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Biomedical subjects

B Hinz

Publications and source records attributed to B Hinz.

At least 37 records · Page 2Linked to original sources
PubMed2000

Salicylate metabolites inhibit cyclooxygenase-2-dependent prostaglandin E(2) synthesis in murine macrophages.

The poor cyclooxygenase (COX) inhibitor and major aspirin metabolite salicylic acid is known to exert analgesic and anti-inflammatory effects by still unidentified mechanisms. In RAW 264.7 macrophages, lipopolysaccharide (LPS)-induced COX-2-dependent synthesis of prostaglandin E(2) (PGE(2)) was suppressed by aspirin (IC(50) of 5. 35 microM), whereas no significant inhibition was observed in the presence of sodium salicylate and the salicylate metabolite salicyluric acid at concentrations up to 100 microM. However, the salicylate metabolite gentisic acid (2,5-dihydroxybenzoic acid; 10-100 microM) and salicyl-coenzyme A (100 microM), the intermediate product in the formation of salicyluric acid from salicylic acid, significantly suppressed LPS-induced PGE(2) production. In contrast, gamma-resorcylic acid (2,6-dihydroxybenzoic acid) as well as unconjugated coenzyme A failed to affect prostanoid synthesis, implying that the para-substitution of hydroxy groups and the activated coenzyme A thioester are important for COX-2 inhibition. Using real-time RT-PCR, none of the salicylate derivatives tested were found to interfere with COX-2 expression. Overall, our results suggest that certain metabolites of salicylic acid may contribute to the pharmacological action of its parent compound by inhibiting COX-2-dependent PGE(2) formation at sites of inflammation.

Acyl Coenzyme A↗
PubMed2000

Prostaglandin E(2) upregulates cyclooxygenase-2 expression in lipopolysaccharide-stimulated RAW 264.7 macrophages.

Prostaglandin E(2) (PGE(2)) has been implicated in the regulation of inflammatory and immunological events. Using RAW 264.7 macrophages, the present study investigates the influence of PGE(2) on the expression of cyclooxygenase-2 (COX-2). Incubation of cells with PGE(2) increased lipopolysaccharide (LPS)-induced COX-2 mRNA levels in a concentration-dependent manner. Upregulation of COX-2 expression by PGE(2) was completely abolished by the specific adenylyl cyclase inhibitor 2',5'-dideoxyadenosine and mimicked by butaprost, a selective agonist of the adenylyl cyclase-coupled PGE(2) receptor subtype 2 (EP(2)), or 11-deoxy PGE(1), an EP(2)/EP(4) receptor agonist. By contrast, the EP(3)/EP(1) receptor agonists 17-phenyl-omega-trinor PGE(2) and sulprostone left LPS-induced COX-2 expression virtually unaltered. Upregulation of LPS-induced COX-2 expression and subsequent PGE(2) synthesis was also observed in the presence of the cell-permeable cAMP analogue dibutyryl cAMP and the adenylyl cyclase activator cholera toxin. Together, our data demonstrate that PGE(2) potentiates COX-2 mRNA expression via an adenylyl cyclase/cAMP-dependent pathway. In conclusion, upregulation of COX-2 expression via an autocrine feed-forward loop may in part contribute to the well-known capacity of PGE(2)/cAMP to modulate inflammatory processes.

Adenylyl Cyclase Inhibitors↗
PubMed2000

Nitric oxide inhibits inducible nitric oxide synthase mRNA expression in RAW 264.7 macrophages.

Using cultured murine RAW 264.7 macrophages, the present study investigates the influence of nitric oxide (NO) on the expression of the inducible NO synthase (iNOS) enzyme at the transcriptional level. Incubation of cells with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) led to a marked increase in iNOS mRNA levels. Inhibition of LPS/IFN-gamma-induced NO synthesis with the L-arginine analogue N(G)-monomethyl-L-arginine (L-NMMA) was accompanied by a significant up-regulation of iNOS mRNA that was reversed in the presence of the NO donor sodium nitroprusside (SNP). Treatment of cells with SNP alone decreased LPS/IFN-gamma-induced iNOS mRNA levels in a concentration-dependent manner. The inhibitory effect of SNP on iNOS mRNA expression was not prevented by 1H-[1,2, 4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ), a selective inhibitor of the soluble guanylyl cyclase. In agreement with this finding, incubation of cells with the membrane-permeable cyclic GMP analogue 8-bromo cyclic GMP left LPS/IFN-gamma-induced iNOS mRNA expression virtually unaltered. Together, our results demonstrate that both iNOS-derived and exogenous NO exert an inhibitory effect on the expression of iNOS by a mechanism independent of the soluble guanylyl cyclase/cyclic GMP pathway. In conclusion, NO may control the extent of iNOS mRNA expression by a negative autoregulatory feedback.

Animals↗
PubMed2000

[Specific cyclooxygenase-2 inhibitors. Basis and options of a pharmacotherapeutic concept].

The therapeutic and unwanted side effects of non-steroidal antiinflammatory drugs (NSAIDs) are attributable to inhibition of the cyclooxygenase (COX) enzyme which catalyzes the first step of the synthesis of prostanoids. In the early 1990s, COX was demonstrated to exist as two distinct isoforms. COX-1 is constitutively expressed as a 'housekeeping' enzyme in most tissues. In contrast, COX-2 can be upregulated by various proinflammatory agents, including endotoxin, cytokines and growth factors. Whereas many of the side effects of NSAIDs (e.g. gastrointestinal ulceration and bleeding, platelet dysfunctions) are due to a suppression of COX-1 activity, inhibition of COX-2-derived prostanoids facilitates the anti-inflammatory, analgesic and antipyretic effects of NSAIDs. Thus, the hypothesis that specific inhibition of COX-2 might have therapeutic actions similar to those of NSAIDs, but without causing the unwanted side effects, was the rationale for the development of specific inhibitors of the COX-2 enzyme as a new class of anti-inflammatory and analgesic agents with improved gastrointestinal tolerability. However, the simple concept that COX-2 is an exclusively proinflammatory and inducible enzyme cannot be supported any longer. Recently, COX-2 was shown to be also expressed under basal conditions in organs such as the ovary, uterus, brain, spinal cord, kidney and bone, suggesting that this isoenzyme may play a more complex physiological role than was expected. The present review assesses concept and molecular mechanism underlying specific COX-2 inhibition as well as indications, pharmakokinetics and unwanted side effects of the recently approved specific COX-2 inhibitors celecoxib and rofecoxib. Moreover, recent advances in COX-2 research, with particular emphasis on new insights into physiological functions of this isoenzyme will be discussed.

Animals↗
PubMed2000

Dexamethasone megadoses stabilize rat liver lysosomal membranes by non-genomic and genomic effects.

PURPOSE: Membrane-stabilizing effects may be part of glucocorticoid action during high-dose glucocorticoid therapy. The present study investigates the mode of action of dexamethasone megadoses on rat liver lysosomal membranes. METHODS: Following intravenous administration of dexamethasone in rats, the release of beta-glucuronidase from liver lysosomes was assessed ex vivo as a marker for lysosomal membrane integrity. RESULTS: Dexamethasone megadoses significantly inhibited beta-glucuronidase release 10 min post-administration by 38% (3 mg/kg dexamethasone) and 33% (10 mg/kg dexamethasone) at corresponding dexamethasone liver concentrations of 3.9 x 10(-5) mol/kg and 15.1 x 10(-5) mol/kg, respectively. Comparable inhibition of beta-glucuronidase release (34% for 3 mg/kg and 38% for 10 mg/kg) was observed 24 h after administration of dexamethasone, although dexamethasone liver concentrations had already declined to 0.09 x 10(-5) mol/kg and 0.19 x 10(-5) mol/kg, respectively. A 2-h oral pretreatment of rats with the glucocorticoid receptor antagonist RU 486 (10 mg/kg) did not alter immediate (10 min) stabilization by dexamethasone (3 mg/kg). but almost completely prevented lysosomal membrane protection 24 h after dexamethasone injection. CONCLUSIONS: Dexamethasone megadoses may preserve lysosomal membrane integrity by a dual action involving both rapid nongenomic effects occurring instantaneously after administration and long-term receptor-dependent genomic events.

Animals↗
PubMed2000

Rapid non-genomic feedback effects of glucocorticoids on CRF-induced ACTH secretion in rats.

PURPOSE: The present study investigates fast negative feedback actions of corticosterone (corticosteroid type I/type II receptor agonist) and RU 28362 (corticosteroid type II receptor agonist) on corticotropin-releasing factor (CRF)-induced adrenocorticotropic hormone (ACTH) secretion in rats. METHODS: To induce fast feedback, glucocorticoids were administered intravenously immediately before injection of the hypophyseotropic stimulus CRF. Plasma ACTH levels, being determined 5 to 30 min thereafter, were used as markers of fast feedback. RESULTS: Fast inhibitory effects on CRF-induced ACTH secretion became evident within 15 min (corticosterone) and 5 min (RU 28362) after steroid administration. Rapid feedback inhibition was also observed in the presence of other corticosteroids (cortisol, dexamethasone, aldosterone), whereas structurally-unrelated steroids (beta-estradiol, progesterone, potassium canrenoate, alphaxalone) were inactive in this respect. Pretreatment of rats with the corticosteroid type II receptor antagonist RU 486 or the transcription inhibitor actinomycin D left fast feedback effects unaltered. CONCLUSIONS: Our results demonstrate that glucocorticoids exert fast negative feedback at the pituitary level via a mechanism that is independent of corticosteroid type II receptor occupation and de novo synthesis of mRNA. In conclusion, corticosteroid-specific nongenomic effects may underly rapid glucocorticoid responses on CRF-induced ACTH secretion.

Adrenocorticotropic Hormone↗
PubMed2000

Actin-dependent lamellipodia formation and microtubule-dependent tail retraction control-directed cell migration.

Migrating cells are polarized with a protrusive lamella at the cell front followed by the main cell body and a retractable tail at the rear of the cell. The lamella terminates in ruffling lamellipodia that face the direction of migration. Although the role of actin in the formation of lamellipodia is well established, it remains unclear to what degree microtubules contribute to this process. Herein, we have studied the contribution of microtubules to cell motility by time-lapse video microscopy on green flourescence protein-actin- and tubulin-green fluorescence protein-transfected melanoma cells. Treatment of cells with either the microtubule-disrupting agent nocodazole or with the stabilizing agent taxol showed decreased ruffling and lamellipodium formation. However, this was not due to an intrinsic inability to form ruffles and lamellipodia because both were restored by stimulation of cells with phorbol 12-myristate 13-acetate in a Rac-dependent manner, and by stem cell factor in melanoblasts expressing the receptor tyrosine kinase c-kit. Although ruffling and lamellipodia were formed without microtubules, the microtubular network was needed for advancement of the cell body and the subsequent retraction of the tail. In conclusion, we demonstrate that the formation of lamellipodia can occur via actin polymerization independently of microtubules, but that microtubules are required for cell migration, tail retraction, and modulation of cell adhesion.

Actins↗
PubMed2000

New insights into physiological and pathophysiological functions of cyclo-oxygenase-2.

During the past few years specific inhibitors of the cyclo-oxygenase-2 enzyme have emerged as important pharmacological tools for treatment of patients with rheumatoid arthritis or osteoarthritis. In comparison to traditional nonsteroidal anti-inflammatory drugs, specific cyclo-oxygenase-2 inhibitors may provide equal efficacy in terms of antiinflammatory and analgesic action, with significantly fewer gastrointestinal side effects. Although cyclo-oxygenase-2 was once regarded as a source of pathological prostanoids, recent studies have indicated that this isoenzyme also fulfills a variety of physiological functions within the organism. The present review assesses recent advances in cyclo-oxygenase-2 research, with particular emphasis on new insights into the biology of this isoenzyme.

Journal Article↗
PubMed1999

Quantifying lamella dynamics of cultured cells by SACED, a new computer-assisted motion analysis.

Formation of lamellipodia and the retraction of ruffles are essential activities during motility and migration of eukaryotic cells. We have developed a computer-assisted stroboscopic method for the continuous observation of cell dynamics (stroboscopic analysis of cell dynamics, SACED) that allows one to analyze changes in lamellipodia protrusion and ruffle retraction with high resolution in space and time. To demonstrate the potential of this method we analyzed keratinocytes in culture, unstimulated or stimulated with epidermal growth factor (EGF), which is known to induce cell motility and migration. Keratinocytes stimulated with EGF exhibited a 2.6-fold increase in their migration velocity, which coincided with enhanced ruffle retraction velocity (144%) and increased ruffle frequency (135%) compared to control cells. We also recorded an enhanced frequency of lamellipodia (135%), whereas the velocity of lamellipodia protrusion remained unchanged. These results on ruffle and lamellipodia dynamics in epidermal cells show that SACED is at least equal to established methods in terms of accuracy. SACED is, however, advantageous concerning resolution in time and therefore allows one to analyze the activity of lamellipodia and ruffles in as yet unknown detail. Moreover, SACED offers two opportunities that render this technique superior to established methods: First, several parameters relevant to cell motility can be analyzed simultaneously. Second, a large number of cells can conveniently be examined, which facilitates the compilation of statistically significant data.

Cell Movement↗
PubMed1999

[Specific COX-2 inhibitors: prospects of therapy with new analgesic and anti-inflammatory substances].

Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit cyclooxygenase (COX) activity, thereby suppressing the synthesis of proinflammatory prostaglandins. The identification and molecular-biological characterization of an inducible COX isoform (COX-2) in inflammatory cells led to the hypothesis that a selective inhibition of COX-2 would result in relief of inflammation and pain without causing the COX-1-dependent side effects (gastrointestinal ulceration, platelet dysfunction, kidney damage) of conventional NSAIDs. On the basis of data obtained in several laboratories by means of the "human whole blood assay" there is now convincing evidence that none of the currently available NSAIDs is a selective COX-2 inhibitor. Meanwhile, the specific COX-2 inhibitors celecoxib and rofecoxib are being tested worldwide in phase III clinical trials on patients with rheumatoid arthritis and osteoarthritis. However, the simple concept of COX-2 being an exclusively proinflammatory inducible enzyme cannot be upheld any longer. In addition, COX-2 is expressed constitutively in brain, spinal cord and kidney, as well as in numerous other organs. In the present review the perspectives and possible risks of specific COX-2 inhibitors are discussed, as well as additional indications for their implementation (e.g. colon cancer).

Analgesics↗
PubMed1999

The nitric oxide donor SIN-1 is free of tolerance and maintains its cyclic GMP stimulatory potency in nitrate-tolerant LLC-PK1 cells.

PURPOSE: Using an established cell culture model, the present study investigates whether linsidomine (SIN-1), a spontaneous donor of nitric oxide and active metabolite of the antianginal drug molsidomine, induces tolerance to its own cyclic GMP stimulatory action or shows a diminished response after tolerance induction with glyceryl trinitrate. METHODS: Incubations with nitric oxide donors were carried out in LLC-PK1 kidney epithelial cells. Intracellular levels of cyclic GMP, the vasodilatory second messenger of nitric oxide, were determined by radioimmunoassay. RESULTS: A 5-h preincubation with glyceryl trinitrate (0.01-100 microM) led to complete inhibition of a subsequent cyclic GMP stimulation by glyceryl trinitrate but left the cyclic GMP response to SIN-1 unaltered. Similarly, cyclic GMP elevations by the spontaneous nitric oxide donors sodium nitroprusside and spermine NONOate were not affected after pretreatment with glyceryl trinitrate. Moreover, pretreatment with SIN-1 (1-1000 microM) had no significant effect on SIN-1-dependent cyclic GMP stimulation. CONCLUSIONS: Our results show that in LLC-PK1 cells, SIN-1 is free of tolerance induction and not cross-tolerant to glyceryl trinitrate. This may be due to the spontaneous nitric oxide release from SIN-1, which in contrast to nitric acid esters does not require enzymatic bioactivation and may therefore be unaffected by nitrate tolerance.

Animals↗
PubMed1998

Pentaerithrityl tetranitrate and its phase I metabolites are potent activators of cellular cyclic GMP accumulation.

Using pig kidney epithelial cells (LLC-PK1), the present study assesses the cyclic GMP stimulatory effect of pentaerithrityl tetranitrate and its metabolites in comparison to other therapeutically used nitric oxide donors. Pentaerithrityl tetranitrate was found to be the most potent activator of cyclic GMP synthesis compared to other clinically relevant organic nitrates (glyceryl trinitrate, isosorbide dinitrate, isosorbide-5-mononitrate). The phase I metabolite pentaerithrityl trinitrate was equipotent with its parent compound in stimulating cyclic GMP. The concentration-response curves of pentaerithrityl dinitrate and isosorbide dinitrate for cyclic GMP accumulation were virtually identical. In contrast, pentaerithrityl mononitrate and the phase II metabolite pentaerithrityl trinitrate glucuronide did not alter basal cyclic GMP levels. It is concluded that the long-term vasodilatory and antiischemic effects of pentaerithrityl tetranitrate are caused to a substantial extent by cyclic GMP-mediated actions of its pharmacologically active phase I metabolites.

Animals↗
PubMed1998

[2-Arylpropionic acids. Role in pain therapy].

The antipyretic analgesics still comprise the most widely used group of analgesic compounds. Until twenty years ago, aspirin, phenazone derivatives and acetaminophen had been used as standard remedies for intercurrent harmless pain conditions in connection with infections, trauma, small surgeries, but also for tension headache and other painful conditions. During the last decades, this group has been supplemented by a variety of 2-arylpropionic acids being primarily developed for the treatment of rheumatic pain and inflammation. Some of them (e.g. ibuprofen, ketoprofen and naproxen) have been given over-the-counter status in some countries because of their relative safety at low doses. Nevertheless, pharmacodynamic and pharmacokinetic differences among the various substances of this group make them suitable to a different degree for the treatment of pain conditions.

English Abstract↗
PubMed1998

Nitrate tolerance is specific for nitric acid esters and its recovery requires an intact protein synthesis.

Using cultured LLC-PK1 cells the present study investigates mechanisms leading to nitrate tolerance and its reversal. A 5-h pretreatment with glyceryl trinitrate (GTN, 0.01-100 microM) resulted in desensitization of the intracellular cyclic GMP response to a subsequent 10-min challenge with GTN (1 microM). The spontaneous donor of nitric oxide (NO) spermine NONOate, which releases NO independently of enzymatic catalysis, did not induce tolerance to its own cyclic GMP stimulatory effect and remained fully effective in GTN-tolerant cells. Tolerant cells regained sensitivity to GTN after a 30-h incubation in media. Recovery of the cyclic GMP response was blocked in the presence of cycloheximide (10 microM) suggesting that de novo protein synthesis is necessary for tolerance reversal. Our results demonstrate that nitrate tolerance is specific for nitric acid esters and possibly due to down-regulation of enzymes involved in bioactivation of, and NO generation from, organic nitrates.

Animals↗
PubMed1998

Vitamin C attenuates nitrate tolerance independently of its antioxidant effect.

In LLC-PK1 kidney epithelial cells, a 5-h pretreatment with glyceryl trinitrate (GTN) resulted in substantial desensitization of the intracellular cyclic GMP response to a subsequent 10-min challenge with GTN (1 microM). GTN-tolerant cells were fully sensitive to the spontaneous nitric oxide (NO) donor spermine NONOate, which does not require enzymatic bioactivation. Cyclic GMP stimulation by GTN was up to 3.1-fold higher when vitamin C (1-10 mM) was present during the pretreatment period. In contrast, other oxygen radical scavengers such as tiron or dimethylsulfoxide and the NO scavenger PTIO left tolerance induction unaltered. Together, our results suggest that reactive oxygen species or NO do not contribute to the development of nitrate tolerance. Tolerance reduction by vitamin C may be due to a stabilizing effect on enzymes involved in the bioconversion of GTN to NO.

Animals↗