Bicarbonate permeability of epithelial chloride channels

To investigate the relation of arachidonate metabolism to the induction of fever by interleukin-1, indomethacin was administered in either an intracerebro-ventricular (icv) or a subcutaneous (sc) route in conscious rabbits. Fever induced by icv administration of recombinant human interleukin-1 (rhIL-1) was depressed by either icv or sc pretreatment with indomethacin. Fever induced by intravenous (iv) administration of rhIL-1 was significantly inhibited, though initial small increase in colonic temperature still remained, and was completely depressed by combination of icv and sc pretreatment with indomethacin. Intracerebroventricularly administered recombinant rabbit IL-1 (rrIL-1) induced dose-dependent increases in colonic temperature, which was depressed by sc pretreatment with indomethacin. There is little species specificity between human and rabbit IL-1, in terms of the pyrogenic potency and the inhibitory effect of sc indomethacin on fever induced by icv IL-1. Further, fever caused by icv administration of sodium arachidonate was significantly depressed by sc pretreatment with indomethacin. These results show that the inhibitory effect of indomethacin, administered either icv or sc, on IL-1-induced fever is similar to that of IL-1-induced fever reported previously [11]. This suggests that the site of arachidonate metabolism significantly involved in the mechanism of fever induction by IL-1 is easily accessible to the brain from the blood.     

Trennung der intestinalen β-Galactosidasen beim lactose-toleranten Erwachsenen durch Ultrazentrifugation im Dichtegradienten

Zusammenfassung Die intestinalen-Galactosidasen von 4 lactose-toleranten, erwachsenen Mitteleuropäern wurden im Saugbiopsie-Gewebe nach Solubilisierung mit Triton X-100 in einem linearen Mannitol-Gradienten (5–20%) auf der Ultrazentrifuge bei 4°C und 44000 U/min getrennt. Bei 12stündiger Zentrifugation fanden sich 3 Fraktionen, von denen die beiden schneller sedimenticrenden Lactose spalten. Alle 3 Fraktionen hydrolysieren p-Nitrophenyl--Galactosid. Die 3 isolierten-Galactosidasen entsprechen wahrscheinlich der neutralen Bürstensaum-Lactase, der sauren lysosomalen Lactase und einer cytoplasmatischen Hetero--Galactosidase.     

Bile acids and their amidates inhibit 11β-hydroxysteroid dehydrogenase obtained from rat kidney

Recently it has ben demonstrated that interaction of corticosteroids with extraadrenal target cells can effectively be modulated by metabolic transformation of the steroid hormone. As far as 11-hydroxylated glucocorticoids are concerned 11-hydroxysteroid dehydrogenase (11-HSD) is the most important enzyme charged with target cell metabolism. Inhibition of 11-HSD function either by genetically transmitted deficiency or by exogenous enzyme inhibitors causes severe pathophysiological derangements, which result in a syndrome of apparent mineralocorticoid excess. In the present paper we have tested whether or not endogenous inhibitors of this enzyme system might exist. The effects of the main naturally occurring mono-, di-, and trihydroxylated bile acids in man on 11-HSD have been studied in in vitro experiments. Using rat renal microsomes it could be demonstrated that unconjugated bile acids of all three classes as well as the corresponding glycine and taurine amidates effectively inhibit oxidative as well as reductive activity of 11-HSD, with lithocholic acid and chenodeoxycholic acid being the most potent compounds. It is concluded that bile acids are potent endogenous inhibitors of 11-HSD and, therefore, could participate in abnormalities of cortisol metabolism observed in liver cirrhosis and extrahepatic biliary obstruction and, possibly, after ingestion of bile acids.Abbreviations CA cholic acid - CDCA chenodeoxycholic acid - DCA deoxycholic acid - GCA glycocholic acid - GCDCA glycochenodeoxycholic acid - GLCA glycolithocholic acid - 11-HSD 11-hydroxysteroid dehydrogenase (EC 1.1.1.146) - IC₅₀ molar concentration of bile acid at 50% inhibition of enzyme activity - LCA lithocholic acid - TDCA taurodeoxycholic acid - TLCA taurolithocholic acid - TUDCA tauroursodeoxycholic acid - UDCA ursodeoxycholic acid Supported by the Deutsche Forschungsgemeinschaft Hi 97/16 1-4. Parts of this study have been presented at the 21st meeting of the Gesellschaft für Nephrologie [23]     

Enzymaktivitätsveränderungen der β-Hexosaminidase im Urin bei Nierenkranken

Zusammenfassung Bei Nierenkranken, nicht renal Erkrankten, essentiellen Hypertonikern und gesunden Probanden wurde die Enzymaktivität der-Hexosaminidase und zum Vergleich der-Galaktosidase im 24 h-Urin bestimmt. Gegenüber allen anderen Gruppen wiesen Nierenkranke eine signifikant höhere Enzymaktivität der-Hexosaminidase bei jedem Patienten auf, während-Galaktosidase nur im Mittelwert erhöhte Werte zeigte. Die Ursache der Enzymerhöhung wird diskutiert.     

Modulation of hematopoietic colony formation of stem cells in peripheral blood by anti-TGF-β in patients with severe immunosuppression

Summary The influence of transforming growth factor- (TGF-) on hematopoiesis has been evaluated by adding blocking antibodies against TGF- to colony forming assays (CFU-c). When optimum concentrations of recombinant growth factors, granulocyte-macrophage colony stimulating factor (GM-CSF), and interleukin-3 (IL-3) were added to stem cells from the peripheral blood of healthy individuals and certain patients with tumors or HIV infection, the anti-TGF- capable of blocking 5 ng/ml of active TGF- had no significant influence on erythroid or myeloid colony formation. However, in certain immunosuppressed individuals, anti-TGF- resulted in a significant decrease of erythroid colony formation and slight suppression of myeloid colony formation. The significant inhibition of hematopoiesis by plasma of HIV patients could be due to the presence of active forms of TGF-. The results of the blocking experiments are consistent with the concept that TGF- in low concentrations is essential for erythropoiesis and myelopoiesis but that higher levels of TGF- primarily inhibit erythropoiesis in vitro. TGF- serves as a coordinating factor when efficient recruitment of granulocytes and monocytes is more essential than erythropoiesis and stem cell growth.Abbreviations BFU-E burst forming unit-erythroid - CFC colony forming cells - CFU-GEMM colony forming unit-granulocyte/erythroid/macrophage/megacaryocyte - CFU-GM colony forming unit-granulocyte/macrophage - EPO erythropoietin - GM-CSF granulocyte/macrophage-colony stimulating factor - HIV human immunodeficiency virus - IL-1 interleukin-1 - IL-3 interleukin-3 - IMDM Iscove's Modified Dulbecco's medium - PBS phosphate buffered saline - TGF- transforming growth factor- - TNF- tumor necrosis factor-     

Myocardial β-adrenergic receptor signaling in vivo: insights from transgenic mice

Heart failure is a problem of increasing importance in cardiovascular medicine. An important characteristic of heart failure is reduced agonist-stimulated adenylyl cyclase activity (receptor desensitization) due to both diminished receptor number (receptor downregulation) and impaired receptor function (receptor uncoupling). These changes in the §-adrenergic receptor (§ AR) system may in part account for some of the abnormalities of contractile function in this disease. Myocardial contraction is closely regulated by G protein coupled -adrenergic receptors through the action of the second messenger cAMP. The -adrenergic receptors themselves are regulated by a set of specific kinases, termed the G protein-coupled receptor kinases. The study of this complex system in vivo has recently been advanced by the development of transgenic and gene targeted (knock out) mouse models. Combining transgenic technology with sophisticated physiological measurements of cardiac hemodynamics is an extremely powerful strategy to study the regulation of myocardial contractility in the normal and failing heart.Abbreviations -AR -Adrenergic receptor - GRK G protein coupled receptor kinase - LV Left ventricular     

Isoenzymes of γ-glutamyl transpeptidase in liver diseases

Summary A new method for the separation of isoenzymes of-glutamyl-transpeptidase is described, using electrophoresis on acetate cellulose gel and a developing solution composed by-glutamyl-naphthylamide, and a colored diazonium compound.The method permits the separation of up to four different isoenzymes, which we called-GT₁,-GT₂,-GT₃,-GT₄, the first two showing an electrophoretic migration similar to that of ₁- and ₂-globulins and the other two to that of-globulins.The present technique has proved its usefulness in detecting isoenzymes in serum with values of total-glutamyl-transpeptidase higher than 80 U/L.The application of this method in 52 patients with different types of biliary obstruction and hepatocellular damage has shown that it provides new possibilities in differential diagnosis.     

Clonidine normalizes low plasmaβ-endorphin concentration and blood pressure in young men with mild essential hypertension

Summary In young men with mild essential hypertension and age-matched normotensive volunteers, plasma concentrations of the endogenous opioid-endorphin were determined hourly from 9:00 p.m. to 2:00 a.m. The hypertensive patients' mean plasma-endorphin concentration was significantly lower in comparison with normotensive controls. After 14 days of treatment with clonidine, systolic and diastolic blood pressure was significantly reduced in both groups. Plasma-endorphin concentration increased in the hypertensive patients, but remained unchanged in the normotensive volunteers. The present findings point to a possible involvement of reduced-endorphinergic activity in blood pressure regulation of young men with essential hypertension.Abbreviation NTS nucleus tractus solitarii Dedicated to Prof. Dr. F. Krück on the occasion of his 65th birthday     

Effect of theophylline onβ-adrenergic receptor density and cAMP content in bovine aortic smooth muscle cells

Activation of vascular-adrenergic receptors prevents an increase in vascular permeability caused by free radicals or inflammatory peptides. Methylxanthines seem to have similar protective effects on vascular endothelium. In the present study we investigated the effect of theophylline on the-adrenergic receptor expression and cAMP concentrations in cultured endothelial and smooth muscle cells from bovine aorta. Comparable values for-receptor density and binding affinity were detected in both cell types. Isoproterenol induced significant downregulation of-receptors in endothelial (BAEC: –60.5%) and smooth muscle cells (BASMC: –52.5%; P < 0.01). Incubation of endothelial cells with theophylline (4 µg/ml and 40 µg/ml) for 24 hours did not affect-receptor expression, whereas in smooth muscle cells the-receptor density was reduced for –31.5% and –28.7, respectively. In endothelial cells a transient effect on cAMP concentrations was observed after stimulation with isoproterenol (1 µM), but no effect was found in theophylline treated endothelial cells. Stimulation of intact smooth muscle cells with isoproterenol and theophylline (4 µg/ml and 40 µg/ml) resulted in a significant increase of cAMP concentrations after 60 and 240 minutes. The present data suggest a novel, celltype specific effect of theophylline on the-adrenergic receptor expression in vascular smooth muscle cells in vitro.     

Antibodies to β2-Glycoprotein-I: Urea Resistance, Binding Specificity, and Association with Thrombosis

The aim of the present study was to evaluate the urea resistance and binding characteristics of anti-2-glycoprotein I (anti-2GPI) antibodies using standard anticardiolipin (aCL) and anti-2GPI enzyme immunosorbent assays (ELISAs). Sera from patients with antiphospholipid syndrome (APS) (n = 22) and non-APS (n = 24), positive in a standard aCL ELISA, were tested in an anti-2GPI ELISA performed in polystyrene-irradiated ELISA plates. Urea resistance aCL and anti-2GPI ELISAs were performed by measuring the ability of antibodies to recognize antigen in the presence of 2 M urea. The serum dilution after urea treatment (D) expressed as a percentage of the serum dilution without urea treatment (D _o) corresponding to the same optical density was defined as residual activity (RA = 100 D/D _o). The higher the RA, the higher the resistance of the antibodies to urea. APS compared to non-APS sera had higher aCL binding (absorbance values ranging between 0.180 and 1.400; median, 0.717 vs 0.120–1.273; median, 0.250, respectively; P < 0.004). Six APS patients' sera had low aCL levels but they expressed RA 30%. Anti-2GPI antibodies were detected in 15 of 22 APS vs 3 of 24 non-APS patients (P < 0.03); RA 30% was detected in 15 of 22 APS vs 1 of 23 non-APS patients (P < 0.004). Using a CL affinity column, antibodies were purified from three APS anti-2GPI negative and three non-APS anti-2GPI-positive patients and tested in a aCL ELISA, using highly purified bovine serum albumin (BSA) as a blocking agent (modified ELISA); reactivity was not detected in two APS and one non-APS sera. On the contrary, the reactivity of the purified antibodies was high when 2GPI was incubated with CL in the ELISA plates; thus some anti-2GPI negative sera from APS patients recognized the CL/2GPI complex, rather than CL or 2GPI alone. In conclusion, anti- 2GPI antibodies are common in the APS patients, but a number of such patients recognize the CL/2GPI complex and not CL or 2GPI. Antibodies to either 2GPI or the CL/2GPI complex derived from APS sera present a high resistance to urea. Anti-2GPI antibodies of low urea resistance exist in a minority of non-APS patients with autoimmune disease.     

β subunits determine the time course of desensitization in rat α3 neuronal nicotinic acetylcholine receptors

Standard two-electrode voltage-clamp techniques were used to investigate some of the pharmacological and functional properties of two types of rat neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes after pairwise injection of 34 or 32 mRNAs. Currents of several A amplitude were elicited by fast application of micromolar concentrations of either acetylcholine (ACh) or 1,1-dimethyl-4-piperazine (DMPP). The activation of either receptor type by DMPP showed cooperativity (Hill coefficient, n1.7) with a half-maximal activation concentration (EC₅₀) of 15–30 M. In 34 receptors, ACh displayed cooperativity (n=1.8) but was less efficacious than DMPP, yet its EC₅₀ was about equal to that of DMPP. Finally, in 32 receptors, ACh was much less efficacious and had a much lower EC₅₀. Desensitization induced by either DMPP or ACh was slow in 34 nicotinic ACh receptors but was rapid and extensive in 32 receptors, causing a significant proportion of the response to wane within the first few seconds of agonist application.     

Comparative investigation of the effects of metoprolol, propranolol, practolol, and verapamil in the acute phase of experimental myocardial infarction

Summary Myocardial infarction in rats was produced by ligation of the left coronary artery. To ensure exact comparison of drug effects, the extent of the myocardial zone excluded from the coronary circulation was determined in each animal, and the experimental data were related to it. For this purpose, the hearts were perfused with Evans blue, and after the photometric determination of the dye content of the hearts the percentage of ischemic myocardium was calculated. With metoprolol, propranolol, and verapamil a significant increase of the survival times was obtained (min/% of non-ischemic myocardium). Metoprolol and propranolol also significantly increased the survival rates. None of the-blockers exerted an antiarrhythmic effect. The arrhythmias were prevented by higher doses of the calcium antagonist verapamil which, however, decreased the survival times. All-blocking agents delayed the typical elevation of the ST-segment in the electrocardiogram, and reduced the increase of the activity of the serum creatine kinase. Propranolol and metoprolol antagonized the blood pH decrease obtained after coronary occlusion. Results concerning heart rate, and arterial and central venous pressures are also reported. — The findings with metoprolol, especially, indicate that the essential mechanism in the therapeutic action of-blockers is their ability to block the cardiac ₁-receptors.Supported by the Deutsche Forschungsgemeinschaft     

Differential induction of human monocyte transforming growth factorβ1 production and its regulation by interleukin 4

We have previously shown that trauma patients' monocytes which arein vivo activated by multiple injury-induced mediators have elevated transforming growth factor-beta (TGF) bioactivity. Interleukin-4 (IL-4), a Th₂ and B lymphocyte stimulatory factor, has been shown to inhibit monocyte production of a number of mediators both after lipopolysaccharide stimulation and after trauma-induced stimulation. However, IL-4 inhibitory effects appears to vary, depending on the mixture of inducing stimuli. Here we describe thein vitro IL-4 inhibition of human monocyte TGF bioactivity using several stimulation induction protocols: muramyl dipeptide stimulation alone, or after FcRI (CD64) cross-linking induction, interferon-gamma (IFN) priming, or trauma-generatedin vivo mediator induction. IL-4 suppressed both muramyl dipeptide-induced TGF bioactivity and TGF mRNA in a dose-dependent fashion and was most effective when IL-4 was administered at initiation of normal monocyte stimulation. Muramyl dipeptide (MDP)-induced increases in trauma patients' monocyte TGF bioactivity were also inhibited by high doses of IL-4 (25 ng/ml). FcRI cross-linking increased MDP-induced normal monocyte TGF bioactivity, but this increase could be consistently inhibited only by very high IL-4 concentrations (50 ng/ml). IL-4 did not consistently downregulate MDP-induced TGF bioactivity in IFN-primed monocytes. IL-4 can suppress monocyte TGF production, as well as other monocyte mediators, but its efficiency depends on the stimuli combination present in the microenvironment.     

Assessment of plasma opioid peptides, β-endorphin and met-enkephalin,at the end of an international nordic ski race

Summary Plasma met-enkephalin, -endorphin, cortisol and lactic acid concentrations were measured in seventeen volunteer male subjects at rest and after a long-distance nordic ski race. Immediately after the race, mean plasma met-enkephalin did not show any significant change, but significant rises in -endorphin, cortisol and lactic acid were noted in all skiers. The change in -endorphin with exercise was significantly related to the change in cortisol (r=0.68;p<0.001) and to the change in plasma lactic acid (r=0.60;p<0.001). Furthermore, the experienced skiers training over 150 km · week^–1 of nordic ski had significantly faster skiing times in this event and showed greater -endorphin, cortisol and lactic acid levels than the recreational skiers who trained for 20 km · week^–1. Our results imply that the changes in plasma -endorphin depend on the intensity of exercise. However the significance of higher levels of skiing training or previous nordic ski experience in the release of -endorphin is expected and cannot be excluded.     

Calcium overload toxicity and functional impairment in peritoneal leukocytes elicited by glycogen or interleukin-1β

Although calcium plays an important role in the activation of leukocytes for such processes as eicosanoid biosynthesis, secretion of granular constituents and superoxide generation, sustained high levels of intracellular calcium ions may be toxic. We have previously found that high concentrations of calcium ionophores induce a rapid-onset calcium overload toxicity in rat peritoneal leukocytes, in which functional responses such as -glucuronidase secretion, superoxide generation and leukotriene B₄ synthesis are greatly attenuated, and some leakage of cytoplasmic LDH occurs. We have now compared this phenomenon in peritoneal leukocytes elicited from animals pretreated in three ways: glycogen, interleukin-1 (IL-1) alone or glycogen plus IL-1. Peritoneal administration of IL-1 caused elicitation of cells which were enriched in eosinophils; however, the functional responses of the cells in all three groups were broadly similar in terms of the ability of the agonists FMLP, PMA and A23187 to initiate superoxide generation, -glucuronidase secretion and leukotriene generation. Cells from all three treatment groups showed diminished responsiveness at 10^–5 M A23187, indicative of calcium overload toxicity. This was most evident for the superoxide and -glucuronidase responses. Some quantitative differences observed between treatment groups may reflect the different sensitivities of the various cells contained in the mixed leukocyte preparations. We conclude that IL-1 induces leukocyte emigration into the peritoneal cavity but that the cell population is different from that induced by glycogen. However, the cells retain susceptibility to calcium overload toxicity.     

C to T transition at the first nucleotide of codon 63 of the β-globin gene corresponding to hemoglobin M-Saskatoon in an Indonesian boy

Summary Hemoglobin (Hb) M-Saskatoon, a variant of methemoglobin, is characterized by mild hemolysis. It is caused by the substitution of a histidine by a tyrosine at the 63rd amino acid residue of the -globin chain. Amplification and sequence analysis of genomic -globin DNA from an Indonesian boy diagnosed as having the more severe disease thalasemia demonstrated the presence of a C to T transition at nucleotide 473 in one of the two -blogin genes resulting in a histidine to tyrosine substitution at 63rd residue. This amino acid change matched with that reported in Hb M-Saskatoon. This nucleotide change abolished a recognition site for the restriction endonucleaseNlaIII.NalIII digestion of the corresponding -globin DNA amplified from the patient's parents indicated that the mutation was inherited through from his mother. This result shows that the world-wide distribution of Hb M-Saskatoon extends to Indonesia, where it was not previously identified. Possible causes of the unusually severe symptoms observed in the case are discussed.     

Glucocorticoids modulate TGF-beta production

TGF- is thought to play a central role in pulmonary fibrosis inducing fibroblast differentiation and extracellular matrix synthesis. In human lung fibroblasts, it is still unclear how various TGB- isoforms affect TGF- production and whether glucocorticoids, commonly used agents to treat fibrotic lung disease, modulate these processes. To this end, human fetal lung fibroblasts (HFL-1) were cultured with various concentrations of glucocorticoids (budesonide, dexamethasone or hydrocortisone) with and without TFG-1, -2, and -3. TGF- mRNA was assessed by real time RT-PCR. Smad 2, 3, and 4 and AP-1 complex (c-fos and c-Jun) cellular localization were evaluated by immunostaining. TGF-2 and -3 stimulated TGF-1 production significantly (p < 0.01 relative to control). TGF-1 stimulated TGF-2 production (p < 0.01 relative to control). TGF-3 was undetectable. Glucocorticoids significantly inhibited TGF-1 and -2 production and reduced expression of the upregulated TGF-1 and -2 mRNA induced by exogenous TGF-1, -2 or -3 (p < 0.01 for each) but had no effect on Smads. Although c-jun-related nuclear staining was not intensified in TGF--stimulated cells, it was reduced by glucocorticoids. Thus, TGF- isoforms may stimulate production of various TGF- isoforms in the lung. Glucocorticoids then may block TGF- production by modulating mRNA levels and c-Jun.     

Expression of calcium channel subunits in the normal and diseased human myocardium

We investigated the expression of ₁ and subunits of the L-type Ca²⁺ channel on the protein level in cardiac preparations from normal human heart ventricles and from the hypertrophied septum of patients with hypertrophic obstructive cardiomyopathy (HOCM). 1,4-Dihydropyridine (DHP) binding and immunorecognition by polyclonal antibodies directed against the C-terminal amino acid sequences of the ₂ and ₃ subunits were used for detection and quantification of ₁, ₂, and ₃ subunits. B_max of high-affinity DHP binding was 35±2 fmol/mg protein in HOCM and 20±2 fmol/mg protein in normal human hearts (P<0.05). In rabbit hearts the anti-₂ subunit antibody immunoprecipitated 80% of the total amount of DHP-labeled Ca²⁺ channels present in the assay. Under identical experimental conditions 25% of labeled Ca²⁺ channels were recovered in the immunoprecipitates of both normal and HOCM ventricles. A similar partial immunoprecipitation was observed in pig hearts. Immunoblot analysis demonstrated that the ₂ subunit was associated with the DHP receptor/Ca²⁺ channel in cardiac muscle of rabbit, pig, and human heart. In neither of these purified cardiac Ca²⁺ channels was the ₃ subunit isoform detected. Our results suggest that both ₁ and ₂ subunit expression is upregulated in HOCM in a coordinate manner.Abbreviations B _max Maximal number of binding sites - DHP 1,4-Dihydropyridine - HOCM Hypertrophic obstructive cardiomyopathy - NH Normal human heart     

Modulation of Nav1.5 by β1- and β3-subunit co-expression in mammalian cells

Cardiac sodium channels (Na_v1.5) comprise a pore-forming -subunit and auxiliary -subunits that modulate channel function. In the heart, 1 is expressed throughout the atria and ventricles, whilst 3 is present only in the ventricles and Purkinje fibers. In view of this expression pattern, we determined the effects of 3 and 1 co-expression alone, and in combination, on Na_v1.5 stably expressed in Chinese hamster ovary cells. The current/voltage relationship was shifted –5 mV with either 1 or 3 co-expression alone and –10 mV with co-expression of both 1 and 3. In addition, 3 and 1/3 co-expression accelerated macroscopic current decay. There were significant hyperpolarizing shifts in equilibrium gating relationships with co-expression of 1 and 3 alone and in combination. Co-expression of 1/3 together resulted in a greater hyperpolarizing shift in channel availability, and an increase in the slopes of equilibrium gating relationships. Co-expression of 3 and 1/3, but not 1, slowed recovery from inactivation at –90 mV. Development of inactivation at –70 and –50 mV was accelerated by -subunit co-expression alone and in combination. -Subunit co-expression also reduced the late Na current measured at 200 ms. In conclusion, -subunits modulate Na_v1.5 gating with important differences between co-expression of 1 and 3 alone and 1/3 together.