Search

Dissociated cells from fetal rat brain areas were grown in culture for up to 4 weeks. They expressed the nervous system-specific induction of cytoplasmic glycerol phosphate dehydrogenase (GPDH; EC 1.1.1.8) by hydrocortisone (HC). HC increased the total and specific activities of GPDH in dissociated cultures from all brain areas examined—cerebrum, cerebellum, and brainstem. HC, however, had no effect on the amount of total or soluble protein or on the specific activity of another soluble enzyme, lactate dehydrogenase (LDH; EC 1.1.1.27). The fully induced level of GPDH in 12-day cerebral cell cultures was about twofold greater than the specific activity of the adult rat cerebrum. The induction of GPDH was greater in cerebral cultures grown in Eagle's Basal Medium than in several other media tested. The inducibility of GPDH over a 48-hr period increased with time in culture until a maximum was reached around 21 days. The induction of GPDH by HC was inhibited by cycloheximide, actinomycin D, and cordycepin but was unaffected by cytosine arabinoside. These results indicate that protein and RNA (in particular mRNA) syntheses are required for the induction but that DNA synthesis is not required.

—Following hypophysectomy or adrenalectomy, glycerolphosphate dehydrogenase (GPDH) (EC 1.1.1.8) activity decreased exponentially in the cerebral hemispheres and brain stem of adult male rats. The latter region was more affected than the former. Malate dehydrogenase (EC 1.1.1.40), isocitrate dehydrogenase (EC 1.1.1.42), lactate dehydrogenase (EC 1.1.1.27) and mitochondrial glycerolphosphate dehydrogenase (EC 1.1.95.5) activities remained unchanged. Injection of adrenocorticotrophic hormone or cortisol in hypophysectomized rats or cortisol in adrenalectomized rats restored GPDH activity. Thyroidectomy and gonadectomy had no effect on GPDH activity. Liver GPDH was not decreased by hypophysectomy or adrenalectomy. Muscle GPDH was diminished slightly by adrenalectomy and as much as brain GPDH by hypophysectomy. In young rats GPDH developmental increase in activity was inhibited by hypophysectomy. These results clearly show that brain GPDH activity is specifically regulated by cortisol (and probably closely related corticosteroids).

—Following head X-irradiation (750 r) of 2-day-old rats the aerobic formation of lactate in the presence or absence of chloral was markedly increased in medulla slices but remained unchanged in cerebral cortex and brain stem slices. The ratio of lactate dehydrogenase (EC 1.1.1.27) to glycerolphosphate dehydrogenase (EC 1.1.1.8) was slightly increased in cortex and brain stem but more than doubled in medulla. The relation of the increased aerobic formation of lactate in irradiated medulla to the lactate dehydrogenase:glycerolphosphate dehydrogenase ratio is discussed.

HORMONAL regulation of enzyme levels in mammalian cells has been known for more than a decade1 and the use of homogeneous populations of glucocorticoid target cells has advanced our understanding of the molecular mechanisms involved2,3. Using a specific antiserum we have now shown that cortisol induces glycerol phosphate dehydrogenase (GPDH, EC 1118) in a rat brain tumour cell line by increasing the number of GPDH molecules rather than by increasing the activity of each molecule. Also, the induction does not involve isozymes since GPDH from induced and uninduced cells is identical with respect to immunological titration, electrophoretic mobility and thermal sensitivity.

The generation of adenosine 3',5'-monophosphate (cyclic AMP) in response to catecholamines in the 2B subclone of RGC6 rat glioma cells previously exposed to norepinephrine and refractory to further norepinephrine addition is substantially increased by addition of inhibitors of RNA and protein synthesis. The time course of the effect of these inhibitors on cyclic AMP concentration suggests that rapid protein synthesis and turnover are involved in catecholamine refractoriness. Norepinephrine induction of cyclic nucleotide phosphodiesterase is demonstrable in RGC6 cells but not in the 2B subclone. Thus, catecholamine refractoriness cannot be attributed to induction of phosphodiesterase. This implies that induction of a protein or proteins, important in catecholamine refractoriness, affects the synthesis rather than the degradation of cyclic AMP.

Glycerol-3-phosphate dehydrogenase ( sn -glycerol-3-phosphate: NAD + 2-oxidoreductase, EC 1.1.1.8) was isolated from rat brain tissue and its physical and kinetic properties investigated. The purification method resulted in the separation of fractions enriched for six other brain proteins and this procedure is presented. The apparent Michaelis constant ( K m ) for dihydroxyacetone phosphate is 0.17 mM whereas the K m for l -glycerol-3-phosphate is 0.30 mM. Under the conditions of the assay, the pH optimum occurs at pH 7.15. The molecular weight determined by Sephadex G-100 chromatography is 72 000 whereas ultracentrifugation in a sucrose gradient yields a molecular weight of 75 000. Furthermore using an antiserum prepared against purified rat brain glycerol-3-phosphate dehydrogenase, it was shown that rat brain glycerol-3-phosphate dehydrogenase is lighter than rabbit muscle glycerol-3-phosphate dehydrogenase ( M r 78 000). The purified enzyme is extremely sensitive to p -mercuribenzoate, being demonstrably inhibited by 10 nM, although it is much less sensitive to N -ethylmaleimide and iodoacetate. The specific activity of the purified enzyme is 152 units/mg of protein, representing a 2800-fold increase in specific activity. Analytical acrylamide gel electrophoresis separates the purified enzyme into two bands of protein, each of which exhibits glycerol-3-phosphate dehydrogenase activity. The isozymes differ from each in charge and neither one represents an oligomeric form of the other.

An affinity column was synthesized and utilized to partially purify glycerolphosphate dehydrogenase (L-glycerol-3-phosphate: NAD+ oxidoreductase, E.C.1.1.1.8) from rat skeletal muscle. The novelty of the column resides in the fact that the ligand used, 6-phosphogluconic acid, is neither an inhibitor nor a substrate of the enzyme when free in solution but when immobilized on an agarose matrix, glycerol phosphate dehydrogenase binds to it with a high degree of specificity. The bound enzyme could be eluted by either increasing the ionic strength or by addition of its natural substrate, α-glycerol phosphate. Using a combination of these methods and ammonium sulfate precipitation GPDH was purified about 250 fold with a 75% yield within 24 hours.

The rat brain glial tumor cell line RGC6 was originally selected for its high content of S100, a nervous tissue-specific protein (Benda et al., 1968). Subsequently, this cell line was shown to have retained other characteristic properties of brain tissue, such as the glycerol phosphate dehydrogenase induction by Cortisol (de Vellis and Inglish, 1969). Although not all tissues have been tested, this induction appears to be brain specific (de Vellis and Inglish, 1968). In addition, de Vellis and Inglish found that catecholamines induced lactic dehydrogenase in RGC6 cells but not in several other cell lines (de Vellis and Inglish, 1969; de Vellis et al., 1971b) Catecholamines, as we shall see later, also cause a large increase in the intracellular concentration of cyclic AMP. The magnitude of the response appears to be unique to nervous tissue, which has the highest level of adenylcyclase and phosphodiesterase (for review, see Robison et al., 1971). The function of cyclic AMP in the nervous system remains largely speculative. More generally, the role of hormones in the acquisition and maintenance of differention and functions of nervous tissue is still far from being understood (de Vellis and Clemente, 1970). In this chapter, we will discuss RGC6 cells as target cells for glucocorticoids and catecholamines, a rather unique opportunity to contrast the mechanisms of action of these two hormones in the same cell line. Furthermore, studies of these hormonal factors and, of course, S100 protein may provide insights into the functions of glial cells, which are essentially unknown except for the role of oligodendrocytes and Schwann cells in myelination of axons.

Myelin has been isolated from brain stems of control rats and their head-irradiated littermates (700 rads at 2 days of age). Rats were sacrificed at 20, 30, 40, and 180 days of age. Much less myelin was found to be deposited in brain stems of head-irradiated rats as compared to controls. However, within experimental error, no differences were ascertained, as functions of either age or irradiation, in major building blocks of myelin. These include total protein, total sterol, phosphatidyl serine, sphingomyelin, phosphatidyl choline + lyso-phosphatidyl choline, phosphatidyl ethanolamine + phosphatidal ethanolamine, and cerebroside + cerebroside sulfate. However, the proportions of all normal unsaturated fatty acids in myelin from irradiated rats were lower with respect to their saturated counterparts than in control myelin. Markedly lower ratios of both monounsaturated C24 acid and monounsaturated C26 fatty acid to their saturated counterparts were noted, and the proportion of total C26 to total C24 fatty a...