Biomedical subjects

D L Severson

Publications and source records attributed to D L Severson.

At least 55 records · Page 3Linked to original sources

PubMed1992

Regulation of lipoprotein lipase activity in cardiac myocytes from control and diabetic rat hearts by plasma lipids.

The objective of this investigation was to test the hypothesis that the diabetes-induced reduction in lipoprotein lipase activity in cardiac myocytes may be due to hypertriglyceridemia. Administration of 4-aminopyrazolopyrimidine (50 mg/kg) to control rats for 24 h reduced plasma triacylglycerol levels and increased the heparin-induced release of lipoprotein lipase into the incubation medium of cardiac myocytes. The acute (3-5 days) induction of diabetes by streptozotocin (100 mg/kg) produced hypertriglyceridemia and reduced heparin-releasable lipoprotein lipase activity in cardiac myocytes. Treatment of diabetic rats with 4-aminopyrazolopyrimidine resulted in a fall in plasma triacylglycerol content and increased heparin-releasable lipoprotein lipase activity. Administration of Triton WR-1339 also resulted in hypertriglyceridemia, but the heparin-induced release of lipoprotein lipase from control cardiac myocytes was not reduced in the absence of lipolysis of triacylglycerol-rich lipoproteins. Treatment with Triton WR-1339 did, however, increase the heparin-induced release of lipoprotein lipase from diabetic cardiac myocytes. Preparation of cardiac myocytes with 0.9 mM oleic acid resulted in a decrease in both total cellular and heparin-releasable lipoprotein lipase activities. These results suggest that the diabetes-induced reduction in heart lipoprotein lipase activity may, at least in part, be due to an inhibitory effect of free fatty acids, derived either from lipoprotein degradation or from adipose tissue lipolysis, on lipoprotein lipase activity in (and (or) release from) cardiac myocytes.

Adenine↗

PubMed1992

Free fatty acids do not release lipoprotein lipase from isolated cardiac myocytes or perfused hearts.

Lipoprotein lipase (LPL), located at the vascular endothelium, catalyzes the hydrolysis of plasma triacylglycerols to fatty acids and 2-monoacylglycerol. In the heart, LPL is synthesized in cardiac myocytes and then translocated to the vascular endothelium. We investigated whether lipolytic products could displace LPL from the cell surface of cardiac myocytes isolated from adult rat hearts. Incubation of myocytes with 0.15-0.9 mM oleic acid or 0.1 mM monoolein did not produce a significant increase in LPL activity in the medium. LPL on the cell surface of intact myocytes hydrolyzed exogenous [3H]triolein, but there was no associated increase in LPL activity measured in the medium. Perfusion of isolated hearts with heparin (5 U/ml) resulted in displacement of LPL from the capillary endothelium. Addition of 0.9 mM oleic acid to the perfusion medium did not increase perfusate LPL activity with perfused hearts from either control or fasted rats. Therefore lipolytic products do not release active LPL from binding sites at the surface of isolated cardiac myocytes or capillary endothelial cells in perfused hearts.

Animals↗

PubMed1992

Lipoprotein lipase release from cardiac myocytes is increased by decavanadate but not insulin.

Streptozotocin-induced diabetes reduced cellular lipoprotein lipase (LPL) activity in cardiac myocytes from rat hearts and decreased the heparin-induced release of LPL into the medium. This effect of diabetes was rapidly reversed by in vivo treatment with insulin (5 U iv for 1 h); administration of insulin in vivo to control rats also increased heparin-releasable LPL activity. In contrast, in vitro addition of insulin to control and diabetic myocytes did not alter either cellular or heparin-releasable LPL activities. Insulin stimulated glucose oxidation and protein synthesis in control and diabetic myocytes. Decavanadate (0.05-1 mM) or vanadyl ion (0.5 mM) enhanced the release of LPL into the medium. Heparin- and decavanadate-induced release of LPL was not additive, and heparin pretreatment reduced the subsequent release of LPL by decavanadate. Decavanadate displaced LPL bound to heparin-Sepharose and increased LPL release into the perfusate of hearts. Therefore, decavanadate can mimic heparin in its effect on LPL. The absence of a direct in vitro effect of insulin on LPL in cardiac myocytes suggests that insulin may require some other in vivo factor or that diabetes-induced changes in LPL activity are secondary to some other metabolic factor.

Animals↗

PubMed1991

Phosphorylation of aortic plasma membranes by protein kinase C.

Incubation of plasma membranes isolated from bovine aorta with either 0.5 mM CaCl2 or with a phorbol ester (1 microM phorbol 12,13-dibutyrate) and phosphatidylserine in an EGTA-containing buffer resulted in the phosphorylation of 10 proteins (Mr of 158, 105, 75, 62, 44, 39, 33, 22, 15 and 9 kDa), presumably due to activation of endogenous protein kinase C (PKC). After heat treatment of the aortic plasma membranes at 80 degrees C for 5 min in order to inactivate all endogenous protein kinase, phosphatase and ATPase activities, membrane phosphorylation was absolutely-dependent upon the addition of an exogenous, partially-purified PKC preparation from bovine aorta. Under these conditions, a total of 17 phosphoproteins could be detected (Mr of 158, 105, 75, 44, 39, 33, 30, 29, 27, 25, 22, 17.5, 16, 15, 11, 10 and 9 kDa). The most prominent phosphoprotein band in native membranes had a molecular weight of 75 kDa (p75); several characteristics suggest that p75 might be autophosphorylated PKC. The phosphorylation of aortic plasma membranes by exogenous PKC required phosphatidylserine and was calcium-dependent (10(-5) to 10(-7) M Ca2+); the addition of diolein resulted in little or no enhancement of phosphorylation. Replacement of phosphatidylserine with oleic acid resulted in the same number of phosphoproteins, but the extent of phosphorylation was diminished. The phosphorylation pattern was altered slightly if the aortic plasma membranes were isolated in the presence of 1 mM Ca2+ instead of EGTA buffers as in the standard procedure. Experiments were performed to determine if the p39 substrate of PKC in aortic plasma membranes was calpactin II (lipocortin I). Immunoblotting established that calpactin II was present in aortic plasma membranes, but there was no corresponding phosphoprotein on the autoradiographs.

Animals↗

PubMed1991

Calmodulin inhibits the protein kinase C-catalysed phosphorylation of an endogenous protein in A10 smooth-muscle cells.

The protein kinase C (PKC) activator phorbol 12,13-dibutyrate stimulated the phosphorylation of a 75 kDa protein (p75) in intact cultured A10 smooth-muscle cells and sonicated cell preparations; p75 was the only major substrate for endogenous PKC in sonicated A10 cells. The Ca(2+)-dependent phosphorylation of p75 in vitro was dramatically decreased in PKC-down-regulated A10 cells; however, p75 from identical sonicated cell preparations was still phosphorylated by an exogenous aortic PKC preparation. Calmodulin inhibited the phosphorylation of p75 by PKC, but not the phosphorylation of other PKC substrates (platelet P47 protein and histone). The addition of calmodulin after the phosphorylation reaction was started prevented further phosphorylation, but did not decrease the extent of phosphorylation of p75 that was reached before the addition of calmodulin. The inhibition of p75 phosphorylation was concentration-dependent, with IC50 values (concn. giving 50% inhibition) ranging from less than 0.5 to 10 micrograms of calmodulin/ml, and was Ca(2+)-dependent, requiring a free Ca2+ concentration of 10 microM or greater. These results suggest that the inhibition of the PKC-catalysed phosphorylation of p75 by calmodulin may be due to its interaction with the substrate, rather than a direct inhibitory effect on the enzyme, and that this inhibition could be regulated by intracellular Ca2+ concentration. Therefore, p75 may be a physiological link between the PKC and Ca2+/calmodulin pathways.

Animals↗

PubMed1991

Diacylglycerol lipase and the contractile action of epidermal growth factor-urogastrone: evidence for distinct signal pathways in a single strip of gastric smooth muscle.

In guinea pig gastric longitudinal muscle preparations, wherein epidermal growth factor-urogastrone (EGF-URO) causes contraction via the generation of arachidonate-derived prostaglandins, the specific diacylglycerol lipase (DG lipase) inhibitor, U57,908 (formerly designated RHC 80267) completely blocked EGF-URO and transforming growth factor-alpha (TGF-alpha)-mediated contraction, without affecting contractions caused by other agonists such as bradykinin, prostaglandin F2 alpha or arachidonic acid (AA). In contrast, the contractile actions of EGF-URO and TGF-alpha on the gastric circular muscle component, present in the same tissue strip as the longitudinal muscle preparation, were unaffected by concentrations of U57,908 that maximally inhibited contraction in the longitudinal muscle preparation. We conclude that in the longitudinal muscle preparation, EGF-URO acts not by the activation of phospholipase A2, but rather via the metabolism of diacylglycerol by DG lipase, thereby liberating arachidonic acid for the synthesis of contractile prostanoids. We also conclude that, even in the same tissue, the effects of EGF-URO on anatomically different components (longitudinal muscle versus circular muscle) can be mediated via two quite distinct signal transduction pathways.

Animals↗

PubMed1991

Diabetes reduces heparin- and phospholipase C-releasable lipoprotein lipase from cardiomyocytes.

Incubation of isolated cardiac myocytes from rat hearts with heparin or phosphatidylinositol-specific phospholipase C (PLC) resulted in the release of lipoprotein lipase (LPL) into the medium. The release of LPL by the combination of heparin and PLC was not additive, and preincubation of cardiac myocytes with heparin eliminated the release of LPL in a subsequent incubation with PLC. This evidence suggests that LPL may be bound ionically to heparan sulfate proteoglycans that are covalently linked to the cell surface of cardiac myocytes by a phosphatidylinositol-glycan membrane anchor; a second pool of LPL may also be bound to proteoglycans attached directly to the myocardial cell surface. The induction of diabetes by the administration of streptozotocin (100 mg/kg for 3-4 days) to rats resulted in a decrease in the initial cellular activity of LPL and a marked reduction in the heparin-induced secretion of LPL into the medium of cardiac myocytes. The intravenous administration of insulin (5 U for 1 h) in diabetic rats reversed the effects of diabetes on cellular and heparin-releasable LPL activities. Diabetes also reduced the PLC-induced release of LPL. The reduction in the release of LPL from diabetic cardiac myocytes could result in a decrease in functional LPL activity at the capillary endothelium of whole hearts.

5'-Nucleotidase↗

PubMed1990

Treatment of cardiac myocytes with 8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate, forskolin or cholera toxin does not stimulate cellular or heparin-releasable lipoprotein lipase activities.

Incubation of isolated cardiac myocytes with 500 microM-8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPT-cAMP) or 100 microM-forskolin for 2 1/2 h did not increase the heparin-induced release of lipoprotein lipase (LPL) into the medium. When LPL activity in cardiac myocytes was depleted by treatment of rats with cycloheximide (2 mg/kg; 2.5 h) and inclusion of the protein-synthesis inhibitor in the isolation solutions, incubation with CPT-cAMP or forskolin did not influence the rate of repletion of LPL activity in cells or the recovery of heparin-releasable LPL activity. Although the administration of cholera toxin (0.5 mg/kg; 16-17 h) to rats increased LPL activity in a low-speed supernatant fraction from heparin-perfused hearts, LPL activity was not increased in cardiac myocytes from cholera-toxin-treated rat hearts, and the heparin-induced release of LPL was unchanged. Incubation of cultured ventricular myocytes with 1 microgram of cholera toxin/ml or 500 microM-CPT-cAMP for 24 h did not increase cellular LPL activity or LPL released into the culture medium after a 40 min incubation with heparin. Therefore interventions that stimulate adenylate cyclase activity (forskolin, cholera toxin) or incubation with CPT-cAMP do not increase cellular LPL activity or promote the translocation of LPL to a heparin-releasable fraction in cardiac myocytes.

Animals↗

PubMed1990

Protein kinase C does not regulate diacylglycerol metabolism in aortic smooth muscle cells.

The effect of a reduction in protein kinase C activity on the metabolism of exogenous [3H]diC8 by freshly isolated smooth muscle cells from rabbit aorta and cultured A10 smooth muscle cells was determined. The metabolism of [3H]diC8 by both smooth muscle cell preparations was predominantly by hydrolysis to yield monoC8 and glycerol (lipase pathway); very little radioactivity was incorporated into phospholipids. Diacylglycerol lipase activity measured in vitro with A10 cell homogenates was much greater than diacylglycerol kinase activity. The addition of the protein kinase C inhibitor H-7 to incubations of isolated aortic smooth muscle cells and cultured A10 cells had no significant effect on the metabolism of [3H]diC8. Protein kinase C activity in cultured A10 cells preincubated for 20 h with a phorbol ester was reduced to 14% of control as a consequence of down-regulation, but diC8 metabolism was not changed. Therefore, protein kinase C does not regulate the metabolism of diacylglycerols in aortic smooth muscle cells.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1990

Inhibition of diacylglycerol metabolism in isolated cardiac myocytes by U-57 908 (RHC 80267), a diacylglycerol lipase inhibitor.

U-57 908 (RHC 80267) inhibited diacylglycerol (DG) lipase activity in soluble and microsomal subcellular fractions from cardiac myocytes isolated from adult rat hearts; half-maximal inhibition was observed at a concentration of 3.5 microM. Monoacylglycerol lipase activity was much less sensitive to inhibition, but U-57 908 reduced lipoprotein lipase activity in cardiac myocytes with the same sensitivity as observed for DG lipase. DG kinase activity was not inhibited by U-57 908. DG metabolism by intact cardiac myocytes was studied in incubations with a cell-permeable DG analog, [3H]-dioctanoylglycerol (diC8). DiC8 was mainly metabolized by conversion to mono-octanoylglycerol (monoC8) and glycerol (lipase pathway); much less radioactivity was incorporated into the triacylglycerol and total phospholipid fractions. U-57 908 reduced the loss of radioactivity from the exogenous diC8 substrate, with a corresponding decline in the formation of radiolabelled monoC8 and glycerol. The incorporation of radioactivity into phospholipids was slightly reduced, but triacylglycerol synthesis from diC8 was increased in the presence of U-57 908. Therefore, U-57 908 is an effective inhibitor of DG metabolism by the lipase pathway in intact cardiac myocytes.

Animals↗

PubMed1990

Influence of exogenous fatty acids and ketone bodies on rates of lipolysis in isolated ventricular myocytes from normal and diabetic rats.

The effect of oleate (0.3 and 1.2 mM) and the combined effect of beta-hydroxybutyrate (4 and 8 mM) and acetoacetate (1 and 2 mM) on rates of lipolysis (glycerol output) was determined with calcium-tolerant myocytes isolated from the hearts of normal rats and hearts from acutely (2-3 days; 100 mg/kg streptozotocin) diabetic rats. In addition, the effect of these exogenous substrates on rates of lipolysis was investigated in triacylglycerol (TG) loaded myocytes prepared from normal hearts by inclusion of oleate in the isolation solutions. Diabetic and TG-loaded myocytes had higher lipolytic rates than normal myocytes. In control myocytes, oleate (1.2 mM) did not affect basal lipolysis, but it reduced isoproterenol-stimulated lipolysis by 30%. In diabetic and TG-loaded myocytes, the addition of 1.2 mM oleate inhibited basal rates of lipolysis by 41 and 40%, respectively, and isoproterenol-stimulated rates of lipolysis by 43 and 53%, respectively. However, lipolytic rates measured in the presence of 1.2 mM oleate with diabetic and TG-loaded myocytes were still higher than lipolysis in normal myocytes incubated in the absence of oleate. Ketone bodies increased both basal and isoproterenol-stimulated lipolysis in normal myocytes. In diabetic myocytes, ketone bodies produced a modest stimulation of basal lipolysis but had no effect on isoproterenol-stimulated rates of lipolysis. These data indicate that mobilization of endogenous TG may play an important role in supplying energy to the heart in the diabetic state. Moreover, accumulation of endogenous TG in diabetic myocardium can only partly be explained by inhibition of lipolysis by exogenous substrates.

Animals↗

PubMed1990

Apolipoprotein VLDL-II inhibits lipolysis of triglyceride-rich lipoproteins in the laying hen.

In the laying hen, very low density lipoprotein (VLDL) particles contain large amounts of apolipoprotein (apo)-VLDL-II in addition to apoB. These triglyceride-rich lipoproteins are transported from the liver primarily to growing oocytes. Since no appreciable hydrolysis of triglyceride occurs during this transport, we have investigated the possibility that apoVLDL-II functions as an inhibitor of lipoprotein lipase (LPL). The presence of LPL in chicken follicular granulosa cells was demonstrated by immunoblotting, and LPL activity with the usual in vitro characteristics could be measured in cultured granulosa cell extracts. ApoVLDL-II inhibited LPL activity in these extracts as well as in the post-heparin medium of rat cardiac myocytes. Half-maximal inhibition in both systems occurred at 40 micrograms/ml, a concentration that is one-tenth of the circulating apoVLDL-II in the laying hen. Much less inhibition was observed with reduced and alkylated apoVLDL-II and with apoA-I. We conclude that the presence of apoVLDL-II on laying hen VLDL ensures efficient delivery of triglyceride to the oocyte for subsequent use as energy source by the embryo.

Animals↗

PubMed1989

Effect of cis-unsaturated fatty acids on aortic protein kinase C activity.

Long-chain cis-unsaturated fatty acids could substitute for phosphatidylserine and activate bovine aortic protein kinase C in assays with histone as substrate. The optimal concentration was 24-40 microM for oleic, linoleic and arachidonic acids. With arachidonic acid, the Ka for Ca2+ was 130 microM and kinase activity was maximal at 0.5 mM-Ca2+. Diolein only slightly activated the oleic acid-stimulated enzyme at low physiological Ca2+ concentrations (0.1 and 10 microM). Oleic acid also stimulated kinase C activity, determined with a Triton X-100 mixed-micellar assay. Under these conditions, the fatty acid activation was absolutely dependent on the presence of diolein, but a Ca2+ concentration of 0.5 mM was still required for maximum kinase C activity. The effect of fatty acids on protein kinase C activity was also investigated with the platelet protein P47 as a substrate, since the properties of kinase C can be influenced by the choice of substrate. In contrast with the results with histone, fatty acids did not stimulate the phosphorylation of P47 by the aortic protein kinase C. Activation of protein kinase C by fatty acids may allow the selective phosphorylation of substrates, but the physiological significance of fatty acid activation is questionable because of the requirement for high concentrations of Ca2+.

Animals↗

PubMed1989

Metabolism of dioctanoylglycerol by isolated cardiac myocytes.

Diacylglycerol (DG) metabolism by intact cardiac myocytes isolated from adult rat hearts and by broken cell preparations from myocytes was investigated in experiments with [3H]dioctanoylglycerol (diC8), a cell-permeable diacylglycerol analog. In a low-speed supernatant fraction, the Km and Vmax for DG kinase activity (formation of phosphatidic acid) was 22 microM and 110 nmol/h/mg, respectively, whereas the Km and Vmax for DG lipase activity (formation of monoacylglycerol) was 80 microM and 1000 nmol/h/mg. At a substrate concentration of 80 microM diC8, lipase activity was 7-fold greater than kinase activity. The majority of DG kinase activity was recovered in the soluble subcellular fraction; DG lipase activity was localized in a microsomal fraction. When [3H]diC8 was incubated with intact cardiac myocytes, 10-fold more radioactivity was incorporated into the products of the lipase pathway (monoacylglycerol and free glycerol) as compared to incorporation into the total phospholipid fraction which contained phosphatidic acid. This predominance of metabolism by hydrolysis through the lipase pathway was observed consistently when the incubation time, content of cardiac myocytes and concentration of exogenous diC8 was varied. Therefore, results from both in vitro determinations of enzyme activities in broken cell preparations and flux studies with intact cells have indicated that the lipase pathway is the principal enzymatic mechanism for the metabolism of diC8 in cardiac myocytes.

Animals↗

PubMed1989

Diacylglycerol metabolism in isolated aortic smooth muscle cells.

The metabolism of the cell-permeable diacylglycerol (DG) analogue, 1,2-dioctanoyl-sn-glycerol (diC8), by isolated intact smooth muscle cells from rabbit aorta was determined. Radiolabeled diC8 was rapidly converted to monoacylglycerol, with smaller amounts of radioactivity recovered as free glycerol and in the total phospholipid fraction that included phosphatidic acid. The greater metabolism of diC8 by the lipase pathway (formation of monoacylglycerol and glycerol) as compared with the kinase pathway (formation of phosphatidic acid and other phospholipids) was observed consistently when experimental conditions were changed by varying the time of incubation (5-30 min), diC8 concentration (0.5-50 microM), and the content of smooth muscle cells in the incubation (0.5-5 X 10(5) cells). These results with the metabolism of exogenous diC8 by intact smooth muscle cells are consistent with in vitro determinations of DG kinase and lipase activities, where lipase activity (especially hydrolysis of the sn-1 position to yield the monoacylglycerol intermediate) was much greater than kinase activity measured with both soluble and particulate subcellular fractions from aortic smooth muscle cells. Both DG kinase and lipase activities were inhibited when 1-monoolein was added to the in vitro assay. Treatment of intact smooth muscle cells with 1-monoolein (200 microM), however, did not reduce the formation of monoacylglycerol from diC8.

Animals↗

PubMed1988

Characterization of bovine aortic protein kinase C with histone and platelet protein P47 as substrates.

A Ca2+- and phospholipid-dependent protein kinase (protein kinase C) was partially purified from the media of bovine aortas by chromatography on DEAE-Sephacel and phenyl-Sepharose. Enzyme activity was characterized with both histone and a 47 kDa platelet protein (P47) as substrates, because the properties of protein kinase C can be modified by the choice of substrate. Both phosphatidylserine and Ca2+ were required for kinase activity. With P47 as substrate, protein kinase C had a Ka for Ca2+ of 5 microM. Addition of diolein to the enzyme assay caused a marked stimulation of activity, especially at low Ca2+ concentrations, but the Ka for Ca2+ was shifted only slightly, to 2.5 microM. With histone as substrate, the enzyme had a very high Ka (greater than 50 microM) for Ca2+, which was substantially decreased to 3 microM-Ca2+ by diolein. A Triton X-100 mixed-micelle preparation of lipids was also utilized to assay protein kinase C with histone as the substrate. Under these conditions kinase activity was almost totally dependent on the presence of diolein; again, diolein caused a large decrease in the Ka for Ca2+, from greater than 100 microM to 2.5 microM. The increased sensitivity of protein kinase C to Ca2+ with P47 rather than histone, and the ability of diacylglycerol to activate protein kinase C without shifting the Ka for Ca2+, when P47 is the substrate, illustrate that the mechanism of protein kinase C activation is influenced by the exogenous substrate used to assay the enzyme.

Animals↗

PubMed1988

Secretion of lipoprotein lipase from myocardial cells isolated from adult rat hearts.

Heparin (5 U/ml) induced the release of LPL into the incubation medium of cardiac myocytes isolated from adult rat hearts. The secretion of LPL occurred in two phases: a rapid release (5-10 min of incubation with heparin) that was independent of protein synthesis followed by a slower rate of release that was inhibited by cycloheximide. The rapid release of LPL induced by heparin likely occurs from sites that are at or near the cell surface. LPL secretion could also be stimulated by heparan sulfate and dermatan sulfate, but not by hyaluronic acid, chondroitin sulfate or keratan sulfate. Heparin-releasable LPL activity measured in short-term incubations represented a large fraction (40-50%) of the initial LPL activity associated with myocytes, but the fall in cellular LPL activity following heparin was less than the amount of LPL activity secreted into the incubation medium. This discrepancy was not due to latency of LPL in the pre-heparin cell homogenates, but in part could be due to a three-fold greater affinity of the heparin-released enzyme for substrate as compared to LPL in post-heparin myocyte homogenates.

Animals↗

PubMed1988

Monoacylglycerol lipase activity in cardiac myocytes.

Monoacylglycerol lipase activity in homogenates of isolated myocardial cells (myocytes) from rat hearts was recovered in both particulate and soluble subcellular fractions. The activity present in the microsomal (100,000 X g pellet) fraction was solubilized by treatment with Triton X-100 and combined with the 100,000 X g supernatant fraction; the properties of monoacylglycerol lipase were investigated with this soluble enzyme preparation. The Km for the hydrolysis of a 2-monoolein substrate was 16 microM. The rates of hydrolysis of 1-monoolein and 2-monoolein were identical, and 1-monoolein was a competitive inhibitor (Ki = 20 microM) of the hydrolysis of 2-monoolein. Monoacylglycerol lipase activity was regulated by product inhibition according to the following order of potency: fatty acyl CoA greater than free fatty acids greater than fatty acyl carnitine.

Animals↗