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Plasmin, a broad spectrum proteinase, is inactivated by an autoproteolytic reaction that results in the destruction of the heavy and light chains of the protein. Recently we demonstrated that a 61-kDa plasmin fragment was one of the major products of this autoproteolytic reaction (Fitzpatrick, S. L., Kassam, G., Choi, K. S., Kang, H. M., Fogg, D. K., and Waisman, D. M. (2000)Biochemistry 39, 1021-1028). In the present communication we have identified the 61-kDa plasmin fragment as a novel four kringle-containing protein consisting of the amino acid sequence Lys(78)-Lys(468). To avoid confusion with the plasmin(ogen) fragment, angiostatin(R) (Lys(78)-Ala(440)), we have named this protein A(61). Unlike angiostatin, A(61) was produced in vitro from plasmin autodigestion in the absence of sulfhydryl donors. A(61) bound to lysine-Sepharose and also underwent a large increase in fluorescence yield upon binding of the lysine analogue, trans-4-aminomethylcyclohexanecarboxylic acid. Circular dichroism suggested that A(61) was composed of 21% beta-strand, 14% beta-turn, 18% 3(1)-helix and 8% 3(10)-helix. A(61) was an anti-angiogenic protein as indicated by the inhibition of bovine capillary endothelial cell proliferation. Plasminogen was converted to A(61) by HT1080 cells and bovine capillary endothelial cells. Furthermore, a plasminogen fragment similar to A(61) was present in the serum of humans as well as normal and tumor-bearing mice. These results establish that plasmin turnover can generate anti-angiogenic plasmin fragments in a nonpathological setting.

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

Effect of Insulin and Epinephrine on the Carbohydrate Metabolism and Adenylate Cyclase Activity of Rhesus Fetal Muscle

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.

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.

Age-Related Changes in Adenylate and Guanylate Cyclases from Rhesus Muscle: Sensitivity to Positive Modulators

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 K m value for the high affinity enzyme averaged 3.9 μM and for the low affinity enzyme 23 μM; the V max 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 K m phosphodiesterase was competitively inhibited, with an average K i of 200 μM; the low K m enzyme was noncompetitively inhibited with an average K i of 110 μM. Experiments on myometrial slices demonstrated that 10 μM indomethacin potentiated the effect of PGE 1 and epinephrine on cyclic AMP levels, presumably by inhibiting the phosphodiesterase 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.

We determined the activities of the adenylatc (homogenate) and guanylate cyclases (100,000 X g supernatant) and the cyclic nucleotide phosphodiesterases (PDEs) (100,000 X g supernatant and particulate) of myometria from: 1) rhesus monkeys spayed for at least 6 months and treated either with estradiol-b7� (E2) for 14 days or with E2 for 14 days and then treated for 5-14 additional days with E2 plus progesterone (P); and 2) 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. The specific activities of the guanylate cyclase and the cAMP- and cGMP-PDE enzymes/mg nitrogen or DNA were decreased in myometria from monkeys in the luteal compared with the follicular phase of both natural and induced menstrual cycles; no difference was observed in adenylate cyclase activity. There was also no difference in the concentrations of nitrogen and of DNA in the follicular and luteal phase myometrium. The average cAMP/cGMP ratio was more than twice as high in the follicular compared with the luteal phase (8.5 vs 3.7). This decrease in the cyclic nucleotide ratio was due mainly to an increase in cGMP levels and suggests that in vivo the activity of the guanylate cyclase enzyme decreased less than did the activity of the cGMP’PDE. These data strongly suggest that it is P that causes the decrease in the activities of the guanylate cyclase and cyclic nucleotide PDE, because enzyme activity is significantly higher in the myometrium during the follicular phases of both the induced and natural menstrual cycles. It appears that cyclic nucleotide metabolism in nonhuman primate myometrium varies during the menstrual cycle.

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).

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.

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 follic...

Effect of thermal injury on tissue metabolism was studied by comparing the glucose metabolism of normal rat diaphragm tissue incubated in plasma from injured (plasma taken 15 min. after injury) and uninjured rats. Glucose concentrations in the plasma from the burned and control rats were made comparable to compensate for the hyperglycemia due to injury. An inhibition of glucose uptake by the diaphragm tissue incubated in plasma from burned rats was demonstrated, whereas the QO2 and the conversion of glucose-U-C14 to C14O2 were not significantly changed. This inhibition of glucose uptake was observed when dialyzed plasma from burned animals or the dialysate was used. Epinephrine under certain circumstances inhibited glucose uptake. These studies indicate that immediately after thermal injury, a disturbance in glucose utilization occurs; this disturbance appears to be other than in the conversion of glucose to CO2; a dialyzable principle is present in the plasma; and epinephrine is not excluded as a causative agent.