Research in experimental medicine. Zeitschrift fur die gesamte experimentelle Medizin einschliesslich experimenteller Chirurgie最新文献

We do not know much about the changes that occur in reduced (GSH) and oxidized (GSSG) glutathione in the development of liver cirrhosis. Therefore, we investigated the glutathione redox system during development of liver cirrhosis after bile-duct ligation in rats. We compared the GSH and GSSG content of liver and plasma between bile-duct-ligated rats and sham-operated controls 6 and 24 h and 5, 15, 23, and 38 days after operation. Compared to controls (x +/- SD: 6.07 +/- 0.52 mumol/g wet wt.), liver GSH significantly increased 24 h (+ 37%) and 5 days (+ 53%) after bile-duct ligation. Thereafter, GSH continuously declined to 4.25 +/- 0.64 mumol/g (-31%; P < 0.001) at the end of the observation period after 38 days. The GSH turnover in 5-day bile-duct-ligated rats with high GSH concentrations was not significantly different than in sham-operated controls (16 nmol/min per g after bile-duct ligation and 15 nmol/min per g in controls). GSSG (211 +/- 42 nmol/g wet wt. in controls) was significantly lower 6 and 24 h after bile-duct ligation (-34% and -43%, respectively). Thereafter, GSSG increased and was about 100% higher than in controls after 23 and 38 days. The relation of GSSG to GSH in liver continuously increased from 3.4 to 20.5% after bile-duct ligation. The course of plasma GSH (9.57 +/- 0.79 mumol/l) paralleled hepatic GSH on a lower level: + 14% at day 5, -41% at day 15 and -51% at the end of the observation period. Plasma GSSG (0.99 +/- 0.31 mumol/l) was inversely related to liver GSSG: there were increased concentrations early after bile duct ligation (day 5: + 91%) and reduced concentrations (-44%) at the end of the observation period. Dynamic changes of the glutathione status occur in the development of liver cirrhosis after bile-duct ligation. These changes are consistent with increased oxidative stress in the liver and a deficit of transporting GSSG from the cells into plasma.

Urinary waste products (UWP) in the amniotic fluid have been held responsible for the intestinal damage (ID) in gastroschisis, based on the fact that the fetus urinates physiologically into the amniotic cavity. However, experimental and clinical evidence suggests that intrauterine defecation is a physiological event; thus gastrointestinal waste products (GWP) may also be responsible for ID in gastroschisis. An experimental study was performed to investigate the effects of intraperitoneal human neonatal urine and diluted meconium on rat intestines. Adult Wistar albino rats were used. Sterile urine and meconium were obtained from newborn humans and 5% meconium suspension was prepared. Histopathological features of the intestines of the rats injected with urine did not differ from the intestines of the untreated rats. The bowel in rats injected with a meconium suspension showed serosal thickening, inflammation, focal fibrin and collagen deposits. Histopathological changes in intestines induced by intraperitoneal diluted meconium were consistent with those described for human gastroschisis specimens. We conclude that GWP, rather than UWP, seems to be responsible for the ID in gastroschisis.

The aim of this study was to investigate whether nitric oxide might be involved in the adaptation of the cardiovascular system in sodium and water loaded organisms. The effects of a semi-chronic (4 day) inhibition of nitric oxide production were studied in normovolemic and per os sodium and water loaded rats. Nitric oxide synthase was inhibited by L-NAME (10 mg/kg in normovolemic and 14 mg/kg in sodium loaded rats) dissolved in the drinking solution. Blood pressure, cardiac output (Stewart-Hamilton's principle) and its regional fractions (Sapirstein's technique using 86Rb isotope as indicator), total peripheral resistance and regional vascular resistances were determined on the 5th day in sodium pentobarbital anaesthesia. The increase in blood pressure following L-NAME pretreatment in both groups was similar, but the elevation of total peripheral resistance was 106% in normovolemic and only 30% in sodium loaded animals. The cardiac output decreased by 44% in normovolemic and 14% in sodium loaded groups after nitric oxide synthase inhibition. The organ vascular resistances increased and organ blood flows decreased after L-NAME administration. These changes were less pronounced in sodium and water load, especially those in the skeletal muscle and intestine. Nitric oxide-induced changes in vascular resistance are more pronounced in normovolemia than in sodium load; sodium load might influence the nitric oxide production. The share of nitric oxide in the setting of vascular tone is different in the various organs.

We tested the hypothesis that the negative metabolic effects of elevating cyclic GMP act through inhibition of L-type calcium channels in quiescent cardiac myocytes. The steady state O2 consumption (VO2) of ventricular myocytes, isolated from hearts of New Zealand white rabbits, was measured in a glass chamber using Clark-type oxygen electrodes. The cellular cyclic GMP levels were determined by radioimmunoassay at baseline with either 0.5 mM or 2.0 mM of Ca2+, sodium nitroprusside at increasing concentration (10(-8),(-6),(-4) M) with and without pretreatment by BAY K8644 10(-5) M (L-type Ca2+ channel activator) in 0.5 mM Ca2+, or nitroprusside with and without pretreatment with nifedipine 10(-4) M (L-type Ca2+ channel blocker) in 2.0 mM Ca2+. In the 0.5 mM Ca2+ medium, basal VO2 was 459 +/- 104 (nl O2/min per 10(5) myocytes) with a corresponding cyclic GMP level of 112 +/- 23 (fmol/10(5) myocytes). With nitroprusside 10(-4) M, VO2 was decreased to 285 +/- 39 and cyclic GMP level was significantly elevated to 425 +/- 128. In the same medium, VO2 was slightly increased by BAY K8644 10(-5) M while the cyclic GMP level did not change. With BAY K8644 10(-5) M, nitroprusside 10(-4) M decreased VO2 and increased cyclic GMP to a level which was similar to cells treated with nitroprusside alone. In the 2.0 mM Ca2+ medium, the basal VO2 and cyclic GMP were 518 +/- 121 and 137 +/- 24. In the presence of nitroprusside 10(-4) M, VO2 was decreased to 295 +/- 49 and cyclic GMP was increased to 454 +/- 116. In the same medium, nifedipine 10(-4) M significantly decreased VO2, while the cyclic GMP level was comparable to the baseline. After nifedipine 10(-4) M, nitroprusside 10(-4) M decreased VO2 and increased cyclic GMP to levels which were similar to control. Therefore, in quiescent cardiac myocytes, the negative metabolic effects associated with cyclic GMP were not primarily mediated through inhibition of L-type Ca2+ channels.

In order to observe the postoperative vertical displacement caused by various open functional stereotaxic procedures in the coordinate of brain target, we produced a similar surgical procedure in 14 cats. On preoperative computed tomography (CT) scans, certain well defined brain structures (the cortical surface of the brain and the floor of the third ventricle) were chosen as anatomical landmarks and their relationship to the skull were determined with the cat in a prone position. Thereafter, a burr hole was opened on the right side of the skull of each animal and a small dural incision was made. All animals were re-examined with CT scanning using the same technique and the distances to the skull were determined. There was a cortical descent in postoperative coronal CT images (range 1.2-3.6, mean 2.1 mm). On the other hand, the vertical coordinate of the floor of the third ventricle was found to be lower than it had been preoperatively, with an average of 0.6 mm lower (range -1.2(-)+0.3 mm). These results indicate that there was a good correlation between the degree of cortical descent and displacement of the vertical coordinate of the floor of the third ventricle (r = 0.70 and r = 0.69, respectively; P < 0.01). Our results also suggest that thalamic targets can be calculated by compensatory adjustment of the vertical coordinate by an additional distance of about half of the vertical descent. However, further experimental and clinical studies will be needed to determine the validity of this result in human beings.

We tested the hypothesis that part of the decreased function and metabolism caused by cyclic guanosine monophosphate (GMP) in beating cardiac myocytes is related to inhibition of L-type calcium channels. The steady state oxygen consumption (VO2) of a suspension of ventricular myocytes isolated from hearts of New Zealand white rabbits was measured using oxygen electrodes. Cellular cyclic GMP levels were determined by radioimmunoassay. Cell shortening was measured with a video edge detector. The VO2 was obtained after: (1) adding sodium nitroprusside (NP 10(-8),(-6),(-4) M), (2) pretreatment by BAY K8644 10(-5) M (BAY, L-type calcium channel activator), nifedipine 10(-4) M (NF, L-type calcium channel blocker) or forskolin 10(-7) M (FK, adenylate cyclase activator), then adding NP 10(-8),(-6),(-4) M, (3) pretreatment with both FK 10(-7) M and NF 10(-4) M and subsequently adding NP 10(-8),(-6),(-4) M. NP 10(-4) M decreased VO2 from 707 +/- 34 to 410 +/- 13 (nl O2/min per 10(5) myocytes), decreased the percentage of shortening (Pcs) from 5.7 +/- 0.6 to 3.7 +/- 0.5 and the rate of shortening (Rs) from 65.5 +/- 4.5 (microns/s) to 46.2 +/- 5.5. NP 10(-4) M also increased cyclic GMP from 264 +/- 70 (fmol/10(5) myocytes) to 760 +/- 283. Both BAY and FK increased VO2, Pcs and Rs without changing cyclic GMP. NF decreased Pcs, Rs and VO2. Similar metabolic and functional effects of NP were observed with pretreatment with these agents separately, compared to NP alone, and the elevation of cyclic GMP level was not different from the control group. With FK alone, NP 10(-4) M decreased VO2 by 51%, Pcs by 44% and Rs by 39%. In the presence of both FK and NF, the negative effects of NP were diminished significantly. NP 10(-4) M decreased VO2 by 37%, Pcs by 25% and Rs 20%. Thus, in beating cardiac myocytes, the negative metabolic and functional effects of cyclic GMP were related to inhibition on L-type calcium channels only when adenylate cyclase was stimulated.

The patch-clamp technique was used to examine the presence of large conductance calcium-activated potassium channels (BKCa) in human endothelial cells and to characterize their properties in terms of voltage dependence, ion conduction and blockade by iberiotoxin (IbTX). Experiments were performed using cell-attached and outside-out configurations on human umbilical vein endothelial cells (HUVEC). For the experiments HUVECs, which were passaged 6-19 times, were used. In early passages channel activities were absent suggesting the appearance of BKCa depending on cell culture time. The inverse logarithmic voltage sensitivity was 10.17 mV (median) for cell-attached recordings and 12.10 mV (median) for outside-out patches (membrane voltage range: 60-120 mV, symmetrical 140 mM K+ solutions). The I/V relationship was quasilinear in the range of 0-80 mV and exhibited a nonlinear behaviour under further depolarization suggesting some kind of saturation mechanism. Using a sigmoid function to fit the data, channel conductance was calculated as 172.9 pS (median) for cell-attached patches and as 262.1 pS (median) for outside-out patches. IbTX, known as one of the most selective blockers of BKCa was perfused to outside-out patches. In two out of three experiments there was complete block of the ion channel after 1 min.

The present investigations were designed to study the hemodynamic effects of different sodium diets in the submandibular gland of rats with or without nitric oxide (NO) synthesis inhibition. Experimental animals were kept on: (1) standard chow and tap water ad libitum (normal group, N), or (2) wheat and distilled water ad libitum for 4 weeks (sodium-depleted animals, SD), or (3) standard chow and saline ad libitum for 4 weeks (sodium-loaded animals, SL). NO synthase was inhibited by N omega-nitro-L-arginine-methyl-ester (L-NAME, 10 mg/kg per day) in the last week. The rats were anesthetized, and blood pressure, cardiac output (Stewart-Hamilton's principle) and blood flow (BF) of the submandibular gland (Sapirstein's technique) were determined. High sodium intake resulted in a 47% increase of glandular BF as compared to BF measured in the control group. In all groups L-NAME decreased BF (ml/min per 100 g gland) as compared to those of rats with no L-NAME treatment (N: 76.4 +/- 15.4 vs. 56.0 +/- 11.6, P < 0.05; SD: 71.0 +/- 17.7 vs. 56.2 +/- 15.1, n.s.; SL: 112 +/- 29.4 vs. 66.9 +/- 18.4, P < 0.001), whereas the vascular resistance (VR, mm Hg x ml-1 x s x kg-1) increased (N: 11.0 +/- 2.3 vs. 17.5 +/- 4.1, P < 0.001; SD: 11.0 +/- 2.7 vs. 17.0 +/- 4.2, P < 0.01; SL: 8.5 +/- 2.4 vs. 14.9 +/- 4.6, P < 0.001). The increase in VR after L-NAME treatment was 64% in normal, 55% in sodium-depleted and 75% in sodium-loaded rats. Our results suggest that NO takes part in the regulation of vascular resistance and BF in the submandibular gland. Sodium load itself increases BF of the submandibular gland and this phenomenon may partly be mediated by NO.

The goal of this paper was the measurement of nitric oxide (NO) production in isolated rat glomeruli using two different techniques. NO production was detected directly by a NO-specific electrode and the results were compared with data measured by the Griess reaction, an indirect index to evaluate NO production. The NO production, determined by both techniques, was dependent on the number of glomeruli. Pretreatment with Nw-nitro-L-arginine methyl ester, an inhibitor of the NO synthesis, reduced the NO concentration detected by the NO-sensor, but increased the NO2-concentration (when both results where compared with glomeruli without treatment). Preincubation with 1 mg/ml of Escherichia coli lipopolysaccharide significantly enhanced both NO and NO2-concentrations. Therefore, the present study provides direct evidence of NO generated in isolated glomeruli under physiological conditions and demonstrates that the measurement of NO2- by the Griess reaction, is not always an adequate technique to evaluate the actual NO production.

Dimensional alteration of hepatic microvessels was demonstrated during reperfusion after normothermic hepatic ischemia. Using a specially designed cover glass, it was possible to relocate selected sites of observation and microvessels repeatedly throughout the whole reperfusion time. Twenty minutes of hepatic ischemia resulted in a decrease of sinusoidal diameter (mean +/- SEM; 10.0 +/- 0.3 microns at baseline, 8.2 +/- 0.2 microns after ischemia) and diameter of postsinusoidal venules (26.4 +/- 1.2 at baseline, 23.0 +/- 1.0 after ischemia). In the control group (no ischemia induced) no changes of these parameters were observed. Thus, the reduction of hepatic microvascular cross section was present during the early phase of reperfusion. Hepatic dysfunction was characterized by increased serum activity of liver enzymes and reduction of bile flow in the ischemia-exposed animals. It has been suggested that postischemic dimensional microvascular changes are involved in postischemic liver dysfunction.