Endothelial tip cells in vitro are less glycolytic and have a more flexible response to metabolic stress than non-tip cells.

Formation of new blood vessels by differentiated endothelial tip cells, stalk cells, and phalanx cells during angiogenesis is an energy-demanding process. How these specialized endothelial cell phenotypes generate their energy, and whether there are differences between these phenotypes, is unknown. This may be key to understand their functions, as (1) metabolic pathways are essentially involved in the regulation of angiogenesis, and (2) a metabolic switch has been associated with angiogenic endothelial cell differentiation. With the use of Seahorse flux analyses, we studied metabolic pathways in tip cell and non-tip cell human umbilical vein endothelial cell populations. Our study shows that both tip cells and non-tip cells use glycolysis as well as mitochondrial respiration for energy production. However, glycolysis is significantly lower in tip cells than in non-tip cells. Additionally, tip cells have a higher capacity to respond to metabolic stress. Finally, in non-tip cells, blocking of mitochondrial respiration inhibits endothelial cell proliferation. In conclusion, our data demonstrate that tip cells are less glycolytic than non-tip cells and that both endothelial cell phenotypes can adapt their metabolism depending on microenvironmental circumstances. Our results suggest that a balanced involvement of metabolic pathways is necessary for both endothelial cell phenotypes for proper functioning during angiogenesis.

Rapid combined light and electron microscopy on large frozen biological samples.

The use of large unfixed frozen tissue samples (10 x 10 x 5 mm(3)) for combined light microscopy (LM) and electron microscopy (EM) is described. First, cryostat sections are applied for various LM histochemical approaches including in situ hybridization, immunohistochemistry and metabolic mapping (enzyme histochemistry). When EM inspection is needed, the tissue blocks that were used for cryostat sectioning and are stored at -80 degrees C, are then fixed at 4 degrees C with glutaraldehyde/paraformaldehyde and prepared for EM according to standard procedures. Ultrastructurally, most morphological aspects of normal and pathological tissue are retained whereas cryostat sectioning at -25 degrees C does not have serious damaging effects on the ultrastructure. This approach allows simple and rapid combined LM and EM of relatively large tissue specimens with acceptable ultrastructure. Its use is demonstrated with the elucidation of transdifferentiated mouse stromal elements in human pancreatic adenocarcinoma explants grown subcutaneously in nude mice. Combined LM and EM analysis revealed that these elements resemble cartilage showing enchondral mineralization and aberrant muscle fibres with characteristics of skeletal muscle cells.

Calvarial osteoclasts express a higher level of tartrate-resistant acid phosphatase than long bone osteoclasts and activation does not depend on cathepsin K or L activity.

Bone resorption by osteoclasts depends on the activity of various proteolytic enzymes, in particular those belonging to the group of cysteine proteinases. Next to these enzymes, tartrate-resistant acid phosphatase (TRAP) is considered to participate in this process. TRAP is synthesized as an inactive proenzyme, and in vitro studies have shown its activation by cysteine proteinases. In the present study, the possible involvement of the latter enzyme class in the in vivo modulation of TRAP was investigated using mice deficient for cathepsin K and/or L and in bones that express a high (long bone) or low (calvaria) level of cysteine proteinase activity. The results demonstrated, in mice lacking cathepsin K but not in those deficient for cathepsin L, significantly higher levels of TRAP activity in long bone. This higher activity was due to a higher number of osteoclasts. Next, we found considerable differences in TRAP activity between calvarial and long bones. Calvarial bones contained a 25-fold higher level of activity than long bones. This difference was seen in all mice, irrespective of genotype. Osteoclasts isolated from the two types of bone revealed that calvarial osteoclasts expressed higher enzyme activity as well as a higher level of mRNA for the enzyme. Analysis of TRAP-deficient mice revealed higher levels of nondigested bone matrix components in and around calvarial osteoclasts than in long bone osteoclasts. Finally, inhibition of cysteine proteinase activity by specific inhibitors resulted in increased TRAP activity. Our data suggest that neither cathepsin K nor L is essential in activating TRAP. The findings also point to functional differences between osteoclasts from different bone sites in terms of participation of TRAP in degradation of bone matrix. We propose that the higher level of TRAP activity in calvarial osteoclasts compared to that in long bone cells may partially compensate for the lower cysteine proteinase activity found in calvarial osteoclasts and TRAP may contribute to the degradation of noncollagenous proteins during the digestion of this type of bone.

Endogenous expression and endocytosis of tartrate-resistant acid phosphatase (TRACP) by osteoblast-like cells.

Tartrate-resistant acid phosphatase (TRACP) is produced by macrophages and other cells of the monohistiocytic lineage. In particular, osteoclasts are characterized for a high expression of this enzyme. Yet, several data suggest that other bone cell types, such as osteocytes and osteoblasts, may also express activity of this enzyme. This is particularly obvious at sites were osteoclasts resorb bone, suggesting that osteoclasts (or their precursors) somehow induce TRACP activity in osteoblasts. In the present study, we investigated this by culturing human osteoblast-like cells with and without conditioned medium (MCM) from human blood monocytes (as a source of osteoclast precursors). High levels of TRACP activity were found in osteoblast-like cells cultured with MCM. Depletion of TRACP from this medium resulted in the absence of its activity in osteoblast-like cells, thus suggesting that the TRACP activity in these cells was the result of endocytosed TRACP that was released by the monocytes in the MCM. Osteoblast-like cells cultured in control (non-conditioned) medium contained very low levels of TRACP-like activity. However, the cells expressed TRACP mRNA and incubation of extracts of these cells with active cathepsin B did induce activity of a TRACP-like enzyme. Inhibition of the activity of cysteine proteinases in general and of cathepsin B in particular, completely blocked TRACP activity of the osteoblast-like cells. This TRACP-like enzyme but not the alleged endocytosed fraction of TRACP was inhibited by fluoride, suggesting that the fractions may be different isoenzymes. Our data seem to indicate that osteoblast-like cells may contain two different fractions of TRACP, one that is released by monocytes and subsequently endocytosed by osteoblast-like cells and a second endogenous fraction that is present in an inactive proform. We hypothesize that the capacity of osteoblast-like cells to endocytose TRACP is important for the removal of this enzyme during or following the bone resorptive activity of the osteoclast.

Haptic after-effect of successively touched curved surfaces.

A flat surface is more often judged to be convex after the touching of a concave surface than after the touching of a convex surface. This haptic after-effect increases with the time of contact with the curved surface till it saturates, and it decreases with the time-lapse between the touching of the first surface and the next one. In this paper, the haptic after-effect of two successively touched spherically curved surfaces is investigated. It is found that both surfaces contribute to the after-effect, but the after-effect is not additive. The time course of the after-effect of two successive surfaces can be described by a first-order integrator with a single time constant of about 7 s and an amplitude equal to the difference between the saturation levels of the after-effects of the two surfaces when measured in isolation. The new saturation level is therefore equal to that of the after-effect of the most recently touched surface.

Influence of shape on haptic curvature perception.

Curvature discrimination of hand-sized doubly curved surfaces by means of static touch was investigated. Stimuli consisted of hyperbolical, cylindrical, elliptical and spherical surfaces of various curvatures. In the first experiment subjects had to discriminate the curvature along a specified orientation (the discrimination orientation) of a doubly curved surface from a flat surface. The curvature to be discriminated was oriented either along the middle finger or across the middle finger of the right hand. Independent of the shape of the surface, thresholds were found to be about 1.6 times smaller along the middle finger than across the middle finger. Discrimination biases were found to be strongly influenced by the shape of the surface; subjects judged a curvature to be more convex when the perpendicular curvature was convex than when this curvature was concave. With the results of the second experiment it could be ruled out that the influence of shape on curvature perception was simply due to a systematic error made by the subject regarding the discrimination orientation.

Dietary omega-3 polyunsaturated fatty acids promote colon carcinoma metastasis in rat liver.

The effects of omega-3 polyunsaturated fatty acids (PUFAs) and omega-6 PUFAs on the development of experimentally induced colon carcinoma metastasis in rat liver were investigated quantitatively in vivo. Rats were kept on either a low-fat diet or on a fish oil (omega-3 PUFAs) or safflower oil (omega-6 PUFAs) diet for 3 weeks before the administration of colon cancer cells to the portal vein, until they were sacrificed at 1 or 3 weeks after tumor transplantation. At 1 week after transplantation, the fish oil diet had induced 7-fold more metastases (in terms of number and size) than had the low-fat diet, whereas the safflower oil diet had not affected the number and total volume of metastases. At 3 weeks after tumor transplantation, the fish oil diet and the safflower oil diet had induced, respectively, 10- and 4-fold more metastases (number) and over 1000- and 500-fold more metastases (size) than were found in the livers of rats on the low-fat diet. These differences were sex independent. Immunohistochemical analysis revealed that the immune system in the liver (Kupffer cells, pit cells, T cells, newly recruited macrophages, and the activation state of macrophages) did not play a significant role in this diet-dependent outgrowth of tumors. In conclusion, omega-3 and omega-6 PUFAs promote colon cancer metastasis in the liver without down-regulating the immune system. This finding has serious implications for the treatment of cancer patients with fish oil diet to fight cachexia.

In situ detection of spontaneous superoxide anion and singlet oxygen production by mitochondria in rat liver and small intestine.

In the present study, the endogenous formation of reactive oxygen species was localized in rat liver and small intestine. The 3,3'-diaminobenzidine (DAB)-Mn2+ technique in which cobalt ions were included in the incubation medium was applied to unfixed cryostat sections of intact tissues. Addition of manganese ions to the DAB-Co(2+)-containing medium greatly increased the amounts of final reaction product formed compared with incubations with only DAB and cobalt ions. In liver, a blue final reaction product was deposited, particularly in hepatocytes surrounding portal tracts. In the small intestine, the DAB-cobalt complex was mainly found at the basal side of enterocytes. Goblet cells remained unstained. Electron microscopical images revealed that an electron-dense reaction product was exclusively present at both inner and outer membranes and at the intermembrane space in mitochondria of liver parenchymal cells and duodenal enterocytes. It was shown that the spontaneous formation of final reaction product was enzymatic and dependent on the presence of oxygen in the medium. Sulphide decreased the reaction, which may indicate that cytochrome c oxidase was partially involved. Benzoquinone and histidine, which are scavengers of superoxide anions and singlet oxygen respectively, reduced the amount of final reaction product considerably. Furthermore, the formation of final reaction product was sensitive to specific inhibitors of NADH:coenzyme Q reductase and aldehyde oxidase, indicating that these enzymes were at least partly responsible for the generation of superoxide anions and singlet oxygen and for the formation of the DAB-cobalt complex.

Investigation into the origin of the haptic aftereffect of curved surfaces.

In haptics, the perceived (phenomenal) flatness of a surface is strongly influenced by a previous surface which has been statically touched. The mechanisms underlying this haptic aftereffect of curved surfaces are investigated. It is shown that the representation of curvature abstracted from the sense of touch, ie a high-level representation, is not affected during the aftereffect. This is concluded because: (1) the aftereffect does not exhibit intermanual transfer; (2) the way in which two successive surfaces are touched can influence the magnitude of the aftereffect; and (3) it is not necessary to touch a surface-active muscular contraction can also result in a shift of the phenomenal flatness. Furthermore, it is suggested that the physiological process involved in the aftereffect is a central process, ie it is located in the brain but it is distinct for each hemisphere. This is supported by the findings that: (1) the decay rate of the aftereffect is not influenced by the degree of peripheral stimulation during the decay; and (2) the aftereffect does not transfer from the adapted hand to the unadapted hand.

Kupffer cells and pit cells are not effective in the defense against experimentally induced colon carcinoma metastasis in rat liver.

The present study was performed to investigate processes involved in circumvention of the immune system by advanced stages of tumor growth in the liver. The efficacy of Kupffer cells and pit cells against cancer cells was tested in vivo in an experimental model of colon carcinoma metastasis in rat liver. Liver tumors were induced by administration of CC531 colon cancer cells into the vena portae. After 3 weeks, livers were obtained and partly fixed for electron microscopic procedures or frozen in liquid nitrogen for enzyme and immunohistochemistry at the light microscope level. The activation status of Kupffer cells was studied by expression of Ia-antigen (MHC class II) and by measurement of glucose-6-phosphate dehydrogenase (G6PDH) activity in the cells in situ as a measure of production of reactive oxygen species. Large numbers of Kupffer cells were found in liver parenchyma surrounding colon carcinomas when compared with levels in control livers, but these cells were not activated. Large numbers of activated monocytes and macrophages, cytotoxic T cells but only a few pit cells were found to be recruited to the boundary between liver parenchyma and tumors or their stroma. In those areas where cancer cells invaded liver parenchyma, only newly recruited macrophages and some Kupffer cells were present but few cytotoxic T cells or pit cells were found. The low activation status of Kupffer cells both in terms of production of reactive oxygen species and Ia-antigen expression and the absence of significant numbers of pit cells at tumor sites suggest that Kupffer cells and pit cells do not play a significant role in advanced stages of tumor growth. High levels of prostaglandin E2 were detected in the parenchyma of livers containing tumors and transforming growth factor beta was detected in the stroma of the tumors, therefore suggest that cytotoxicity of newly recruited monocytes, macrophages and cytotoxic T cells may be limited in these stages because of local production of these immunosuppressive factors.

Haptic aftereffect of curved surfaces.

A haptic aftereffect of curved surfaces is demonstrated. Two spherical surfaces were presented sequentially to human subjects. They rested one hand on the first (conditioning) surface. After a fixed conditioning period they transferred their hand to the second (test) surface and judged whether the test surface was convex or concave. In experiment 1 the curvature of the conditioning surface was varied; the subject's judgment of convexity or concavity of the test surface was strongly shifted in the direction opposite to the curvature of the conditioning surface (negative aftereffect). Therefore, subjects judged a flat surface to be concave after being exposed to a convex surface. After a conditioning period of 5 s the shift was about 20% of the curvature of the conditioning surface. In experiment 2 the duration of the conditioning period was varied; the magnitude of the aftereffect could be described by a first-order integrator with a time constant of 2 s. In experiment 3 the time interval between the conditioning period and the touching of the second surface was varied; the magnitude of the aftereffect could be described by an exponential decay with a time constant of 40 s. It is concluded that the haptic aftereffect of curved surfaces is an important effect that occurs almost instantaneously and lasts for an appreciable period.

Ectopic mineralized cartilage formation in human undifferentiated pancreatic adenocarcinoma explants grown in nude mice.

Mineralized as well as nonmineralized cartilage-like structures enclosing cells resembling chondrocytes were found in human-derived undifferentiated but not in poorly differentiated pancreatic adenocarcinoma explants grown in nude mice. The structures reacted with anti-mouse IgG but not with antibodies against human cytokeratin 19, indicating that the newly formed tissue was of mouse origin. High activity of alkaline phosphatase was found in cell layers surrounding the structures and in cells embedded in the matrix. The extracellular matrix was strongly positive after Sirius red staining, reacted with anti-collagen type II antibodies, and the presence of proteoglycans was demonstrated with Alcian blue staining and by metachromasia after Giemsa staining. Electron microscopic inspection revealed the presence of bundles of both thick collagenous fibrils with low levels of fine filamentous material and thin collagenous fibrils with high concentrations of filamentous components. The majority of both types of matrices was found to be partially or completely calcified. The mean area density of the cartilage-like structures in the undifferentiated tumors was 0.31%. The frequent formation of the cartilage-like structures in the rapidly growing undifferentiated explants and its absence in the slowly growing, more differentiated explants suggest that low oxygen tensions in combination with altered levels of growth factors, such as members of the transforming growth factor beta superfamily, create conditions that induce differentiation of fibroblasts to chondrocytes. It is concluded that these human tumors grown in nude mice can be used as an in vivo model to study ectopic formation of mineralized cartilage.

Effects of partial hepatectomy, phenobarbital and 3-methylcholanthrene on kinetic parameters of glucose-6-phosphate and phosphogluconate dehydrogenase in situ in periportal, intermediate and pericentral zones of rat liver lobules.

Glucose-6-phosphate dehydrogenase (G6PDH) and phosphogluconate dehydrogenase (PGDH) are heterogeneously distributed in liver lobules of female rats. The maximum activity of both enzymes is approximately twice higher in intermediate and pericentral zones than in periportal zones. Enzyme activities and their distribution patterns were manipulated by partial hepatectomy and treatment with phenobarbital (PB) or 3-methylcholanthrene (3-MC). Vmax values of G6PDH for glucose-6-phosphate decreased mainly in intermediate and pericentral zones after partial hepatectomy, whereas they increased after PB treatment. Vmax values of PGDH for phosphogluconate decreased after partial hepatectomy in both zones, whereas other treatments did not have any effect. The affinity of G6PDH for glucose-6-phosphate was similar in all zones and it was decreased 2-3 fold by PB and 3-MC treatment. The affinity of PGDH for phosphogluconate was 1.4-2.3 times lower in intermediate and pericentral zones than in periportal zones of all livers tested and was not affected by treatment. From these data it can be concluded that not only the maximum activity of enzymes may differ in periportal, intermediate and pericentral zones of the liver lobule but also the affinity of enzymes for their substrates. The implication of these findings is that metabolic flux rates as they occur in vivo in these different metabolic compartments may be significantly different from predictions on the basis of maximum enzyme activities as detected immunohistochemically, microchemically or cytophotometrically.

Adaptive sex-dependent changes in the zonation of carbohydrate and lipid metabolism in rat liver lobules after partial hepatectomy.

To evaluate changes in metabolic heterogeneity in rat liver lobules after partial hepatectomy, we measured parameters of carbohydrate and lipid metabolism cytophotometrically in periportal and pericentral zones of livers of mature female and male rats. Glycogen content was shown to be always higher in pericentral zones than in periportal zones. After a rapid depletion of glycogen stores during the first 8 hr after partial hepatectomy, the levels were restored to normal after 24 hr, but a significant depletion was found again at 48 hr after operation. These fluctuations were similar in female and male rat livers. The lipid content in control rat livers was low and was mainly localized in periportal zones. Partial hepatectomy caused a significant increase in lipid content after 24 to 48 hr in periportal zones only, which was distinctly higher in female than in male rat livers. Activity of NADPH-producing glucose-6-phosphate dehydrogenase was heterogeneously distributed in lobules of female control rats with highest activity in pericentral zones, whereas a lower but evenly distributed activity was found in lobules of control male rats. The activity was not affected by partial hepatectomy in male rats, whereas the activity in female rat livers decreased to levels found in male rats at 24 to 48 hr after operation. Another NADPH-producing enzyme, malate dehydrogenase, showed the highest activity pericentrally in female rats, and a low activity was evenly distributed in male rats. The activity did not change significantly after partial hepatectomy. The ketogenic enzyme beta-hydroxybutyrate dehydrogenase showed the highest activity in pericentral zones of control livers. The activity in male rat livers was almost twice as high as in female rat livers in both zones. Partial hepatectomy caused a distinct reduction in activity in both zones and both sexes, but the strongest reduction was found periportally. Alkaline phosphatase activity, which is linked with bile acid secretion by hepatocytes, was low in control male and female rats and was mainly found periportally. The activity was increased dramatically at 24 to 48 hr after partial hepatectomy in both zones and particularly in male rat livers. The index for the Krebs cycle, succinate dehydrogenase activity, was highest in periportal zones. At 24 to 48 hr after partial hepatectomy, this preferential zonation was lost, and the activity was slightly higher in pericentral zones. This reversal of zonation was found in all livers of female and male rats investigated.(ABSTRACT TRUNCATED AT 400 WORDS)

Use of frozen biologic material for combined light and electron microscopy.

A simple and rapid method that enables the use of unfixed frozen material for light and electron microscopic purposes is described. At the light microscopic (LM) level, unfixed cryostat sections were used for enzyme histochemistry. When electron microscopic (EM) inspection was needed, tissue blocks, which were stored at -80 degrees C, were fixed at 4 degrees C and prepared for EM according to standard procedures. Ultrastructural analysis of this material demonstrated that most morphologic aspects of normal (human pancreas and rat liver) and pathologic (human pancreatic adenocarcinoma and rat colon carcinoma metastases in liver) tissue were rather well retained. Cryostat sectioning at -25 degrees C did not appear to have damaging effects on the morphology. The method was applied to correlate enzyme histochemical (LM) data with ultrastructural (EM) aspects of mineralization of stroma in explants of human pancreatic adenocarcinoma grown in nude mice and of nonparenchymal cells around metastases of colon carcinoma in rat liver.

Experimentally induced colon cancer metastases in rat liver increase the proliferation rate and capacity for purine catabolism in liver cells.

Metastases in rat liver were generated experimentally by intraportal injection of colon cancer cells to investigate the effects of cancerous growth on the metabolism of surrounding liver tissue. Maximum activities (capacity) of glucose-6-phosphate dehydrogenase, phosphogluconate dehydrogenase, lactate dehydrogenase, succinate dehydrogenase, alkaline phosphatase, 5'-nucleotidase, xanthine oxidoreductase, purine nucleoside phosphorylase and adenosine triphosphatase have been determined. Two types of metastases were found, a small type surrounded by stroma and a larger type in direct contact with hepatocytes. Both types affected the adjacent tissue in a similar way suggesting that the interactions were not mediated by stroma. High capacity of the degradation pathway of extracellular purines released from dead cells of either tumours or host tissue was found in stroma and sinusoidal cells. Metastases induced both an increase in the number of Kupffer cells and proliferation of hepatocytes. The distribution pattern in the liver lobulus of most enzymes investigated did not change distinctly. However, activity of alkaline phosphatase, succinate dehydrogenase and phosphogluconate dehydrogenase was increased in hepatocytes directly surrounding metastases. These data imply that the overall metabolic zonation in liver lobuli is not dramatically disturbed by the presence of cancer cells despite the fact that various metabolic processes in liver cells are affected.

Quantitative changes in acid phosphatase, alkaline phosphatase and 5'-nucleotidase activity in rat liver after experimentally induced cholestasis.

Acid phosphatase, alkaline phosphatase and 5'-nucleotidase activities were analyzed cytophotometrically in cryostat sections of rat liver up to 8 weeks after ligation and transsection of the common bile duct. Ligation resulted in cholestasis and induced alterations in both localization and activity of the enzyme investigated. The cellular distribution but not the activity of acid phosphatase changed in liver parenchyma. In control liver, the final reaction product was localized as discrete granules in the bile canalicular region of hepatocytes. The final reaction product was precipitated more diffusely within the cytoplasm after induction of cholestasis, most probably due to increased fragility of lysosomal membranes. In control liver, alkaline phosphatase activity was low and localized in the bile canalicular plasma membranes only. The total parenchymal activity increased threefold after the induction of cholestasis and is considered to be a compensatory mechanism in order to enhance the excretion of bile salts from hepatocytes. 5'-Nucleotidase was present at the bile canalicular and sinusoidal surfaces of plasma membranes of hepatocytes in control liver; total activity in pericentral areas was significantly higher than in periportal areas. Induction of cholestasis resulted in higher total activity and redistribution of the activity over all three surfaces of the plasma membranes, whereas heterogeneity over the different zones of the acinus disappeared. The appearance of the enzyme at lateral plasma membranes is suggested to be related to the formation of new sites for bile salt transport out of the hepatocytes. With respect to all three enzymes studied, alterations of liver parenchymal cells due to a disturbed bile transport were already established during the first week of cholestasis.

Human epidermal Langerhans cells undergo profound morphologic and phenotypical changes during in vitro culture.

Morphology, phenotype, and enzyme activity of highly enriched (80%) unlabeled human epidermal Langerhans cells (LC) have been studied, with emphasis on changes during a short-term culture of three days in vitro. All freshly isolated LC contained Birbeck granules and expressed high levels of CD1a, CD1c, and MHC class II molecules HLA-DR, -DP, and -DQ. They have a weak to moderate expression of RFD1, C3biR, Fc gamma R, p 150/95, MHC class I molecules HLA-ABC, and of the adhesion molecules LFA-3 and ICAM-1, whereas no expression of LFA-1 and several monocyte/macrophage markers were detected. Human LC undergo profound changes during in vitro culture. Birbeck granules, C3biR, Fc gamma R, and p 150/95 were completely lost and the expression of CD1a and CD1c was markedly decreased or lost. Expression of molecules that have essential functions in antigen presentation remained present at the same level (MHC class II molecules and ICAM-1) or was markedly enhanced (LFA-3 and MHC class I). Highly remarkable was the dramatically enhanced expression of interdigitating cell marker RFD1. The monocyte/macrophage markers initially absent remained absent and the enzyme activity initially present (including ATPase and nonspecific esterase) remained present. In conclusion, the results in this report stress rapid alterations of human LC during in vitro culture, resulting in transformation into cells that have phenotypical characteristics of potent antigen presenting cells that resemble interdigitating cells.

Cytophotometric analysis of reaction rates of succinate and lactate dehydrogenase activity in rat liver, heart muscle and tracheal epithelium.

Reaction rates of succinate and lactate dehydrogenase activity in cryostat sections of rat liver, tracheal epithelium and heart muscle were monitored by continuous measurement of formazan formation by cytophotometry at room temperature. Incubation media contained polyvinyl alcohol as tissue protectant and Tetranitro BT as final electron acceptor. Control media lacked either substrate or substrate and coenzyme. Controls were also performed by adding malonate (a competitive inhibitor of succinate dehydrogenase), pyruvate (a non-competitive inhibitor of lactate dehydrogenase), oxalate (a competitive inhibitor of lactate dehydrogenase) or N-ethylmaleimide (a blocker of SH groups). A specific malonate-sensitive linear test minus control response for succinate dehydrogenase activity was obtained in liver (1.6 mumol H2cm-3 min-1) and tracheal epithelium (0.8 mumol H2cm-3 min-1) but not in heart muscle. All variations in the incubation conditions tested did not result in a linear test minus control response in the latter tissue. Because the reaction was sensitive to malonate, it was concluded that the initial reaction rate was the specific rate of succinate dehydrogenase activity in heart muscle (9.1 mumol H2 cm-3 min-1). Test minus control reactions for lactate dehydrogenase activity were distinctly non-linear for all tissues tested. This appeared to be due to product inhibition by pyruvate generated during the reaction and therefore it was concluded that the appropriate control reaction was the test reaction in the presence of 20 mM pyruvate. The initial rate of the test minus this control was the true rate of lactate dehydrogenase activity. The lactate dehydrogenase activity thus found in liver parenchyma was 5.0 mumol of H2 generated per cm3 liver tissue per min.