bluuebunny/crossref_metadata_2025 huggingface dataset:
N-Aralkylated derivatives of 1-aminobenzotriazole as isozyme-selective, mechanism-based inhibitors of guinea pig hepatic cytochrome P-450 dependent monooxygenase activity
Authors: Woodcroft Kimberley J. , Bend John R.
Published: April 2011

The mechanism-based inactivation of hepatic cytochrome P-450 by the suicide inhibitor 1-aminobenzotriazole and two of its derivatives, N-benzyl-1-aminobenzotriazole and N-α-memylbenzyl-1-aminobenzotriazole, was investigated in microsomes from untreated, phenobarbital-induced, and β-naphthoflavone-induced guinea pigs. Microsomal 7-ethoxyresorufin O-deethylase, 7-pentoxyresorufm O-dealkylase, and benzphetamine N-demethylase activities, and cytochrome P-450 content were determined following incubation with 1-aminobenzotriazole and its analogues. The loss of hepatic cytochrome P-450 content and monooxygenase activity was dependent on inhibitor concentration and required NADPH. N-Benzyl-1-aminobenzotriazole and N-α-methylbenzyl-1-aminobenzotriazole were more potent inhibitors of monooxygenase activity than the parent compound in microsomes from untreated and phenobarbital-induced guinea pigs. In microsomes from phenobarbital-induced guinea pigs, N-α-methylbenzyl-1-aminobenzotriazole (10 μM) was highly selective for the inactivation of the major cytochrome P-450 isozyme catalyzing 7-pentoxyresorufin O-dealkylation (the guinea pig ortholog of P-450IIB1) compared with those isozymes catalyzing 7-ethoxyresorufin O-deethylation or benzphetamine N-demethylation (88 ± 3% loss of activity vs. 35 ± 11 and 13 ± 7%, respectively). N-Benzyl-1-aminobenzotriazole was also selective for the inactivation of 7-pentoxyresorufm O-dealkylase activity, but to a lesser degree (56 ± 6 vs. 31 ± 8 and 21 ± 8%, respectively). In hepatic microsomes from untreated guinea pigs, the two N-substituted analogues were selective for the inhibition of 7-pentoxyresorufin O-dealkylation compared with benzphetamine N-demethylation, but not 7-ethoxyresorufin O-deethylation. The spectrally assayed loss of cytochrome P-450 caused by 1-aminobenzotriazole paralleled the inhibition of enzyme activity in all three treatment groups; however, the loss of cytochrome P-450 caused by N-benzyl-1-aminobenzotriazole and N-α-methylbenzyl-1-aminobenzotriazole was never greater than 45% even when monooxygenase activity was virtually 100% inhibited. In general, N-benzyl-1-aminobenzotriazole and N-α-methylbenzyl-1-aminobenzotriazole were more potent inhibitors of cytochrome P-450-dependent monooxygenase activity in hepatic microsomes from untreated compared with induced guinea pigs (for example, 100 μM N-benzyl-1-aminobenzotriazole inhibited 93 ± 3, 81 ± 1, and 61 ± 7% of the 7-ethoxyresorufin O-deethylase activity in hepatic microsomes from untreated, phenobarbital-induced, and β-naphthoflavone-induced guinea pigs, respectively). These latter data are consistent with the facile inactivation of guinea pig P-450IA1 but not P-450IA2 by N-benzyl-1-aminobenzotriazole.Key words: cytochrome P-450, guinea pig, isozyme selective, suicide inhibitors.
Biliary excretion of proteins in the rat during dehydrocholate choleresis
Authors: Marinelli Raúl A. , Carnovale Cristina E. , Garay Emilio A. Rodríguez
Published: April 2011

Choleresis induced by dehydrocholate (DHC) stimulates the discharge into bile of lysosomes, which are implicated in the biliary excretion of proteins. Contrary to taurocholate-induced choleresis, DHC choleresis is not affected by microtubule (mt) inhibition. Therefore, the role of mt's in the biliary protein excretion during bile salt choleresis was analyzed in this study. Normal rats and rats treated with the mt poisons colchicine or vinblastine or with the acidotropic agent chloroquine (Cq) were used. The analysis of the protein component in bile was made on SDS–polyacrylamide gel, and the individual polypeptides were quantitated by densitometry. The excretion of bile polypeptides was compared with that of lysosomal acid phosphatase. Bile flow and bile salt output did not show changes on account of treatments. The biliary excretion of acid phosphatase was stimulated by DHC, and it was not affected by mt inhibitors but was markedly diminished by Cq. DHC choleresis produced different effects on the bile polypeptides. The biliary excretion of polypeptides of high molecular mass (84–140 kDa) was stimulated by DHC. Cq treatment increased their basal biliary excretions, whereas DHC-induced secretion was qualitatively and quantitatively similar to that of controls. The 69-kDa polypeptide (albumin) also increased during DHC-induced choleresis, but it showed a different excretory pattern. Cq treatment inhibited such an increase but no correlation with the excretory pattern of the lysosomal marker was found. The biliary excretion of polypeptides of low molecular mass (down to 14 kDa) suffered a transitory decrease and then a subsequent increase over basal values during the DHC choleresis. Cq treatment diminished their biliary excretions during basal and DHC-induced choleresis. Treatment with the mt inhibitors markedly diminished the biliary excretions of all polypeptides. The results indicate that DHC-induced choleresis influenced the biliary protein excretion in a way different to that induced by taurocholate. Thus, the biliary excretion of proteins that did not require lysosomes to reach the bile was stimulated, while that requiring such organelles was transiently diminished. In addition, the biliary excretion of proteins was dependent on mt's excepting those of lysosomal source.Key words: dehydrocholate choleresis, biliary proteins, microtubule inhibitors, chloroquine.
Inactivation of rat liver microsomal steroid hydroxylations by 4-alkyl analogues of 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine: evidence for selectivity among steroid-inducible cytochrome P450IIIA forms
Authors: Riddick D. S. , McGilvray I. , Marks G. S.
Published: April 2011

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 7α-hydroxylase, AD 16β-hydroxylase, and PG 21-hydroxylase, selective markers for P450IIA1/2, IIB1, and IIC6, respectively. In contrast, DDC analogues were effective inactivators of PG 2α-hydroxylase and steroid 6β-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 6β-hydroxylases. However, in microsomes from dexamethasone-treated female rats, which express P450IIIA1 but not IIIA2, no inactivation of the steroid 6β-hydroxylases was observed. Thus, 4-ethyl DDC appears to be a potentially valuable tool for differentiating between P450IIIA forms.Key words: 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine, cytochrome P-450, mechanism-based inactivation, rat hepatic microsomes, steroid hydroxylations.
Cytochrome P-450 mediated biotransformation of organic nitrates
Authors: McDonald Bernard J. , Bennett Brian M.
Published: April 2011

The vascular biotransformation of organic nitrates appears to be a prerequisite for their action as vasodilators. In the current study, we assessed the involvement of cytochrome P-450 in the denitration of glyceryl trinitrate and the enantiomers of isoidide dinitrate. Denitration of organic nitrates by the microsomal fraction of rat liver was NADPH dependent and followed apparent first-order kinetics. Under aerobic conditions, the t1/2 of D-isoidide dinitrate was significantly shorter than that of L-isoidide dinitrate (11.9 vs. 14.1 min, p ≤ 0.05), which is consistent with the greater potency of the D-enantiomer for vasodilation. Under anaerobic conditions, the denitration of glyceryl trinitrate was very rapid (t1/2 approximately 30 s). Organic nitrate biotransformation was inhibited by carbon monoxide, SKF 525A, and dioxygen. This suggests that the biotransformation of organic nitrates can occur through the direct interaction with the heme moiety of cytochrome P-450. The biotransformation of glyceryl trinitrate was catalyzed preferentially by those isoenzymes induced by phenobarbital. The biotransformation of glyceryl trinitrate was regioselective for 1,3-glyceryl dinitrate formation except in phenobarbital-induced microsomes under aerobic conditions, in which preferential formation of 1,2-glyceryl dinitrate occurred. These data suggest that cytochrome P-450 is involved in the biotransformation of organic nitrates and raises the possibility that vascular cytochrome P-450 may play a role in the mechanism-based biotransformation of organic nitrates, the result of which is vascular smooth muscle relaxation.Key words: cytochrome P-450, glyceryl trinitrate, isoidide dinitrate, biotransformation, liver.
Effect of subchronic administration of ethanol and methylmercury in combination on the tissue distribution of mercury in rats
Authors: Turner C. J. , Bhatnagar M. K. , Speisky H.
Published: April 2011

The effect of oral administration for 14 weeks of 8 g∙kg−1∙day−1 ethanol and 0.5 mg∙kg−1∙day−1 methylmercuric chloride in combination to rats fed isocaloric diets has been investigated. Ethanol, in contrast to published studies, failed to influence the tissue distribution of methylmercury and its inorganic mercury metabolite in brain and kidney, and did not inhibit the increase in kidney weight induced by methylmercury. Ethanol and methylmercury, in combination and individually, reduced the renal but not the hepatic activity of γ-glutamyltransferase, but did not affect the renal and biliary concentration of reduced glutathione. Further study is required to determine the circumstances under which ethanol can influence the tissue distribution of methylmercury and its inorganic mercury metabolite.Key words: toxicology, ethanol, methylmercury.
Effect of protein synthesis Inhibition on brain corticotropin-releasing factor and plasma adrenocorticotropin
Authors: Haas Daniel A. , Sturtridge William C. , George Susan R.
Published: April 2011

The effect of inhibiting protein synthesis on concentrations of corticotropin-releasing factor (CRF) in rat brain and plasma adrenocorticotropin (ACTH) was assessed following the administration of the general protein synthesis inhibitor anisomycin. Compared with vehicle-injected controls, protein synthesis inhibition resulted in significantly reduced CRF immunoreactivity (CRF-ir) in median eminence within 1 h (p < 0.01), remained decreased after 4 h (p < 0.025), and was nonsignificantly decreased after 24 h. Plasma ACTH levels were greatly increased within 1 h posttreatment (p < 0.0005), continued elevated after 4 h (p < 0.01), and returned to normal levels after 24 h. CRF-ir measured in other brain areas 24 h after anisomycin showed decreased levels in medulla–pons (p < 0.025) and neurointermediate lobe of pituitary (p < 0.05), with no change noted in frontal cortex, hippocampus, midbrain–thalamus, or cerebellum. Overall these data show that blockade of normal protein synthesis with anisomycin can elicit changes in CRF-ir and ACTH content.Key words: corticotropin-releasing factor, adrenocorticotropin, anisomycin.
Receptors for Arg8-vasopressin, angiotensin II, and atrial natriuretic peptide in the mesenteric vasculature of pregnant rats
Authors: Parent Angèle , Schiffrin Ernesto L. , St-Louis Jean
Published: April 2011

Blood pressure and sensitivity of blood vessels to vasoconstrictors are decreased in term-pregnant rats (20–21 days). To determine if changes in receptors for vasoactive peptides could account for these observations, receptor kinetics were measured for Arg8-vasopressin (AVP), angiotensin II (Ang II), and atrial natriuretic peptide (ANP) in the mesenteric vascular bed of the rat throughout pregnancy. Receptors for AVP were statistically similar in the five groups of animals (nonpregnant; pregnant 9, 15, and 21 days; and postpartum). The dissociation constant (KD) for [3H]AVP varied from 0.41 to 0.52 nmol/L (NS), while receptor density (Bmax) varied from 310 ± 110 to 455 ± 135 fmol/mg protein for six experimental measurements. Similar observations were made for Ang II receptors where KD of 125I-labelled Sar1, Ile8-Ang II was between 0.60 and 0.97 nmol/L and Bmax between 215 ± 30 and 250 ± 40 fmol/mg protein in the different groups. 125I-labelled ANP (101–126) receptors were markedly modified in terms of number of sites. Bmax was significantly increased during pregnancy (9 days, 429 ± 86; 15 days, 541 ± 54; 20 days, 438 ± 72) and decreased in the postpartum period (133 ± 21) by comparison with the nonpregnant group (245 ± 35 fmol/mg protein), while KD was similar in the different experimental groups (57 to 82 pmol/L). Despite these increases in receptor density, the vasorelaxant effects of ANP was only increased at 9 days of pregnancy. These results indicate that receptors for vasoconstrictor peptides (AVP and Ang II) are not altered during pregnancy and that receptor homospecific downregulation does not explain the decrease in sensitivity to vasoconstrictors at the end of pregnancy in the rat. Receptors for ANP are, however, markedly increased in number, but this did not result in an increase in the vascular action of the peptide throughout pregnancy.Key words: pregnancy, receptors, angiotensin, vasopressin, atrial natriuretic peptide.
Synergic activity of combined prostacyclin: dimethyl sulfoxide in experimental brain ischemia
Authors: Torre J. C. de la
Published: April 2011

We examined whether drugs that block calcium, prostaglandins, free radicals, and endorphin release could modify cerebral blood flow or nerve tissue pathology following a focal cerebrovascular lesion. Cats were randomly divided into six groups and were subjected to standard middle cerebral artery occlusion (MCAO) performed using a transorbital approach. One hour after MCAO, cats received the following compounds intravenously: (i) saline (CS), 1.5 mL/kg or polyethylene glycol, 300 μg (CP); (ii) naloxone (NX), 2 mg/kg; (iii) nimodipine (NM), 1 μg∙kg−1∙min−1 × 60 min; (iv) dimethyl sulfoxide (DS), 0.9 g/kg in a 40% solution; (v) prostacyclin (PGI2), 200 ng∙kg−1∙min−1 for 60 min; or (vi) DS–PGI2 combined. At 1-h intervals, local CBF was recorded from the cortical tissue proximal and distal to the MCAO site using the hydrogen clearance method. Five hours after MCAO, cortical tissue was removed for catecholamine histofluorescence or perfused for tyrosine hydroxylase immunoreactive axon examination. Treatment with NX, NM, CP, or CS had no effect on either CBF or cortical tissue neurotransmitter morphology. PGI2 showed a transiently modest but significant increase of CBF, while DS provided moderate protection of catecholaminergic fibers and increased CBF by 27% after MCAO. The combination of DS–PGI2 resulted in significant cytoprotection of cortical catecholaminergic fibers and generated a sustained CBF increase of 68% of control values. These findings suggest that combining DS with PGI2 can yield a synergic effect with respect to cortical neurotransmitter and CBF protection after MCAO.Key words: dimethyl sulfoxide, prostacyclin, cerebral ischemia, nimodipine, naloxone.
The effects of ethylenediamine in the rat small intestine: a powerful relaxant of the muscularis
Authors: McKay Allison , Krantis Anthony
Published: April 2011

The pharmacology of ethylenediamine (EDA) actions in the rat small intestine was examined using isolated gut-bath preparations of proximal segments of the duodenum, jejunum, and ileum. EDA evoked concentration-dependent tetrodotoxin-insensitive relaxations of the intestine, evidently by direct action on the muscularis. Such actions were simultaneous on the longitudinal and circular muscle layers. Investigation of EDA actions on the circular muscle showed that EDA actions were unrelated to any intrinsic GABAergic mechanisms. Moreover, EDA interacted with muscle sites distinct from ATP, histamine, bradykinin, muscarinic, and adrenergic receptors. The ability of EDA to relax the intestinal musculature was generally greater than the smooth muscle relaxant papaverine and substantially better than nicotinic stimulation of the intrinsic inhibitory neurones. It would appear that EDA may be useful as a direct acting smooth muscle relaxant for the study of the physiology–pharmacology of the rodent small intestine.Key words: ethylenediamine, rat intestine, muscle relaxant, circular muscle.
Hypothyroidism increases serotonin turnover and sympathetic activity in the adult rat
Authors: Henley W. N. , Chen X. , Klettner C. , Bellush L. L. , Notestine M. A.
Published: April 2011

Adult, male Sprague–Dawley rats underwent surgical thyroidectomy (Tx) or sham surgery. In all three experiments from which data are reported, a 3-week recovery period was allowed. In experiments I and II, baseline measurements of colonic temperature (Tc) and urinary norepinephrine excretion (NE) were obtained, and both variables were monitored daily for the duration of the studies. After baseline measurements, half of each surgical group was given either triiodothyronine (T3) or vehicle injections subcutaneously; in experiment I replacements continued for 1.5 days, while in experiment II T3 replacement continued for 3.5 days. Rats were decapitated at the end of each experiment and serotonin (5-HT) turnover was measured in brainstem. Serotonin turnover in rostral and caudal brainstem was increased with Tx (p < 0.05). Increased turnover in caudal brainstem was normalized by T3 only in experiment II. Similarly, decreased Tc and elevated NE with Tx were normalized in experiment II but not in experiment I. In experiment III, NE measurements normalized on a creatinine excretion basis indicated that increased NE is evident with Tx, irrespective of normalization procedure. Significant correlations between 5-HT in caudal brainstem and metabolic correlates of sympathetic function, concurrent normalization of NE and 5-HT in caudal brainstem, plus work from other laboratories describing sympathoexcitatory serotonergic neurons located in me caudal brainstem suggest that the central and peripheral changes in me hypothyroid rat are causally related.Key words: triiodothyronine, norepinephrine, serotonin, brainstem, metabolism.
Free radicals may contribute to oxidative skeletal muscle fatigue
Authors: Barclay J. K. , Hansel M.
Published: April 2011

We used mouse soleus in vitro (n = 30) and canine gastrocnemius–plantaris preparations (n = 20) pump-perfused at the animal's blood pressure to establish if free radicals contribute to fatigue in oxidative skeletal muscle. The soleus from each leg contracted for 200 ms (70 Hz) once every minute for 60 min in Hepes buffer gassed with 100% oxygen at 27 °C. When contracting in Hepes alone, both muscles fatigued at 0.9 mN/mm2∙min over the 60 min. The addition of purines to the bath increased the rate to 1.4 mN/mm2∙min and the addition of xanthine oxidase to generate free radicals increased the rate again to 1.9 mN/mm2∙min. Thus free radicals appeared to attenuate oxidative skeletal muscle function. Each canine muscle contracted isometricaliy at 4 Hz for 30 min and then rested for 45 min before contracting for a second 30 min at 4 Hz. In each experiment, we infused saline at 0.76 mL/min into resting muscle and at 1.91 mL/min during the first contraction period. During the remainder of the experiment, we infused, at the same rates, saline (n = 4), 10 μM dimethyl sulfoxide (DMSO) (n = 4) to identify the effect of scavenging hydroxyl radicals, 1 mM allopurinol to establish the effect of blocking xanthine oxidase (n = 4), or 200 μM desferoxamine to determine the effect of chelating iron (n = 4). With saline, the fatigue rate over the 30 min of contractions increased from 5.0 ± 0.2 to 6.3 ± 0.5 N/kg∙min from the first to the second stimulation period. The fatigue rate was slower in the second period with each of the three experimental substances (DMSO, 5.9 ± 0.8 to 3.2 ± 0.3; allopurinol, 7.3 ± 1.1 to 4.6 ± 0.6; desferoxamine, 6.8 ± 0.8 to 4.4 ± 0.8 N/kg∙min). The fatigue rate was the same as control when DMSO was infused only during the second contraction period. Therefore, free radicals appeared to contribute to fatigue in oxidative skeletal muscle.Key words: twitch and tetanic contractions, desferoxamine, allopurinol, dimethyl sulfoxide, xanthine oxidase, pump-perfused muscles.
Muscle metabolism in older subjects using 31P magnetic resonance spectroscopy
Authors: McCully Kevin K. , Forciea Mary Ann , Hack Laurita M. , Donlon Eileen , Wheatley Roger W. , Oatis Carol A. , Goldberg Todd , Chance Britton
Published: April 2011

We used phosphorus magnetic resonance spectroscopy to study the calf muscles of elderly normal (mean ± SD) (80.0 ± 5.12 years), elderly impaired (80.7 ± 0.58 years), old normal (66.8 ± 1.92 years), and young normal people (24.6 ± 4.72 years). Relative levels of inorganic phosphate (Pi), phosphocreatine (PCr), and adenosine triphosphate were measured with a 1.9-tesla, 30-cm bore magnet at rest and following plantra flexon exercise. No differences were found at rest or during recovery from exercise in the elderly normal subjects with respect to gender or the presence of stable medical problems treated with medication. At rest there was an age-related decrease in the ratio of PCr/Pi. After exercise, the time constant of PCr recovery increased with age. A mild 7-week exercise regimen consisting of plantar flexion had no effect on time constant of PCr recovery in the elderly subjects. Four elderly impaired subjects had lower PCr/Pi ratios at rest and slower time constant of PCr recovery after exercise than normal elderly subjects. We conclude that gender and the presence of stable medical problems had no effect on muscle metabolism in the elderly and that the elderly recovered slower than young controls. This slower recovery was not corrected with a mild exercise program.Key words: human muscle, aging, exercise, nuclear magnetic resonance, gastrocnemius.
The 1990 Merck Frosst Award. Ischemic and reperfusion injury in the heart. Cellular mechanisms and pharmacological interventions
Authors: Karmazyn Morris
Published: April 2011

Reperfusion in the heart represents an important form of tissue injury, particularly in view of the emerging importance of reperfusion protocols aimed at salvaging the ischemic myocardium. Both the manifestations and the causes of reperfusion injury are multifold. With respect to the former, reperfusion injury can be characterized by various abnormalities including development of arrhythmias, contractile dysfunction, ultrastructural damage as well as various defects in intracellular biochemical homeostasis. The mechanisms underlying myocardial reperfusion injury are equally complex, but most likely involve numerous processes acting in concert resulting in eventual cell death. In this review, a description of various such potential mechanisms, which represent primary interests of the author, are presented. An understanding of these mechanisms has led to novel pharmacological approaches towards the protection of the reperfused myocardium. For instance, several lines of evidence implicate enhanced eicosanoid, and in particular prostaglandin, synthesis in reperfusion injury, since (1) such injury is involved with enhanced prostaglandin biosynthesis, (2) inhibition of prostaglandin synthesis with various nonsteroidal anti-inflammatory drugs attenuates injury, and (3) exogenous prostaglandins increase injury. Another intracellular process that is emerging as an important contributor to reperfusion injury in the heart is the Na+/H+ exchanger, which is most likely activated upon reperfusion. Such activation would lead to numerous intracellular disturbances including the increased synthesis of prostaglandins and elevated intracellular Ca2+ concentrations. Indeed, inhibitors of Na+/H+ exchange such as amiloride have been shown to effectively inhibit reperfusion injury. Reperfusion is also associated with depressed mitochondrial function, particularly in subsarcolemmal mitochondria which are rapidly injured as a result of both ischemic and reperfusion conditions. Preservation of mitochondrial function with dissimilar approaches such as carnitine or phosphatidylcholine administration markedly reduces reperfusion injury. A nonpharmacological novel approach towards the protection of the reperfused myocardium represents the induction of so-called stress or heart shock proteins in the heart prior to initiation of ischemia and reperfusion. The salutary effect of the heat shock response may be dependent not on the heat shock proteins themselves, but through the concomitant elevation of tissue catalase content resulting in enhanced detoxification of intracellular hydrogen peroxide. Thus reperfusion injury represents numerous complex events such that manipulations aimed at limiting such injury can be initiated to prevent specific defects with the ultimate goal of an overall reduction in cell damage.Key words: heart, ischemia, reperfusion, prostaglandins, leukotrienes, Na+/H+ exchange, subsarcolemmal mitochondria, interfibrillar mitochondria, heat shock proteins, tissue protection.
Calcium dependence of effects of endothelin on rat mesenteric microvessels
Authors: Deng Li Yuan , Schiffrin Ernesto L.
Published: April 2011

We investigated the calcium dependence of the effects of endothelin (ET) on resistance vessels (less than 300 μm lumen diameter) from the mesenteric vascular bed of the rat, mounted on a wire myograph. ET-1 induced a potent sustained contraction with an ED50 of 12 nmol/L. The response to ET-3 and big ET at the maximum concentrations used (100 nmol/L) was less than 40% of that to ET-1, with an estimated ED50 of 45 nmol/L. Relaxation of the ET-1-induced contraction was slow, and resulted in a reduction of the maximum response to a second challenge with ET-1 to 60% of the initial contraction after 3 h. Long-lasting tachyphylaxis to arginine vasopressin (AVP) induced contraction also occurred. The response to 100 nmol/L ET-1 produced an active tension 88% greater than that induced by 124 mmol/L KCl, and similar to that produced by norepinephrine and AVP. The response to 100 nmol/L ET-1 in the absence of calcium + 1 mmol/L EGTA in the medium for 30 min resulted in a maximum contraction of 43% of the response in the presence of calcium, followed by a faster relaxation rate. The addition of calcium produced a further contraction, and stimulation with 100 nmol/L ET-1 at this point did not result in further response. The calcium channel blocker nitrendipine in concentrations of 1–10 μmol produced increasing reductions of the responses to 100 nmol/L ET-1 to 35% at the higher concentration. Nitrendipine (3 μmol/L) partially blocked the response to calcium after ET-1 was added in the absence of calcium. We conclude that resistance microvessels of the rat mesentery are more sensitive to ET-1 than ET-3 or big ET, which were also much less potent. These microvessels have a sensitivity to AVP 20 times greater than that to ET-1. The response to ET-1 may be mediated in part by the release of intracellular calcium, but the influx of calcium from the extracellular fluid through plasma membrane voltage-dependent channels is necessary for completing approximately 60% of the initial contraction and for the persistence of a sustained response.Key words: mesenteric arterioles, vasopressin, norepinephrine, nitrendipine, calcium channel blockers.
Effects of retinoic acid on the concentrations of radioactive metabolites of retinol in tissues of rats maintained on a retinol-deficient diet
Authors: Bhat Pangala V. , Lacroix André
Published: April 2011

The effect of feeding retinoic acid for 2 and 6 days on the metabolism of labeled retinol in tissues of rats maintained on a vitamin A deficient diet was studied. The metabolites of retinol were analyzed by high performance liquid chromatography. Feeding retinoic acid for 2 days significantly reduced the blood retinol and retinyl ester levels without affecting the vitamin A content of the liver. In intestine and testis the content of labeled retinoic acid was decreased significantly by dietary retinoic acid. Addition of retinoic acid to the diet for 6 days resulted, in addition to decreased blood retinol and retinyl ester values, in an increase in the retinyl ester values in the liver. The accumulation of retinyl ester in the retinoic acid fed rat liver was accompanied by an absence of labeled retinoic acid. Kidney tissue was found to contain the highest levels of labeled retinoic acid, retinol, and retinyl esters; dietary retinoic acid did not alter the concentrations of these retinoids in the kidney during the experimental period. Since kidney retained more vitamin A when the liver vitamin A was low and also dietary retinoic acid did not affect the concentrations of radioactive retinoic acid in the kidney, it is suggested that the kidney may play a major role in the production of retinoic acid from retinol in the body.Key words: retinol, retinoic acid, vitamin A deficiency, tissue metabolites, rat.
The correlation between the increase in slow outward current and in contraction induced by caffeine, ryanodine, and rapid cooling in voltage-clamped frog muscle fibers
Authors: Nasri-Sebdani Mohammad , Traoré Flavien , Mouzou Aklesso , Cognard Christian , Potreau Daniel , Raymond Guy
Published: April 2011

The effects of caffeine, ryanodine, and rapid cooling were tested on the depolarization-induced contraction and the apamin-insensitive slow outward current (Iso) of voltage-clamped (double mannitol gap) single frog muscle fibers. Subthreshold caffeine concentrations (0.5–2 mM) induced a monotonic increase in contractile and Iso amplitude. Whatever the concentration, the increase in contraction was roughly twice the one in current. Similar results were obtained upon rapid cooling (20–4 °C) in the presence of 0.5 mM caffeine. In the absence of external Na+ (choline-substituted) 10−5 M ryanodine induced a delayed increase (≈30 min) in contraction and in current, shortly before the development of a drastic and irreversible contracture. Here again, the increase in contraction was twice that in current. In the presence of 5 mM tetraethylammonium (TEA) and (or) 25 nM charybdotoxin, 2 mM caffeine still induced a strong facilitating effect on contraction but the parallel increase in current was strongly reduced. The linear relationship between the increase in current and contractile amplitude has a slope ≈0.5 (whatever the drug used to increase contractility); it is ≈0.1 in the presence of TEA and (or) charybdotoxin. In conclusion, provided the changes in contractile amplitude are caused by parallel changes in depolarization-induced sarcoplasmic reticulum Ca2+ release, about 50% of the apamin-insensitive Iso is controlled by internal Ca2+ release. The main part of this current corresponds to the TEA- and charybdotoxin-sensitive component of Iso.Key words: skeletal muscle, voltage clamp, Ca2+-dependent K+ current, sarcoplasmic reticulum calcium release, caffeine, ryanodine, charybdotoxin.
The vasodilator effect of thiamylal in dog mesenteric artery: contribution of intracellular action
Authors: Nakamura Kumi , Hatano Yoshio , Nishiwada Makoto , Mori Kenjiro
Published: April 2011

The effects of thiamylal on contractions induced by various mechanisms were investigated in mesenteric arteries isolated from dogs. Thiamylal (10−4 to 10−3 M) significantly inhibited contractions induced by KCl (20 mM) in normal media, and those induced by norepinephrine (10−5 M) in normal and Ca2+-free media. Caffeine-induced contraction was significantly inhibited by thiamylal in the concentrations greater than 3 × 10−5 M in intact fibers and 10−5 M in chemically skinned fibers. Chemically skinned fibers that were precontracted with Ca2+ were relaxed by thiamylal in concentrations lower than those required to relax intact fibers that were precontracted with KCl (20 mM); the ED50 was 1.52 × 10−5 M in skinned fibers and 5.50 × 10−4 M in intact fibers. These results suggest that intracellular mechanisms are involved in thiamylal-induced vasodilatation of dog mesenteric artery.Key words: barbiturates, thiamylal, mesenteric artery, vascular smooth muscle.
Comparative effects of dopamine and dobutamine on glucoregulation in a rat model
Authors: Keck F. S. , Foldenauer A. , Zeller G. , Wolf Chr.-F. , Pfeiffer E.-F.
Published: April 2011

The influence of dopamine as compared with dobutamine on glucose homeostasis has been assessed in thyroidectomized euthyroid rats. Both sympathomimetic agents were given intravenously over 6 h at four dosages, varying from 2 to 30 μg∙kg−1∙min−1. Immediately before the end of the infusion period, serum concentrations of glucose and insulin as well as plasma glucagon concentrations were measured. Dobutamine infusions did not exert any influence on these parameters. At a dose of 7.5 μg∙kg−1∙min−1, dopamine infusion caused a decrease in glucose concentrations, accompanied by a rise of glucagon and insulin levels. Glucose levels were significantly increased in the presence of unaltered insulin and decreasing glucagon levels at higher dopamine doses. The rise in glucose levels was reversed by 8 μg∙kg−1∙min−1 and inverted to a decrease by 12 μg∙kg−1∙min−1 of the α-adrenergic blocking agent phentolamine, simultaneously infused with 15 μg∙kg−1∙min−1 dopamine, while the insulin levels were increased and glucagon levels remained elevated. These findings demonstrate that dopamine acts on glucoregulation divergently, according to the dosage applied. The data suggest that dopamine rather than dobutamine treatment may disturb glucose homeostasis.Key words: dopamine, dobutamine, glucose regulation, thyroidectomy, rats.
Pattern-and age-dependency of the antiepileptic effects induced by valproic acid in the rat hippocampus
Authors: Fueta Yukiko , Avoli Massimo
Published: April 2011

The effects induced by the antiepileptic drug valproic acid were studied in the CA3 subfield of in vitro hippocampal slices obtained from young (16- to 27-day-old) and adult (over 60-day-old) rats. Spontaneous epileptiform discharges were induced by the addition of the convulsant 4-aminopyridine to the medium. Valproic acid (0.5 mM) selectively blocked the ictal epileptiform discharges in slices obtained from young rats. Interictal epileptiform discharges disappeared during perfusion with higher doses of valproic acid (2 mM). This blockade of interictal epileptiform activity was not observed when valproic acid (0.5–5 mM) was tested in hippocampal slices from adult rats. Thus, in the hippocampus of young rats, 4-aminopyridine-induced ictal activity is more sensitive to valproic acid than are interictal discharges. Moreover, valproic acid is effective in controlling interictal discharges in the young, but not in the adult rat hippocampus.Key words: valproic acid, epilepsy, 4-aminopyridine, hippocampus, rat.
Activation and function of prorenin: different viewpoints
Authors: Osmond D. H. , Sealey J. E. , McKenzie J. K.
Published: April 2011

This Symposium includes 15 presentations and an editorial review dealing with prorenin activation and function. It comes 20 years after prorenin was first reported in various contexts and attracted attention because of its connection with renin–angiotensin, its high concentration relative to renin in the blood, and its presence in extrarenal, as well as renal, tissues. Intriguing changes in plasma prorenin have been reported after treatment with antihypertensive and other drugs, following various physiological stimuli, and in pathophysiological states such as Wilms' tumor, Bartter's syndrome, and diabetic nephropathy. Lately, very high prorenin concentrations have been found in human and animal ocular fluid, ovarian follicular fluid, and in association with angiogenesis and microangiopathy. High circulating prorenin concentrations and fulminant hypertension have been reported in rats harbouring the mouse Ren-2 gene. However, what prorenin does in all these extrarenal fluids, tissues, and conditions is not well understood. Among the reasons for this lack of understanding are the difficulties in measuring prorenin and in establishing good animal models. We have not answered the critical question as to whether prorenin itself is bioactive like a hormone, and if so, what its action(s) might be. Nor have we established the main alternative, i.e., whether the function of prorenin is indirect, through renin–angiotensin, be it in the circulation or in the extrarenal tissues. This Symposium provides only partial methodological advances and answers, but we hope it will stimulate the breakthrough work needed to supply more complete answers.Key words: hormonal function, renin–angiotensin, hypertension.
Biochemical and immunological differences between plasma inactive renin from normal and nephrectomized rats
Authors: Kim Shokei , Hosoi Masayuki , Nakajima Kiichiro , Yamamoto Kenjiro
Published: April 2011

Using immunological techniques, we have demonstrated that about half the trypsin-activatable renin in normal rat plasma is prorenin, while the other is not, and that inactive renin in nephrectomized rat plasma is not prorenin. In the present study, the trypsin-induced angiotensin I generating activity not related to prorenin from normal rat plasma disappeared after HPLC on G3000SW. HPLC analysis of trypsin-treated plasma showed the generation of active renin by trypsin for normal rat plasma, while it did not for nephrectomized rat plasma. These results indicate that trypsin treatment of crude plasma results in the generation of angiotensin I generating activity not due to prorenin, as well as activation of prorenin. HPLC on G3000SW is a useful tool for the determination of plasma prorenin.Key words: prorenin, antibody against prorenin prosegment, trypsin treatment, molecular weight, nephrectomy.
Rate of fatigue during repeated submaximal contractions of human quadriceps muscle
Authors: Dolmage T. , Cafarelli E.
Published: April 2011

Our purpose was to determine the effect of eight different combinations of contraction intensity, duration, and rest on the rate of fatigue in vastus lateralis muscle. A single combination consisted of contractions at 30 or 70% maximal voluntary contraction (MVC), held for 3 or 7 s with 3- or 7-s rest intervals. Contractions were repeated until the subject could not hold the force for the requisite duration. At regular intervals during each experiment, a brief MVC, a single twitch, and the response to eight stimulation pulses at 50 Hz were elicited. The rate of fatigue was the rate of decline of MVC calculated from regression analysis. Mean rate of fatigue (n = 8) ranged from 0.3 to 25% MVC/min and was closely related (r = 0.98) to the product of the relative force and the duty cycle. Force from 50 Hz stimulation fell linearly and in parallel with MVC. Twitch force was first potentiated and then fell twice as fast as 50 Hz stimulation and MVC (p < 0.05). Differentiated twitch contraction and relaxation rates were higher at potentiation and lower at the limit of endurance, compared with control values (p < 0.05). The maximal electromyogram decreased 25% and the submaximal EMG increased to maximal by the end of the protocol, indicating that the entire motor unit pool had been recruited. The close relation between rate of fatigue and the force × time product probably reflects the off-setting interaction of contraction amplitude, duration, and rest interval. This occurs despite the changes in twitch characteristics and the apparent recruitment of fast fatiguing motor units.Key words: fatigue, surface EMG, limit of endurance, force × time product, twitch interpolation.
Protective effects of ceruloplasmin against electrolysis-induced oxygen free radicals in rat heart
Authors: Chamine Ramez , Mateescu Mircea Alexandru , Roger Stéphane , Yamaguchi Nobuharu , Champlain Jacques de , Nadeau Réginald
Published: April 2011

The potentially injurious effects of oxygen-derived free radicals (OFR) on the myocardium can be prevented in part by pretreatment with OFR scavengers or antioxidants. Since ceruloplasmin (CP) has been shown to possess potent antioxidant activity and scavenge a variety of OFR in vitro, we have undertaken to study its protective effects against myocardial injury induced by OFR. CP was freshly purified by a fast method that minimized proteolytic enzyme degradation. Free radicals were generated by the electrolysis (10 mA DC current for 1 min) of a Krebs–Henseleit solution perfusing an isolated rat heart preparation under constant pressure conditions. CP (0.25 μM) afforded 80 and 63% protection (n = 8, p < 0.05), respectively, against the deleterious effects of electrolysis-induced OFR on left ventricular pressure and coronary flow. The increase in left ventricular end diastolic pressure used here as an index of heart failure did not occur in the presence of 0.25 μM CP. Moreover, CP significantly reduced the increase of norepinephrine washout in the effluent perfusate after electrolysis suggesting a protection against free radical-induced injury to sympathetic nerve endings.Key words: oxygen free radicals, heart, ceruloplasmin, superoxide dismutase, norepinephrine.
Molecular mechanisms of ANP inhibition of renal sodium transport
Authors: Stanton Bruce A.
Published: April 2011

ANP, a hormone secreted by the atria of mammalian hearts in response to volume expansion, increases urinary sodium excretion in part by inhibiting sodium reabsorption across the inner medullary collecting duct. A number of nephron segments may contribute to the ANP-induced natriuresis; however, this review will focus on the cellular mechanisms of ANP inhibition of electrogenic sodium reabsorption by the inner medullary collecting duct. Patch-clamp studies conducted on rat inner medullary collecting duct cells in primary culture revealed that ANP, via its second messenger cGMP, inhibits electrogenic sodium reabsorption by reducing the open probability of a cation channel located in the apical membrane. Cyclic GMP inhibits the cation channel and thereby sodium reabsorption by two mechanisms. First, cGMP inhibits the channel by a phosporylation-independent mechanism, by binding either to an allosteric modifier site on the channel or to a regulatory subunit. Second, cGMP inhibits the channel by activating cGMP-dependent protein kinase, which by a sequential pathway involving the GTP-binding protein, Gi inhibits the channel. These cGMP-dependent mechanisms inhibiting sodium reabsorption across the inner medullary collecting duct account for a substantial component of the natriuresis following a rise in ANP levels.Key words: inner medullary collecting duct, G proteins, cyclic GMP-dependent protein kinase, 3′,5′-cyclic GMP, signal transduction, papillary collecting duct.
Effects of hydrogen peroxide and hypochlorite on membrane potential of mitochondria in situ in rat heart cells
Authors: Konno Noburu , Kako K. J.
Published: April 2011

Hydrogen peroxide (H2O2) and hypochlorite (HOCl) cause a variety of cellular dysfunctions. In this study we examined the effects of these agents on the electrical potential gradient across the inner membrane of mitochondria in situ in isolated rat heart myocytes. Myocytes were prepared by collagenase digestion and incubated in the presence of H2O2 or HOCl. Transmembrane electrical gradients were measured by distribution of [3H]triphenylmethylphosphonium+, a lipophilic cation. The particulate fraction was separated from the cytosolic compartment first by permeabilization using digitonin, followed by rapid centrifugal sedimentation through a bromododecane layer. We found that the mitochondrial membrane potential (161 ± 7 mV, negative inside) was relatively well maintained under oxidant stress, i.e., the potential was decreased only at high concentrations of HOCl and H2O2 and gradually with time. The membrane potential of isolated rat heart mitochondria was affected similarly by H2O2 and HOCl in a concentration- and time-dependent manner. High concentrations of oxidants also reduced the cellular ATP level but did not significantly change the matrix volume. When the extra-mitochondrial free calcium concentration was increased in permeabilized myocytes, the transmembrane potential was decreased proportionally, and this decrease was potentiated further by H2O2. These results support the view that heart mitochondria are equipped with well-developed defense mechanisms against oxidants, but the action of H2O2 on the transmembrane electrical gradient is exacerbated by an increase in cytosolic calcium. Keywords: ATP, calcium, cardiomyocyte, cell defense, mitochondrial membrane potential, oxidant, triphenylmethylphosphonium.
Cardiac hypothermia: 31P and 1H NMR spectroscopic studies of the effect of buffer on preservation of human heart atrial appendages
Authors: Lareau Sylvain , Keon Wilbert J. , Wallace Julia C. , Whitehead Kristine , Mainwood Graham W. , Deslauriers Roxanne
Published: April 2011

31P and 1H nuclear magnetic resonance spectroscopy has been used to follow noninvasively the time course of energetic metabolite levels in human heart atrial appendages preserved under various temperatures and buffer conditions. From sample harvest up to the normal 5-h time limit for heart preservation, ATP levels in human atrial appendages are much better maintained in 0.9% saline and PIPES-buffered preservation solutions at 12 °C than at 4 °C. Furthermore, preservation at 12 °C can be improved considerably by using high extracellular buffer concentrations. The increased buffer concentration allows better maintenance of the intracellular pH and leads to a faster glycolytic rate as measured by lactate production. At 4 °C, ATP levels decline rapidly during the first 5 h but reached a stable plateau, which is well maintained over 15–20 h. At this temperature, the rate of lactate production is similar at all buffer concentrations (20, 60, and 100 mM PIPES). As a consequence of these observations, we postulate that the mechanisms of ATP production and utilization at 4 °C and at 12 °C are different. At 4 °C, the rate of glycolysis is temperature limited whereas at 12 °C, low intracellular pH inhibits glycolysis.Key words: NMR, spectroscopy, heart, preservation, buffer.
Effects of reduced external sodium concentration and multivalent cations on caffeine contractures in young ferret atrial trabeculae
Authors: Noireaud J. , Baudet S. , Huchet C. , Leoty C.
Published: April 2011

The characteristics of caffeine (1.25–80 mM) transient contractures have been examined in small atrial trabeculae (diameters 50–250 μm) isolated from young (1 – 1.5 months) ferret hearts. In the control medium, the half-saturation constant and the maximum contracture strength (at infinite caffeine concentration) were 37.8 ± 10.2 mM and 0.9 ± 0.2 kN∙kg−1 (n = 11), respectively. The contractile response to caffeine was markedly enhanced following reduction of external sodium (70–0 mM). The perfusion of young ferret trabeculae with the sodium-free medium (up to 3 min) decreased the half-saturation constant by a factor of three (12.4 ± 1.6 mM, n = 8) with an increase in maximum contracture strength (1.09 ± 0.3 kN∙kg−1, n = 8). The effects of various divalent and trivalent cations have been tested on the 10 mM caffeine contracture in trabeculae perfused with Na-containing (140 mM) solution. The order of cation effectiveness is Gd3+ (half effect 0.04–0.07 mM) > Cd2+ (0.15–0.25 mM) > Ni2+ (2–2.5 mM) > Co2+ (7–7.5 mM) [Formula: see text] Mn2+. In conclusion, the present work has shown that in atrial trabeculae isolated from young ferret hearts, the strength of the caffeine contracture was markedly affected by the activity of the sarcolemmal Na–Ca exchange.Key words: caffeine, cardiac muscle, sarcoplasmic reticulum, sodium–calcium exchange, atrium, ferret.