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R Köhler

Publications and source records attributed to R Köhler.

At least 37 records · Page 2Linked to original sources
PubMed1998

Pressure-activated cation channel in intact rat endocardial endothelium.

OBJECTIVE: The endocardial endothelium (EE) regulates myocardial performance in response to humoral and mechanical stimuli. In vascular endothelium mechanosensitive ion channels (MSC) act as mechanosensors for hemodynamic changes. In the present study we examined whether MSC are present in intact EE of rat papillary muscle segments and characteristics of MSC are altered in experimental hypertension. METHODS: MSC were investigated by the use of standard patch-clamp technique. For a comparative study, ion channel characteristics were determined in EE of two-kidney-one-clip rats and sham-operated controls. RESULTS: We identified a new class of MSC with a mean conductance of 21.8 +/- 4.4 (s.d.) pS for K+ and Na+ and of 4.1 +/- 1.5 pS for Ca2+. Channel activity was initiated by positive pipette pressure and blocked by negative pipette pressure. Channel open probability (Po) was characterized by its pressure sensitivity. Po increased from 0.06 at 10 mmHg to 0.37 and 0.55 at 20 mmHg and 30 mmHg, respectively. Gadolinium (20 microM), a blocker of MSC, completely inhibited channel activity. In some experiments activation of this pressure-activated channel (PAC) was followed by the opening of a Ca2(+)-dependent non-selective cation channel (NSC). This indicates that Ca2+ influx through PAC may be sufficient to increase intracellular Ca2+ concentration and thereby to activate neighboring NSC. In renovascular hypertension (2K1C), channel density of PAC was significantly increased compared to sham-operated controls. Channel density of NSC was not changed in 2K1C compared to sham-operated controls. CONCLUSION: A novel type of Ca2+ permeable MSC in intact EE of rat ventricular papillary muscle was identified, which is regulated by membrane pressure. PAC might be implicated in EE mechanotransduction by inducing an intracellular Ca2+ signal. Up-regulation of PAC density in EE from 2K1C might contribute to an altered mechanotransduction in hypertension.

Animals↗
PubMed1998

Mechanosensitive Ca2+ oscillations and STOC activation in endothelial cells.

Activation of ion channels and the increase in intracellular Ca2+ concentration [Ca2+]i play a key role in endothelial responses to hemodynamic forces and subsequent vasoregulation. In bovine aortic endothelial cells subjected to shear stress in a parallel flow chamber, we demonstrate shear stress activation of hyperpolarizing K+ currents that occur simultaneously with oscillating increases of [Ca2+]i. Oscillating K+ currents, also known as spontaneous transient outward currents (STOC), were regulated in frequency and amplitude by the rate of shear stress in a range from 5 to 18 dyn/cm2. Activation of STOC depended on Ca2+ influx; current depended on the extracellular Ca2+ concentration and was blocked by 50 microM Gd3+. Emptying of Ca2+ stores by BHQ abolished current responses to shear stress. STOC activation was significantly reduced by cell dialysis with ryanodine (20 microM), but not heparin (200 microg/ml). Shear stress-induced STOC activation was also observed in the intact endothelium. The endothelial response to shear stress involves oscillating [Ca2+]i increase and STOC activation, which depend on Ca2+ influx-induced Ca2+ release from ryanodine-sensitive stores, demonstrating a new signaling pathway in endothelial mechanotransduction.

Animals↗
PubMed1998

Stretch-activated cation channel in human umbilical vein endothelium in normal pregnancy and in preeclampsia.

OBJECTIVE: To determine whether stretch-activated cation channels (SAC) are present in intact human umbilical vein endothelium (HUVE) and in an endothelial cell line (EA.hy) and whether they act as endothelial mechanosensors, and to determine whether endothelial SAC in HUVE from women with pregnancies complicated by preeclampsia undergo functional changes compared with those in HUVE from women with normotensive pregnancies. METHODS AND RESULTS: By use of the patch-clamp technique we identified a SAC in intact HUVE and in an endothelial cell line. The SAC had mean conductances of 29+/-5 pS (n = 38) for K+ and 12+/-2 pS (n = 4) for Ca2+. Administration of 50 micromol/I gadolinium, a blocker of mechanosensitive ion channels, completely blocked activity of this channel. We found from single-channel recordings that influx of Ca2+ through SAC directly activated high-conductance Ca2+-dependent potassium channels, proving that a significant influx of Ca2+ through SAC occurs at physiologic concentrations of Ca2+. In a comparative study, apparent channel density of SAC (percentage of patches with SAC activity) in HUVE from women with pregnancies complicated by preeclampsia (36.2 +/- 4.3%) was twofold higher than that in HUVE from women with normal pregnancies (17.9+/-2.9%, P< 0.01). Channel conductance and sensitivity to stretching of SAC were not altered by preeclampsia. CONCLUSIONS: Since SAC are capable of acting as endothelial mechanosensors, the greater than normal density of SAC associated with preeclampsia might reflect an alteration of mechanotransduction.

Adult↗
PubMed1997

Mechanosensitive cation channels in aortic endothelium of normotensive and hypertensive rats.

In response to humoral and hemodynamic stimuli, vascular endothelium regulates vascular tone by releasing endothelium-derived vasoactive factors. Stretch-activated cation channels have been postulated to act as endothelial mechanosensors that respond to changes in hemodynamic forces. We report the presence of a nonselective (n=98) and K+-selective (n=53) stretch-activated channel in rat intact aortic endothelium and isolated aortic endothelial cells. The nonselective channel showed a permeability ratio for Na+, K+, and Ca2+ of 1:0.95:0.23 and was completely blocked by 50 micromol/L gadolinium, a blocker of stretch-activated channels. The K+-selective channel was selectively permeable for K+, with a K+-Na+ permeability ratio of 10.9:1. In whole-cell current recordings, hyposmotic cell swelling induced an increase in cell conductance. The swelling-induced current was completely blocked by 50 micromol/L gadolinium, showing that stretch-activated channels were activated by cell swelling and carry macroscopic cell currents. In a comparative study with normotensive Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR), the K+-selective stretch-activated channel was observed in a 4.4-fold higher density in adult SHR compared with WKY. Also, in adult SHR, the stretch sensitivity of the nonselective channel was nearly twice as high as in WKY. In contrast, channel properties were unchanged in young SHR (5 to 6 weeks old) compared with age-matched WKY. These data suggest that stretch-activated channels are regulated in their sensitivity and density when subjected to increased hemodynamic forces such as in hypertension. Since the channels are capable of acting as endothelial mechanosensors, the altered channel properties might contribute to an altered mechanoreception in hypertension.

Age Factors↗
PubMed1996

Up-regulation of pressure-activated Ca(2+)-permeable cation channel in intact vascular endothelium of hypertensive rats.

In endothelial cells, stretch-activated cation channels have been proposed to act as mechanosensors for changes in hemodynamic forces. We have identified a novel mechanosensitive pressure-activated channel in intact endothelium from rat aorta and mesenteric artery. The 18-pS cation channel responded with a multifold increase in channel activity when positive pressure was applied to the luminal cell surface with the patch pipette and inactivated at negative pipette pressure. Channel permeability ratio for K+, Na+, and Ca2+ ions was 1:0.98:0.23. Ca2+ influx through the channel was sufficient to activate a neighboring Ca2(+)-dependent K+ channel. Hemodynamic forces are chronically disturbed in arterial hypertension. Endothelial cell dysfunction has been implicated in the pathogenesis of arterial hypertension. In two comparative studies, density of the pressure-activated channel was found to be significantly higher in spontaneously hypertensive rats and renovascular hypertensive rats compared with their respective normotensive controls. Channel activity presumably leads to mechanosensitive Ca2+ influx and induces cell hyperpolarization by K+ channel activity. Both Ca2+ influx and hyperpolarization are known to induce a vasodilatory endothelial response by stimulating endothelial nitric oxide (NO) production. Up-regulation of channel density in hypertension could, therefore, represent a counterregulatory mechanism of vascular endothelium.

Animals↗
PubMed1996

Anti-peptide immunoglobulins from rabbit and chicken eggs recognise recombinant human dihydroorotate dehydrogenase and a 44-kDa protein from rat liver mitochondria.

Mitochondrially bound dihydroorotate dehydrogenase catalyses the fourth sequential step in the de novo synthesis of uridine monophosphate. 312-bp and 983-bp regions of the human dihydroorotate dehydrogenase sequence (1496 bp) were amplified by the polymerase chain reaction, and subcloned into the expression vector pQE 32. The identity of the PCR products was verified by dideoxynucleotide sequencing. Transformation of Escherichia coli strain M15 resulted in expression of 13-kDa and 36-kDa proteins with an affinity tag consisting of six consecutive histidine residues; these proteins could be purified by solubilisation in 8 M urea and by chromatography on a Ni2+-chelating resin. In immunoblotting analyses, the fusion proteins were recognised by polyclonal avian and mammalian anti-peptide immunoglobulins. These were generated against synthetic peptides corresponding to two amino acid sequences deduced from human and rat cDNA of dihydroorotate dehydrogenase. The peptides were synthesized as multiple copies on a branching lysyl matrix. Rabbits and laying hens were immunized with these peptides without conjugation to a carrier protein. Comparison of the anti-peptide immunoglobulins produced from egg yolk and rabbit serum demonstrated that avian anti-(dihydroorotate dehydrogenase) immunoglobulins may be considered a superior alternative to the mammalian equivalent; antibodies from both sources were applicable for all immunochemical purposes. Here, these antibodies were applied for identification of a 44-kDa protein from rat liver mitochondria, which was correlated with dihydroorotate dehydrogenase activity.

Amino Acid Sequence↗
PubMed1995

The effects of desipramine on thyroid hormone concentrations in rat brain.

The effects of the antidepressant desipramine on the tissue concentrations of thyroxine and triiodothyronine in 9 different regions of the brain and also in the pituitary and liver were investigated in male rats. The investigations were carried out at three different times of the light/dark cycle: 5 a.m., 1 p.m. and 11 p.m. After fourteen days' treatment with 20 mg/kg/day desipramine by gavage the concentrations of triiodothyronine in the frontal and parieto-occipital cortex were significantly higher than in the saline-treated controls, those in the hippocampus lower and those in the 6 remaining brain regions the same. In 8 areas of the brain the concentrations of thyroxine were lower in the desipramine-treated rats and the tissue ratios of triiodothyronine to thyroxine were enhanced in 6 regions. These effects are most likely the result of the action of desipramine on the activity of the isoenzyme 5'II deiodinase. This enzyme catalyzes the deiodination of thyroxine to triiodothyronine in rat brain and its activity has recently been reported to be enhanced by desipramine. The observed effects were dose-dependent and also strongly dependent upon the time within the 24 h light/dark cycle at which the hormone concentrations were measured. No effects of desipramine were seen in the pituitary or liver after 14 days' treatment, or in various areas of the central nervous system 24 h after administration. In view of the psychotropic properties of thyroid hormones, it seems possible that the observed increases in triiodothyronine concentrations, particularly in cortical areas, are involved in the mechanisms of action of desipramine.

Administration, Oral↗
PubMed1995

Monomeric and dimeric forms of soluble receptors can differ in their neutralization potential.

Recombinant soluble forms of transmembrane receptors can be produced in monomeric and dimeric versions. Binding affinity and neutralization potential of these different forms of soluble receptors depend on the quaternary structure of their ligands. Monomeric ligands will be bound with equal affinity by both forms, whereas trimeric ligands, e.g. members of the tumor necrosis factor family of ligands, interact with much higher affinity with dimeric soluble receptors than with monomeric ones.

Amino Acid Sequence↗
PubMed1994

Hypothalamic-pituitary-thyroid axis in chronic alcoholism. II. Deiodinase activities and thyroid hormone concentrations in brain and peripheral tissues of rats chronically exposed to ethanol.

Thyroxine (T4), triiodothyronine (T3) concentrations, and the activities of the three deiodinase isoenzymes were measured in different brain regions and peripheral tissues of rats. According to an animal model of alcohol addiction, "behaviorally" dependent rats having lost control over their intake of ethanol were compared with alcohol-naive controls and ethanol-experienced, but "controlled" consumers. The two kinds of alcohol-experienced rats were investigated either 24 hr or 3 months after ethanol withdrawal. The results of these four groups were compared with those of an ethanol-naive control group. During withdrawal, the activities of type II 5'-deiodinase (which catalyzes deiodination of T4 and T3 in the CNS) in both the "behaviorally dependent" rats and the "controlled drinkers" were significantly lower than in the alcohol-naive controls in the frontal cortex, parieto-occipital cortex, hippocampus, and striatum, but not in the cerebellum or pituitary. Probably as a result, the tissue concentrations of T4 were higher in areas of the CNS in the groups exposed to alcohol. However, the T3 concentrations were normal. No relevant differences were seen between the activities of type III 5-deiodinase (which catalyzes the further deiodination of T3) observed in these groups. After 3 months of abstinence, the type II 5'-deiodinase activities had almost returned to normal in both "controlled drinkers" and "behaviorally dependent" animals, whereas type III 5-deiodinase activity was inhibited, possibly to maintain physiological concentrations of T3 during abstinence. Indeed, the tissue levels of T3 were normal in the areas of the CNS, and the T4 levels were still elevated. However, the liver concentrations of T3 and T4 were significantly lower in the "behaviourally dependent" animals than in the "controlled" drinkers after 3 months of abstinence, whereas no differences were found between the T4 and T3 concentrations in the areas of the CNS investigated in the two groups exposed to ethanol. These results suggest that chronic administration of ethanol affects intracellular thyroid hormone metabolism in both rat CNS and liver in the highly complex manner. No direct evidence of ethanol-induced enhancement of tissue uptake or concentrations was obtained. However, taking into account the numerous similarities between the clinical picture of hyperthyroidism and the symptomatology of alcoholism, it may be hypothesized that ethanol may directly influence any step in the as yet unknown biochemical cascade of thyroid hormone function.

Alcoholism↗
PubMed1994

The influence of desipramine on thyroid hormone metabolism in rat brain.

The effect of the antidepressant desipramine (DMI) on the activities of the three iodothyronine deiodinase isoenzymes involved in the central metabolism of thyroid hormones were investigated in 11 brain regions and 3 peripheral tissues in the rat. The investigations were carried out at three different times during the light/dark cycle: 5 A.M., 1 P.M. and 11 P.M. Interest is focused on changes in the two enzymes that catalyze: i) the 5'deiodination of T4 to the biologically active T3, i.e., type II 5'deiodinase (5'D-II), and ii) the 5 (or inner-ring) deiodination of T3 to the biologically inactive 3,3'T2, i.e., type III 5 deiodinase (5D-III). Fourteen days' treatment with 20 mg/kg DMI, but not with 5 mg/kg DMI, induced significant increases in 5'D-II in eight different areas of the CNS. The regions affected were identical to those that receive noradrenergic input from the locus coeruleus. Even control animals showed a circadian rhythm of 5'D-II activity in some brain regions, and the effects of DMI also depended on the time of death within the 24-hr rhythm. 5D-III was not affected. Serum T4 were lower after administration of DMI, most probably because of enhanced tissue uptake of T4. This is in line with the corresponding finding in depressed patients, indicating that similar changes in both central and peripheral thyroid hormone metabolism may occur after antidepressant pharmacotherapy in both humans and rats. These data support the hypothesis that interactions with the CNS metabolism of the thyroid hormones may be involved in the mechanisms of action of DMI.

Animals↗
PubMed1993

The influence of sleep deprivation on thyroid hormone metabolism in rat frontal cortex.

The effects of 24 h sleep deprivation (SD) on central thyroid hormone metabolism were investigated in rat frontal cortex. SD induced a significant rise in the activity of iodothyronine type II 5'-deiodinase (5'D-II), which catalyzes the conversion of thyroxine (T4) to triiodothyronine (T3) in the rat central nervous system (CNS). Tissue concentrations of T4 remained unchanged, whereas levels of T3 increased to more than 150% of the corresponding levels measured in control rats. Serum concentrations of T4 and T3 were also significantly enhanced by SD--an effect that has previously been described in depressed patients having undergone the same procedure. These results suggest that SD can dramatically increase T3 concentrations (and possibly function) in rat CNS. Whether or not these findings are of relevance in regard to the well-known antidepressant effect of SD in psychiatric patients with major depressive disorders remains to be established.

Animals↗
PubMed1993

Exogenous and endogenous contributions to nitrogen fluxes in the digestive tract of pigs fed a casein diet. III. Recycling of endogenous nitrogen.

The aim of the present study was to measure the incorporation of infused 15N in blood fractions, urine, digesta, faeces and in the exocrine pancreatic and biliary secretions, in order to estimate the endogenous part of nitrogen in the ileal digesta and in the faeces of pigs fed a casein diet and to calculate the total endogenous nitrogen secretion as well as its recycling in the digestive tract. For 8 d 11 Large White female pigs (50.1 +/- 1.8 kg) received a continuous infusion of L-[15N]leucine via a catheter in the jugular vein. The 15N-enrichment was measured in several fractions. The 15N-level of the pancreatic juice was higher than that in the biliary secretion, TCA-blood fractions, and urine during the whole experimental period. Using the 15N-isotope dilution method it was found that casein was completely digested up to the terminal ileum and that all the nitrogen in the ileal digesta was of endogenous origin. The total endogenous secretion was estimated at approximately 11 g N/d. The reabsorption of endogenous nitrogen amounted to 79% up to the end of the small intestine and 88% over the whole digestive tract.

Absorption↗
PubMed1993

Effects of selenium and iodine deficiency on type I, type II and type III iodothyronine deiodinases and circulating thyroid hormones in the rat.

The effects of nutritional selenium (Se) deficiency over a period of three generations and of a combined selenium and iodine deficiency on hepatic and cerebrocortical iodothyronine deiodinases and on circulating thyroid hormone levels were examined in the rat. Se deficiency strongly decreased hepatic type I iodothyronine 5'- and 5-deiodinase to 6-13% of that in controls. Iodine depletion had only a marginal decreasing effect on the type I activity. Cerebrocortical type II 5'-deiodinase was decreased in Se-deficient, iodine-replete rats. Its 5-6-fold elevation in iodine-deficient rats was not reversed by additional selenium deficiency. Cortex type III 5-deiodinase was modestly decreased in all groups with insufficient trace element supply. Long-term Se deficiency has only limited effects on serum T4 and T3 levels. Two months of iodine deficiency decreased serum T4 to less than 10% of that in controls, but did not significantly affect serum T3 levels. The strong decrease of hepatic outer- and inner-ring deiodination of T4 in Se deficiency obviously reflects the reduced tissue concentration of the type I deiodinase which was recently identified as a selenoenzyme. The maintenance of increased cerebrocortical type II deiodinase in iodine-depleted animals irrespective of adequate or deficient selenium supply suggests that the type II isoenzyme does not contain selenium in its catalytic site. Further studies are necessary to clarify whether the weak, but repeatedly confirmed decrease of cortex type III deiodinase is the direct effect of Se deficiency or the indirect consequence of the multilevel change in thyroid hormone metabolism.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

[Determination of endogenous N-excretion and nitrogen balance in the range of low N-intake in rats].

The investigations were carried out in order to develop foundations for a complex growth model including the effect of N-efficiency mechanism first of all for the rat. Therefore N-balance trials with rats of different live weights were made using diets of equal protein quality. The curve of the regressively calculated N-efficiency function courses relatively linear in its lower part, i.e. for low N-intakes. The protein quality value "A.b" was proved to be constant for all investigated live weight ranges. In similar trials with different protein qualities was also found a linear course for low N-intakes. The regressively estimated N-maintenance requirement (for N-Intake = 0) related to metabolic live weight (NMR/LWkg0.67) amounts to 180 mg/d. In addition endogenous N-excretions were determined in 15N tracer experiments on animals of different live weight fed graduated amounts of a quality constant protein. The NMR-values (sum of endogenous excretions from faeces and urea) depend on the level of N-intake, i.e. they can not be considered as constant. The endogenous faecal N-loss related to metabolic live weight is relatively constant indeed, whereas the amount of endogenous urea N-loss is dependent on the level of N-intake. Therefore calculations based on the N-efficiency curve, e.g. amino acid requirement dependent on performance and prediction of N-deposition respectively, should not be carried out on the basis of "N-retention" (N-balance + NMR) but only on the basis of N-balance.

Animals↗