Martha K. Young

Effect of Indomethacin on cyclic AMP phosphodiesterase activity in myometrium from pregnant rhesus monkeys.

Our results indicate that indomethacin inhibits cyclic AMP phosphodiesterase in the myometrium of the pregnant rhesus monkey under in vitro as well as in vivo conditions. Kinetic data on extracts of myometrium from pregnant rhesus monkeys indicated two cyclic AMP phosphodiesterase activities. The apparent Km value for the high affinity enzyme averaged 3.9 muM and for the low affinity enzyme 23 muM; the Vmax values averaged 0.56 and 1.4 nmoles cyclic AMP hydrolized per mg protein min-1 respectively. When indomethacin was added to the myometrial extracts, the activity of the high Km phosphodiesterase was competitively inhibited, with an average Ki of 200 muM; the low Km enzyme was noncompetitively inhibited with an average Ki of 110 muM. Experiments on myometrial slices demonstrated that 10 muM indomethsacin potentiated the effect of PGE1 and epinephrine on cyclic AMP levels, presumably by inhibiting the phophodiesterase activity. The uterine relaxing effect of indomethacin is generally attributed to the inhibition of prostaglandin synthetase activity. However, treatment of pregnant rhesus monkeys with therapeutic doses of indomethacin resulted in a significant inhibition of myometrial cyclic AMP phosphodiesterase activity in association with uterine relaxation and prolongation of gestation.

Effect of insulin and epinephrine on the carbohydrate metabolism and adenylate cyclase activity of rhesus fetal muscle.

Extract: Carbohydrate metabolism of skeletal muscle from rhesus fetuses at 58% of gestation (95 days) is sensitive to epinephrine in vitro. Epinephrine increased lactate production and decreased glucose uptake, 14C-lactate production, glycogen content, and 14C-glycogen formation as well as 14CO2 production. These responses to epinephrine are similar to those in adult muscle. However, in some cases the magnitude of these responses appears lower in fetal muscle. The content of cyclic 14C-adenosine 3‘,5’-monophosphate (cyclic 14C-AMP) was about 2.5-fold higher in the 100-day fetal muscle than in the adult. Epinephrine stimulated adenylate cyclase activity almost threefold in fetal and fourfold in adult muscle. When incubated with muscle from 85-day fetal monkeys, insulin increased glucose uptake, lactate and lactate-14C production, and 14CO2 production; the greatest effect was found in the increased incorporation of labeled glucose into glycogen. Both synthetase I and phosphorylase a activities were present at 78–80 days of fetal age. Our data show that as early as about 58% of term the carbohydrate metabolism of fetal rhesus muscle in vitro is sensitive to epinephrine and that the hormone probably acts through the adenylate cyclase and the “second messenger” system of cyclic AMP as it does in adult muscle.Speculation: Our data offer indirect evidence that insulin and epinephrine mediate glycogen metabolism via cyclic AMP in rhesus fetal muscle as early as 85 days of gestational age (52% of term) and that these hormones operate through similar enzyme systems in fetal and adult muscle. It is possible that glycogen synthetase and phosphorylase, or other enzymes mediating the cyclic AMP response, are not identical in fetal and adult muscle; fetal isoenzyme patterns for muscle lactate dehydrogenase differ markedly from those of the adult. However, it is difficult to reconcile the existence of major differences in the enzyme milieu in fetal and adult muscle with the similar overall actions of epinephrine and insulin on glycogenesis and glycogenolysis demonstrated in our experiments.

Aging: Effects on the prostaglandin production by skeletal muscle of male rhesus monkeys (Macaca mulatta)

Prostaglandin (PG) production from [1-14C] arachidonic acid was measured in homogenates of sartorius muscle of aged (more than 20 years) and young adult (7 to 11 years) rhesus monkeys. Total production by the aged series (3.21 +/- 0.31 (S.E.) nmol PG per g N per min) was about two times that of the young series (1.76 +/- 0.14 (S.E.) nmol per g N per min). Epinephrine-stimulated PG production was also twice as great in the aged muscle series as in the young adult series (7.22 +/- 0.74 (S.E.) and 3.54 +/- 0.52 (S.E.) nmol per g N per min). This difference was mainly due to greater production of 6-keto-PGF1 alpha and PGF2 alpha. In both series, the addition of 500 microM epinephrine to the assay media significantly increased production of all types of PGs measured (6-keto-PGF1 alpha, PGF2 alpha, PGE2 plus thromboxane B2 [TXB2], the stable breakdown product of TXA2, and PGD2). The absolute increases in all types of PGs studied in the presence of epinephrine were significantly greater in the aged muscle, with the exception of PGF2 alpha. The distribution patterns of the various types of PGs studied were similar in all series, except for 6-keto-PGF1 alpha; this PG accounted for a greater percentage of the total production by aged muscle than by young adult muscle (p less than 0.05).

Glucose Utilization of Cardiac and Skeletal Muscle Homogenates from Fetal and Adult Rhesus Monkesy

Extract: The oxygen consumptions and CO2 productions were similar for the homogenates of adult and 113-day fetal muscle from rhesus monkeys but higher in the 155-day fetal series (Table I). The percentages of total CO2 and the micromoles of CO2 arising from glucose plus glycogen (carbohydrate) were the same in homogenates of fetal and adult skeletal muscle. No difference was observed between the metabolism of homogenates of adult and neonatal muscle.In homogenates of cardiac muscle, the oxygen consumption and CO2 production in fetal muscle were higher at 113 days than in adult muscle; by 155 days, values in the fetal and adult muscle were similar (Table I). The percentage of total CO2 arising from carbohydrate and the micromoles of CO2 produced from the oxidation of carbohydrate were higher in both fetal series than in the adult. The oxygen consumption, CO2 production, and micromoles of CO2 arising from carbohydrate per gram protein per 30 min were two- to sevenfold higher in cardiac than in skeletal muscle, the greatest difference occurring in the 113-day fetal series. However, the percentages of total CO2 arising from carbohydrate were remarkably similar; the only difference was seen in the adult series where skeletal muscle was relatively more dependent on carbohydrate as a substrate than cardiac muscle. All these parameters were also measured in homogenates of cardiac muscle from neonates, and no difference in metablism was observed between the neonatal and adult series.In the fetal homogenates from both types of muscle, a major portion, 65 to 73%, of the total CO2 produced originated from a source or sources other than glucose and glycogen. Thus, both skeletal and cardiac homogenates of fetal, neonatal, and adult muscle are potentially able to obtain a large portion of their energy from the oxidation of substrates other than glucose and glycogen.Speculation: In vitro experiments from our laboratory demonstrate that 65% or more of the total CO2 produced by skeletal and cardiac muscle homogenates originated from the oxidation of substrates other than glucose and glycogen in the fetal as well as in the adult series; the data from broken cell preparations (homogenate) and previous data from intact cell preparations (muscle fiber groups) are similar. The possibility that other tissues and organs of the fetus utilize noncarbohydrates as an energy source should be investigated. Tsoulos et al. have reported that fetal lambs obtain a maximum of 50% of their metabolic requirements from glucose utilization under presumably normal conditions.

Estradiol-17 beta and progesterone: effects on guanylate cyclase activity in the myometrium of macaques.

Abstract Guanylate cyclase activity was determined in various subcellular fractions of myometrium (1) from rhesus monkeys spayed for at least 6 months and treated either with estradiol-17β (E2) for 14 days or with E2 for 14 days and then for 5 to 14 additional days with E2 plus progesterone (P) and (2) from cynomolgus monkeys during the follicular and luteal phases of natural menstrual cycles. Plasma levels of E2 and P were similar in the spayed rhesus monkeys treated with hormones and the naturally cycling cynomolgus monkeys. In the 100,000g supernatant and particulate fractions of macaque myometrium before and after treatment with Triton X-100, the specific activities (per milligram nitrogen or DNA) of the guanylate cyclase were less in the luteal than in the follicular phases of both natural and induced menstrual cycles. The hormonal effects appear specific for myometrial smooth muscle since there was no difference in the guanylate cyclase activity of intestinal smooth muscle (taenia coli) in the follicular and luteal phases. The effect of varying concentrations of Mn2+ on myometrium at different phases of an induced menstrual cycle was studied after homogenization with 1 mM EDTA. Guanylate cyclase activity in the 100,000g supernatant and particulate were higher in the “follicular” than in the “luteal” phase at all levels of Mn2+ studied. At the lower levels of Mn2+ the addition of 3 mM Ca2+ increased guanylate cyclase activity in the 100,000g supernatant fraction and decreased this activity in the 100,000g particulate fraction. The Ca2+-induced increment in guanylate cyclase activity was greater in “luteal” than “follicular” myometrium, whereas the Ca2+-induced decrement in the activity of the particulate fraction was greater in “follicular” than in “luteal” myometrium. It appears that cyclic nucleotide metabolism in nonhuman primate myometrium varies significantly during the menstrual cycle.

Control of Glycolysis in Skeletal Muscle from Fetal Rhesus Monkeys

Extract: In our studies of metabolic control mechanisms in skeletal muscle from rhesus fetus we have determined the tissue levels of the metabolic intermediates and cofactors of the glycolytic pathway and have calculated the mass-action ratios for each reaction. Skeletal muscle from rhesus fetuses (Macaco mulatto), 90–155 days of gestational age, and from adult rhesus monkeys was used in these experiments.The apparent equilibrium constants for hexokinase and phosphofructokinase (PFK) in these tissues were over 1,000 times larger than the massaction ratios at all ages studied; the corresponding values for pyruvate kinase were more than 800 times different. The data suggest that these three enzymes are rate-limiting for fetal skeletal muscle as early as 54% of gestation. The next step was to study some of the numerous factors that modify these nonequilibrium reactions. Increasing the ATP concentration had a marked effect on the PFK activity of both fetal and adult muscle, first increasing and then inhibiting enzyme activity. At maximum PFK activity, the amount of fructose-6-PO4 (F6P) phosphorylated per mg of protein was 2–3 times greater in the two fetal than in the adult series. At a concentration of 0.3 mM, citrate decreased PFK activity of the 100-day fetal muscle; a further decrease occurred at 1.2 mM citrate. At a citrate level of 0.3 mM, the addition of inorganic phosphate (P1) or cyclic AMP returned PFK activity to the uninhibited levels (pH 7.0). Relief of ATP inhibition of F6P phosphorylation with P1 and cyclic AMP was also observed at pH 7.0 in extracts of 100-day fetal skeletal muscle.Speculation: Control of carbohydrate metabolism in the fetus and newborn (30, 31) has recently become a point of considerable interest. For example, it is now known that the mammalian fetus possesses an endocrino~ogic autonomy and many hormones have been identified in the fetal circulation early in gestation. The mere presence of a hormone, however, does not necessarily imply that it is physiologically or biochemically active since tissue responsiveness may not have developed. It is generally agreed that in the adult, one of the primary effects of many hormones is to change the intracellular concentration of cyclic AMP (26). However, for the hormone to have its effect the cell must be capable of responding to such a change. In an earlier paper (7), we suggested that insulin and epinephrine affect carbohydrate metabolism in rhesus fetal muscle by changing the level of cyclic AMP. But even the simplest hormonemediated action has its origin in a chain of events, every link of which must be intact if the system is to work. These considerations are relevant to the perinatal period, for the enzymes in the adult cell are not necessarily present in the corresponding fetal cell (30). Our data provide evidence that as early as midterm the enzyme, PFK, which is generally agreed to be rate-limiting for glycolysis in adult tissues, is also rate-limiting in fetal muscle and is sensitive to changes in the level of effector molecules such as cyclic AMP. These results provide support for the hypothesis that in the overall regulation of glycolysis and the action of such hormones as epinephrine there is no major difference between fetal and adult muscle.

Erythrocyte glucose metabolism in duchenne muscular dystrophy

We compared glucose metabolism by erythrocytes from patients with Duchenne muscular dystrophy and by erythrocytes from control individuals. There was a significant decrease in the rate of lactate production at pH 7.2 in the dystrophic group. When the pH of the incubation medium was changed to 8.0, we found that the increase in the rate of lactate production for the dystrophic group was significantly larger. We measured the concentrations of key glycolytic metabolites and adenine nucleotides and determined the values of the energy charge during these incubations. We also determined the concentrations of polyol pathway intermediates, the activities of the oxidative portion of the pentose phosphate pathway and the activities and kinetics of phosphofructokinase from both cell groups. There were no significant differences between groups for any of these variables.

Cyclic AMP in Developing Muscle of the Rhesus Monkey: Effect of Prostaglandin E2

Cyclic AMP levels, measured by a competitive protein-binding assay and by a prelabeling technique with adenine-14 C, were determined on fetal, infant, and adult heart, diaphragm, and skeletal muscle of the rhesus monkey (Macaca mulatta); the effect of PGE on cyclic AMP accumulation was also studied. The levels of cyclic AMP were higher in the 3 types of muscle from 150-day fetuses (91% of term) than in adult muscles and even higher at 78-100 days fetal age, and histological evidence indicated that by 65 days gestation, the majority of the rhesus skeletal muscle cells are in the myotube stage. These data agree with the observation in cell cultures that a decrease in cyclic AMP is correlated with fusion of myoblasts to form myotubes. PGE (2.8 MUM) stimulated cyclic AMP accumulation in all the fetal series, even in the earliest series (47% of term), as well as in all the adult series.

Effect of prostaglandins and epinephrine on the level of cyclic AMP-14C of smooth muscle from the rhesus monkey.

Abstract The effects of prostaglandins (PG) and epinephrine on the level of cyclic AMP-14C (adenylate cyclase activity) of myometrium from pregnant rhesus monkeys (145–160 days) were measured. Adenylate cyclase activity was determined from the formation of cyclic AMP-14C from ATP labeled by incubation with adenine-8-14C. PGE2 (2.8 μM) increased cyclic AMP-14C levels both with and without caffeine (2.5 μmM). When 2.5 mM caffeine was present the effects of PGE1 and PGE2 (2.8 μM), like those of PGF1α and PGF2α, were similar; the PGE series, however, had a greater effect on cyclic AMP-14C. Similar differences between the series E and F prostaglandins were observed in concentrations from 0.28 to 5.6 μM. Epinephrine also increased cyclic AMP-14C levels of myometrium; the addition of propranolol (33 μM) decreased the effect of epinephrine but did not alter the effect of PGE2. The basal level of cyclic AMP-14C in the myometrium of pregnant rhesus monkeys was 2.5-fold higher than that of taenia coli from nonpregnant monkeys. The addition of caffeine to the medium increased cyclic AMP-14C concentrations slightly in taenia coli and, on a percentage basis, the addition of PGE2 in the presence of 2.5 mM caffeine caused similar increases in cyclic AMP-14C levels in taenia coli and in myometrium. Epinephrine (10 μM) also increased the cyclic AMP-14C concentration of the taenia coli.

Age-related changes in adenylate and guanylate cyclases from rhesus muscle: sensitivity to positive modulators.

Summary: The basal adenylate cyclase activities, per g wet wt, of homogenates of adult skeletal muscle (4 ± 0.4 S.E. nmoles/10 min · g wet wt−1) were lower than those values in 150- and 80-day fetal muscle (18 ± 1.2 and 23 ± 1.6 S.E. nmoles/10 min · g wet wt−1). This difference in enzyme activity relative to growth was also apparent in the 100,000 × g particulate fractions of adult muscle (2.5 ± 0.2 S.E. nmol/10 min·g wet wt−1) compared to the 150-and 80-day fetal muscle (12 ± 1.5 and 14 ± 1.9 S.E. nmoles/10 min·g wet wt−1). The fluoride-stimulated adenylate cyclase activities of the homogenates and particulates and the increases in enzyme activity with fluoride were also at least four-fold greater in 80- and 150-day fetal than in adult muscle. When basal adenylate cyclase activities of homogenates were compared on the basis of the nitrogen content of the fraction analyzed rather than on the wet weight of the original samples, the differences in enzyme activities of the adult compared to the fetal series were even greater because of the lower nitrogen content of fetal muscle (1.8 ± 0.2 S.E. nmoles/10 min·10 mg N−1 for the adult series and 10.2 ± 0.7 and 22.2 ± 1.2 S.E. nmoles/10 min·10 mg N−1 for the 150-and 80-day series). The difference in activity with age was again apparent in the 100,000 × g particulate fractions (1.9 ± 0.2 S.E. nmoles/10 min·g wet wt−1 for the adult series and 9.8 ± 0.9 S.E. and 24 ± 2.2 nmoles/10 min·g wet wt−1 for the 150- and 80-day series). Because the nitrogen content of the 80-day fetal muscle was less than that of the 150-day series, the adenylate cyclase activities per mg N were highest in the younger fetal series. In the presence of guanylylimidodiphosphate the percent increase in adenylate cyclase activity was two to three times greater in the fetal particulate preparations than in the adult and was concentration-dependent in both series.The basal guanylate cyclase activities for both the 100,000 × g supernatant and particulate fractions were higher in 80-day fetal than in adult muscle; two- to three-fold higher in terms of wet wt and over six-fold higher in terms of nitrogen. Both the absolute and percentage increases in activity of the fetal particulate enzyme with Triton were at least twice those of the adult. Triton had no effect on the enzyme activity of the 100,000 × g supernatant fraction of either series. The concentration of cyclic adenosine 3′: 5′-monophosphate was 8 to 10 times higher in rapidly growing than in adult muscle, and the concentration of cyclic guanosine 5′-monophosphate was 26 to 62 times greater.Speculation: There is a striking difference in the degree of maturation at birth of skeletal muscle in different species of animals. Skeletalmuscle from the rhesus fetus is particularly suitable for study if one is interested in human fetal muscle metabolism because the maturation rate of this tissue (on the basis of percent of gestation) is similar in the two species. Because fetal tissues must proliferate and differentiate, they must perform synthetic processes at a rapid rate, and synthetic processes are expensive in terms of energy. Therefore, it is not surprising that a number of fetal enzymes are more active than are those of the adult. In addition, a greater sensitivity of fetal enzymes to effector molecules may further magnify the activity of these enzymes. It would be of interest to measure the cyclic nucleotide-dependent protein kinase activities in fetal and adult rhesus muscle. Recent evidence suggests possible differences in the functions of types I and II cAMP-dependent protein kinases in relation to cellular growth and differentiation. Studies of these kinases in rhesus fetal muscle during the predominantly proliferative phase (earlier than 85 days gestation) and during the period when differentiation is predominant (85 to 110 days) should be of value in studying the biologic significance of these kinases.