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Biomedical subjects

L Merkel

Publications and source records attributed to L Merkel.

14 recordsLinked to original sources
PubMed2000

Preconditioning of human smooth muscle cells via cyclopentenone prostaglandins protects against toxic effects of oxidized low-density lipoprotein.

Human vascular smooth muscle cells (SMC) exhibit upregulation of inducible heat shock protein 70 (Hsp70), upon exposure to oxidized low-density lipoproteins (LDL(ox)). The presence of Hsp70 is thought to protect the cell against the toxic effects of the modified lipoprotein. In order to test this hypothesis, Hsp70 in SMC was upregulated by exposure to Delta(12) prostaglandin J(2) (Delta(12)PGJ(2)) before cells were exposed to LDL(ox). Hsp70 levels were measured after exposure to Delta(12)PGJ(2) and before exposure to LDL(ox). Cell protection was monitored after LDL(ox) exposure by determination of cell toxicity measured by cell lactate dehydrogenase (LDH) release into the medium. Cells treated with Delta(12)PGJ(2) exhibited a 23-fold increase in Hsp70 levels and 56% lower LDH activity release after exposure to LDL(ox) when compared to cells that were not pretreated with Delta(12)PGJ(2). In addition, cells pretreated with prostaglandins that did not induce Hsp70 did not exhibit increased tolerance against the toxic effects of LDL(ox). The results support a protective role for Hsp70 against the toxic effects of LDL(ox) and hint at the potential for the use of small molecules for the prevention of deleterious effects of LDL(ox) through heat shock protein upregulation.

Cyclopentanes↗
PubMed2000

Induction of heat shock protein 70 by herbimycin A and cyclopentenone prostaglandins in smooth muscle cells.

This study characterizes Hsp70 induction in human smooth muscle cells (SMC) by herbimycin A and cyclopentenone prostaglandins. The magnitude of Hsp70 induction by cyclopentenone prostaglandins was 8- to 10-fold higher than induction by herbimycin A. Hsp70 induction by delta12PGJ2 was first observed at 10 microM, rose to 4000-5000 ng/mL within one log unit and a maximum response was not observed; concentrations of delta12PGJ2 higher than 30 microM were toxic to the cells. A maximum response with herbimycin A (500 ng/mL) was reached at 0.05 microM and maintained to 1 microM without toxicity. Both, delta12PGJ2 and herbimycin A, were inhibited by dithiothreitol (DTT, 100 microM) at lower concentrations and became less sensitive to inhibition at higher concentrations. Hsp70 induction after incubation of SMC with delta12PGJ2 followed by addition of herbimycin A was significantly higher than Hsp70 induction after incubation with herbimycin A followed by addition of delta12PGJ2. When cells were incubated with [3H]-PGJ2, followed by protein denaturation, substantial radioactivity remained protein-bound suggesting that the prostaglandin must be covalently bound. Covalent binding was largely insensitive to DTT. Maximal Hsp70 induction was observed after 5 minutes of exposure of the cells to herbimycin A followed by a 20 hour recovery period in agent-free medium. Cells required 3-4 hours of exposure to delta12PGJ2 followed by a 20 hour recovery period in order to see high Hsp70 induction. Binding of the heat shock factor (HSF) to the heat shock element (HSE) in the presence of herbimycin A or delta12PGJ2, and the effects of DTT, mirrored the results of Hsp70 induction. The results suggest that probable differences between the 2 agents are at the level of the signal transduction prior to HSF activation.

Anti-Bacterial Agents↗
PubMed1999

Differential effects of the adenosine A(1) receptor allosteric enhancer PD 81,723 on agonist binding to brain and adipocyte membranes.

The benzoylthiophene analog, PD 81,723, has been shown to allosterically enhance agonist binding and functional activation of the mammalian adenosine (ADO) A(1) receptor subtype by putatively maintaining the receptor in a high affinity state. The present studies were conducted to evaluate the ability of PD 81,723 to enhance the binding of [3H]cyclohexyladenosine ([3H]CHA) to A(1) receptors of neural (cerebral cortex) and non-neural (adipocyte) origin in three different species; rat, guinea pig and dog. PD 81, 723 (0.3-100 microM) produced a concentration-dependent enhancement of [3H]CHA binding to rat brain A(1) receptors. These effects were also species-dependent with larger enhancements (150-200% of control) observed in guinea pig and dog brain membranes as compared to the rat (120% of control). In contrast, PD 81,723 did not produce any enhancement of [3H]CHA binding to A(1) receptors in adipocyte membranes from any of the species examined. Additional binding studies were conducted using pharmacological manipulations that have previously been shown to enhance the allosteric effects of PD 81,723. In the presence of 1 mM GTP, the allosteric effects of PD 81,723 (15 microM) were increased in rat, guinea pig and dog brain membranes, however, in adipocyte membranes from each species, no significant alteration in agonist binding was observed. Similarly, the A(1) receptor selective antagonist 8-cyclopentyl-1, 3-dipropylxanthine (added to effectively reduce the intrinsic antagonist properties of PD 81,723) was found to enhance the allosteric effects of PD 81,723 (15 microM) in brain, but produce no alteration of agonist binding in adipocyte membranes from each species. Examination of the dissociation kinetics of [3H]CHA binding from rat brain and adipocyte membranes revealed that PD 81,723 (15 microM) differentially slowed agonist dissociation from brain, but not adipocyte, membranes. Taken together, the present data support the hypothesis that in tissues that are sensitive to PD 81,723, this benzyolthiophene functions to maintain the A(1) receptor in a high-affinity state and that the relative proportions of high-affinity A(1) receptors present in specific tissues may contribute, at least in part, to the apparent differential effects of PD 81,723 on agonist binding. The tissue specific modulation of A(1) receptor function by PD 81,723 also illustrates the possibility that the locus of allosteric modulation by PD 81,723 may be manifest via a specific, but indirect and tissue-dependent, interaction with the A(1) receptor.

Adenosine↗
PubMed1999

Restenosis following angioplasty in the swine coronary artery is inhibited by an orally active PDGF-receptor tyrosine kinase inhibitor, RPR101511A.

BACKGROUND: Platelet-derived growth factor (PDGF), a purported mediator of arterial response to injury, stimulates proliferation, chemotaxis, and matrix production by activation of its membrane receptor tyrosine kinase. Because these activities underlie restenosis, inhibition of the PDGF-receptor tyrosine kinase (PDGFr-TK) is postulated to decrease restenosis. METHODS AND RESULTS: RPR101511A is a novel compound which selectively and potently inhibits the cell-free and in situ PDGFr-TK and PDGFr-dependent proliferation and chemotaxis in vascular smooth muscle cells (VSMC). To evaluate the effect of RPR101511A (30 mg. kg-1. d-1 BID for 28 days following PTCA) on coronary restenosis, PTCA was performed in hypercholesterolemic minipigs whose left anterior descending (LAD) coronary artery had been injured by overdilation and denudation, yielding a previously existing lesion. Angiographically determined prePTCA minimal lumen diameters (MLD) were similar in vehicle and RPR101511A-treated pigs (1.98+/-0.09 versus 2.01+/-0.08 mm) and increased to the same extent in the 2 groups following successful PTCA (2.30+/-0.06 versus 2.52+/-0.13). At termination, there was an average 50% loss of gain in the vehicle-treated group but no loss of gain with RPR101511A (2.16+/-0. 05 versus 2.59+/-0.11, P<0.001). Morphometric analysis of the LAD showed that RPR101511A caused a significant decrease in total intimal/medial ratio (0.96+/-0.58 versus 0.67+/-0.09, P<0.05). CONCLUSIONS: RPR101511A, which acts by inhibition of the PDGFr-TK, completely prevented angiographic loss of gain following PTCA and significantly reduced histological intimal hyperplasia.

Administration, Oral↗
PubMed1998

Electroencephalographic and psychometric differences between boys with and without attention-deficit/Hyperactivity disorder (ADHD): a pilot study.

Attention-Deficit/Hyperactivity Disorder (ADHD) is reported to have an incidence of 3-5%, and is associated with a variety of interpersonal, academic, and social problem behaviors. There is controversy as to whether ADHD is a learned behavioral or brain dysfunction. Research has explored a variety of measures to assess behavioral and brain dysfunctions in this population, with no consistent and clearly diagnostic results. We investigated whether a new psychometric and a new electroencephalographic procedure would clearly differentiate ADHD. The psychometric was based on DSM-IV criteria and the EEG measure was based on the assumption that ADHD interferes with cognitive transition from one discrete task to another. Parents of four ADHD boys (ages 8-12) and four age- and interest-matched non-ADHD boys completed the ADHD Symptom Inventory, while their sons' EEG was monitored during viewing of a video and reading of a book. For the ADHD boys, this was repeated a second time, 3 months later, to assess test-retest reliability. Both the psychometric and the EEG measures clearly differentiated the two samples (p's < .01) with no overlap in scores, were reliable over 3 months (r = .87), and were significantly correlated with one another (r = .85). While a small sample size, these robust, related and reliable findings suggest that both the psychometric and the psychophysiological EEG measures deserve further replication and exploration.

Attention Deficit Disorder with Hyperactivity↗
PubMed1995

Enzyme inhibition during the conversion of squalene to cholesterol.

Two separate enzymatic assays were developed in order to test the selectivity of inhibitors in cholesterol biosynthesis. One assay detects inhibition of delta 5.7-sterol delta 7-reductase, the enzyme involved in the conversion of 7-dehydrocholesterol to cholesterol. Delta 5.7-Sterol delta 7-reductase was inhibited by both RPR 101821, a protonated cyclohexylamine, and BM 15.766, a piperazine derivative, with IC50 values of 1 microM. The second assay detects accumulation of any of five intermediates (squalene oxide, squalene dioxide, lanosterol, desmosterol, and 7-dehydrocholesterol) upon inhibition of enzymes catalyzing reactions in the conversion of squalene to cholesterol. In this assay, inhibition data were most accurate when control assays exhibited a conversion of squalene to cholesterol in the order of 50%. The time required to attain 50% conversion of squalene to cholesterol was 6 h. Given a high inhibitor to substrate concentration ratio and the possible values of Ki, kon, and koff for the reaction between enzymes and inhibitor to form enzyme-inhibitor complexes, it was predicted that in the presence of inhibitors, intermediate accumulation could still be observed after 6 h incubation. The experimental results were in agreement with this prediction.

Animals↗
PubMed1990

Dissociation between myosin phosphorylation and shortening velocity in canine trachea.

The relationship between glycogen phosphorylase activity (an index of cytosolic Ca2+ content), myosin light-chain phosphorylation, isotonic shortening velocity, and isometric tension was examined in canine trachealis. Responses were measured in tracheal strips contracted with various concentrations of methacholine or K+. Both agonists produced prolonged and concentration-dependent increases in isometric tension that reached 90% of the plateau level within 1 (methacholine) to 5 (K+) min and remained stable over 60 min. In contrast to the monotonic increase in isometric tension, shortening velocity reached a maximum almost immediately (12-48 s) after the addition of either methacholine or K+ and then declined over time to a steady-state level that was 25-40% of the peak. Phosphorylase activity also increased transiently, reaching a maximum 1-2 min after the addition of either agonist before declining to near-basal levels over the 60-min observation period. Unlike the increases in shortening velocity and phosphorylase activity, agonist-induced myosin phosphorylation was not markedly transient. Moreover, regardless of the contractile agonist used, no correlation was found between myosin phosphorylation and shortening velocity when these parameters were compared at corresponding time points. This suggests that myosin phosphorylation is not the sole determinant of shortening velocity in canine trachealis.

Animals↗
PubMed1989

Interaction of smooth muscle tropomyosin and smooth muscle myosin. Effect on the properties of myosin.

Several techniques were used to investigate the possibility that smooth muscle tropomyosin interacts with smooth muscle myosin. These experiments were carried out in the absence of actin. The Mg2+-ATPase activity of myosin was activated by tropomyosin. This was most marked at low ionic strength but also occurred at higher ionic strength with monomeric myosin. For myosin and HMM, the activation of Mg2+-ATPase by tropomyosin was greater at low levels of phosphorylation. There was no detectable effect of tropomyosin on the Mg2+-ATPase activity of S1. The KCl dependence of myosin viscosity was influenced by tropomyosin, and in the presence of tropomyosin, the 6S to 10S transition occurred at lower KCl concentrations. From the viscosity change, an approximate stoichiometry of 1:1 tropomyosin to myosin was estimated. The phosphorylation dependence of viscosity, which reflects the 10S-6S transition, also was altered in the presence of tropomyosin. An interaction between myosin and tropomyosin was detected by fluorescence measurements using tropomyosin labeled with dansyl chloride. These results indicate that an interaction occurs between myosin and tropomyosin. In general, the interaction is favored at low ionic strength and at low levels of phosphorylation. This interaction is not expected to be competitive with the formation of the actin-tropomyosin complex, but the possibility is raised that a direct interaction between myosin and tropomyosin bound to the thin filament could modify contractile properties in smooth muscle.

Animals↗
PubMed1986

Cyclic GMP-dependent protein kinase relaxes skinned fibers from guinea pig taenia coli but not from chicken gizzard.

The effect of cGMP and cGMP-dependent protein kinase (cG-PK) on contraction and relaxation was studied in skinned smooth muscle fibers from guinea pig taenia coli and chicken gizzard. At a fixed [Ca2+] relaxation was significantly enhanced by activated cG-PK in fibers from guinea pig taenia coli, but not in those from chicken gizzard. The Ca2+-requirement for half maximal tension maintenance was shifted to the right. Relaxation was associated with a decline in phosphorylated myosin light chain-2 from 34% to 25%. Similarly to relaxation activated cG-PK inhibited tension development only in fibers from taenia coli. These results suggest that mammalian and chicken smooth muscle fibers respond differently to cG-PK.

Animals↗
PubMed1985

Prolonged relaxation of detergent-skinned smooth muscle involves decreased endogenous phosphatase activity.

Since contraction of smooth muscle involves Ca2+-dependent phosphorylation of the 20 Kd myosin light chains, changes in endogenous phosphatase activity may participate in regulating smooth muscle contractility. We found that detergent-skinned fibers from 7 of 10 chicken gizzards studied were characterized by relatively high endogenous light chain phosphatase activity (23 mU/mg protein) and rapid relaxation (t1/2 = 1-3 min) in the absence of Ca2+ (less than 10(-8) M). In contrast, skinned fibers from 3 of the gizzards exhibited very low phosphatase activity (3 mU/mg protein) and markedly prolonged relaxation (t1/2 = 50-200 min). However, such slow relaxing fibers were converted to a form resembling rapidly relaxing fibers (t1/2 = 4-10 min) when an aortic polycation-modulable phosphatase was included in the incubation medium. Moreover this phosphatase-enhanced relaxation was associated with dephosphorylation of the light chains. Maximal isometric force (1 mN) and light chain phosphorylation (0.8 mol PO4/mol light chain) were similar in slowly and rapidly relaxing fibers. Thus, the two populations of skinned fibers, though dramatically different with respect to phosphatase activity and relaxation time, appeared to be very similar in terms of Ca2+-dependent contraction. These findings strongly suggest that prolonged relaxation of smooth muscle of the kind noted in this study, and perhaps in hypertensive or aging vascular smooth muscle, may reflect decreased endogenous phosphatase activity.

Animals↗
PubMed1984

The variable relation between myosin light-chain phosphorylation and actin-activated ATPase activity in chicken gizzard smooth muscle. Modulation by tropomyosin.

Using pure myosin light-chain kinase and myosin from chicken gizzard and rabbit skeletal actin, we have established the relationship between myosin phosphorylation and actin-activated ATPase activity by varying the concentration of Ca2+ or calmodulin (CaM). By increasing Ca2+ or CaM the extent of myosin phosphorylation increased from 10% to about 100%, the requirements for half-maximal activation being 0.17 microM Ca2+ and 1 nM CaM. ATPase was activated only when a phosphorylation 'threshold' of about 60% was surpassed. By increasing phosphorylation from 60% to 100%, ATPase activity was further stimulated, the relationship between myosin phosphorylation and ATPase activity being curvilinear. Addition of 1 microM tropomyosin lowered the Ca2+ requirement for half-maximal ATPase activity in the prescence of 0.05 microM CaM from 0.4 microM to 0.2 microM but did not affect the maximal ATPAse activity. In addition, the CaM requirement of ATPase activity was also lowered by gizzard tropomyosin at 9 microM Ca2+. The Ca2+/CaM requirement for myosin phosphorylation remained unaffected by tropomyosin. Thus, tropomyosin altered the relationship between myosin phosphorylation and ATPase activity: the phosphorylation 'threshold' was lowered from 60% to about 10%, hence causing a leftward shift of the relationship which becomes similar to that in the case of native actomyosin.

Actins↗