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Binding of ricin and viscumin to paraformaldehyde fixed cell surface has been studied by confocal laser scanning microscopy (CLSM). Both toxins were labeled with different fluorochromes to allow for their identification after being applied jointly. The experiments indicated that viscumin and ricin bind to different cell receptors. Viscumin bound to the very periphery of the cells including lamellapodia and cell contact regions. Labeled ricin became localized in surface clusters located close to the cell body. The binding of toxins to the cell membrane was completely inhibited by 100 mmol/l lactose and in the presence of unlabeled homological toxins 500 times in abundance of the fluorochromed toxins. The experiments indicate that uptake and intracellular transport of ricin and viscumin starts from different membrane sites.

Excessive right heart hypertrophy was investigated under additional acute hypoxic stress to find out a possible contribution of mitochondrial dysfunction to sudden heart failure. Severe right heart hypertrophy in rats was induced by exposure to hypobaric pressure (46,663 Pa) for 4 weeks. Heart rate, isovolumic pressure and coronary flow were determined in the Langendorff mode of perfusion. After normoxia, the hearts were subdued to acute hypoxia/reoxygenation. Mitochondrial membrane potential was measured at the heart surface by fluorometry using 2-(dimethylaminostyryl)-l-ethylpyridinium iodide (DASPEI). At the end of each experiment mitochondria were isolated and ATP synthesis, ATPase, as well as creatine kinase activity were determined. Compared to normal hearts the heart rate is decreased in the hypertrophied group whereas right ventricular systolic and (end)diastolic pressure (adjusted to isovolumetric maxima) are increased. Coronary flow is decreased. Cytosolic creatine phosphate ATP levels and ATP/ADP ratios are significantly (p < 0.01) decreased. Furthermore, ATP synthesis and creatine kinase activities are diminished. At high ADP, respiration is loosely coupled or partially uncoupled. Acute hypoxia is particularly deleterious to hypertrophied hearts: Mitochondrial membrane potential as measured by heart surface fluorometry decreases extensively and is only very incompletely restored during reoxygenation. Rate-pressure product decreases precipitously and is restored during reoxygenation only to a very low extent. The results indicate an insufficient energy metabolism of mitochondria during acute hypoxia/reoxygenation which adds to the earlier described shifted isozyme pattern of myosin and decreased activities of myosin and sarcoreticular Ca2+ ATPase, leading to myocardial failure in right heart hypertrophy.

Rat hearts hypertrophied by exposure of the animals to low oxygen pressure were perfused by the Langendorff technique. After oxidative stress induced by hypoxia/reoxygenation, functional recovery of the hypertrophied right heart was insufficient when compared to non-hypertrophied controls. Accordingly, mitochondrial membrane potential did not recover sufficiently. There was a positive trend for improvement of the rate-pressure product during reoxygenation in lipoic acid (CAS 1077-28-7; 0.8 mumol/l) treated hearts which was also verified for membrane potential. Adenosine 5'-triphosphate and creatine phosphate contents as well as the ATP/ADP ratio in hypertrophied right ventricle were significantly increased after reoxygenation in hearts treated with lipoic acid. With lipoic acid, there was a significantly higher content of glutathione (oxidized form) after reoxygenation, Ca2+ uptake was significantly increased in mitochondria isolated from hypertrophied right ventricles and treated by 12 nmol/mg protein of lipoic acid. The results reveal a distinct improvement of mitochondrial structure/function by lipoic acid and suggest for therapy a combination with the synergistic free radical scavenging properties of tocopherol (CAS 10191-41-0).

The effects of 0.1 nmol/l ouabain (CAS 630-60-4) and digitoxin (CAS 71-63-6) on oxygen consumption rate (OCR) and the contractility of cultured chick embryo cardiomyocytes were investigated using a perfusion system which allowed microscopic observation during the experiment. contractility was assessed by a novel image analysis procedure. During substrate free perfusion the OCR was increased on application of 0.1 nmol/l ouabain or digitoxin for 60 min, whilst contractility was not affected. Digitoxin (0.1 nmol/l) elicited no change in the OCR in the presence of glucose, pyruvic acid, lactic acid and caprylic acid. Ouabain (0.1 nmol/l) did not affect the OCR or contractility in the presence of glucose, pyruvic acid and lactic acid, however together with caprylic acid the OCR was increased significantly. These results demonstrate a hitherto unknown stimulation of fatty acid utilization by low concentrations of ouabain.

The mode of topical action of minoxidil (U-10,858, CAS 38304-91-5) ist not entirely clear. The influence of minoxidil on the ultimate differentiation of keratinocytes was investigated. Human keratinocytes (HaCaT-cells) were incubated with minoxidil containing culture medium (10-250 micrograms/ml). Minoxidil inhibited in a concentration dependent manner cell mobility whereas the adhesion area and the cell circumference were increased. The minoxidil treated cultures had a higher desmosome density compared to parallel control cultures and they expressed the suprabasal keratins 1 and 10 (indicating progress in differentiation) earlier and to a larger extent than the controls. Keratins were revealed immunohistochemically and by two-dimensional polyacrylamide gel electrophoresis. The results suggest that minoxidil supports the differentiation of human keratinocytes.

The effect of digitoxin (CAS 71-63-6) and ouabain (g-strophantin, CAS 630-60-4) on respiration, morphology and beating activity of cardiomyocytes in culture derived from embryonic chick hearts has been investigated. The drugs were applied in a perfusion system using a protein- and substrate-free perfusion medium (BSS) at two concentration of K+ (5.4 and 4 mmol/l). In either K+ concentration oxygen consumption was 0.13 +/- 0.05 nmol O2 h-1 per 1000 cells. During 3 h of perfusion with BSS oxygen consumption declines only slightly to 79 +/- 15% of the initial value. No relation was found between beating frequency and oxygen consumption. Increase in respiration ranged from 5 to 45% and lasted between 5 and 120 min. At concentrations being inhibitory to the Na+/K(+)-ATPase (greater than or equal to 1 mumol/l) ouabain stimulated respiration by about 20% at 4 mmol/l K+ and 10% at 5.4 mmol/l K+ while digitoxin was effective at 5.4 mmol/l only (a transient increase of 20%). At 0.1 nmol/l, (a concentration below the KD of the high affinity binding site of the Na+/K(+)-ATPase) ouabain caused a long lasting activation of respiration by about 30%, digitoxin induced a transient rise of up to 20% at 5.4 mmol/l K+. At 4 mmol/l K+ digitoxin did not affect respiration while ouabain caused a transient increase. The lowest concentration of ouabain inducing a reproducible activation of oxygen consumption was 0.1 pmol/l. At this concentration digitoxin was no longer effective. At 1 fmol/l respiration was stimulated only occasionally.(ABSTRACT TRUNCATED AT 250 WORDS)

Coal tar and glucocorticosteroids are among the preparations used for the treatment of hyperproliferative inflammatory dermatosis. Coal tar generally seems to have a better effect on psoriasis capillitii than betamethasone-17-valerate. This finding is confirmed by investigations with human skin fibroblasts. The coal tar preparation Berniter has a strongly inhibitory effect on cell proliferation. The action of the preparation with tar can be clearly distinguished from the effect of the vehicle. At the ED50 concentration of the substance with tar, the vehicle without tar causes no inhibition. Based on weight equivalents Berniter has a greater inhibitory effect on proliferation of human skin fibroblasts than clobetasol-17-propionate. In the case of both substances protein biosynthesis is only affected at concentrations higher than those needed for the inhibition of proliferation. Berniter and the vehicle without tar have an equally strong effect on protein synthesis. Berniter affects the nucleoside triphosphatase bound to the nuclear membrane, and thus influences the nucleocytoplasmic transport of mRNA. The effect of the vehicle and the preparation containing tar on this parameter are very similar. Berniter has a positive effect on cell mobility. In preconfluent cultures the mobility of the cell is slightly increased and in confluent cultures significantly. Our results show that Berniter inhibits proliferation in cell cultures of human skin fibroblasts to a higher degree than clobetasol-17-propionate. It is to be assumed that neither the inhibition of protein synthesis nor the inhibition of mRNA transport play a causal role in the antiproliferative effect of coal tar. Inhibition of proliferation as a result of general damage to the cell can also be practically ruled out.