Rose Mary Bocek

COMPARISON OF RED AND WHITE VOLUNTARY SKELETAL MUSCLES OF SEVERAL SPECIES OF PRIMATES

There was a direct correlation between the qualitative histochemical classification by staining intensity for succinic dehydrogenase and the quantitative measurements of succinic dehydrogenase activity for the quadratus femoris (red), soleus (red), sartorius (predominantly red) and the superficial portion of the brachioradialis (predominantly white) muscles of the rhesus monkey. The relative succinic dehydrogenase activities were quadratus femoris > soleus > sartorius > brachioradialis, the quadratus femoris having 7 times more enzyme activity than the brachioradialis. The sartorius of male rhesus monkeys had a higher enzyme activity than that of the female. Muscle samples were stained with sirius red and graded for amounts of connective tissue as follows: soleus < sartorius < brachioradialis. These histochemical results were verified by chemical analyses. The soleus, sartorius and brachioradialis from 10 other species of primates had the same relative succinic dehydrogenase activities and histochemical staining patterns as the rhesus.

GLYCOGEN SYNTHETASE AND PHOSPHORYLASE IN RED AND WHITE MUSCLE OF RAT AND RHESUS MONKEY

Homogenates of red and white muscles from rats and monkeys were assayed for total phosphorylase and phosphorylase a and for the total and independent forms of glycogen synthetase. Total and phosphorylase a activities were higher in the supernatant fraction of homogenates of white as compared with red muscle from both rats and monkeys. Both forms of phosphorylase were higher in white muscle from rats when assayed on whole homogenates. The total and d form of glycogen synthetase activities were higher in red muscle from both species of animals. The ratio of I/total synthetase was 2- to 3-fold higher in muscle from monkeys as compared with that from rats. These results support histochemical evidence that phosphorylase is higher in white muscle fibers and glycogen synthetase is higher in red muscle fibers.

DIFFERENTIATION OF RED AND WHITE FIBERS IN MUSCLE FROM FETAL, NEONATAL AND INFANT RHESUS MONKEYS

The hydroxyproline (collagen) content of muscle in terms of wet weight was lowest at 90 days gestational age (rhesus monkey) and increased until birth. Histologic techniques also demonstrated a progressive increase in staining density for connective tissue with increasing fetal age. However, when expressed in terms of nitrogen content of muscle, the hydroxyproline concentrations were similar from the 90-day fetal through the 2-6-week infant series, and were consistently higher in rapidly growing as compared to adult muscle. The succinic dehydrogenase activity (soleus), on the basis of wet weight or of nitrogen, was lowest in the 90-day fetal series and reached a plateau by 120 days. The histochemical data indicated an increase in staining density for succinic dehydrogenase in muscle as the fetus matured. By 90 days gestational age, the hydroxyproline concentration of red muscle was lower as compared to that of white muscle, and this difference was also apparent histologically. The succinic dehydrogenase activity was higher in red as compared to white muscle at all ages, and histochemical differentiation was apparent at 120 days fetal age.

Pentose cycle activity in muscle from fetal, neonatal and infant rhesus monkeys☆

Abstract The glucose-14C uptakes (mg/gm/hour wet weight) for muscle fiber groups from 90-, 120-, and 150–160-day rhesus fetuses were similar. However, the glucose uptakes of muscle fiber groups from 2- to 6-week-old rhesus monkeys were slightly higher than those of the fetal series. Incubation under anoxic conditions increased glucose uptake by muscle from both fetal and infant monkeys. From the data on glucose uptakes and specific yields of lactate under aerobic and anaerobic conditions it is concluded that the ability of the fetus and neonate to survive anoxia is not explained by a greater response of the glycolytic enzymes of fetal and neonatal as compared to adult muscle during anaerobiosis. In all 3 fetal series the average ratios of specific yields for 14CO2 from glucose-1- and glucose-6-14C were significantly greater than 1.0, under both aerobic (1.5–2.0) and anaerobic conditions (3.8–4.0). In muscle from infant monkeys the ratios of specific yields for 14CO2 for the 2 substrates were not significantly greater than 1.0 under aerobic conditions, but under anaerobic conditions this ratio rose to 10. This data indicate that pentose cycle activity is more important in muscle from the fetal as compared with muscle from the infant monkey. However, when pentose cycle activity was estimated in relation to the total amount of glucose metabolized, less than 0.4% of the total glucose uptake was metabolized via this route.

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.

Glycogen Synthetase, Phosphorylase, and Glycogen Content of Developing Rhesus Muscle

Extract: Total glycogen synthetase and syntheatse I (glucose 6-PO4 indendependent) and phosphorylase and phosphorylase a activites determined in muscle from fetal,neonatal, infant, and adult rhesus monkeys (Macaca mulatta) and correlated with glycogen concentrations. On the basis of nitrogen content, glycogen levels in fetuses in the 55- to 65-day series where higher than those in fetuses in the 100-day series, but similar to those in the fetuses near term (table I). Both glycogen synthetase and phosphorylase activities where demonstrated histochemically in the immature muscle fibers from fetuses at 55, 62, and 65 days of gestation. Total synthetase activity (assayed with 10 mM glucose 6-PO4) was demonstrated quantitatively at 78 days of gestation; this activity was highest at term (table II). No synthetase I activity could be found in the 78- and 90-day series (table II); hence, it is suggested that in fetal muscle, synthetase Dis responsible for the levels of glycogen present early in gestation. No correlatin between the glycogen content and the percentage of synthetase I present coulf be found in fetal muscle (fig.1). Total phosphorylase, lowest at 78 days of gestation, increased almost 10-fold by term (table II). No singnificant phosphorylase a activity could be demonstrated at 78 days; however, by 90–100 days, phosphorylase a activity was present and remained at about the same level (2–7%) thereafter.It is concluded that the enzymes for glycogen formation and degradation are present in immature muscle fibers of the rhesus fetus as early as the first third of gestation (55 days). The ratio of totao synthetase to phosphorylase activites facors lycogen deposition in the younger fetus (78–125 days) and glycongen breakdown in the fetus at term.Speculation: In adult animals, control of muscle glycogen metabolism depends greatly on hormonal activity; insulin promotes glycogen formation by increasing synthetase D to I conversion, and epinephrine causes glycogenolysis by promoting phosphorylase a formation while depressing synthetase activity. The role of hormonal control of glycogen metabolism may be less critical in fetal muscle than it is in adult muscle. If synthetase D is the active from of the enzyme in glycogen formation in fetal muscle, it would appear that at the cellular level, insulin is a less important regulator of glycogen synthesis than a metabolite control.

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.