in a concentration-dependent manner the proton pump (H(+)-K(+) ATPase) activity in isolated hog gastric mucosal microsomes, therefore, confirming its classification as a proton pump inhibitor. The inhibitory efficacy of YJA20379-2 on the proton pump was about eight times higher than that of omeprazole at pH 7.4. The activity of the inhibited enzyme was not restored by dilution and washout method, so this implied that the inhibition of YJA20379-2 is not reversible. YJA20379-2, given intraduodenally or orally, potently suppressed acid secretion in pylorus-ligated rats, with ED50 values of 3.6 and 7.7 mg.kg(-1), respectively. Pretreatment with YJA20379-2 dose dependently protected the gastric mucosa from damage induced by absolute ethanol, water-immersion stress, indomethacin, and the duodenal mucosa from damage induced by mepirizole in rats, with ED50 values of 11.0, 21.0, 0.5, and 18.7 mg.kg(-1), respectively. Repeated administration of YJA20379-2 also dose dependently accelerated spontaneous healing of acetic acid induced gastric ulcers. These results suggest that YJA20379-2 has potent antisecretory and antiulcer effects, which are exerted by suppression of H(+)-K(+) ATPase activity in gastric parietal cells, such that YJA20379-2 may be useful for the clinical treatment of peptic ulcer diseases.
 
Article
Toxicity for the chick of 1,2-dihydro-6-ethoxy-2,2,4-trimethylquinoline (EQ) was significantly greater when the diet was low in protein. When fat supplementation of the diet enhanced EQ toxicity the effect appeared to result from an increase in the energy:protein ratio of the diet rather than from the fat itself. The concentration of EQ in the livers of chicks fed 0.25% EQ for 6 weeks was significantly higher when the dietary level of protein was 17% than when the level was 23%.
 
Article
Colon cancer incidence is higher in developed countries than in developing countries. We determined the effect of oregano (Origanum vulgare L.) on fecal bacterial enzyme activities in 1,2-dimethylhydrazine (DMH)-induced experimental colon carcinogenesis in rats. Male Wistar albino rats were divided into 6 groups and all animals were fed with a high-fat diet (20% fat in the diet). Group 1 served as control and group 2 animals received 60 mg.kg(-1) body weight (b.w.) oregano daily for 15 weeks. To induce colon cancer, DMH (20 mg.kg(-1) b.w.) was injected subcutaneously once a week for the first 4 weeks (groups 3-6). In addition, oregano was administered at 20, 40, or 60 mg.kg(-1) b.w. each day orally for the entire 15 weeks (groups 4-6). We analyzed the fecal bacterial enzyme activities and found it to be significantly higher in the group treated with DMH alone than in the control group. Oregano supplementation at all 3 doses significantly suppressed the bacterial enzyme activities and modulated oxidative stress significantly compared with the unsupplemented DMH-treated group. Results of our present investigation therefore revealed that oregano markedly inhibited DMH-induced colon carcinogenesis and that the optimal dose of 40 mg.kg(-1) b.w. was more effective than either the higher or lower doses.
 
Article
Carbon monoxide (CO), a vasodilator, has been implicated as an activator of soluble guanylyl cyclase (sGC) to effect smooth muscle relaxation; however, this idea has not received universal support. The purpose of this study was to examine the effects of the sGC inhibitor 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ) on relaxation of rabbit aortic rings (RARs) induced by CO. Administration of 10 microM ODQ completely abolished relaxation of RARs by CO (30 microM), whereas only a partial attenuation of NO-induced relaxation was achieved by the same concentration of ODQ. The results of this study suggest that CO-mediated relaxation of RARs is mediated by sGC and indicate that ODQ may serve as a useful tool in the investigation of the actions of CO. Furthermore, these observations support the idea that ODQ is less potent in inhibiting relaxations by NO, thereby implicating a component of NO-induced relaxation that is independent of sGC/cGMP.
 
Article
In the present study, we investigated the in vitro effects of D-myo-inositol 1,2,6-trisphosphate (PP-56) on platelets from normal and streptozotocin-induced diabetic rats. PP-56 markedly inhibited aggregation, in a dose-related manner, when added in vitro, more efficiently with thrombin- than with ADP-induced aggregation and, after 90 min incubation, more in diabetic than in normal platelets. The PP-56 platelet inhibitory effect seems to be related to its phosphate content. PP-56 blocked the release of malondialdehyde from erythrocytes, but to the same extent in normal and diabetic rats. PP-56, after 20 min incubation, restored the platelet phosphoinositide turnover, which was significantly modified in diabetic rats. This last observation could explain at least part of the specificity of PP-56 for normalizing platelet aggregation in diabetic animals after long-term administration in vivo.
 
Article
The effects of gradually increasing doses of 1,25(OH)2D3 on plasma calcium and 45Ca radioactivity were studied in young dogs that had been extensively prelabelled with 45Ca. The effects of orally and intravenously administered 1,25(OH)2D3 were evaluated in normal and thyroparathyroidectomized dogs fed a normal diet. In normal dogs when 1,25(OH)2D3 increased the plasma calcium within the normal range (2.9-3.1 mmol/L) there was no significant increase in plasma 45Ca. In thyroparathyroidectomized dogs, oral or intravenous 1,25(OH)2D3 increased the low blood calcium to a normal level (1.8-2.9 mmol/L) without significantly increasing plasma 45Ca. In normal and thyroparathyroidectomized dogs, any 1,25(OH)2D3-induced increase in plasma calcium above the normal range was associated with a significant increase in 45Ca, indicating mobilization of bone calcium. Intravenous administration of 1,25(OH)2D3 in the normal or thyroparathyroidectomized dogs had a much larger effect than oral doses in mobilizing bone 45Ca when inducing a similar level of hypercalcemia. The major physiological effect of 1,25(OH)2D3 in the low or normal range of plasma calcium is on intestinal absorption of calcium without a significant effect on mobilizing bone calcium. The pharmacological effect of 1,25(OH)2D3 in vivo is to mobilize bone calcium as well as dietary calcium into blood.
 
Article
In view of the established biological role for 1,25-dihydroxycholecalciferol (DHCC) in calcium mobilization and the significance of calcium availability in platelet activation, it was of interest to investigate the potential for DHCC to act as a proaggregating agent. The present results provide evidence that DHCC at a concentration of 0.01 micrograms/microL can both aggregate human platelets and potentiate ADP-induced platelet aggregation. DHCC was also capable of aggregating rat platelets.
 
Article
The hepatic uptake of the hormone 1,25-dihydroxyvitamin D3 has been studied, in vivo, using the multiple indicator dilution technique. The fractional uptake of 1,25-dihydroxyvitamin D3 during a single circulatory passage across the dog liver has been estimated at 34.4 +/- 3.3% while its hepatic clearance was estimated at 364.3 +/- 94.1 mL/min. The hepatic uptake of 1,25-dihydroxyvitamin D3 is discussed in relation to its systemic bioavailability following intravenous or oral administration as well as in relation to the hepatic uptake of other vitamin D sterols; it is postulated that the hepatic uptake of vitamin D sterols does not seem to be mediated by specific receptors on the liver plasma membrane; it seems, however, that the hepatic uptake of vitamin D sterols may be inversely related to their relative affinity for the circulating carrier, the vitamin D binding protein.
 
Article
The effect of diaminopropanol, an inhibitor of polyamine synthesis, on the metabolic response of liver to insulin was studied in streptozotocin-diabetic rats. Insulin elicited a prompt and very marked increase in ornithine and S-adenosylmethionine decarboxylase activities and in putrescine concentration. Pretreatment of rats with diaminopropanol prevented the increase in the decarboxylases and resulted in decreased spermidine and spermine content of liver. The insulin-induced increase in glycogen content was depressed by 50% and the increase in the rate of lipogenesis in vivo was completely prevented by prior injection of diaminopropanol. These studies implicate altered polyamine metabolism in the metabolic response of liver of streptozotocin-diabetic rats to insulin.
 
Specific binding of [ 125 I]ET-1 to ET receptors versus con- A centration of HJP272. Binding of 224 pmol/L [ 125 I]ET-1 to ET re- A ceptors was inhibited by HJP272 from 10 3 –10 8 pmol/L. Each point 
Effect of HJP272 on time of delivery of the first pup in low- dose (10 mg/kg) LPS-treated mice delivering prematurely. Percentage of total mice delivering prematurely over time is shown for ( ^ ) control mice ( n = 10) and ( & ) group 1 mice treated with 50 mg/kg HJP272 ( n = 7). Difference between groups at 24 h significant at p < 0.004. 
Effect of HJP272 on premature delivery of pups in low-dose (10 mg/kg) LPS-treated mice. Number of premature pups delivered over time is shown for ( ^ ) control mice, as a percentage of the total number of pups delivered to control mice ( n = 65) and for ( & ) mice treated with 50 mg/kg HJP272, as a percentage of the total number of pups delivered to HJP272-treated mice ( n = 43). The difference in the total number of pups delivered preterm at 24 h in group 5 (control) compared with that in group 1 (50 mg/kg HJP272) was significant at p < 0.001. 
Effect of HJP272 on high-dose (50 mg/kg) LPS-treated mice delivering prematurely. Percentage of mice delivering preterm over time is shown for ( ^ ) control mice ( n = 11) and ( ! ) mice treated with 50 mg/kg HJP272 ( n = 8). Difference at 24 h significant at p < 0.001. 
Effect of HJP272 on premature delivery of pups in high- dose (50 mg/kg) LPS-treated mice. Number of premature pups as a percentage of the total pups delivered over time is shown for ( * ) control mice ( n = 63) and ( ! ) mice treated with 50 mg/kg HJP272 ( n = 44). Difference at 24 h significant at p < 0.001. 
Article
Preterm birth (PTB), defined as any birth occurring before 37 weeks of gestation, occurs in only 12% of all births, yet accounts for nearly half of long-term neurological morbidity, and 60%-80% of perinatal mortality. The single most common cause of PTB is intrauterine infection. Endothelin-1 (ET-1) is a potent vasoconstrictor peptide that is both upregulated by inflammatory cytokines and capable of increasing myometrial smooth muscle tone. We hypothesized, therefore, that ET-1 is a critical component of the parturition cascade in the setting of infection-associated PTB. In our previous work, we have shown that blockade of ET-1 synthesis through the use of the metalloproteinase inhibitor phosphoramidon results in control of preterm labor. In the current work, we showed that blockade of ET-1 action with 5-50 mg/kg i.p. 3-(3-carboxybenzyl)-1-((6-ethylbenzo[d][1,3]dioxol-5-yl)methyl)-6-hydroxy-4-oxo-1,4-dihydroquinoline-2-carboxylic acid (HJP272), a putative novel selective ETA-receptor antagonist (IC50, 70 nmol/L), prevents PTB induced with up to 50 mg/kg of i.p. lipopolysaccharide in a mouse model. This is the first report, to our knowledge, of control of infection-associated PTB with a specific ETA-receptor antagonist. The identification of a novel effective therapy for PTB could have important clinical implications.
 
Article
The ferrochelatase-lowering activity of 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine (DDC) analogues in chick embryo hepatocyte culture has been assumed to be due to the formation of an N-alkylprotoporphyrin IX. This assumption required confirmation. For this reason the 4-ethyl analogue of DDC was administered to phenobarbital-pretreated 19-day-old chick embryos. This resulted in hepatic accumulation of a green pigment with ferrochelatase-inhibitory activity. The green pigment was identified as an N-alkylprotoporphyrin IX by comparison of the electronic absorption spectra of its dimethyl ester and Zn complex with the corresponding spectra obtained from synthetic N-ethylprotoporphyrin IX.
 
Article
3-Ethoxycarbonyl-1,4-dihydro-2,4-dimethylpyridine (EDP) was shown to lack the ferrochelatase-lowering activity of 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine (DDC) in chick embryo liver cells in culture. This was attributed to the inability of EDP to cause destruction of the heme moiety of cytochrome P-450 with concomitant formation of N-methylprotoporphyrin IX. EDP was less potent as a porphyrinogenic agent than DDC and caused the accumulation of uroporphyrin, heptacarboxylic porphyrin, and coproporphyrin in contrast with DDC which caused primarily protoporphyrin to accumulate. The inactivity of EDP as a ferrochelatase-lowering agent and its low porphyrinogenic potency was explained, at least in part, by its rapid transformation in aqueous solution to other nondihydropyridine products. The two ethoxycarbonyl substituents of DDC are therefore essential for N-methylprotoporphyrin formation, ferrochelatase-lowering activity, and optimal porphyrin-inducing activity.
 
Article
Cytochrome P450- and heme-destructive effects of the 4-nonyl and 4-dodecyl analogues of 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine (DDC) were determined using hepatic microsomal preparations obtained from untreated, beta-naphthoflavone-treated, and phenobarbital-treated chick embryos. The 4-nonyl analogue of DDC was less efficacious than 4-ethyl DDC and 4-hexyl DDC, but more efficacious than 4-dodecyl DDC with respect to cytochrome P450-destructive activity. In all hepatic microsomal preparations, cytochrome P450 destruction by 4-nonyl DDC was accompanied by loss of microsomal heme. In contrast, 4-dodecyl DDC caused loss of heme only in hepatic microsomal preparations obtained from phenobarbital-treated chick embryos. The ability of 4-nonyl DDC and 4-dodecyl DDC to lower ferrochelatase activity was compared with that of 4-ethyl DDC and 4-hexyl DDC in cultured chick embryo hepatocytes. As the length of the 4-alkyl group was increased, the ferrochelatase-lowering efficacy and potency of the DDC analogue decreased. The 4-dodecyl DDC analogue was unable to lower ferrochelatase activity, which accorded with the finding that the administration of 4-dodecyl DDC to phenobarbital-treated rats did not lead to the accumulation of an N-alkylprotoporphyrin. The ability of 4-nonyl DDC to lower ferrochelatase activity was attributed to the formation of N-nonylprotoporphyrin IX following the administration of 4-nonyl DDC to phenobarbital-treated rats.
 
Article
Using progesterone 21-hydroxylase as a selective substrate for P450 2C6 in phenobarbital-treated male rats, and androstenedione and progesterone 6 beta-hydroxylases as well as erythromycin N-demethylase as selective markers for P450 3A1 in dexamethasone-treated female rats, we have shown that these P450 isozymes undergo mechanism-based inactivation after in vivo administration of 3,5-diethoxycarbonyl-1,4-dihydro-2,6-dimethyl-4-ethylpyridine (4-ethyl DDC). These results differ from our previous studies where no inactivation was observed after in vitro administration of 4-ethyl DDC to rat hepatic microsomes. We show that the differences between the in vivo and in vitro effects of 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine (DDC) analogues are due to the presence of residual 4-ethyl DDC in the in vitro experiments causing time-independent competitive inhibition and obscuring observation of mechanism-based inactivation.
 
Article
The actions of 2,6-dimethyl-3,5-dicarbomethoxy-4-(2-isothiocyano)phenyl-1,4- dihydropyridine (o-NCS-DHP), a nifedipine analog bearing a reactive group, have been characterized in vitro by pharmacological and radioligand binding techniques in a number of smooth muscles and in vivo by blood pressure and radioligand binding. o-NCS-DHP exhibits persistent, but slowly reversible, antagonism in guinea pig ileal longitudinal smooth muscle, guinea pig bladder, taenia coli, rat portal vein, and rat tail artery to receptor responses (muscarinic and alpha-adrenoceptor) and K+ depolarization initiated responses. Duration of response was significantly longer than that of equivalent concentrations of nifedipine. In many tissues a component of antagonism produced by o-NCS-DHP was not reversed by repeated washing over the duration of the experiment (up to 2 or 7 h). A comparison of the actions of o-NCS-DHP and its isomers m-NCS-DHP and p-NCS-DHP revealed the former to be significantly longer lasting in rat tail artery against K+ depolarization induced responses. A similar profile was exhibited when the Ca2+ channel activator Bay K 8644 was employed as the stimulant, but the antagonism produced by all three compounds was fully reversed with sufficiently prolonged washing. In vivo administration of o-NCS-DHP (5-25 mg/kg) produced a persistent reduction of [3H]nitrendipine binding in rat brain, gut, and heart characterized as Bmax, but not KD, changes. No effects on [3H]dihydroalprenolol or [3H]quinuclidinyl benzilate binding were detected. Binding site recoveries were characterized by t1/2 values of 35-50 h, and these were significantly prolonged to 91-107 h in animals treated with cycloheximide. Recovery of [3H]nitrendipine binding sites correlated with blood pressure restoration in spontaneously hypertensive rats. These data suggest that o-NCS-DHP possesses both reversible and irreversible actions. The reversible actions are unusually persistent compared with nifedipine and other 1,4-dihydropyridine analogs. This persistent, but reversible component, may be accompanied by an irreversible action particularly at the higher concentrations employed in the in vivo experiments.
 
Article
Various 4-alkyl analogues of 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6- trimethylpyridine (DDC) cause mechanism-based inactivation of cytochrome P-450 (P-450) via destruction of the heme prosthetic group. This is an important component of these compounds' porphyrinogenic mechanism. In an attempt to map the P-450 isozyme selectivities of DDC analogues, we have examined the effects of these compounds on the regioselective and stereoselective hydroxylation of androstenedione (AD) and progesterone (PG) in rat liver microsomal systems. In microsomes from phenobarbital-treated male rats, DDC analogues did not cause time-dependent inactivation of AD 7 alpha-hydroxylase, AD 16 beta-hydroxylase, and PG 21-hydroxylase, selective markers for P450IIA 1/2, IIB1, and IIC6, respectively. In contrast, DDC analogues were effective inactivators of PG 2 alpha-hydroxylase and steroid 6 beta-hydroxylases, selective markers for P450IIC11 and IIIA forms, respectively. We conclude that differences in porphyrinogenicity observed with various DDC analogues are not likely to be due to the selective destruction of different P-450 isozymes by different analogues, but rather to properties of the DDC analogues themselves. 4-Ethyl DDC was found to be capable of discriminating between P450IIIA subfamily forms. In microsomes from untreated male rats, which express P450IIIA2 but not IIIA1, 4-ethyl DDC inactivated both AD and PG 6 beta-hydroxylases. However, in microsomes from dexamethasone-treated female rats, which express P450IIIA1 but not IIIA2, no inactivation of the steroid 6 beta-hydroxylases was observed. Thus, 4-ethyl DDC appears to be a potentially valuable tool for differentiating between P450IIIA forms.
 
Article
Binding of [3H]nitrendipine, [3H]nimodipine, and (+)[3H]PN 200-110 to microsomal preparations of guinea pig smooth and cardiac muscle and brain synaptosomes revealed high affinity interaction with KD values in the sequence, (+)PN 200-110 greater than nitrendipine greater than nimodipine. Bmax values for a particular tissue were independent of the 1,4-dihydropyridine employed in radioligand binding at 25 degrees C. The temperature dependence of [3H]nitrendipine binding in cardiac and smooth muscle microsomal preparations and brain synaptosomes was measured from 0 degrees to 37 degrees C and for skeletal muscle preparations from 0 degrees to 30 degrees C. Bmax values increased with temperature for cardiac membranes, but did not vary in other tissues. van't Hoff plots were nonlinear in all tissues, enthalpy and entropy changes becoming increasingly negative with increasing temperature. Competition binding of the activator-antagonist enantiomeric 1,4-dihydropyridine pairs of Bay k 8644 and PN 202-791 for [3H]nitrendipine in smooth muscle did not reveal significant thermodynamic differences between activator and antagonist molecules.
 
Article
Contraction of canine ventricular trabeculae were recorded stimulation at a frequency of 0.5 Hz and after rest periods of 2 and 8 min to analyze the effect of the Ca channel agonist BAY k 8644, on sarcoplasmic reticular function. Short periods of rest interposed between steady trains of stimuli caused a potentiation of the postrest beat. This is believed to be due to the mobilization of activator Ca from the sarcoplasmic reticulum (SR). Racemic BAY k 8644 and its Ca channel agonist enantiomer, (-) BAY k 8644, both produced an increase in contraction in response to a steady train of stimuli but converted rest potentiation into rest depression. This has been interpreted as increased loss of Ca from the SR during diastole. Addition of Ca channel antagonists, (+) BAY k 8644, nitrendipine, or nifedipine, to reverse the agonistic effect of (-) and racemic BAY k 8644 on the Ca channel did not convert the rest depression into rest potentiation. In the presence of stimuli but converted rest potentiation into rest depression. This has been interpreted as increased loss of Ca from the SR during diastole.(ABSTRACT TRUNCATED AT 250 WORDS)
 
Article
Chick neural retina cells contain functional L-type voltage-dependent Ca2+ channels sensitive to 1,4-dihydropyridines. To investigate the effects of chronic depolarization, cells were grown in medium containing elevated K+. After 4-h to 4-day treatments with elevated K+ (12-73 mM), there was a concentration-dependent decrease in high affinity [3H]PN200-110 binding. Saturation analysis of cells treated for 4 days with 40 mM K+ showed a reduction in maximum ligand binding with no change in affinity. Control and experimental Bmax values were 70.7 +/- 6.4 and 42.2 +/- 4.5 fmol/mg protein, respectively, and control and experimental KD values were 70.2 +/- 7.4 and 68.6 +/- 7.4 x 10(-12) M. The effect of chronic depolarization was time-dependent, reversible, and without effect on cellular protein content. Reduction in 45Ca2+ uptake following chronic depolarization correlated well with the reduction in [3H]PN200-110 binding. The calcium ionophore A23187, 10(-6) M for 24 h, also decreased the binding site density. The calcium channel antagonist D600 had no effect alone on [3H]PN200-110 binding; however, D600 blocked the down-regulation of calcium channels induced by chronic depolarization. The mechanism for Ca2+ channel down-regulation may involve calcium entry, since the effect was blocked by D600 and mimicked by the calcium ionophore A23187. Chronic depolarization with either elevated K+ or veratridine, or chronic treatment with A23187 had no effect on calcium channels in rat neonatal ventricular myocytes, although these cells express functional channels of the 1,4-dihydropyridine-sensitive class.(ABSTRACT TRUNCATED AT 250 WORDS)
 
Article
The voltage-sensitive calcium channel in cultured chick neural retina cells was characterized by the actions of the enantiomers of Bay K 8644 and 202-791 and other 1,4-dihydropyridines. These cells showed time- and voltage-dependent Ca2+ uptake that was stimulated by K+ depolarization and blocked by the inorganic calcium channel blockers Cd2+ and Co2+. A small fraction only (15% maximum) of the uptake was inactivated by predepolarization of the cells with 80 mM K+. Ca2+ uptake was sensitive to the 1,4-dihydropyridine calcium channel antagonists and activators. (S)-Bay K 8644 and (S)-202-791 stimulated the Ca2+ uptake, and (R)-Bay K 8644 and (R)-202-791 as well as nitrendipine and PN 200-110 inhibited Ca2+ uptake stimulated by K+ depolarization or channel activators. The K+ depolarization-stimulated uptake was inhibited by 90%, but the activator-stimulated uptake was completely blocked by the 1,4-dihydropyridine antagonists. The potencies of these agents as inhibitors of Ca2+ uptake were significantly lower than the binding affinities in membrane preparations from the same cells or their binding and pharmacologic affinities in vascular smooth muscle. K+ depolarization or (S)-Bay K 8644 induced 45Ca2+ uptake was not observed in a glial cell culture. [3H]Nitrendipine and [3H]PN 200-110 bound to membrane preparations of the cells consistent with the presence of a single type of high affinity binding site.(ABSTRACT TRUNCATED AT 250 WORDS)
 
Article
Various rat liver cytochrome P-450 (P-450) isozymes are targets for mechanism-based inactivation by 3,5-diethoxycarbonyl-2,6-dimethyl-4-ethyl-1,4- dihydropyridine (4-ethyl DDC). Unlike rat liver, which contains multiple P-450 isozymes, rabbit lung contains only three major isozymes referred to as forms 2, 5, and 6. We have examined the ability of 4-ethyl DDC to destroy P-450 heme in hepatic and pulmonary microsomes from untreated and beta-naphthoflavone (beta NF)-treated rabbits. This compound destroyed 31% of the P-450 in either hepatic microsomal preparation, but was ineffective at lowering P-450 and heme levels in pulmonary microsomes when examined at a range of concentrations (0.45-5.0 mM). These data suggest that rabbit pulmonary P-450 forms 2, 5, and 6 are not targets for destruction by 4-ethyl DDC, despite the ability of this compound to inactivate rat liver P-450c, the orthologue of rabbit lung form 6.
 
Article
Soluble polymers of rat (or human) albumin and alpha-1,4-glucosidase are prepared using the cross-linking agent glutaraldehyde. The resulting polymer has an average molecular weight of 800 000 indicating an average composition of 12 albumin molecules for each enzyme molecule. Compared with an equivalent amount of free enzyme, the enzyme-albumin polymer has an increased resistance to heat denaturation (half-life of 15 h compared with 1 h for free enzyme at 37 degrees C) and to proteolysis by trypsin (half-life of 180 min compared with 10 min). The high degree of resistance to bioinactivation of the enzyme-albumin polymer is discussed in relation to requirements for enzyme replacement therapy in a range of metabolic diseases including type II glycogenosis (Pompe's disease) where alpha-1,4-glucosidase is the defective enzyme.
 
Article
Intracellular applications of a fixed amount (0.2 to 8 nmol) of inositol 1,4,5-trisphosphate (InsP3) over a brief period (2 s) into barnacle muscle fibers induced vigorous contractures. Peak tension attained during the first application depended on [InsP3]: the maximum tension evoked by the injection of 8 nmol was 1.6 kg/cm2. Peak tension during a second application of a high dose of InsP3 (greater than 10 microM) was always smaller than that during the first application. Extracellular Ca2+ could be omitted with no measurable effects on either the amplitude or time course of the contractures evoked by InsP3. Aequorin was used to measure InsP3-evoked Ca2+ release from intracellular stores in minced muscle fibers from lobster and in skinned muscle fibers from barnacle. Provided the sarcoplasmic reticulum was preloaded with Ca2+, application of InsP3 induced a transient Ca2+ release that was [InsP3] dependent. During each transient, [Ca2+] rose rapidly to a peak value (t1/2 less than 5 s) and then slowly returned (t1/2 less than 100 s) to a basal level. Maximum Ca2+ release was obtained at [InsP3] less than 100 microM and amounted to 4 nmol Ca2+/g of muscle, enough to increase [Ca2+]i from 0.1 to 8 microM had the Ca2+ release occurred in the intact fiber. Successive applications of a fixed amount of InsP3 elicited successive transient increases in Ca2+. The effects of [Ca2+] on the incorporation of [3H]inositol into the pools of phosphatidylinositol, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bisphosphate pools were measured.(ABSTRACT TRUNCATED AT 250 WORDS)
 
Article
Inositol 1,4,5-trisphosphate (InsP3) is an intracellular messenger generated from the hydrolysis of phosphatidylinositol 4,5-bisphosphate by phospholipase C in response to Ca(2+)-mobilizing stimuli. InsP3 interacts with a specific receptor responsible for the release of sequestered Ca2+ from an intracellular store. The purpose of the present study was to evaluate the relative affinities of the naturally occurring D-isomer of InsP3 and that of its L-stereoisomer for the InsP3 receptor and the InsP3 metabolizing enzymes from bovine adrenal cortex. The InsP3 receptor recognized D- and L-isomers with respective affinities of 4.8 nM and 7.3 microM. This high degree of selectivity was also reflected in the capacity of both isomers to mobilize Ca2+ from the microsomal preparation. The partially purified InsP3 kinase preparation was also able to discriminate between the two stereoisomers. The activity of the kinase was half-maximally inhibited in the presence of 11 microM L-InsP3, a value much higher than the Km of the kinase for D-InsP3 (0.4 microM). Both stereoisomers exhibited equipotent affinities (around 17 microM) for the particulate preparation of InsP3 phosphatase. The enzyme, however, appeared to hydrolyze L-InsP3 at a much slower rate. These results demonstrated that the different recognition sites for InsP3 were expressing distinct levels of stereoselectivity. This property, which is an important aspect of ligand-receptor interaction, could be exploited for the design of new selective drugs interfering with InsP3 action and metabolism.
 
Article
Inositol 1,4,5-trisphosphate (InsP3) is an important second messenger that interacts with a specific intracellular receptor and triggers a release of Ca2+ from intracellular stores. InsP3 is preferentially metabolized by two enzymes. A specific 5-phosphatase (InsP3 phosphatase) produces an inactive metabolite, inositol 1,4-bisphosphate, while a specific 3-kinase (InsP3 kinase) produces an active metabolite, inositol 1,3,4,5-tetrakisphosphate. With the goal of developing selective ligands of the diverse InsP3 recognition sites, we have studied the effects of some chemical dyes on the binding of InsP3 to its receptor and on the activity of its metabolic enzymes. Although these dyes possess similar chemical structures, they showed varied selectivities towards the three recognition sites. Thymol Blue was the most potent inhibitor of InsP3 binding activity, with an IC50 of 105 microM. Phenol Red demonstrated a higher selectivity for InsP3 phosphatase inhibition, with an IC50 of 100 microM. 3',3",5',5"-Tetraiodophenolsulfonephthalein showed its most potent inhibitory effect on InsP3 kinase activity, with an IC50 of 35 microM. Tetrabromophenol Blue potently inhibited InsP3 phosphatase and InsP3 kinase activities, with respective IC50 values of 25 and 12 microM. Phenolphthalein Diphosphate and Phenolphthalein Carbinol Disulfate demonstrated weak inhibitory effects towards the three recognition sites for InsP3. These results reveal certain structural clues that should help in the development of more selective inhibitors.
 
Article
Inositol 1,4,5-trisphosphate (IP3) was injected iontophoretically into cat spinal motoneurons in pentobarbital-anaesthetized cats and nonanaesthetized, decerebrate cats. Injections of IP3 induced a long-lasting, reproducible hyperpolarization without consistent change in input resistance. The peak amplitude of post-spike afterhyperpolarization (AHP) was significantly increased by IP3 when the membrane potential was adjusted to the control level. Intracellular injections of Ca2+ chelators, which depressed the Ca2(+)-activated AHP, prevented the IP3-induced long-lasting hyperpolarization, suggesting that IP3 acts by a Ca2(+)-dependent mechanism. Intracellular injections of myo-inositol did not consistently induce hyperpolarizations. Also intracellular injections of Li+, which blocks IP3 catabolism, did not prevent the IP3-evoked hyperpolarization. These data suggest that IP3 itself, rather than its breakdown product myo-inositol, is mainly responsible for the hyperpolarizing effect. Possible mechanisms for the IP3-induced hyperpolarization are discussed.
 
Article
Diltiazem, a 1,5-benzothiazepine, has demonstrated efficacy in the treatment of numerous cardiovascular diseases. TA-3090, a newly synthetized 1,5-benzothiazepine compound was studied in open-chest anesthetized dogs to characterize its hemodynamic properties, to compare it with diltiazem, and finally to correlate hemodynamic properties and plasma level concentrations. Anesthetized open-chest dogs were instrumented with electronic devices and fluid-filled catheters to monitor cardiac, coronary, and peripheral hemodynamic changes. A cumulative intravenous bolus administration of TA-3090 (n = 16) or diltiazem (n = 15) (15, 50, 200, and 400 micrograms/kg) was carried out, and blood samples were taken before and 5 min following each dose administration. Hemodynamic changes were followed for 30 min after each administration, at which time most hemodynamic parameters were back to baseline levels. The results indicate that both TA-3090 and diltiazem elicit slight peripheral and coronary vasodilator properties at low doses (15 and 50 micrograms/kg). With higher dosage, hemodynamic effects were maximal: coronary blood flow increased by 75%, arterial pressure decreased by 25%, and reflex positive inotropic effects were also observed. Heart rate was significantly reduced (10%). Comparison between TA-3090 and diltiazem indicates that both drugs elicit coronary vasodilator selectivity and TA-3090 has a prolonged duration of action compared with that of diltiazem. A straightforward relationship is demonstrated between vasodilator properties and plasma levels of either TA-3090 or diltiazem. Our data suggest that with plasma levels between 40 and 80 ng/mL, significant hemodynamic changes were observed with TA-3090. Changes of heart rate were not correlated with plasma levels.(ABSTRACT TRUNCATED AT 250 WORDS)
 
Maximal decreases in mean aortic pressure (DMAP max ) elicited by increasing bolus doses (0.3–10 mg/kg, i.v.) of 1,8- cineole in conscious, freely moving, vehicle-pretreated rats and in pentobarbital-anesthetized rats with or without bilateral vagotomy. Values are means of changes expressed as a percentage of baseline and vertical bars indicate SE (n = 6). In all groups studied, maximal decreases in MAP were significantly related to the dose of 1,8-cineole (P < 0.001, one-way ANOVA). The dose – hypotensive-response curve for conscious rats pretreated with vehicle (from Fig. 2) was included here for comparison with that for intact, anesthetized rats. Bilateral vagotomy had no significant effect on 1,8-cineole-induced hypotension. *, P < 0.05; **, P < 0.001 (Tukey's tests with respect to preinjection values).  
Maximal decreases in mean aortic pressure (DMAP max ) elicited by increasing bolus doses (0.3–10 mg/kg, i.v.) of 1,8- cineole in conscious, freely moving rats subjected to i.v. pretreatment with vehicle (1 mL/kg), hexamethonium (30 mg/kg), atenolol (1.5 mg/kg), or methylatropine (1 mg/kg). Values are means of changes expressed as a percentage of baseline and vertical bars indicate SE (n = 6–7). In all groups studied, maximal decreases in MAP were significantly related to the dose of 1,8- cineole (P < 0.001, one-way ANOVA). Pretreatment with i.v. hexamethonium, atenolol, or methylatropine had no effects on the magnitude of 1,8-cineole-induced hypotension. *, P < 0.01; **, P < 0.001 (Tukey's tests with respect to preinjection values).  
Time course of the changes in mean aortic pressure (DMAP) elicited by an i.v. injection of 1,8-cineole (10 mg/kg) and the positive reference drug acetylcholine (5 mg/kg) in conscious , freely moving rats. Values are means of changes expressed as a percentage of baseline, and vertical bars indicate SE (n = 6). The time course of the 1,8-cineole-induced changes in MAP was significantly different from that obtained with acetylcholine (P < 0.05, two-way ANOVA); *, P < 0.05; **, P < 0.001 by Tukey's tests with respect to preinjection values.  
Effects of increasing concentrations (0.006–2.6 mM) of 1,8-cineole on the contraction induced by 60 mM KCl in rat isolated endothelium-containing thoracic aortae. Vertical bars indicate SE (n = 6). *, P < 0.05; **, P < 0.01 (Dunnett's tests with respect to control values).  
Effects of increasing concentrations (0.01–3 mM) of the positive reference drug verapamil on the contraction induced by KCl (60 mM) in rat isolated endothelium-containing thoracic aortae. Vertical bars indicate SE (n = 5). *, P < 0.05; **, P < 0.01 (Dunnett's tests with respect to control values).  
Article
The cardiovascular effects of i.v. treatment with 1,8-cineole, a monoterpenic oxide present in many plant essential oils, were investigated in normotensive rats. This study examined (i) whether the autonomic nervous system is involved in the mediation of 1,8-cineole-induced changes in mean aortic pressure (MAP) and heart rate (HR) and (ii) whether the hypotensive effects of 1,8-cineole could result from its vasodilatory effects directly upon vascular smooth muscle. In both pentobarbital-anesthetized and conscious, freely moving rats, bolus injections of 1,8-cineole (0.3-10 mg/kg, i.v.) elicited similar and dose-dependent decreases in MAP. Concomitantly, 1,8-cineole significantly decreased HR only at the highest dose (10 mg/kg). Pretreatment of anesthetized rats with bilateral vagotomy significantly reduced the bradycardic responses to 1,8-cineole (10 mg/kg) without affecting hypotension. In conscious rats, i.v. pretreatment with methylatropine (1 mg/kg), atenolol (1.5 mg/kg), or hexamethonium (30 mg/kg) had no significant effects on the 1,8-cineole-induced hypotension, while bradycardic responses to 1,8-cineole (10 mg/kg) were significantly reduced by methylatropine. In rat isolated thoracic aorta preparations, 1,8-cineole (0.006-2.6 mM) induced a concentration-dependent reduction of the contraction induced by potassium (60 mM). This is the first physiological evidence that i.v. treatment with 1,8-cineole in either anesthetized or conscious rats elicits hypotension; this effect seems related to an active vascular relaxation rather than withdrawal of sympathetic tone.
 
Article
The aim of this study was to investigate the anxiolytic potential of a series of novel carboxylic acid based 1,8 naphthyridines as 5-HT3 receptor antagonists. The pA2 values of all the compounds were determined against agonist 2-methyl-5-hydroxytryptamine in longitudinal muscle myenteric plexus preparations from guinea pig ileum. Compounds with higher pA2 values, particularly those greater than ondansetron, a standard 5-HT3 receptor antagonist, and optimal log P values were screened in mice by using behavioral tests such as a light-dark (L/D) aversion test, elevated plus maze (EPM) test, and an open field test (OFT). In the L/D test, compounds 7a, 7b, 7d, 7e, and 7i (2 mg/kg body mass, intraperitoneal) significantly (P < 0.05) increased the latency time to leave the light compartment, total time spent in the light compartment, and the number of transitions between the light and dark compartments. Compounds 7a, 7d, 7f, 7h, and 7i (2 mg/kg, i.p.) significantly (P < 0.05) increased the time spent in the open arms and the number of entries into the open arms in the EPM test. In addition, compounds 7a, 7d, 7e, 7f, and 7h (2 mg/kg, i.p.) significantly (P < 0.05) increased the ambulation scores and the frequency of rearing in the OFT.
 
Article
The effects of EDTA on rat uterine horns were studied and found to consist of contraction followed by relaxation, and inhibition of subsequent contraction in response to acetylcholine. An hypothesis was formulated to explain these actions. The contraction is attributed to complexing and consequent decrease in the activity of magnesium in or near the membrane, accompanied by unstabilization of the membrane following removal of calcium from sites at which it exerts a stabilizing action. The relaxation is attributed to similar complexing of membrane calcium bound to other sites from which it is released during excitation to cause contraction. Na4EDTA was much more effective in evoking contractions than Na2EDTA; this is attributed to the competition between hydrogen and magnesium.Experiments in which ions in the bathing medium were varied supported this general hypothesis. Furthermore, MgEDTA was found to produce only relaxation, while CaEDTA was found to produce little or no effect; mixtures had intermediate effects. The calcium-depleted membrane was found to compete effectively with CaEDTA for its calcium. Similar studies were carried out with DTPA (diethylenetriaminepentaacetic acid) and similar results obtained. DTPA was more dependent than EDTA on a high pH, as expected from the greater affinity of this material for hydrogen ions. EGTA (ethylene bisglycol (β-aminoethylether) tetraacetic acid), a substance with equivalent ability to complex calcium and less ability to complex magnesium relative to EDTA or DTPA, was ineffective in evoking contraction unless magnesium was omitted from the medium; however, it did inhibit responses to subsequent acetylcholine. None of these complexing agents produced significant contractions when strontium was substituted for calcium.Other complexing agents unrelated to EDTA were studied as well. Desferrioxamine (DFO) caused contractions when added at a pH of 9 or more. It had little inhibitory action on acetylcholine-evoked contractions. Oxalate was relatively ineffective in evoking contraction or inhibition of responses to acetylcholine. Both these sets of findings are compatible with the hypotheses for EDTA when considered in the light of the complexometric characteristics of DFO and oxalate. 1,10-Phenanthroline, which complexes neither magnesium nor calcium effectively, evoked no contractions, but did inhibit contractions in response to other agents by a mechanism apparently unrelated to calcium.
 
Article
In rats, spironolactone and ethylestrenol, like phenobarbital, enhance the NADPH-dependent hydroxylation of benzo(a)pyrene in the hepatic microsomal plus supernatant fraction and increase liver weight and microsomal phospholipid content as well as NADPH cytochrome c reductase and diaphorase activities, but only ethylestrenol and phenobarbital influence the microsomal protein content and cytochrome P-450 level.Neither spironolactone, ethylestrenol, nor phenobarbital affects NADH cytochrome c reductase and diaphorase activities, and only phenobarbital alters the cytochrome b5 level.These findings indicate that, while both the steroids and phenobarbital stimulate microsomal mixed-function oxidation, there are qualitative and quantitative differences between their action.
 
shows representative examples of K +-induced contraction and of ACh-and NA-evoked dose-response curves recorded in the control group. Results of contractions induced by K + at each resting tension are shown in Table 1. For each value of resting tension (i.e. 1.25, 1.50, 1.75, and 2.00 g), there were no significant differences in the strength of the responses to K +-induced depolarization between acclimatized and non-acclimatized samples. We also compared the strength of the maximal contraction developed by each vessels in response to K + , regardless of the basal tension value; acclimatisation to 10.1 MPa 
Representative recordings of tension changes induced in non-acclimatized eel ventral aorta by both high KCl and cumulative concentrations of either acetylcholine or noradrenaline.
Article
We examined in vitro vascular reactivity of eels previously acclimatized to 10.1 MPa hydrostatic pressure (HP) for 21 days. The isometric tension developed by ventral aortic rings was measured at atmospheric pressure. Dose-response curves for either acetylcholine (ACh) or noradrenaline (NA), as well as contractions evoked by 80 mM K+, were compared with time-matched experiments conducted on rings obtained from control eels. Results showed that neither the optimal tension nor the maximal force of the K+-evoked contraction were significantly modified, suggesting that acclimatization to high HP did not change the vascular smooth muscle contractile machinery. The dose-response curve to ACh was not significantly changed. Conversely, although NA always relaxed aortic rings, the response of acclimatized eels was significantly reduced over the entire range of the agonist concentration tested (10(-8) to 10(-3) M), except for the lowest one (10(-9) M). The maximal amplitude of the NA-induced relaxation was significantly reduced in aortic rings from acclimatized eels as compared with non-acclimatized samples (339.3 +/- 86.5 vs. 744.3 +/- 72.1 mg x mg(-1) dry weight, P < 0.005). Our results suggest that acclimatization to high HP could selectively alter the control of vascular tone by catecholamines.
 
Article
Nonalcoholic steatohepatitis (NASH) is a common and potentially severe form of liver disease. This study aimed to determine the effect of ursodeoxycholic acid and its NO-releasing derivative NCX-1000 alone or in combination with antioxidants on cultured mouse hepatocytes treated with amiodarone to mimic certain aspects of hepatocyte injury found in NASH. Isolated mouse hepatocytes were incubated with ursodeoxycholic acid or NCX-1000 (0-100 micromol/L) combined or not combined with the hydrophilic antioxidants butylated hydroxytoluene and ascorbic acid (0-100 micromol/L) or with the lipophilic antioxidant alpha-tocopherol (0-100 micromol/L) 15 min before adding amiodarone (50 micromol/L) to the culture medium. Twenty hours later, necrosis, apoptosis, superoxide anion production, and malondialdehyde levels were assessed in cultured cells. Amiodarone led to a dose-dependent decrease in cell viability with an LD50 of 50 micromol/L and increased production of superoxide anion and lipid peroxidation. NCX-1000 showed a better protective potential than ursodeoxycholic acid against the toxic effects of amiodarone. The hydrophilic antioxidants had no effect on the toxicity of amiodarone, whereas alpha-tocopherol at a concentration >100 micromol/L almost completely suppressed it. Ursodeoxycholic acid and NCX-1000 protection was additive only when they were combined with alpha-tocopherol, not with butylated hydroxytoluene or ascorbic acid. In addition, all the antioxidants tested reduced the superoxide anion detected, but only alpha-tocopherol prevented lipid peroxidation induced by amiodarone. The combination of lipophilic antioxidants with ursodeoxycholic acid or NCX-1000 enhances their protective potential and could represent an interesting therapeutic approach to explore for the treatment of NASH.
 
Body mass and body composition.
Skeletal muscle fiber cross sectional area for type I and type II muscle fibers in the soleus (A) and extensor digitorum longus (B). *, significantly different from CON (P < 0.05). 
Article
Corticosteroids are used as chemotherapeutic agents in many medical conditions, despite many common and potentially serious side effects. Supplementation with creatine monohydrate (CrM) can increase strength and lean body mass in humans and, therefore, may be a viable countermeasure to the side effects of corticosteroids. Therefore, the purpose of this study was to determine if CrM could prevent the attenuation of growth associated with corticosteroid administration. Forty male Sprague-Dawley rats were randomized to the following groups: control (CON, n = 10), 7 mg methylprednisolone x kg(-1) x week(-1) (PRED, n = 10), 2% CrM in diet (CD, n = 10), or CrM and methylprednisolone (CD-PRED, n = 10). Animals received either a weekly sham injection (saline; CON and CD) or an injection of methylprednisolone (PRED and CD-PRED) for 6 weeks. At the completion of the 6th week, body composition was determined and skeletal muscles were collected. Weight gain was attenuated in PRED as compared with all other groups (P < 0.05). Muscle total creatine and phosphocreatine were greater in the extensor digitorum longus in the CD and CD-PRED groups as compared with the CON and PRED groups (P < 0.05); however, total creatine and phosphocreatine in the soleus were not different. Mean fiber area was greater in type II fibers from the extensor digitorum longus in the CD and CD-PRED groups as compared with the CON and PRED groups (P < 0.05); no treatment effect was seen in the soleus. In conclusion, CrM supplementation prevented the attenuation of growth associated with corticosteroids and also increased type II muscle fiber area. These results could have important clinical implications for several patient populations commonly treated with corticosteroids, and further work is required to determine the specific mechanisms underlying the physiological effects that were observed.
 
Top-cited authors
Barbara C Vanderhyden
  • Ottawa Hospital Research Institute
Táňa Ravingerová
  • Slovak Academy of Sciences
Nader Shahrokhi
  • Physiology and Neuroscience Research Center, kerman, iran
Mohammad Khaksari Haddad
  • Kerman University of Medical Sciences
Zahra Soltani
  • Kerman University of Medical Sciences