Local hemostasis in subarachnoid hemorrhage

Fibrinopeptide A (FPA), platelet-secreted protein, platelet factor 4 and beta-thromboglobulin were determined in the cerebrospinal fluid of patients who had suffered from subarachnoid hemorrhage and were treated with 6 g tranexamic acid or 4 million KIU aprotinin to prevent rebleeding. Platelet-secreted proteins and FPA were cleared from the cerebrospinal fluid within 3 days after bleeding. Their vasoactive and thrombotic capability is limited to the initiation period of vasospasm that usually comes to clinical observation 3-8 days after bleeding. Increased thrombotic activity of the cerebrospinal fluid, as reflected by high levels of FPA and platelet-secreted protein, seemed to promote the occurrence of neurological deficits.     

Atrial natriuretic peptide and verapamil can prevent gentamicin induced acute renal failure in the rat

Summary: Calcium channel blockers are able to improve renal function in acute renal failure (ARF) and natriuretic peptides can also exert beneficial effects. At present it is unknown whether administration of atrial natriuretic peptide (ANP) and a calcium channel blocker given before a toxic lesion can prevent gentamicin induced ARF. the mechanisms of action of natriuretic peptides and calcium channel blockers are different and, as yet, it has not been clarified if combined administration can augment the effects on renal function. After a basal period we investigated the effects of verapamil (VER, 0.66 mg/kg), ANP, (30 μg/kg) and a combination of both (identical doses as described individually). the drugs were given intravenously for a period of 40 min (infusion period) before gentamicin (15 mg/kg, i.v.) was administered for induction of ARF. Basal values for glomerular filtration rate (GFR, mL/min) were around 1.8 with no differences between the groups. At the end of the infusion period (before application of gentamicin) GFR was significantly elevated with VER + ANP (3.13 ± 0.51), ANP (2.70 ± 0.59) and VER (2.34 ± 0.47) compared to controls (saline, 1.7 ± 0.48). After application of gentamicin GFR significantly dropped in the control group (0.77 ± 0.21, 0.75 ± 0.19, respectively), indicating development of ARF. In contrast with VER + ANP, ANP and VER GFR could be maintained for 30 min (2.47 ± 0.39, 2.28 ± 0.33, 2.22 ± 0.43, respectively) and 130 min (2.11 ± 0.32, 1.86 ± 0.29, 2.11 ± 0.28, respectively) after gentamicin. Moreover ANP and VER revealed natriuretic activity and, due to their vasorelaxing potency, also influenced arterial blood pressure. We conclude that both VER and ANP are able to prevent early gentamicin induced ARF when given before the toxic lesion. Both drugs induce hyperfiltration while infused, in particular when administered in combination.     

Adenosine triphosphate-dependent K currents activated by metabolic inhibition in rat ventricular myocytes differ from those elicited by the channel opener rilmakalim

Adenosine triphosphate (ATP) dependent potassium channels (K_ATP channels) in heart ventricular muscle cells can be activated by depletion of intracellular ATP stores as well as by channel openers. In the present study we examined whether properties of K_ATP channels are dependent on the mode of activation. Whole-cell and single-channel currents were investigated by use of the patch-clamp technique in isolated ventricular rat myocytes. The channel opener rilmakalim dose dependency activated whole-cell currents [concentration for half-maximal activation (EC₅₀) = 1.1 M, Hill coefficient = 3.1, saturation concentration 10 M]. Metabolic inhibition with 2-deoxy-d-glucose (10 mmol/l) also activated K_ATP currents after a time lag of several minutes. These currents were about two-fold higher than the rilmakalim-activated currents (rilmakalim-activated current 3.9 ±0.2nA, 2-deoxy-d-glucose-activated current 8.1±0.9 nA; both recorded at 0 mV clamp potential). While the rilmakalim-activated current could be blocked completely and with high affinity by the sulphonylurea glibenclamide [concentration for half-maximal inhibition (IC₅₀) = 8 nM, Hill coefficient = 0.7] the 2-deoxy-d-glucose-activated current could only be blocked partially (by maximally 46%) and higher glibenclamide concentrations were needed (IC₅₀ = 480 nM, Hill coefficient = 0.8). The partial loss of blocking efficiency after metabolic inhibition was not restricted to glibenclamide but was also observed with the sulfonylureas glimepiride and HB 985, as well as with the non-sulfonylureas HOE 511 and 5-hydroxydecanoate. Single-channel studies were in accordance with these whole-cell experiments. Both rilmakalim and metabolic inhibition with the uncoupler carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) activated single channels in the attached mode, where the number of current levels was significantly higher in the case of FCCP. Rilmakalim-activated channels were completely blocked by 10 M glibenclamide, whereas several single-channel levels appeared in the presence of 100 M glibenclamide after metabolic inhibition. In conclusion, after metabolic inhibition the amplitude of the activated K_ATP current is about twice as high as under saturating concentrations of the opener rilmakalim. Moreover, channels activated by metabolic inhibition lost part of their sensitivity to known channel blockers.     

Piretanide-dextran and piretanide-polyethylene glycol interact with high affinity with the Na+ 2 Cl− K+ cotransporter in the thick ascending limb of the loop of Henle

Piretanide blocks the Na⁺ 2Cl^– K⁺ cotransporter protein in the thick ascending limb (TAL) of the loop of Henle reversibly. When tested from the luminal side in isolated perfused cTAL segments it leads to a half maximal inhibition (IC₅₀) of the equivalent short circuit current (I_sc) at a concentration of 10^–6 mol/l. From the basolateral side it has no effect on I_sc up to 10^–4 mol/l. The present study was designed to search for high affinity blockers of the Na⁺ 2Cl^– K⁺ cotransporter with large molecular weight in an attempt to use these macromolecules for antibody-labelling or affinity separation of this transport-protein. Amino-ethyl-dextran or amino-ethyl-polyethylene glycol (M.W. 5kd) were coupled to isothiocyanato-piretanide (ISO-PIR) at room temperature in DMSO. The resulting compounds dextran-sulfonylurea-piretanide (PIR-DEX) and polyethylene glycol-sulfonylurea-piretanide (PIR-PEG) (M.W. 5.38kd) were purified and tested in isolated perfused cTAL segments. IC₅₀ values for ISO-PIR, PIR-DEX and PIR-PEG were estimated from dose response curves after their addition to the lumen or bath perfusate, respectively. ISO-PIR, PIR-DEX and PIR-PEG acted from the lumen side at 3·10^–6, 6·10^–6 and 2·10^–6 mol/l. The inhibitory effect was easily reversible. From the basolateral side no effect for any compound was seen at up to 10^–4 mol/l. In clearance experiments PIR-DEX was given to female Wistar rats as an i.v. bolus (25 mol/kg) and the diuretic urine was collected. After dialysis (exclusion limit 2.5kd) the dialysed urine and the dialysate were tested in isolated perfused cTAL segments. The dialysates had no effect on I_sc, but the dialysed urine inhibited I_sc by 35% from the luminal side. The present data show: High molecular derivatives of piretanide with dextran or polyethylene glycol moieties block the Na⁺ 2Cl^– K⁺ cotransporter in cTAL segments at roughly the same low concentration as piretanide itself. Our data exclude a metabolism of these piretanide compounds in the kidney. Since these macromolecular probes can probably not enter the cell their inhibitory effect indicates that the binding site for piretanide diuretics on the Na⁺ 2Cl^– K⁺ cotransporter is exposed on the surface of the luminal cell membrane.This study was supported by Deutsche Forschungsgemeinschaft Gr 480/9     

Correlation between fluorescence in-situ hybridization analyses and in-vitro development to blastocyst stage of embryos from Robertsonian translocation (13;14) carriers

BACKGROUND: Little is known about the extent and timing of selection against the embryos that are carriers of unbalanced translocations. METHODS: Fluorescence in-situ hybridization (FISH) with probes for chromosomes 13, 14 and 18 was performed, mostly on day 3, on 69 human embryos which were then allowed to develop further in culture to day 5, from five carriers of Robertsonian translocation (RT) t(13;14). RESULTS: Twelve normal/balanced blastocysts were replaced in seven consecutive cycles (day 5). Three cycles resulted in clinical pregnancies. The proportion of blastocysts displaying a normal/balanced karyotype was 56%, while only the 20% of blocked embryos were normal/balanced (chi(2): P < 0.05). All the embryos analysed on day 5, except one, displayed mosaicism. The percentages of diploid cells for chromosomes 13 and 14 were significantly lower than for chromosome 18 (chromosome 13: 49.0 +/- 28.0; chromosome 14: 53.0 +/- 31.8; chromosome 18: 75.7 +/- 20.4; Mann-Whitney test: P < 0.01). The embryos displaying vertical line 62% of diploid cells for at least two of the three chromosomes analysed, more frequently reached the blastocyst stage (blocked embryos: blastocysts chromosome 13: 43.1 +/- 30.3, 64.9 +/- 29.0; chromosome 18: 64.9 +/- 29.0, 83.0 +/- 12.9; Mann-Whitney test: P < 0.01). CONCLUSIONS: Normal/balanced embryos developed better but the proportion of abnormal blastocysts was still high. Preimplantation genetic diagnosis is recommended to select normal/balanced embryos from RT t(13;14) carriers.     

Immunofluorescent evidence for exocytosis and internalization of secretory granule membrane in isolated chromaffin cells

Cultured bovine adrenal medullary chromaffin cells were stimulated with the secretogogues Ba2+ or carbamyl choline plus Ca2+ in the presence of a monospecific rabbit IgG fraction directed against bovine dopamine beta-hydroxylase. The anti-dopamine beta-hydroxylase was labeled either with fluorescent protein A or with a fluorescent second antibody to rabbit IgG. Stimulation produced a patchy cell surface distribution of fluorescence. There was no noticeable internalization of the fluorescence for up to 2 h. In similar experiments using fluorescent monovalent fragments (Fab) of the same monospecificidopamine-beta-hydroxylase IgG, a more uniform distribution of the fluorescence was observed. A few min after a 5 min period of stimulation with Ba2+, the fluorescence appeared to be on or near the cell surface; however, after 20 min or more it was distributed throughout the cytoplasm except that the cell nuclei were not labeled. Thus, dopamine beta-hydroxylase which appeared on the cell surface as a consequence of exocytosis was internalized in the presence of monovalent antibody fragments, but not in the presence of the divalent (polyclonal) antibody, presumably because endocytosis of dopamine beta-hydroxylase was inhibited by crosslinking of the dopamine beta-hydroxylase molecules. The internalized anti-dopamine beta-hydroxylase Fab fragments were found to reappear on the cell surface during a second secretory response. It is concluded that the interior of the chromaffin granule membrane, for which dopamine beta-hydroxylase is a marker, becomes exposed on the surface of the cell during secretion and that the membrane is then retrieved back into the cell where it can be re-used in a further secretory cycle.