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C A Watkins

Publications and source records attributed to C A Watkins.

At least 19 recordsLinked to original sources
PubMed1990

Metabolic effects of isoflurane on rat lungs perfused in situ.

1. The current experiments studied the effects of the inhalation anesthetic, isoflurane, on 5-hydroxytryptamine (5-HT) metabolism, protein synthesis, and angiotensin-converting enzyme activity in perfused rat lungs. 2. Under first order reaction conditions, isoflurane decreased the accumulation of tissue 5-hydroxyindoleacetic acid, the principle metabolite of 5-HT in a concentration-related, competitive, and reversible manner, indicating inhibition of endothelial 5-HT uptake. 3. In apparent contrast, isoflurane appeared to stimulate uptake of 5-HT by an imipramine-sensitive process, into a cell type unable to metabolize the parent amine. 4. Isoflurane increased absolute angiotensin-converting enzyme activity only at an inspired concentration of 5%. The anesthetic did not affect lung protein synthesis.

Animals↗
PubMed1989

Effects of ketamine and fentanyl on lung metabolism in perfused rat lungs.

The effects of ketamine and fentanyl on serotonin (5-hydroxytryptamine; 5-HT) metabolism, angiotensin-converting enzyme (ACE), and protein synthesis (PS) were investigated in an isolated lung model. Rat lungs were perfused in situ with a blood-free physiological salt solution. The pulmonary vasculature was exposed to ketamine (0.005-2.1 mM) or fentanyl (1.8-4.5 microM) for up to 2 h. After 1 h, accumulation of 5-[14C]hydroxyindoleacetic acid (5-HIAA) by the lung was monitored as an index of 5-HT metabolism. ACE activity was estimated from hydrolysis of [3H]benzoylphenylalanyl-alanyl-proline, a synthetic substrate for the enzyme. [3H]phenylalanine was added to the perfusate after 1 h, and its incorporation into acid-precipitable lung protein was measured over the subsequent hour. Ketamine inhibited 5-HT uptake in a concentration-related manner. The inhibition was characterized as competitive and reversible. Fentanyl had no effect on lung 5-HIAA accumulation. Neither drug altered ACE activity or protein synthesis over the concentration ranges tested. The results indicate an action by ketamine that inhibits the 5-HT membrane-transport process. The different effects observed by ketamine and fentanyl on this process could contribute to the diverse pharmacological properties of these two drugs.

Animals↗
PubMed1988

Anesthetic effects on 5-hydroxytryptamine uptake by rat brain synaptosomes.

As a neurotransmitter involved in modulating central nervous system nociception and awareness, 5-hydroxytryptamine (5-HT) may play an important role in the clinical sequelae of certain anesthetic compounds. Anesthetic agents are known to affect peripheral, non-neuronal 5-HT uptake but little is known about their effects on 5-HT metabolism in the central nervous system. The effects of several anesthetic compounds on 5-HT uptake were examined in synaptosomes isolated from rat brain cortex. Inhibition of this uptake process was observed by exposure to clinically relevant concentrations of the volatile anesthetics halothane, isoflurane, and enflurane. The non-volatile agent, ketamine also inhibited uptake while the narcotic fentanyl had an effect only at the highest concentrations tested. Non-volatile agents which had neither a consistent nor significant effect on synaptosomal 5-HT uptake included pentobarbital, sufentanil, and etomidate. These alterations of 5-HT metabolism could represent a mechanism that contributes to anesthetic action.

Anesthetics↗
PubMed1988

Inhibition of synaptosomal serotonin uptake by Ketalar.

The effects of the clinical preparation of ketamine, Ketalar and its preservative, benzethonium chloride on [3H]5-hydroxytryptamine uptake were studied using rat brain synaptosomes. Ketalar caused a concentration-dependent inhibition of substrate uptake by the high affinity transport site (I50 = 20.2 +/- 2.75 microM) while benzethonium chloride had no effect. Kinetic analysis indicated the inhibition to be competitive with serotonin; the apparent km (54 nM) was increased nearly two-fold at 10 microM ketamine. This action may represent a mechanism involved in ketamine anesthesia.

Animals↗
PubMed1987

Effects of reduced pulmonary flow and hypoxia on metabolism of serotonin by rat lungs perfused in situ.

To investigate the extent to which reduced pulmonary flow may affect non-ventilatory functions of the lung, pulmonary artery pressures were altered systematically in an in vitro perfused lung preparation. Metabolic integrity of the tissue was assessed at two levels: disposition of exogenous serotonin (5-hydroxytryptamine; 5-HT) was monitored as a specific indicator of endothelial cell metabolism; and whole-tissue rates of protein synthesis and levels of ATP were evaluated as indices of general metabolic activity and energy availability. Rat lungs were perfused with recirculating cell-free buffer (37 degrees C) for 1 or 3 h at high (36) or low (3 ml.min-1.g-1) pulmonary flow; initial rates of 5-HT metabolism were measured over a subsequent 2-min interval of single-pass perfusion. Metabolism of 5-HT was inhibited and protein synthesis decreased 35% at low pulmonary flow. These changes did not appear to result directly from hypoxia, nor from the associated fall in tissue ATP. The effects of low flow were not reversed at high PO2, nor was 5-HT metabolism inhibited by restricted oxygen availability at high flow rates. After as long as 3 h exposure to a combination of low flow, ventilation (V = 0), and temperature (27 degrees C) and to the volatile anesthetic, halothane, inhibitory effects on both amine and protein metabolism were rapidly reversible. Reductions in the rate of 5-HT metabolism at reduced flow involved a decrease in the maximal velocity (Vmax: 8.0 to 2.2 nmol.min-1.g-1), without change in the apparent Km (2.6-3.2 microM) of the pathway for amine metabolism.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Effects of halothane on transport of 5-hydroxytryptamine by platelet membranes.

Na+-dependent uptake of 5-HT (5-hydroxytryptamine) into plasma membrane vesicles derived from bovine blood platelets and ATP-dependent 5-HT uptake into storage vesicles in platelet lysates were measured. Na+-dependent uptake was temperature-dependent, inhibited by imipramine and exhibited Michaelis-Menten kinetics (apparent Km, 0.12 +/- 0.02 microM; Vmax. 559 +/- 54 pmol/min per mg of protein. Halothane had no effect on Na+-dependent transport of 5-HT in plasma-membrane vesicles. ATP-dependent 5-HT transport into storage granules also exhibited Michaelis-Menten kinetics (apparent Km 0.34 +/- 0.03 microM; Vmax. 34.3 +/- 1.7 pmol/min per mg of protein) and was inhibited by noradrenaline (norepinephrine), but not by imipramine. Exposure of the granules to halothane resulted in a progressive decrease in Vmax. The results demonstrate a possible site for disruption of platelet function by anaesthetics.

Adenosine Triphosphate↗
PubMed1986

Protein synthesis in perfused rat lungs: determinations based on incorporation of radioactive proline.

Functional compartmentation and metabolism of radioactive proline was evaluated to define conditions under which synthesis of lung proteins could be measured accurately based on proline incorporation. Rat lungs were perfused with Krebs-Henseleit bicarbonate buffer equilibrated with O2/N2/CO2 (20:75:5) and containing 4.5% (w/v) bovine serum albumin, 5.6 mM glucose and amino acids at plasma levels. Intracellular proline increased linearly as perfusate proline concentration was increased from 108 microM, the plasma level, to 540 or 1080 microM. At each concentration, the pool of proline which provided precursors to protein synthesis rapidly reached a steady-state specific radioactivity, but when extracellular proline was 108 microM, this pool was diluted significantly by proline from endogenous sources. At 540 or 1080 microM extracellular proline, the specific radioactivities of perfusate and intracellular proline approached equality and rates of protein synthesis calculated based on the specific radioactivity of extracellular proline compared favorably with those calculated from the specific radioactivity of phenylalanyl-tRNA. Similar results were obtained in lungs of two groups of rats in which intracellular proline concentration differed 3-fold. Thus, the contribution of endogenous proline to the pathway of protein synthesis was minimized when extracellular proline was present at high concentration. Under this condition, calculations of protein synthesis based on proline incorporation were most accurate.

Animals↗
PubMed1985

Lung growth in response to unilateral pneumonectomy in rapidly growing rats.

The rapidity with which lung growth is initiated and completed after pneumonectomy was examined in young rats (4 wk of age; 82 g). After left pneumonectomy, the remaining lobes of the right lung grew to equal the weight of both lungs of control animals by day 7 and within 14 days increased from 366 to 968 mg. The tissue concentrations of RNA, DNA phosphate, collagen, and noncollagen proteins did not increase during the growth response. In contrast, total amounts of these constituents increased significantly in the remaining lung of pneumonectomized animals during the 1st postoperative wk and approached levels found in both lungs of sham-operated and unoperated controls by the end of the 2nd wk after pneumonectomy. Although cell size increased in control lungs during the experimental period, there was little evidence of additional cellular hypertrophy associated with compensatory lung growth. The character of the response to pneumonectomy in these rats was similar to that observed previously in older animals (320 g). Thus in spite of the higher basal rate of lung growth in the younger rats, the pattern and rapidity of compensation after pneumonectomy was similar in both age groups.

Animals↗
PubMed1985

Plasmalemmal metabolic activities in cultured calf pulmonary arterial endothelial cells.

We have determined kinetic characteristics of angiotensin converting enzyme, 5'-nucleotidase and transmembrane serotonin uptake and metabolism in cultured calf pulmonary arterial endothelial cells. Angiotensin converting enzyme activity was 2.8 +/- 0.03 Units/10(6) cells (N = 19; 1 Unit: amount of enzyme required to metabolize 1% of substrate, benzoyl-Phe-Ala-Pro, in 1 min under conditions of first order reaction kinetics) in confluent monolayers and 2.31 +/- 0.06 Units/10(6) cells (N = 20) in homogenates. 5'-Nucleotidase activity (substrate: 5'-AMP) was 0.25 +/- 0.01 Units/10(6) cells (N = 19) in monolayers and 0.26 +/- 0.01 Units/10(6) cells (N = 20) in homogenates. Kinetic constants for angiotensin converting enzyme were: Km = 7.6 microM, Vmax = 5.2 nmol/hour/10(6) cells and for 5'-nucleotidase: Km = 52.6 microM, Vmax = 6.3 nmol/hour/10(6) cells. These data confirm that both angiotensin converting enzyme and 5'-nucleotidase are ectoenzymes with no cytoplasmic activity. Serotonin uptake exhibited both a saturable (Km = 0.27 microM, Vmax = 17 pmol/hour/10(6) cells) and a non-saturable component.

Animals↗
PubMed1984

Effect of age on the accumulation of lung protein following unilateral pneumonectomy in rats.

The effects of unilateral pneumonectomy (PNX) on the net synthesis of right lung protein were investigated in vivo using three groups of rats with body weights (BW) ranging from 85 to 330 g. These data were compared to those from sham-operated and normal growing control animals. After PNX, both the 2-day lag prior to the compensatory increase in right lung mass (LW) and the subsequent rate of increase in LW and LW/BW ratio were independent of two-fold differences in the basal rate of lung growth. In all PNX groups, both right LW and LW/BW reached control values for both lungs, but in the older rats the time required for complete compensation was extended from 5 days to 12 days. The rate of net accumulation of right lung protein increased two-fold in the youngest PNX rats and 6 to 8-fold in the older animals, but when these changes were normalized to the protein content of the remaining tissue, the older rats appeared to respond to PNX less efficiently. Increased tissue levels of RNA and the resulting increased capacity of the lungs for protein synthesis could account for the accelerated rate of gain in right lung protein following PNX in both adult and young animals.

Aging↗
PubMed1983

Reversible inhibition of protein synthesis in lung by halothane.

Alterations in the synthesis and degradation of proteins were investigated in intact lungs exposed to the volatile anaesthetic halothane. In rat lungs perfused in situ with Krebs-Henseleit bicarbonate buffer containing 4.5% (w/v) bovine serum albumin, 5.6 mM-glucose, plasma concentrations of 19 amino acids and 690 microM-[U-14C]-phenylalanine and equilibrated with O2/N2/CO2 (4:15:1), protein synthesis, calculated based on the specific radioactivity of aminoacyl-tRNA, was inhibited by halothane. The anaesthetic did not affect degradation of lung proteins. The inhibition of protein synthesis was rapid in onset, dose-dependent, and quickly reversible. It did not appear to be associated with overall energy depletion, with non-specific changes in cellular permeability, or with decreased availability of amino acids as substrates for protein synthesis.

Adenosine Triphosphate↗
PubMed1983

Identification of specific proteins synthesized by type II pneumocytes in primary culture.

Proteins from primary cultures of type II granular pneumocytes have been examined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis to identify type II cell-specific proteins. The distribution of Coomassie Blue-stained bands in preparations of cellular proteins, culture medium, lavage and lamellar bodies have been compared. The most prominent stained band in the serum-free medium from type II cell cultures (HS1; Mr 39900) corresponds to a major protein in acellular sedimentable (20000 g for 30 min) crude surfactant obtained from rat lungs by saline (0.9% NaCl) lavage. A second protein (HS2; Mr 12000) is also found both in type II cell-conditioned medium and in lavage. Neither rat serum nor donor calf serum (used in the isolation of the type II cells) contains a protein co-migrating with HS1 or HS2 proteins. HS1 is also found in Coomassie Blue-stained gels of cellular proteins and of lamellar bodies isolated from whole lungs. Cultures of type II cells incorporate [14C]phenylalanine into HS1 and HS2 as shown by autoradiography of sodium dodecyl sulphate/polyacrylamide gels of culture medium. Rat lungs perfused in situ incorporate [35S]methionine into HS1 in the lamellar body fraction. A third protein (HS3; Mr 47000) is observed only in autoradiographs of cell culture medium; no corresponding Coomassie Blue-stained band can be identified in medium, in cells or in lung lavage. No protein bands corresponding to HS1, HS2 or HS3 are found in conditioned media from pulmonary alveolar macrophages, rat fibroblasts or bovine aorta endothelial cells. Two-dimensional gel electrophoresis of HS1 shows a single polypeptide with an isoelectric point of 6.3; HS3 appears as a chain of spots with a range of isoelectric points from 6.3 to 6.6. HS2 has not been identified on two-dimensional gels. The amino acid composition of HS1 does not differ significantly from that of surfactant apoproteins studied previously; however, HS1 is not detected by glycoprotein stains, nor does it appear to be a subunit of a thiol-linked multimer.

Animals↗
PubMed1983

Effect of halothane on metabolism of 5-hydroxytryptamine by rat lungs perfused in situ.

The effect of halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) on the uptake of 14C-labelled 5-hydroxytryptamine (5-HT) and its metabolism to 5-hydroxyindol-3-ylacetic acid (5-HIAA) was investigated in rat lungs perfused in situ. The rate of accumulation of 14C-labelled 5-HIAA in the tissue, monitored as an index of 5-HT metabolism, was linear with time, displayed saturation kinetics and remained stable for at least 180 min of perfusion. Exposure of the lungs to halothane (4%) for 60 min reversibly reduced production of 5-HIAA through an increase in the apparent Km for metabolism of the amine from 1.45 to 3.52 microM (P less than 0.001); the anaesthetic had no effect on the Vmax. of the process. The magnitude of the inhibition increased with time of exposure to the anaesthetic. Halothane exposure did not alter the distribution of [3H]sorbitol or [14C]5-HT, pulmonary vascular resistance, levels of ATP or the kinetics of amino acid transport in the tissue. Inhibition of protein synthesis by cycloheximide did not mimic the effect of the anaesthetic. These observations, together with those made in lungs exposed to inhibitors of 5-HT uptake and metabolism, were consistent with a halothane-mediated inhibition of 5-HT uptake, which did not appear to involve non-specific changes in membrane permeability.

Animals↗
PubMed1983

Regulation of protein synthesis in lung by amino acids and insulin.

Acute effects of amino acid availability and insulin on protein synthesis were investigated in rat lungs perfused in situ with buffer containing either 4.5% fraction V bovine serum albumin (FrV BSA), 4.5% essentially fatty acid-free (FAF) BSA, or 4.5% dextran to maintain colloid osmotic pressure. In the presence of FrV BSA, protein synthesis was unaffected by perfusion for 1 or 3 h with buffer containing no added amino acids (0X), as compared with amino acids at concentrations one (1X) or five (5X) times those in rat plasma. Regardless of the amino acid concentration, addition of insulin was without effect. Likewise, in lungs perfused for 1 h with either FAF BSA or dextran, protein synthesis was insensitive to amino acid availability or to insulin. After 3 h, however, protein synthesis decreased 34 and 37%, respectively, when these lungs were perfused in the absence of both amino acids and insulin. In both cases, the inhibition was prevented by addition of insulin to the perfusate; addition of the hormone to perfusate containing 1X amino acids or elevating perfusate amino acids to 5X did not affect protein synthesis. The deficit in protein synthesis observed in the absence of both amino acids and insulin was not accompanied by ATP depletion or by lower intracellular concentrations of amino acids. Similarly, the effect of insulin was not associated with a general elevation in intracellular amino acid concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed1982

The measurement of protein synthesis in biological systems.

Attempts to quantitate metabolism in the lung and other tissues using radioactive precursors may be subject to significant errors arising from inappropriate assumptions regarding precursor metabolism, compartmentation and specific radioactivity. This article reviews the type and magnitude of error which may complicate such measurements by presenting specific data from experiments using radioactive amino acids to estimate the rate of protein synthesis. The applicability of these observations to other metabolic systems is discussed briefly in order to develop a more general awareness of the errors which may result from incomplete validation of experimental measurements using radioisotopes.

Amino Acids↗
PubMed1982

Additive effects of pentobarbital and halothane to inhibit synthesis of lung proteins.

The effect of pentobarbital on synthesis of lung proteins was investigated, both when administered alone and in combination with halothane. When rat lungs perfused in situ with Krebs-Henseleit bicarbonate buffer containing plasma levels of 19 amino acids, 690 microM phenylalanine, 5.6 mM glucose, and 4.5 per cent fraction V bovine serum albumin were exposed to pentobarbital, a dose-related inhibition of [14C]phenylalanine incorporation into protein was observed, with a maximal inhibition (74 per cent) at a pentobarbital concentration of 324 micrograms/ml. Halothane (1-4 per cent equilibrated with O2/N2/CO2, 4:15:1) also rapidly inhibited synthesis of lung proteins in a dose-dependent manner. At the maximally effective concentration of pentobarbital, exposure of the lungs to halothane enhanced the inhibition of protein synthesis; halothane concentrations ranging from 1 to 4 per cent were equally effective. Furthermore, when lungs were exposed to a combination of pentobarbital (100 micrograms/ml) and halothane (1 per cent) at doses which had no effect when given alone, protein synthesis was inhibited 35 per cent (P less than 0.001). Thus, the metabolic effects of the anesthetics were potentiated when the drugs were administered in combination. The inhibition of protein synthesis by pentobarbital (324 micrograms/ml), with or without 4 per cent halothane, was fully reversible. A similar inhibitory effect of pentobarbital was observed in perfused rat hearts.

Adenosine Triphosphate↗
PubMed1982

Effect of diabetes on metabolism of 5-hydroxytryptamine by rat lungs perfused in situ.

The effect of diabetes induced by treatment of rats with streptozotocin on metabolism of circulating 5-hydroxytryptamine (5HT) was investigated using an in situ lung perfusion preparation. Tissue uptake of 5HT and production of its metabolite, 5-hydroxyindolacetic acid, were unaffected in lungs of diabetic animals provided 2 or 20 microM exogenous 5HT. At constant perfusion pressure, pulmonary flow was not altered by substrate concentration or by streptozotocin treatment. Thus, in the experimental models of diabetes used, metabolism of circulating 5HT by the pulmonary endothelium remained unaffected.

Animals↗