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Biomedical subjects

C J Limas

Publications and source records attributed to C J Limas.

At least 55 records · Page 3Linked to original sources
PubMed1985

Phorbol ester- and diacylglycerol-mediated desensitization of cardiac beta-adrenergic receptors.

There are specific phorbol ester receptors on cardiac myocytes which may be identical with the calcium/phospholipid-dependent protein kinase (protein kinase C). Incubation of enzymatically dissociated rat cardiac myocytes with biologically active phorbol esters (such as 4 beta-phorbol-12, 13-dibutyrate and 12-O-tetradecanoyl phorbol-13-acetate) leads to a time- and concentration-dependent loss of beta-adrenergic receptors detectable with the hydrophilic ligand [3H]-CGP-12177. This loss is attributable to a reduction in both maximal beta-receptor numbers and their affinities. The synthetic diacylglycerol, 1-oleyl-2-acetyldiglycerol, which is known to activate protein kinase C, also induces desensitization of beta-receptors. Both phorbol dibutyrate and 1-oleyl-2-acetyldiglycerol have additive effects to isoproterenol, suggesting a separate site of action in promoting beta-receptor desensitization. The effects of phorbol dibutyrate and 1-oleyl-2-acetyldiglycerol are prevented by colchicine (but not its inactive analog, trimethylcolchicinic acid), indicating a microtubule dependence. The loss of membrane-bound beta-receptors after phorbol dibutyrate- or 1-oleyl-2-acetyldiglycerol preincubation is accompanied by an increase in beta-receptors associated with a cytosol-derived vesicular fraction devoid of plasma membrane markers, a finding consistent with an internalization process. These results suggest that protein kinase C activation by diacylglycerols derived from receptor-linked phosphoinositide hydrolysis may be a novel mechanism of cardiac beta-receptor desensitization.

Animals↗
PubMed1984

Prostaglandin receptors in rat kidney.

The physiological effects of prostaglandins (PGs) are mediated through their interactions with specific binding sites (receptors) on effector cells. Since such receptors potentially regulate the action of PGs on the kidney, the distribution and properties of renal PG receptors in the rat were examined. The distribution of PGE2, PGE1, and PGF2 alpha receptors along the nephron was not uniform; the outer medulla had by far the greatest density of sites, followed by the inner medulla and cortex. Receptors were found exclusively in the particulate fractions, of which the 40,000g pellet had the highest specific activity. In the outer medulla, receptor density calculated from Scatchard plots was 2.12 pmol/mg for PGE2, 1.12 for PGE1, and 0.44 for PGF2 alpha; the KD's were similar for all prostaglandins. The conditions for optimal in vitro binding of PGE2 and PGF2 alpha by outer medullary membranes were investigated. In vivo administration of 16,16'-dimethyl-PGE2 resulted in a dose-dependent "down" regulation of PGE2 binding to outer medullary membranes due to changes in both the number and affinities of receptors. Changes in the numbers and/or properties of PG receptors may be an important mechanism for regulating the effects of PGs and renal function under normal and pathologic conditions.

Alprostadil↗
PubMed1984

Rapid recovery of cardiac beta-adrenergic receptors after isoproterenol-induced "down"-regulation.

Desensitization of beta-receptor-linked adenylate cyclase occurs after prolonged occupancy of the beta-receptors by their agonists. We have followed the development and recovery from "down"-regulation of beta-receptors in enzymatically dissociated cardiac myocytes by using the hydrophilic antagonist [3H]-CGP-12177 to identify surface-bound beta-receptors. After in vitro incubation with (-)-isoproterenol, almost 50% of the beta-receptors are lost within 10 minutes. Isoproterenol-mediated cyclic adenosine monophosphate accumulation by isolated myocytes was also decreased after a 15-minute preincubation with isoproterenol. "Lost" beta-receptors can, however, be recovered when isoproterenol-pretreated, washed cardiac myocytes are incubated at 37 degrees C, 85 +/- 7% of the lost beta-receptors have returned to the cell surface after 20 minutes of incubation. The requirements for such recycling were investigated. Receptor recovery does not depend on de novo protein synthesis, since it is unaffected by prior exposure to cycloheximide. It is, however, dependent on cellular energy, because it is prevented by adenosine triphosphate depletion and involves a lysosomal step since it is inhibited by the lysomotropic agent, chloroquine. In addition, the Golgi apparatus and the microtubules are involved in the beta-receptor recycling to the cell surface, as evidenced by the inhibitory effects of monensin and colchicine, respectively. The mechanism of isoproterenol-induced down-regulation of cardiac beta-receptors involves a rapid, reversible cycling to and from the cytosol and the cell membrane. This intracellular receptor traffic is energy dependent, requires several structures, including lysosomes and microtubules, and may be modified by pathological processes involving the heart.

Adenosine Triphosphate↗
PubMed1984

Comparative effects of hydralazine and captopril on the cardiovascular changes in spontaneously hypertensive rats.

Vascular changes that develop during the course of blood pressure rise in spontaneously hypertensive rats (SHRs) can be modified by antihypertensive therapy. It is not known, however, whether there is selectivity in the structural response to specific antihypertensive drugs. This issue was examined by comparing the effects of a direct vasodilator (hydralazine) and a converting enzyme inhibitor (captopril) on morphologic aspects of the cardiovascular system. Male SHRs, 21 weeks of age, were given either hydralazine (HCl plus hydrochlorothiazide, captopril plus hydrochlorothiazide, or hydrochlorothiazide alone. Untreated age-matched SHRs and Wistar-Kyoto normotensive rats (WKYs) were used as controls. Animals were sacrificed at 27-28 weeks of age. Both hydralazine and captopril lowered significantly the blood pressure of SHRs, whereas hydrochlorothiazide alone was ineffective. The heart/body weight ratios were dramatically reduced in captopril-treated SHRs to below the level of WKYs; hydralazine induced only a very modest (5%) reduction, whereas the diuretic alone was ineffective. The morphology of the aortic intima improved dramatically in response to captopril and hydralazine and, to a lesser extent, hydrochlorothiazide alone. This effect becomes apparent within 6 weeks of treatment, and the only evidence of preexisting disease is the persistence of collagen in the subendothelium. Captopril and hydralazine, but not hydrochlorothiazide alone, reduce the thickness of the aortic media below that of normotensive controls. In addition, captopril and hydralazine improve the structure of small intrarenal vessels. There was a strong correlation between the relative effectiveness of the three pharmacologic agents in lowering blood pressure and in improving the changes of intrarenal vessels. These results highlight the capacity of antihypertensive therapy to arrest or reverse the structural sequelae of hypertension. In addition, they underscore the heterogeneity in the response of different components of the cardiovascular system, which, in part, reflects selectivity in the action of antihypertensive agents.

Animals↗
PubMed1983

Involvement of microtubules in the isoproterenol-induced 'down'-regulation of myocardial beta-adrenergic receptors.

The number of cardiac beta-adrenergic receptors identified by [3H]dihydroalprenolol binding decreases in a concentration-dependent manner during prolonged administration of isoproterenol. Loss of membrane beta-receptors is paralleled by the appearance of [3H]dihydroalprenolol binding activity in the cytosol. This redistribution of receptors is prevented by colchicine and vinblastine but not lumicolchicine. Cardiac receptor desensitization is, therefore, dependent on microtubules and may be influenced by agents interfering with tubulin polymerization.

Animals↗
PubMed1983

Phospholipid metabolism in the rat renal inner medulla.

In view of the importance of phospholipids as a source of precursor fatty acids for the high prostaglandin synthesis in the renal inner medulla, we studied pathways of phospholipid esterification and degradation in the rat inner medulla. De novo acylation of [14C]arachidonate occurred predominantly in position 2 of phosphatidylcholine in the microsomal fraction. This newly esterified [14C]arachidonate was accessible to deacylation by a microsomal phospholipase A2 (EC 3.1.1.4) with alkaline optimum which was Ca2+-dependent and resistant to 0.1% deoxycholate. No phospholipase A1 (EC 3.1.1.32) activity against endogenous labeled phosphatidylcholine could be demonstrated in the microsomal fraction. When exogenous phosphatidylcholine labeled at position 2 was deacylated by renomedullary homogenates, labeled free fatty acid but no labeled lysophosphatidylcholine was recovered in the reaction products. This could be attributed to further degradation of generated lysophosphatidylcholine by a cytosolic lysophospholipase (EC 3.1.1.5). Sodium deoxycholate at a concentration of 0.1% or higher inhibited the lysophospholipase and allowed the demonstration of both A2 and A1 alkaline phospholipase activities in the homogenate. The major in vitro pathway of lysophosphatidylcholine disposition is further degradation by a cytosolic lysophospholipase, while reutilization for phosphatidylcholine synthesis through the action of a predominantly microsomal acyltransferase appears to be a minor pathway. In the presence of several acyl-CoAs, reutilization of lysophosphatidylcholine is significantly increased by an acyl-CoA:lysophosphatidylcholine acyltransferase (EC 2.3.1.23) but there is no preferential transfer of arachidonyl-CoA compared to other acyl-CoAs.

1-Acylglycerophosphocholine O-Acyltransferase↗
PubMed1983

Biochemical aspects of cardiac hypertrophy.

Cardiac hypertrophy is a basic adaptive response to increased functional demands on the heart. The mechanisms regulating protein synthesis and energetic efficiency in the hypertrophying myocardium have important implications for the pump performance of the heart. Although our knowledge of the initial steps in the transduction of hypertrophic stimuli to increased RNA and protein synthesis is scanty, progress is being made in elucidating the control of nuclear RNA synthesis in the normal and hypertrophied myocardium. It is expected that application to the myocyte of recently acquired knowledge about growth control in other cell systems will lead to new concepts with potential clinical relevance.

Animals↗
PubMed1983

Effect of antihypertensive therapy on the vascular changes of spontaneously hypertensive rats.

The effect of antihypertensive therapy on vascular morphologic characteristics was studied in spontaneously hypertensive rats (SHRs). Starting at either 7 or 22 weeks of age, SHRs were given a combination of hydralazine, chlorothiazide, and reserpine, which reduced the blood pressure significantly below the level of untreated animals. Rats were sacrificed at either the 22nd or the 42nd week of age along with age-matched untreated SHRs and normotensive Wistar-Kyoto controls. The following treatment groups were thus obtained: a) treated from the 7th to the 22nd or 42nd week, b) treated from the 22nd to the 42nd week, and c) treated from the 7th to the 22nd and left untreated until the 42nd week. Ultrastructural and morphometric studies were carried out on the aorta and intrarenal vessels. The expansion of the subendothelial space and medial thickening in the aorta as well as the increase in external to luminal diameter ratios and wall thickness of intrarenal vessels were prevented in SHRs treated continuously from the 7th week on. The severity of vascular lesions was markedly reduced in animals treated from the 22nd to 42nd week. Discontinuation of therapy resulted in rapid reestablishment and progression of both aortic and renal vascular disease to a degree identical to that of untreated SHRs. The results indicate that hypertensive vascular changes are preventable by early treatment and, depending on their nature, can be arrested or reversed by delayed treatment.

Animals↗
PubMed1982

Age-dependency of vascular phospholipid deacylation-reacylation in spontaneously hypertensive rats.

We have studied the temporal relation of phospholipid turnover and prostaglandin synthesis to the evolution of hypertensive vascular disease in the spontaneously hypertensive rat. The incorporation of arachidonate into aortic phospholipids, its release by phospholipase A2 and its utilization for prostaglandin synthesis were compared in spontaneously hypertensive and Wistar-Kyoto rats aged 7, 20 and 42 weeks. When expressed per mg of protein in the assay medium, arachidonate incorporation into aortic phospholipids decreased, while prostaglandin synthesis increased, with age in both rat strains. No significant differences were noted between hypertensive and normotensive animals at 7 weeks of age whereas both enhanced phospholipid turnover and prostaglandin synthesis was demonstrated in hypertensive rats at 20 and 42 weeks of age. The higher phospholipase activity in hypertensive aortas was associated with a significant increase in the capacity for exogenous lysophosphatide hydrolysis. Transacylation and reacylation of lysolecithin, however, were not significantly enhanced in hypertensive aortas. These biochemical changes accompany, and may be related to, structural modifications of the aortic wall in the course of hypertension.

Acylation↗
PubMed1982

Enhanced myocardial RNA synthesis in spontaneously hypertensive rats possible role of high-mobility group non-histone proteins.

Cardiac hypertrophy in spontaneously hypertensive rats is associated with increased nuclear RNA polymerase activity. In order to explore mechanisms facilitating the interaction of the enzyme with its endogenous template, we compared the structure of nuclear chromatin from myocytes of 20-week-old spontaneously hypertensive rats and normotensive Wistar-Kyoto controls. Enhanced RNA synthesis in hypertensive rats was accompanied by increased susceptibility to digestion by deoxyribonuclease I. Nick translation of nuclei also resulted in higher nucleotide incorporation in hypertensive rats. Salt-extraction abolished the differences in deoxyribonuclease I sensitivity between the two animal groups. Reconstitution with either 0.35 M NaCl-extract or high mobility group (HMG) non-histone proteins restored digestion susceptibility but did not equalize SHR and WKY cells. SDS-polyacrylamide gel electrophoresis of 0.35 M NaCl-extracts and supernatants from deoxyribonuclease I digestion revealed the presence of HMG proteins which were preferentially released in hypertensive rats. There was a small but statistically significant increase in nuclear HMG protein content in hypertensive rats (0.12 +/- 0.02 mg/mg DNA vs. 0.09 +/- 0.02 mg/mg DNA in Wistar-Kyotos, P less than 0.05) but no difference in their electrophoretic appearance. These results indicate that chromatin structure is altered in the hypertrophied myocardium with resultant increase in deoxyribonuclease I susceptibility. This increase appears to be partly dependent on the high-mobility group non-histone proteins.

Animals↗
PubMed1982

Nulceoprotein changes in the hearts of cardiomyopathic turkeys.

The possible involvement of nuclear proteins in the pathogenesis of a spontaneously occurring model of congestive cardiomyopathy in turkeys was examined. This model is characterised by cardiac hypertrophy and dilatation, reduced cardiac output and depressed contractility. The protein composition of myocardial nuclei was compared in normal (n = 9) and cardiomyopathic (n = 18) turkeys, 70 to 140 days old. Myopathic hearts as a group have a higher histone content (1.75 +/- 0.09 (SD) mg . mg DNA-1 vs 1.65 +/- 0.07 in controls, P less than 0.01) and histone/nonhistone protein (NHP) ratio (1.07 +/- 0.07 vs 0.95 +/- 0.02 in controls, P less than 0.01). The latter was independent of age and correlated well with the degree of cardiac dilatation. The electrophoretic patterns of chromatin proteins was decreased in myopathic hearts. This decrease was primarily accounted for by lower NHP phosphorylation (5.78 +/- 1.38 pmol 32P . mg prot-1 . 15 min-1 vs 8.33 +/- 0.81 in controls, P less than 0.01). DEAE-Sephacel chromatography separated cyclic AMP-dependent and -independent nuclear protein kinases with similar substrate specificities but lower specific activities in myopathic hearts. SDS-polyacrylamide similar substrate specificities but lower specific activities in myopathic hearts. SDS-polyacrylamide gel electrophoresis of phosphorylated nucleoproteins revealed differences in the lower molecular species of NHPs between control and myopathic hearts. There was a significant correlation between NHP phosphorylation and degree of cardiac dilatation (r = -0.78) or contractility as reflected by left ventricular systolic time intervals (r = -0.57). These results suggest that development of this model of spontaneous cardiomyopathy is associated with, and may, in part, be secondary to changes in the composition and function of myocardial nucleoproteins.

Animals↗
PubMed1982

Contrasting effects of spontaneous and induced cardiomyopathy on the nucleoproteins of turkey hearts.

The possible involvement of nuclear proteins in the pathogenesis of cardiomyopathy was studied in a spontaneously occurring and a furazolidone-induced model of turkey cardiomyopathy. Both models are characterised by cardiac hypertrophy and dilatation, systemic hypotension and depressed contractility. The protein composition of myocardial nuclei was compared in normal (n = 9) and cardiomyopathic (spontaneous n = 6, furazolidone-induced n = 21) turkeys. Cardiac nuclei from spontaneously myopathic animals had a higher histone content (1.827 +/- 0.058 (mean +/- SD) mg . mg DNA-1 vs 1.688 +/- 0.187 in controls, P less than 0.05) and histone/nonhistone protein ratio (1.122 +/- 0.020 vs 0.882 +/- 0.128 in controls, P less than 0.01). Nuclear protein phosphorylation was lower in spontaneously cardiomyopathic turkeys primarily because of decreased nonhistone protein phosphorylation (5.100 +/- 0.759 pmol 32 P . mg prot-1 .15 min-1 vs 8.456 +/- 0.886 in controls, P less than 0.01). In contrast, furazolidone-induced cardiomyopathy of similar severity to the spontaneously occurring model was not associated with changes in nucleoprotein composition or degree of phosphorylation. These results indicate that development of spontaneous cardiomyopathy in turkeys may be related to the composition and function of nuclear nonhistone proteins. These changes are not secondary to the cardiac hypertrophy/dilatation accompanying the myopathic process.

Animals↗
PubMed1982

Aortic morphology in salt-dependent genetic hypertension.

Excessive salt intake is an important determinant of human essential hypertension. Hypertension resulting from genetically determined salt sensitivity can be studied by the used of the salt-sensitive (S) and -resistant (R) rat strains developed by Dahl. A longitudinal morphometric and ultrastructural study of S and R Dahl rats fed different amounts of salt (0.6%, 4.0%, and 8.0% NaCl) for 2-14 weeks was undertaken. Only S rats responded to high-salt (4.0% and 8.0%) diets with an increase in blood pressure, and the rate of hypertension development was proportional to the daily amount of salt consumed. Likewise, S but not R rats fed high-salt diets showed thickening of the aortic media which paralleled the rise of blood pressure. Intimal lesions were characterized by the accumulation of an amorphous, electron-dense substance in the subendothelial space (SES), adherence or penetration of lymphoid cells, and subendothelial fibrin deposition. The extent and severity of SES expansion correlated more closely with the duration of salt feeding than with the level of blood pressure. Fibrin deposition was noted only in severely hypertensive animals and was not related to the salt concentration in the diet. Morphologic abnormalities in endothelial cells were noted in hypertensive animals by transmission and scanning electron microscopy as well as by en face preparation, but endothelial denudation and junctional disruptions were notably absent. In contrast to the large numbers of lymphoid cells, neither platelets nor fibrin were seen adherent on the endothelium. These results, in conjunction with previous studies in other hypertensive models, indicate that the nature and extent of vascular lesions depend not only on the severity of hypertension but also on its rate of development, duration, and pathophysiologic characteristics.

Age Factors↗
PubMed1980

Effect of cyclic AMP-dependent phosphorylation on the reactivity of sulphydryl groups in cardiac sarcoplasmic reticulum.

Phosphorylation of cardiac sarcoplasmic reticulum by cyclic AMP-dependent protein kinase results in enhanced Ca2+ transport even though Ca2+-dependent ATPase is not a substrate for the kinase. The mechanisms involved in this enhancement are not clear. In the present study, we used the reactivity of sulphydryl groups in the Ca2+-dependent ATPase as an index of conformational change during the Ca2+ transport cycle and examined the effects of protein kinase-catalysed phosphorylation. N-Ethylmaleimide alkylation allowed the distinction of several thiol groups with variable functional significance for the ATPase. A sulphydryl group involved in the formation of the phosphorylated intermediate (EP) of the Ca2+-dependent ATPase was protected by adenosine 5'-[beta, gamma-imido]triphosphate. Reactivity of an additional thiol group was related to EP dephosphorylation and was dependent on Ca2+. The Ca2+ concentration for change in the reactivity of this sulphydryl group and ATPase inhibition occurred within the range for Ca2+ binding to the high-affinity sites. Phosphorylation of cardiac sarcoplasmic reticulum by cyclic AMP-dependent protein kinase resulted in decreased N-ethyl[1-14C]-maleimide binding and the ATPase inhibition; the thiol groups involved in EP dephosphorylation were selectively protected. The results indicate that protein kinase-catalysed phosphorylation results in conformational changes of the ATPase, which renders certain thiol groups inaccessible to N-ethylmaleimide. This conformational change may facilitate functional movements involved in Ca2+ transport.

Animals↗