Triton X-100 promotes the accumulation of phosphatidic acid and inhibits the synthesis of phosphatidylcholine in human decidua and chorion frondosum tissues in vitro.

Triton X-100 is known to affect phospholipid metabolism and the generation of various signal molecules from cellular phospholipids. In the present work the effect of Triton X-100 on phospholipid metabolism of human decidua and of the primordial placenta (chorion frondosum) was studied. Triton X-100 (0.05%, v/v) added to tissue mince 30 min before the end of a 60 min incubation stimulated 2-4-fold (decidua) and 4-6-fold (placenta) the incorporation of [32P]phosphate ([32P]Pi) into phosphatidic acid, while markedly decreasing the labeling of phosphatidylcholine. Triton X-100 had no effect on the labeling of phosphatidylinositol in the decidua, and only a slight increase was observed in the placenta. When labeled glucose was used to assess phospholipid synthesis, the addition of Triton had no effect on phosphatidic acid, while decreasing the synthesis of phosphatidylcholine. Incorporation of [32P]Pi into phosphatidic acid was not accelerated by a submicellar concentration (0.01%) of Triton, whereas the synthesis of phosphatidylcholine was decreased irrespective of detergent concentration. Anionic or cationic detergents could not mimic the action of Triton on phosphatidic acid synthesis. Although Triton inhibited the synthesis of ATP in a dose-dependent manner, this could not account for the above results. Instead, it is suggested that diacylglycerol kinase and phosphocholine:CTP cytidylyltransferase are possible targets of the action of Triton X-100.

Conversion of arachidonic acid to prostanoids in the avian uterus.

In an effort to obtain information on the possible source of prostaglandins which have been shown to play an important role in oviposition we examined the metabolism of arachidonic acid in microsomal preparations of both the muscular and the glandular tissue of the hen uterus. We found that adrenaline and tryptophan (but not hydroquinone) were effective stimulators of prostanoid synthesis. On incubation with [3H]arachidonic acid we identified, using TLC radiochromatography and several solvent systems, prostaglandins F2 alpha and E2 and, predominantly, thromboxane B2 which could not be attributed to platelet contamination. Addition of reduced glutathione increased prostaglandin E2 formation at the expense of thromboxane B2 and at 1 mM concentration suppressed adrenaline-promoted prostanoid synthesis. While the former effect has been documented in many other systems and could be ascribed to the activation of prostaglandin H2 to prostaglandin E2 isomerase, the latter effect is postulated to be due to an inhibition of cyclooxygenase. Interestingly, this inhibitory effect was shared by a number of reducing agents. Although the subcellular preparations were derived from structurally and functionally different tissues, there was no qualitative difference with respect to prostanoid synthesis. Our data support the role of locally produced primary prostaglandins in the regulation of oviposition and raise the question of a potential role for thromboxane in this process.

The relation between thromboxane and prostaglandin synthesis in human decidua tissue: a comparison of eicosanoid synthesis in minced tissue with that in a cell-free preparation.

Radiotracer studies and radioimmunoassay measurements demonstrate that minced tissues of human decidua produce chiefly thromboxane B2 (TxB2) (70% of total eicosanoids) and small amounts of prostaglandin F2 alpha (PGF2 alpha) (13%) PGD2 (8%), 6-keto-PGF1 alpha (5%) and PGE2 (4%). Inhibition of thromboxane synthesis with a specific inhibitor (OKY-1581: sodium (E)-3-[4(-3-pyridylmethyl)-phenyl]-2-methyl propenoate) increased prostaglandin formation in general, with the main product being PGF2 alpha (38%), a nonenzymic derivative of PGH2. Crude particulate fractions prepared from the same tissue synthesized two major products from [3H]arachidonate, TxB2 and 6-keto-PGF1 alpha (54 and 30%, respectively) and some PGF2 alpha and PGE2 (8-8%). However, in the presence of reduced glutathione (GSH), PGE2 became the main product (81%) (TxB2, 15%; PGF2 alpha, 2%; and 6-keto-PGF1 alpha, 2%). Half-maximal stimulation of PGE2 synthesis occurred at 46 microM GSH. The GSH concentration of tissue samples was found to be 110 +/- 30 microM. We conclude that human first trimester decidua cells possess the key enzymes of prostaglandin and thromboxane synthesis. Apparently, the production of these compounds is controlled by a specific mechanism in the tissue, which keeps PGE and prostacyclin synthesis in a reversibly suppressed state, whereas the formation of thromboxane is relatively stimulated.

Studies on growth hormone secretion. VII. Effects of somatostatin on plasma GH, insulin, and glucagon in sheep.

To determine whether synthetic somatostatin originally isolated from sheep hypothalamus can inhibit hormone secretion in the same species, we measured plasma levels of GH, insulin, glucagon, and glucose of normal sheep under a variety of experimental conditions in the presence and absence of somatostatin infusion. An oral dose of 2.5 mg./kg. 3,5-dimethypyrazole increase plasma GH from 10.9 to 376.9 ng. per milliliter, which was suppressed by 50 per cent and 80 per cent with 0.5 and 1 mg. synthetic cyclic somatostatin, respectively. Linear somatostatin (0.5 mg.) was without effect in two animals tested. Propionate (0.5 mmole per kilogram) and arginine (10 gm.) induced a rise in plasma insulin and GH, and glucagon was effectively blocked by cyclic somatostatin (0.5 mg.). Similarly, somatostatin inhibited glucose, and glucagon provoked GH and insulin secretory responses without affecting glucose or FFA levels. Somatostatin had no effect on the disappearance of injected glucagon. Finally, addition of somatostatin to incubation media prevented PGE promoted GH release, and suppressed cyclic AMP accumulation, although to a lesser extent, in sheep anterior pituitary pieces. In view of the large amounts required to suppress stimulated hormone release and the general lack of specificity of somatostatin, it is suggested that this peptide may have a functional role only in the release of hormones of the pituitary, where it could occur in relatively high local concentrations. Its inhibition of extrapituitary hormone secretion may be purely a pharmacologic effect that, nevertheless, suggests an interference with a step common to the secretory process of hormones.

Selective binding of prostaglandin F2 alpha to membrane fractions enriched in 5-nucleotidase, Ca2+-ATPase, and Mg2+-(Na+ + K+)ATPase.

Homogenates of uterine smooth muscle of laying hens were fractionated by differential centrifugation. Three of the five fractions thus obtained were further separated on sucrose gradient into four subfractions. By employing various enzyme markers, such as 5'-nucleotidase, Mg2+-(Na+ + K+)-ATPase, alkaline phosphatase, Ca2+-ATPase, cytochrome oxidase, acid phosphatase, and N-beta-acetyl glucosaminidase, the major subcellular fractions have been tentatively identified. Using gel filtration to separate bound and free prostaglandin F2 alpha (PGF2 alpha) we found that the specific uptake of PGF2 alpha was highest in the subfractions which also exhibited the highest enrichment in enzymes viewed generally as markers for cell membrane. The results suggest that PGF2 alpha-induced contractile activity in this tissue is initiated by the specific interaction of this agonist with discreet receptors in the sarcolemma, rather than by binding of PGF2 alpha to intracellular organelles or to a cytosolic receptor.

Luteinizing hormone-induced intracellular calcium mobilization in granulosa cells: comparison with forskolin and 8-bromo-adenosine 3',5'-monophosphate.

Digitonin-permeabilized avian granulosa cells were used as a model to study the effects of LH, 8-bromo-cAMP (8-Br-cAMP), and forskolin on intracellular calcium mobilization. LH caused a biphasic calcium efflux. There was a small but significant (P less than 0.05) calcium release within 1-2 min (rapid phase) with an ED50 of 35 ng/ml, followed by a larger one after 5 min (slow phase) with an ED50 of 10 ng/ml. The intracellular calcium antagonist TMB-8 inhibited the action of LH in both phases. Forskolin had no effect on calcium efflux during the rapid phase. However, it stimulated intracellular calcium efflux in a dose-dependent manner in the slow phase with an ED50 of 30 microM. Low doses of 8-Br-cAMP caused a small but significant increase in calcium uptake corresponding to the rapid phase, although high concentrations of 8-Br-cAMP caused efflux in the slow phase (ED50, 0.7 mM). Both LH and 8-Br-cAMP mobilized calcium from a nonmitochondrial pool, whereas forskolin induced calcium efflux from a nonendoplasmic reticular site. Apparently receptor-mediated (LH) and nonreceptor-mediated (forskolin and 8-Br-cAMP) intracellular calcium mobilization are qualitatively different, suggesting that rapid intracellular calcium mobilization may represent one of the initial steps in the mechanism of action of LH.

Clomiphene and tamoxifen inhibit progesterone synthesis in granulosa cells: comparison with estradiol.

The actions of clomiphene, tamoxifen, and 17 beta-estradiol were studied in an isolated avian granulosa cell system during short term incubations. A dose-dependent inhibition of ovine LH-enhanced progesterone production was observed for all three compounds, with an IC50 of 3 X 10(-6) M and maximal inhibition (95%) at 5 X 10(-5) M. A similar inhibition was found when cells were stimulated by either 8-bromo-cAMP or forskolin. Neither clomiphene nor tamoxifen had an inhibitory effect on the conversion of pregnenolone to progesterone, a step that was blocked by 17 beta-estradiol and isoxazole , a known inhibitor of 3 beta-hydroxysteroid dehydrogenase. On the other hand, cells exposed to clomiphene failed to use [6-3H]25-hydroxycholesterol, while estradiol significantly increased the conversion of labeled substrate to pregnenolone at the expense of progesterone (30% vs. 7% of the total radioactivity). By comparison, in control cells, progesterone represented the major metabolite with 36% of the total radioactivity; pregnenolone had only 2.8%. The results demonstrate that these triphenylethylene compounds, which are used clinically as antiestrogens, inhibit steroidogenesis at a step before pregnenolone formation, probably at the site of the cholesterol side-chain cleavage enzyme complex, while 17 beta-estradiol inhibits 3 beta-hydroxysteroid dehydrogenase.

Somatostatin inhibits urinary cyclic AMP excretion in diabetic rats.

Short term (30 min) infusion of cyclic somatostatin (50 microgram/rat), insulin (1 U/rat) or the two together significantly suppressed urinary cyclic AMP excretion in streptozotocin-diabetic rats. While somatostatin tended to increase cyclic GMP excretion, insulin had an opposite effect in diabetic but not in normal rats. It is suggested that somatostatin suppresses cyclic AMP excretion by inhibiting directly adenylate cyclase in liver and perhaps in other organs. The possibility that suppression of urinary cyclic AMP is due to inhibition of glucagon secretion is also considered.

Regulation of intracellular free calcium in human myometrial cells by prostaglandin F2 alpha: comparison with oxytocin.

The effects of prostaglandin F2 alpha (PGF2 alpha) on intracellular Ca2+ concentration ([Ca2+]i) and inositol phosphate (IP) generation in human myometrial cells were evaluated and compared to the effects of oxytocin. Basal [Ca2+]i levels were 146 and 153 nM in the absence and presence of 1 mM extracellular Ca, respectively. In Ca-containing medium, both PGF2 alpha and oxytocin significantly (P less than 0.01) increased [Ca2+]i over control values, eliciting half-maximal stimulation (ED50) at 4 and 1 nM, respectively. In Ca-free medium the potency of PGF2 alpha to raise [Ca2+]i was drastically reduced (ED50, 2 microM), whereas that of oxytocin remained the same, although maximal responses were markedly decreased. PGF2 alpha had no effect on total IP production in the concentration range that significantly raised [Ca2+]i. However, at a 100 times higher concentration (10 microM), PGF2 alpha produced a maximum 48% increase in total IP, with a rapid (15-30 s) rise in IP3 and IP2, followed by IP1. A similar increase in IP production was obtained when [Ca2+]i levels were raised by A23187 to the same level as that obtained with 10-50 microM PGF2 alpha. The effect of PGF2 alpha was dependent on extracellular Ca and could be suppressed by verapamil, but not by pertussis toxin, or phorbol ester. In contrast, the potencies of oxytocin to raise IP and [Ca2+]i were similar and independent of extracellular Ca2+, and could be suppressed by pertussis toxin and phorbol ester, but not by verapamil. These data provide evidence that in isolated human myometrial cells, PGF2 alpha and oxytocin trigger an increase in [Ca2+]i by different mechanisms. The action of PGF2 alpha depends on extracellular Ca2+, whereas oxytocin activates the G-protein-dependent phospholipase-C-IP3-Ca2+ signal-transducing pathway, complemented by the influx of extracellular Ca2+.

Regulation of myometrial smooth muscle functions.

Regulation of myometrial functions during gestation, labor and birth are in the forefront of research in reproductive sciences. The complexity of the problem is reflected by our scant understanding of the intimate cellular and molecular events underlying these phenomena, despite extensive efforts spanning several decades. Unlike other smooth muscles, the myometrium is, to a large extent, under hormonal control. Of these, the steroid hormones, progesterone and estrogen, play dominant roles in terms of uterine growth, the maintenance of quiescence during gestation and the preparation of the uterus for labor and delivery. In addition to steroid hormones, there are a number of factors that modulate myometrial contractility (oxytocin, prostaglandins, endothelin, platelet activating factor) and relaxation (corticotropin releasing hormone, prostacyclin, nitric oxide). Although notable advances have been made towards understanding some of the key steps in receptor signaling that define the actions of these factors, a good deal of new information is needed to fully understand this fundamental life process. Pharmaceutical agents have been used extensively to induce labor or to prolong pregnancy in the case of preterm labor that represents the major cause of perinatal morbidity and mortality. Because preterm labor is a syndrome of multiple etiologies, pharmacologic agents will have to be targeted accordingly. This review attempts to present a critical overview of these topics.

Calcium-dependent nitric oxide synthesis is potently stimulated by tetrahydrobiopterin in human primordial placenta.

Homogenized first trimester human placenta exhibits both Ca(2+)-dependent (90-95 per cent) and Ca(2+)-independent (5-10 per cent) nitric oxide (NO)-synthesizing activities. Addition of tetrahydrobiopterin (BH4) to homogenates containing Ca2+ in maximally activating concentrations (> 0.5 microM) results in a further 2-2.5-fold activation of NO synthesis, with half-maximal stimulation observed at 26 +/- 8.2 microM BH4 (mean +/- SEM, n = 4). Chelation of Ca2+ in the medium abolishes the stimulatory effect, indicating that only a Ca2(+)-dependent NO-synthase (NOS) isoform is activated by BH4. Based on our previous findings, we suggest that this isoform is the endothelial or Type III NOS. Importantly, BH4 has no significant effect on the Ca2(+)-dependency of NOS activity, the apparent Km values for Ca2+ are comparable in the absence (1.8 +/- 0.4 microM, mean +/- SEM, n = 6) or presence (2.5 +/- 0.6 microM, mean +/- SEM, n = 6) of 50 microM BH4. The BH4 content of these placentae is 207.4 +/- 86.7 pmol/g wet tissue (mean +/- s.d., n = 9), therefore, BH4 added to the homogenate does not simply restore the concentrations that occur endogenously. The results provide the first evidence that in the early human placenta, a constitutively expressed CA 2(+)-dependent NOS isoform is stimulated by exogenous BH4, raising the possibility that BH4 is an important regulator of NOS activity in this tissue. This novel aspect of the NO-generating pathway may have implications in the aetiology and treatment of pregnancy-induced hypertension and pre-eclampsia.

Dual action of arachidonic acid on calcium mobilization in avian granulosa cells.

The primary aim of this study was to evaluate the effects of arachidonic acid (AA) on calcium mobilization from intracellular compartments in digitonin-permeabilized granulosa cells isolated from the largest preovulatory follicles of laying hens. At low concentrations (ED50 0.2 microM) AA released 35% 45Ca from the endoplasmic reticulum (ER), whereas at higher concentrations (ED50 16 microM) it stimulated 45Ca efflux from mitochondria. These effects of AA were mimicked at 10-20 times lower concentration by the calcium ionophore A23187. Inositol 1,4,5-trisphosphate (IP3) also stimulated 45Ca efflux from the ER, with a markedly lower potency than AA (ED50 6.2 microM), as well as exhibiting a biphasic response. Heparin abolished the effect of IP3 and luteinizing hormone (LH), but it had no influence on AA-promoted 45Ca efflux. Moreover, the actions of IP3 and AA were additive, indicating that AA and IP3 access different Ca pools in the ER by different mechanisms. Several other unsaturated fatty acids also stimulated 45Ca mobilization from both ER and mitochondria but, with the exception of eicosapentaenoic acid, were significantly less effective than AA. It is concluded that free AA, at submicromolar concentrations that might be viewed as physiological, is a potent calcium mobilizing agent and thus may play an important role in signal transduction in avian granulosa cells, akin to that of IP3. At high (greater than 10 microM) concentrations AA removes Ca2+ from the mitochondria, an action that may be responsible for its reported inhibitory effects on steroidogenesis and other cellular functions.

Proposed signaling role of arachidonic acid in human myometrium.

The objective of this study was to test the hypothesis that, in human myometrial cells (HMC), PGF2 alpha and oxytocin promote the release of arachidonic acid (AA) which, in turn, acts to mobilize intracellular Ca2+. Primary monolayer cultures of HMC were labeled with [3H]arachidonic acid ([3H]AA) to isotopic equilibrium before exposure to PGF2 alpha or oxytocin. Radiolabeled phospholipids were separated on thin layer chromatography and quantitated by scintillation counting. Prostanoids were analyzed by high performance liquid chromatography. Calcium release was quantitated in digitonin-permeabilized myocytes preloaded with 45Ca, in the presence of ATP and ruthenium red. PGF2 alpha (10(-7) M) caused a rapid (peaking at 2 min), and significant (P < 0.01) increase in [3H]AA release that was derived selectively from phosphatidylethanolamine (PE), indicative of phospholipase A2 activation. Oxytocin caused a rapid (30 s) and significant increase in diacylglycerol, concomitant with a drop in phosphoinositides, as well as an increase in [3H]AA and a fall in PE and phosphatidylcholine. Exogenous AA caused a rapid and dose-related efflux of 45Ca2+, which was not inhibited by blockers of AA metabolism, or by heparin that abolished inositol 1,4,5-trisphosphate-induced 45Ca2+ release. It is concluded that PGF2 alpha and oxytocin promote, by different mechanisms, the release of AA, which in turn may amplify their action by enhancing Ca2+ mobilization from the sarcoplasmic reticulum, thereby fulfilling the role of intracellular signaling molecule in human myometrium.

Signal transduction in rat myometrial cells: comparison of the actions of endothelin-1, oxytocin and prostaglandin F2 alpha.

The objectives of this study were to evaluate and compare the actions of endothelin-1 (ET-1), oxytocin, prostaglandin F2 alpha (PGF2 alpha) and inositol 1,4,5-trisphosphate (IP3) on 45Ca2+ mobilization in permeabilized rat myometrial cells and to examine the activation of the inositol lipid cycle in intact myocytes. Cells were isolated from late pregnant rat myometrium and used as confluent monolayers after a single passage. All four agonists caused a biphasic release of 45Ca2+ from non-mitochondrial pool(s), with the rank order of potency: oxytocin > PGF2 alpha > ET-1 > IP3. Inhibitors of phospholipase C blocked ET-1- and oxytocin-promoted but not PGF2 alpha-promoted 45Ca2+ efflux. Similarly, heparin, an IP3 receptor blocker, failed to inhibit PGF2 alpha-induced 45Ca2+ release while inhibiting the action of the other agonists. Endothelin-1 and oxytocin stimulated inositol phosphate accumulation at concentrations similar to those that promoted 45Ca2+ efflux, whereas about 100 times higher concentrations of PGF2 alpha were needed to activate this signaling pathway in intact cells. It is concluded that the primary action of PGF2 alpha in myometrial cells is to enhance Ca2+ influx, whereas oxytocin and ET-1 receptors are coupled to phospholipase C, generating IP3 and raising the intracellular concentration of free Ca2+ from intracellular as well as extracellular sources.

Metabolism of progesterone by avian granulosa cells in culture.

Previous studies have demonstrated that progesterone is the primary product of steroidogenesis in avian granulosa cells during short-term incubation. However, during more prolonged culture, lasting several days, the progesterone content in the medium was found to decrease progressively, indicating in vitro metabolic conversion. In the present study we have isolated and identified a number of progesterone metabolites. Granulosa cells, isolated from mature ovarian follicles of laying hens, were cultured in medium 199 supplemented with fetal calf serum and containing [14C]progesterone. After 4 days in culture, cells + media were extracted and the radioactive metabolites separated and identified by TLC, HPLC and GC-MS. Several of the metabolites were further characterized by derivatization and crystallization to constant specific activity. A total of 24 radioactive substances was detected. Of these, 15 have been positively identified, 5 tentatively and the remaining 4 are unidentified. The principal metabolite, representing more than 45% of the total radioactivity, was identified as 3 alpha-hydroxy-5 beta-pregnan-20-one. In addition, significant amounts of 3 alpha-hydroxy-5 alpha-pregnan-20-one (5.76%), 5 beta-pregnane-3,20-dione (3.05%), and 5 alpha-pregnane-3,20-dione (2.95%) were detected and identified. The results indicate that avian granulosa cells possess 3 alpha-hydroxy-steroid dehydrogenase (3 alpha-HSD), 17 beta-HSD, 20 alpha-HSD, 20 beta-HSD, 17 alpha-hydroxylase, C17-20-lyase and 5 alpha- and 5 beta-reductase activities. These enzyme activities may convert progesterone to biologically inactive or less active metabolites. However, a functional role for some of these metabolites cannot be ruled out.

Adipose tissue metabolism in essential fatty acid deficienty. Effects of prostaglandin e1, epinephrine, and ACTH.

In an effort to better define some of the metabolic changes that accompany essential fatty acid deficiency (EFAD), we studied glucose metabolism in adipose tissue of EFAD and normal mice under basal conditions and in the presence of prostaglandin E1 (PGE1), epinephrine, and ACTH1-18. Isolated fat cells were incubated in Krebs-Ringer bicarbonate medium containing glucose 1(-14C) or 6(-14C), and the incorporation of radioactive carbon into CO2, total fat, fatty acids, and glyceride-glycerol was determined. It was found that EFAD increased glucose uptake over controls which could be attributed to increased oxidation to CO2 and fatty acid synthesis. The contribution of the pentose cycle to glucose oxidation was 50-80% higher in EFAD adipocytes as compared to controls. ACTH1-18 (0.1 mug/ml) suppressed this by 18 and 30% in the control and EFAD groups, respectively, while epinephrine decreased pentose cycle activity by 83 and 55% in the two groups, respectively. PGE1 alone had no significant effect, but in combination with epinephrine it abolished the inhibitory action of the catecholamine in both groups. It is suggested that, although EFA serve as prostaglandin precursors, the effects of EFAD on the metabolism of fat cells cannot be reversed by PGE1, in vitro. However, these fat cells retain their responsiveness to the action of both lipolytic (ACTH and epinephrine) and antilipolytic (PGE1) agents.

Pressor responsiveness to endothelin is not attenuated in gravid rats.

The aim of this study was to evaluate the effect of synthetic human/porcine endothelin (ET-1) on mean arterial blood pressure (MAP) in pregnant and non-pregnant rats and to compare this to the effects of two well characterized agonists, angiotensin II (AII), and vasopressin (VP). On day 14 of gestation (parturition day 22) polyethylene catheters were chronically implanted in the abdominal aorta for monitoring of MAP and in the vena cava for administration of drugs. Pressor responsiveness was measured in conscious freely moving animals on day 20 and again on the 7th day post-partum. All three agonists increased MAP in a dose related manner. However, whereas the sensitivity of pregnant rats (P) to AII and VP was significantly blunted compared to postpartal (PP) measurements, the MAP responses to ET-1 were the same in both groups. Moreover, the combined administration of ET-1 at a subpressor dose (0.05 pmol/100 g bw) and AII or VP at effective doses significantly potentiated (particularly in P) the pressor effects of AII and VP. These results demonstrate that ET-1 and possibly other vasoactive substances of endothelial origin, override the compensatory mechanism of normal pregnancy with respect to the blunted responsiveness to AII and VP. Such a mechanism may be of particular relevance in the evolution of pregnancy-induced hypertension.

Some effects of medroxyprogesterone acetate on intermediary metabolism in rat liver.

PIP: This study examines the effects of MPA (medroxyprogesterone acetate) on some of the hepatic enzymes of carbohydrate and lipid metabolism in the rat, and compares these with the effects of cortisol and saline. Levels of reduced nicotinamide adenine dinucleotide phosphate (NADPH) were also measured. Intact mature female Wistar rats with average initial weight of 200 gms were injected with MPA (mO mg/kg IM) once a week for 4 weeks and were sacrificed 3 to 5 days after the last injection. Hydrocortisone (Solu-Cortef [R]) 40 mg/kg IM were given to cortisol-treated animals twice daily for 7 days. The animals were sacrificed 2-4 hours after the last dose was given. Normal saline (0.2 mg. IM) was injected in control animals twice a day. The method of Jellinek, Amako, and Willman was used to analyze NADPH. Liver samples were assayed for various enzymatic activities such as phophofructokinase (PFK); pyruvate kinase (PK), glycerol-3-phosphate dehydrogenase (G3PD), "malic" enzyme (ME), and glucose-6-phosphate dehydrogenase (G6PD). The methods of Colowick and Kaplan were used in enzymatic analyses. Lipogenic stimulation by MPA is indicated by increased levels of G3PD and ME, both of which are implicated in lipogenesis, as well as by NADPH. PFK, PK, and G6PD were all unaffected by the MPA regimen, suggesting that elevation of ME and NADPH activities may reflect increased amino acid conservation. The enzymatic pattern of MPA treatment shows lipogenesis and protein conservation, while that of cortisol regimen shows significantly lower levels of ME, G3PD, and PRK.^ieng