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Biomedical subjects

L H Botelho

Publications and source records attributed to L H Botelho.

At least 19 recordsLinked to original sources
PubMed1991

Cytoprotective function of nitric oxide: inactivation of superoxide radicals produced by human leukocytes.

The oxygen-derived free radical superoxide anion (.O2-) plays an important role in the pathogenesis of various diseases. Recent demonstrations that .O2- inactivates the potent vasodilator endothelium-derived relaxing factor (EDRF) and that EDRF is probably nitric oxide (NO) suggest that EDRF(NO) may act as an endogenous free radical scavenger. This hypothesis was tested in an in vitro system by analyzing the effect of authentic NO (dilutions of a saturated aqueous solution) on .O2- production (detected spectrophotometrically as reduction of cytochrome c) by fMet-Leu-Phe-activated human leukocytes (PMN). NO depressed the rate of reduction of cytochrome c by .O2- released from PMN's or generated from the oxidation of hypoxanthine by xanthine oxidase. This effect was concentration-dependent and occurred at dilutions of the saturated NO solution (1:250 to 1:10) which inhibited platelet aggregation. NO had no direct effect on cytochrome c or on xanthine oxidase. These observations indicate that NO(EDRF) can be regarded as a scavenger of superoxide anion and they suggest that EDRF(NO) may provide a chemical barrier to cytotoxic free radicals (.O2-).

Adenosine Diphosphate↗
PubMed1991

Endothelins.

The discovery of endothelium-derived relaxing (prostacyclin, EDRF) and contracting factors (EDCF) in the past decade opened up new vistas not only for basic and clinical research, but revolutionized our thinking about regulation and control of the cardiovascular system in health and disease. One of the most exciting developments in recent years was the discovery of a peptidergic EDCF and its isolation and identification as a unique 21-amino-acid peptide, endothelin (ET). This review summarizes the state-of-the-art in some areas of this fast-moving field, including the biosynthesis, tissue-specific expression, and binding of ET isoforms. Recent information about the nature of endothelin-converting enzyme (ECE) and the cloning, sequencing, and expression of ET receptor subtypes will be discussed. Based on current knowledge of the wide variety of biological actions of ETs, working hypotheses are presented about the possible autocrine, paracrine, and humoral actions of ETs and their potential role in modulating cardiovascular functions. In addition, the proposed significance of ETs in human cardiovascular diseases is summarized. In spite of the abundance of studies generated over the past 3 years and the postulated working hypotheses based on these findings, the true significance of ETs in short- and long-term regulation/modulation of tissue function remains to be determined. This will be the task of future investigations, using more sensitive detection methods and selective inhibitors of the biosynthesis and actions of ETs.

Amino Acid Sequence↗
PubMed1991

Smooth muscle cells contain a factor responsible for decreasing big endothelin and endothelin-1 produced by cultured endothelial cells.

The production of big endothelin-1 (big ET-1) and its processing to endothelin (ET-1) were examined in cocultures of bovine pulmonary artery endothelial cells (BPECs) and human bronchiolar smooth muscle cells (HBSMCs). The BPECs produced immunologically reactive ET-1 (ir-ET-1) and big ET-1 (ir-big ET-1) that increased linearly in the cell culture media over a 72 h time course when confluent monolayers were incubated at 37 degrees C in serum-free media. HBSMCs maintained in serum-free media over a 72-h period did not produce any detectable ir-big ET-1 or ir-ET-1. After coculture of these two cell types for 24 h (the monolayers were physically separated), the culture medium contained no detectable ir-big ET-1 or ir-ET-1. When medium conditioned for 24 h with the HBSMCs was transferred to the BPEC monolayers, the accumulation rates of ET-1 and big ET-1 were attenuated 70 and 10%, respectively. When medium conditioned for 24 h with the BPECs was transferred to the HBSM cell monolayers, the amount of ir-ET-1 and ir-big ET-1 in the medium decreased greater than 90% over the next 24 h. Cellular binding and uptake could not account for this decrease as ascertained by quantitation of cellular levels of ET-1 and big ET-1. When conditioned medium from the HBSMCs was added to conditioned medium from the BPECs in a 1:1, 2:1, 3:1, 1:2, or 1:3 ratio (v/v) in the absence of cells, the amount of ET-1 or big ET-1 present in the EC medium did not decrease significantly over the following 24 h. These data imply that HBSMCs contain a factor that is responsible for decreasing the accumulation of ir-big ET-1 and ir-ET-1 in the cell culture media of BPECs. The data also suggest that the most likely mechanism for this effect is a factor that degrades BET-1 and ET-1 at different rates.

Animals↗
PubMed1991

Evidence of glycosylated sites on the endothelin-1 receptor in Swiss 3T3 cells.

The effects of incubation of intact cells with six different lectins on the specific binding of [125I]endothelin-1 (ET-1) were determined in Swiss 3T3 fibroblasts. ET-1 binding was unaffected by pretreatment of cells for 1 h at 37 degrees C with concanavalin A, soybean agglutinin, Ulex europaeus agglutinin I, peanut agglutinin, or Galanthus nivalis agglutinin. However, preincubation of cells with 300 micrograms/ml of wheat germ agglutinin resulted in a 70% decrease in specific binding of ET-1 to cell-surface receptors. The inhibitory effects of wheat germ agglutinin were diminished by brief incubation of lectin-treated cells with 100 mM N-acetylglucosamine, a monosaccharide specifically recognized by wheat germ agglutinin. Neither glucose nor mannose had any effect on wheat germ agglutinin-mediated inhibition of the specific binding of ET-1. These results suggest that the ET-1 receptor on 3T3 cells is a glycoprotein that contains one or more N-acetylglucosamine residues at or near the ligand binding site.

3T3 Cells↗
PubMed1989

Synergistic inhibition of hepatic ketogenesis in the presence of insulin and a cAMP antagonist.

The separate or combined effects of insulin and the cAMP antagonist, the Rp-diastereomer of adenosine cyclic 3',5'-phosphorothioate (Rp-cAMPS), were examined on fatty acid-stimulated ketogenesis in hepatocytes from normal fasted rats. Addition of 0.4 mM oleic acid or 0.4 mM octanoic acid resulted in a linear increase in ketone production measured over 60 min. When oleic acid was the substrate, incubation with 1 to 30 microns Rp-cAMPS alone or 0.1 to 10 nM insulin alone caused a variable decrease in the production of ketones which did not exceed an average value of 30% in any one experiment. The simultaneous addition of Rp-cAMPS and insulin resulted in a greater than additive inhibition which reached average values between 47-60% when the theoretical combined inhibitory effect of the insulin alone plus the Rp-cAMPS alone was less than 18%. No significant effects of either insulin or Rp-cAMPS, alone or in combination, were seen when octanoic acid was the substrate. These data imply that Rp-cAMPS can potentiate insulin inhibition of hepatic ketogenesis through inhibition of a cAMP-mediated process.

3-Hydroxybutyric Acid↗
PubMed1989

Myofibrillar Ca++ activation and heart failure--Ca++ sensitization by the cardiotonic agent APP 201-533.

Certain forms of cardiac failure appear to be associated with a decrease in the Ca++ sensitivity of the contractile structures, possibly due to troponin I phosphorylation. Interference of cardiotonic drugs with myofibrillar Ca++ activation instead of enhancement of Ca++ influx may therefore provide a more causal therapeutic concept in the treatment of cardiac insufficiency. APP 201-533 (3-Amino-6-methyl-5-phenyl-2(1H)-pyridinone) (the structure of which is shown below) is a novel cardiotonic agent acting neither via beta adrenoceptor stimulation nor inhibition of Na+/K+ ATPase. In the 100 microM concentration range, it increases the Ca++ sensitivity and the Ca++ affinity of functionally isolated cardiac contractile structures. This coincides with an inhibitory effect on the cAMP-dependent protein kinase from rat liver. A possible relation with the regulation of troponin I phosphorylation is discussed.

Animals↗
PubMed1989

Endothelin-1 stimulates phosphatidylinositol hydrolysis and calcium uptake in isolated canine coronary arteries.

The effects of synthetic endothelin-1 (ET-1) (10(-10)-3 x 10(-7) M) on isometric force, 45Ca2+ uptake, and phosphatidylinositol (PI) hydrolysis were determined in isolated canine coronary artery rings. ET-1 caused contraction and stimulated 45Ca2+ uptake and PI hydrolysis (determined as inositol monophosphate accumulation) in a concentration-dependent manner with EC50 values of 6.3 x 10(-9), 2 x 10(-9), and 3 x 10(-9) M, respectively. Maximal responses were obtained with 3 x 10(-8) M ET-1 for all three parameters. At the maximally effective concentration, ET-1 caused a 1.8-fold increase in the rate of 45Ca2+ uptake following a 1-min exposure (the shortest time point tested) while the contractile response reached maximum only after 6 min. ET-1 (3 x 10(-8) M) stimulated a biphasic accumulation of inositol monophosphate with an initial rapid 1.4-fold increase detectable between 30 and 60 s followed by a secondary 11.9-fold increase at 30 min. These data show that PI hydrolysis and Ca2+ uptake are early events in the action of ET-1 on coronary artery vascular smooth muscle that precede the maximal contractile response. It is suggested that all of these responses are triggered by the interaction of ET-1 with a cell-surface receptor.

Animals↗
PubMed1988

Inhibition of cAMP-dependent protein kinase by adenosine cyclic 3'-, 5'-phosphorodithioate, a second cAMP antagonist.

A single sulfur substitution for either the axial or the equatorial exocyclic oxygen of adenosine cyclic 3', 5'-phosphate (cAMP) results in diastereometric phosphorothioate analogs of cAMP with agonist versus antagonist properties towards activation of cAMP-dependent protein kinase. Sulfur substitutions for both of the exocyclic oxygens of cAMP results in a dithioate analog of cAMP, adenosine cyclic 3', 5'-phosphorodithioate (cAMPS2), which has antagonist properties. cAMPS2 displaced [3H]cAMP from the binding sites on bovine heart Type II cAMP-dependent protein kinase as demonstrated by equilibrium dialysis experiments with an apparent Kd of 6.3 microM. The addition of 10, 30, or 100 microM cAMPS2 when measuring cAMP-induced activation of pure porcine heart Type II cAMP-dependent protein kinase resulted in a concentration-dependent increase in the amount of cAMP required to produce half-maximal activation (EC50). A plot of the EC50 values as a function of the cAMPS2 concentration resulted in a straight line from which a KI value of 4 microM was derived. cAMPS2 had no significant effect on the degree of cooperativity (n) of cAMP activation of the holoenzyme. These data suggest that the most important structural requirement for the dissociation of the holoenzyme is an equatorial exocyclic oxygen.

Algorithms↗
PubMed1987

A study of the mechanism of glucagon-induced protein phosphorylation in isolated rat hepatocytes using (Sp)-cAMPS and (Rp)-cAMPS, the stimulatory and inhibitory diastereomers of adenosine cyclic 3',5'-phosphorothioate.

Maximal doses of glucagon increase the phosphorylation state of 12 cytosolic proteins in isolated hepatocytes from fasted rats (Garrison, J. C., and Wagner, J. D. (1982) J. Biol. Chem. 257, 13135-13143). Incubation of hepatocytes with lower concentrations of glucagon indicates that a hierarchy of substrates exists with the concentration of glucagon required for half-maximal increases in phosphorylation varying 5-15-fold. The proteins whose phosphorylation state is most sensitive to low concentrations of glucagon are pyruvate kinase and 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, both of which play key roles in the regulation of gluconeogenesis. Treatment of hepatocytes with (Sp)-cAMPS, the stimulatory diastereomer of adenosine cyclic 3',5'-phosphorothioate, mimics the response seen with glucagon. When hepatocytes are pretreated with the cAMP antagonist, (Rp)-cAMPS, the phosphorylation response is abolished at low concentrations of glucagon, and the dose of glucagon required for half-maximal stimulation of phosphorylation is increased 5-10-fold. The (Sp)-cAMPS-stimulated increases in phosphorylation state are also blunted by (Rp)-cAMPS. These results provide direct pharmacological evidence for the activation of the cAMP-dependent protein kinase in response to glucagon in the intact cell. Although low doses of glucagon appear to stimulate protein phosphorylation via the cAMP-dependent protein kinase, high doses of glucagon also cause a small increase in the concentration of free intracellular Ca2+ in hepatocytes. The glucagon-stimulated increases in the level of Ca2+ can be mimicked by (Sp)-cAMPS and inhibited by pretreatment with (Rp)-cAMPS. These results suggest that glucagon can elevate intracellular Ca2+ via cAMP and the cAMP-dependent protein kinase.

Animals↗
PubMed1986

Synergistic inhibition of glucagon-induced effects on hepatic glucose metabolism in the presence of insulin and a cAMP antagonist.

Inhibition of hepatic glycogenolysis by an intracellular inhibitor of cAMP-dependent protein kinase in glucagon-stimulated hepatocytes was potentiated by insulin. When hepatocytes isolated from fed rats were treated with 0.3 nM glucagon, which activates glycogen breakdown half-maximally, the Rp diastereomer of adenosine cyclic 3',5'-phosphorothioate [Rp-cAMPS), a cAMP antagonist, inhibited glucose production half-maximally at 3 microM. A 10-fold lower concentration of antagonist was required to half-maximally inhibit glucose production in the presence of 10 nM insulin, which alone produced only 15% inhibition. Under the same experimental conditions, the maximal effect of (Rp)-cAMPS was also potentiated. In addition, the increase in the concentration of glucagon required for half-maximal activation of phosphorylase activity and inactivation of glycogen synthase activity in the presence of minimally effective concentrations of insulin and (Rp)-cAMPS were clearly synergistic. It is postulated that the synergism observed is a consequence of action at several enzymatic sites leading to, and including, alteration of the phosphorylation state of the two rate-limiting enzymes in glycogen metabolism.

Animals↗
PubMed1986

Inhibition of hepatic gluconeogenesis by the Rp-diastereomer of adenosine cyclic 3',5'-phosphorothioate.

The specific intracellular cyclic AMP-dependent protein kinase antagonist, the Rp-diastereomer of adenosine cyclic 3',5'-phosphorothioate (Rp-cAMPS), inhibited both basal and cyclic AMP-agonist-induced rates of gluconeogenesis in hepatocytes isolated from fasted rats. Incubation of the cells in the presence of pyruvate and lactate and either the Sp-diastereomer of adenosine cyclic 3',5'-phosphorothioate (Sp-cAMPS) or glucagon produced a concentration-dependent increase in the rate of gluconeogenic glucose production which was shifted to higher concentrations of Sp-cAMPS or glucagon in the presence of Rp-cAMPS. Incubation of the cells with Rp-cAMPS in the absence of agonist produced no increase in the rate of glucose production and, in most cases, 100 microM-Rp-cAMPS resulted in 14-20% decrease in the substrate-stimulated rate of glucose production. Sp-cAMPS-induced gluconeogenesis was inhibited half-maximally at 1 microM-Rp-cAMPS and glucagon-induced gluconeogenesis was inhibited half-maximally at 12 microM-Rp-cAMPS. Approx. 10-15% of the inhibition of gluconeogenesis observed in the presence of Rp-cAMPS was due to conversion of glucose 6-phosphate to liver glycogen, consistent with Rp-cAMPS-induced reactivation of glycogen synthase. The remaining 85-90% inhibition of gluconeogenic glucose production resulted from the action of Rp-cAMPS on the cyclic AMP-sensitive enzymes controlling the rate of gluconeogenesis.

Animals↗
PubMed1986

Synergistic inhibition of hepatic glycogenolysis in the presence of insulin and a cAMP antagonist.

The effects of insulin on the ability of the specific intracellular cAMP-dependent protein kinase antagonist, the Rp diastereomer of adenosine cyclic 3',5'-phosphorothioate, to inhibit glycogenolysis induced by the Sp diastereomer was studied in hepatocytes isolated from fed rats. Addition of the cAMP agonist, (Sp)-cAMPS, to hepatocytes resulted in a concentration-dependent increase in glycogenolytic glucose production concomitant with the cAMP-dependent activation of phosphorylase and inhibition of glycogen synthase. Activity curves were shifted to the right in the presence of the cAMP antagonist, (Rp)-cAMPS. Preincubation of the hepatocytes with a maximally effective concentration of insulin did not affect the concentration of (Sp)-cAMPS required for half-maximal activation of phosphorylase but did result in a 10-fold shift in the concentration of (Sp)-cAMPS required for half-maximal inactivation of glycogen synthase. Preincubation of hepatocytes with a combination of the cAMP antagonist, (Rp)-cAMPS, and insulin resulted in synergistic inhibition of (Sp)-cAMPS-induced phosphorylase activation, glycogen synthase inactivation, and glycogenolytic glucose production. Since neither phosphorothioate diastereomer was hydrolyzed significantly during the course of the experiments, the synergistic effects of insulin are postulated to be working through a mechanism subsequent to the phosphodiesterase activation step.

Animals↗
PubMed1985

Inhibition of cyclic AMP-dependent protein kinase-induced changes in the kinetic properties of hepatic pyruvate kinase by the specific cyclic AMP antagonist, the (Rp)-diastereomer of adenosine cyclic 3',5'-phosphorothioate.

The effects of the diastereomers of adenosine cyclic 3',5'-phosphorothioate, (Sp)- and (Rp)-cAMPS, on the kinetic properties of pyruvate kinase were studied in hepatocytes isolated from fed rats. Incubation of the cells with the cAMP-dependent protein kinase agonist, (Sp)-cAMPS, produced a concentration-dependent increase in S0.5 for phosphoenolpyruvate, but had no effect on Vmax. The (Sp)-cAMPS-treated enzyme was more sensitive to inhibition by alanine and ATP and, under the same conditions, was less responsive to activation by fructose-1,6-bisphosphate when assayed at a subsaturating phosphoenolpyruvate concentration. Incubation of the hepatocytes with only the cAMP-dependent protein kinase antagonist, (Rp)-cAMPS, produced no change in any kinetic parameters, but did suppress the (Sp)-cAMPS- or glucagon-induced increase in the S0.5 for phosphoenolpyruvate with IC50 values of 10 microM and 5 microM (Rp)-cAMPS. (Rp)-cAMPS is exerting an effect on the kinetic properties of pyruvate kinase through inhibition of cAMP-dependent protein kinase.

Adenosine Triphosphate↗
PubMed1984

Effects of the specific cAMP antagonist, (Rp)-adenosine cyclic 3',5'-phosphorothioate, on the cAMP-dependent protein kinase-induced activity of hepatic glycogen phosphorylase and glycogen synthase.

The cAMP-dependent protein kinase-induced effects on phosphorylase and glycogen synthase activities and glucose production were studied in hepatocytes isolated from fed rats in the presence of the diastereomers of adenosine cyclic 3',5'-phosphorothioate, (Sp)-cAMPS and (Rp)-cAMPS. Incubation of hepatocytes with (Sp)-cAMPS or glucagon, both of which lead to cAMP-dependent protein kinase activation, resulted in a concentration-dependent increase in glycogen phosphorylase activity and a decrease in glycogen synthase activity. Incubation of hepatocytes with the cAMP-dependent protein kinase antagonist, (Rp)-cAMPS, in the absence of an agonist, had no significant effect on phosphorylase or glycogen synthase activities. Incubation of hepatocytes with a half-maximally inhibitory concentration of (Rp)-cAMPS shifted the agonist-induced activation curves for phosphorylase and the agonist-induced inhibition curves for glycogen synthase to 5-fold higher concentrations for both (Sp)-cAMPS and glucagon. Phosphorylase activity was very sensitive to the rapid, concentration-dependent inhibition by (Rp)-cAMPS of agonist-induced activation of cAMP-dependent protein kinase. The effects on phosphorylase activity were observable in 30 s and were concentration-dependent with half-maximal inhibition at 10 microM, similar to that observed for cAMP-dependent protein kinase. In contrast, glycogen synthase activity was less sensitive to (Rp)-cAMPS inhibition of agonist-induced activation of cAMP-dependent protein kinase. The effects on glycogen synthase activity lagged behind those on phosphorylase activity and the concentration dependence did not parallel the cAMP-dependent protein kinase effect, but was shifted to higher concentrations of (Rp)-cAMPS with half-maximal inhibition at 60 microM. Glucose (10 to 40 mM) increased the sensitivity of glycogen synthase to (Rp)-cAMPS inhibition of cAMP-dependent protein kinase over a narrow range of agonist concentration, but had no significant effect throughout most of the agonist-induced activation range. Thus, the diastereomers, (Sp)- and (Rp)-cAMPS, influence glycogen metabolism and the glycogenolytic enzymes through their modulation of cAMP-dependent protein kinase levels.

Animals↗
PubMed1984

Inhibition of glucagon-induced glycogenolysis in isolated rat hepatocytes by the Rp diastereomer of adenosine cyclic 3',5'-phosphorothioate.

The ability of the Rp diastereomer of adenosine cyclic 3',5'-phosphorothioate (Rp cAMPS) to inhibit glucagon-induced glycogenolysis was studied in hepatocytes isolated from fed rats. Preincubation of the cells for 20 min with progressively higher concentrations of Rp cAMPS followed by a 1 X 10(-9) M glucagon challenge resulted in a 50% inhibition of glucose production over a 30-min period at 2-3 X 10(-6) M Rp cAMPS. A maximal inhibition of 50-74% was achieved, the actual value depending upon the length of preincubation with Rp cAMPS. The inhibitory effect did not increase when the concentration of Rp cAMPS was increased from 3 X 10(-6) to 3 X 10(-4) M. Addition of 1 X 10(-5) M Rp cAMPS to the cells followed by 10(-11) to 10(-6) M glucagon shifted the glucagon concentration required for half-maximal glucose production measured at 10 min to 6-fold higher glucagon concentrations and the concentration of glucagon required for apparent maximal glucose production measured at 10 min to greater than 10-fold higher glucagon concentrations. The cAMP-dependent protein kinase activation curve was similarly shifted to higher concentrations of glucagon. These data show that Rp cAMPS acts as a cAMP antagonist capable of opposing the glucagon-induced activation of cAMP-dependent protein kinase and the concomitant activation of the glycogenolytic cascade.

Animals↗
PubMed1983

Inhibition of glycogenolysis in isolated rat hepatocytes by the Rp diastereomer of adenosine cyclic 3',5'-phosphorothioate.

The diastereomeric forms of adenosine cyclic 3',5'-phosphorothioate, Rp cAMPS and Sp cAMPS, were studied in isolated hepatocytes from fed rats for their ability to interact with the intracellular cAMP-dependent protein kinase and to affect the phosphorylase kinase-phosphorylase glycogenolytic cascade. Incubation of the cells with increasing concentrations of Sp cAMPS produced a concentration-dependent activation of cAMP-dependent protein kinase with a concomitant increase in the glycogenolytic rate. Half-maximal and maximal velocities of glycogenolysis were reached at 8 X 10(-7) and 1 X 10(-5) M Sp cAMPS, respectively. Incubation of the cells with 10(-9) to 10(-4) M Rp cAMPS had no effect on basal glucose production or on cAMP-dependent protein kinase activity. Incubation of the cells simultaneously with 3 X 10(-6) M Sp cAMPS and increasing concentrations of Rp cAMPS produced half-maximal inhibition of glycogenolysis at 1 X 10(-5) M Rp cAMPS and maximal inhibition at 1 X 10(-4) M. The concentrations of Sp cAMPS required for half-maximal and maximal activation of glycogenolysis were increased 10-fold when 1 X 10(-5) M Rp cAMPS was present. These data imply that Sp cAMPS is a cAMP-agonist while Rp cAMPS is a cAMP-antagonist.

Animals↗