Biomedical subjects
Publications and source records attributed to C M Clark.
Use was made of the differential DNase I assay to estimate the relative amounts of polymerised and unpolymerised actin in Walker 256 carcinoma cells from solid or ascitic tumours. The concentration of actin per unit DNA and the relative amount of actin present in a polymerised form were both greater in ascitic tumour cells than in cells from solid tumours.
The evolution of clinical knowledge about the management of a common chronic disease was determined by applying analysis of variance and multiple discriminant analysis to responses on two patient management problems by groups of junior medical students and internal medicine residents. Differences between junior and resident groups were found on both problems for management decisions and therapeutic actions and on one problem for information-gathering. Discriminant analyses of original responses clearly identified patterns of choices contributing to group differences. The utility of patient management problems when analyzed under these two empirical models is discussed.
The effect of tumor growth on the amount, state of polymerization, and synthesis of liver actin was investigated in fed and fasted rats bearing a Walker 256 carcinoma. The increase in liver size in the tumor-bearing animal was accompanied by a rise in the total amount of actin and protein, although the amounts present per g liver fell in both cases. Soluble actin increased in both concentration and total amount in the tumor-bearing animals. While the ratio of total actin to total protein in liver was unaltered by tumor growth, the ratio of soluble actin to total actin was increased. The incorporation of [3H]leucine into liver actin relative to that into liver protein, in vivo, was not affected by tumor growth, but the radioactivity incorporated into soluble actin relative to total actin in the livers of the tumor-bearing rats was increased. Liver polysome preparations from tumor-bearing rats showed an increased ability to synthesize actin and total protein, whereas polysomes from skeletal muscle of tumor-bearing rats exhibited a decreased synthesis of actin and total protein. These results suggest that, in the liver of the tumor-bearing rat, while there is an increase in actin synthesis in parallel with a net increase in protein synthesis, there is a decrease in the polymerization of action. In livers of both control and tumor-bearing rats, the consumption of a meal was accompanied by a decrease in soluble actin relative to total actin and an increase in the synthesis of action relative to total protein.
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Guanyl nucleotides are effective activators of rat heart adenylyl cyclase activity from the earliest age studied, 16 fetal days, through adulthood. Both guanosine triphosphate and guanylyl-imidodiphosphate (GMP-PNP) significantly enhance epinephrine-stimulated adenylyl cyclase activity at all fetal and postnatal ages. The dose-response curves for epinephrine alone, GMP-PNP alone, or for the combination of epinephrine and GMP-PNP are similar for the newborn and adult heart preparations. Our results indicate that the epinephrine-guanyl nucleotide-adenylyl cyclase system is "mature" as a functional unit in the rat heart even prior to birth. The rat heart adenylyl cyclase system shows a dramatic increase in responsiveness to epinephrine and to guanyl nucleotides just before birth, a responsiveness which rapidly falls by 3 days of neonatal life. With increasing age, the absolute guanosine triphosphate or GMP-PNP activated rate rises in parallel with the basal rate of adenylyl cyclase activity in the rat heart. Speculation. The activation of adenylyl cyclalse systems by guanyl nucleotides may be a general feature of fetal as well as of adult tissues. This activation by guanyl nucleotides may magnify tissue hormonal responses at some stages of development.
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Heart cells from neonatal rats have been cultured. The ability of 10(-5)M glucagon to stimulate adenylyl cyclase activity, and to increase the cAMP concentration and the beating rate in these cells was followed as a function of time in culture. The cultured cells show no response to 10(-5)M glucagon until 5 weeks. By contrast, the cells do respond to 10(-5)M epinephrine with an increase in beat rate, adenylyl cyclase activity and cAMP levels when freshly prepared or after 1 week in culture. Previous studies on the newborn rat heart, acutely isolated, have also shown that the neonatal rat heart is insensitive to glucagon until 4-5 weeks after birth. We conclude that the cultured neonatal rat heart cells can also mature in the same time frame with respect to a glucagon response.
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Previous studies of the ability of the immature heart to respond to glucagon have yielded conflicting results. To test the possibility that the apparent discrepancies might be explained in part by species variability, isolated hearts of fetal mice and rats (13-22 days' gestational age) were studied under identical conditions in vitro. Changes in atrial rate and ventricular contractility were measured in spontaneously beating hearts exposed to glucagon, and activation of adenylate cyclase was assayed in cardiac homogenates. In mice of 16 days' gestational age or less, there was no change in heart rate in response to glucagon; at 17-18 days, minimal responsiveness was present; and after 19 days, 10muM glucagon caused an increase in spontaneous atrial rate of 30 +/- 4% (SEM) (P less than 0.001). Measurement of the extent and speed of volume displacement of the isotonically contracting hearts with a specially constructed capacitance transducer revealed that ventricular inotropic responsiveness also appeared after 17-19 days. Cardiac stores of glycogen were reduced in older hearts exposed to glucagon, but not in those aged less than 16 days. In contrast, glucagon failed to activate adenylate cyclase in homogenates of hearts of fetal mice at any age. Furthermore, glucagon failed to elicit an increase in the concentration of cyclic AMP in spontaneously beating hearts that developed tachycardia. Responses in hearts of fetal rats were distinctly different from those in mouse hearts: at no age was there any change in heart rate, strength of contraction, glycogen content, or adenylate cyclase activation. Thus, there are major species differences in cardiac pharmacological maturation. Although the mouse heart develops the ability to increase its rate and strength of contraction and to undergo glycogenolysis in response to glucagon well before birth, the rat heart does not. In addition, there is an apparent disparity in late fetal mouse hearts between the ability of glucagon to induce functional responses and its ability to stimulate adenylate cyclase and increase cyclic AMP levels. It is impossible, of course, to rule out absolutely the possibility that localized increases in a critical cyclic AMP pool were present but too small to measure in the entire tissue. Nevertheless, the most obvious interpretation of our results is that they are compatible with the hypothesis that glucagon may exert some of its hemodynamic effects independently from the adenylate cyclase-cyclic AMP system in the late-fetal mouse heart.
The ontogenesis of the hepatic glucagon-sensitive adenylate cyclase system has been studied in the rat. With a partially purified liver membrane preparation, fetal adenylate cyclase was less responsive to glucagon than the enzyme from neonatal or adult livers. Similar results were obtained in gently prepared liver homogenates, suggesting that destruction of essential components of the fetal liver membrane did not account for the relative unresponsiveness of the adenylate cyclase enzyme to glucagon. Investigation of other factors that might account for diminished fetal hepatic responsiveness to glucagon indicate (a) minimal glucagon degradation by fetal membranes relative to 8-day or adult tissue; and (b) available adenylate cyclase enzyme, as suggested by a 13-fold increase over basal cyclic AMP formation with NaF in fetal liver membranes. These results indicate that neither enhanced glucagon degradation nor adenylate cyclase enzyme deficiency accounts for the relative insensitivity of the fetal hepatic adenylate cyclase system to glucagon. In early neonatal life, hepatic adenylate cyclase responsiveness to glucagon rapidly developed and was maximal 6 days after birth. These changes were closely paralleled by a fivefold increase in glucagon binding and the kinetically determined Vmax for cyclic AMP formation. These observations suggest that (a) fetal hepatic unresponsiveness to glucagon may be explained by a limited number of glucagon receptor sites; (b) during the neonatal period, the development of glucagon binding is expressed primarily as an increase in adenylate cyclase Vmax; (c) the ontogenesis of hepatic responsiveness to glucagon may be important in the resolution of neonatal hypoglycemia.
The basal adenylate cyclase activity of the rat heart increases with the age of the animal. By itself, 10(-5) M glucagon activates only adenylate cyclase activity from adult rat hearts. In contrast, 10(-5) M glucagon in the presence of 10(-4)M 5'-guanylylimidodiphosphate (GMP-PNP) clearly activates adenylates cyclase activity in the 14-day-old rat heart, with some activation being evident in hearts of 7-day-old animals. GMP-PNP, 10(-4) M, activates adenylate cyclase activity by itself at ages of 14 days and older, but to a far lesser degree than in combination with 10(-5) M glucagon. Activity elicited by NaF increases throughout the neonatal period. The ratio of NaF-stimulated activity to basal activity increases from 6.3 at 2 days to 10.0 in the adult, a change which is not statistically significant. We conclude that a cardiac receptor for glucagon is present early in neonatal period of the rat, but this receptor cannot effect activation of adenylate cyclase and an increase in heart rate, or depletion of glycogen. Even in the presence of 10(-4) GMP-PNP, the response to glucagon by cardiac adenylate cyclase depends on the age of the rat. In heart cells from a 7-day-old rat, the response is barely measurable but the magnitude of the response increases each week.
The effects of glucagon on the adult mouse heart have been studied. Glucagon (1 mg/kg) increased heart rate in the adult mouse. This effect was enhanced in animals which had pretreated with reserpine to deplete catecholamines. No change in the cardiac cyclic AMP concentration after the injection of glucagon was seen in the hearts of reserpinized animals. Moreover, adenylyl cyclase activity, measured in two different laboratories in whole homogenates, 600 X g pellets or washed particles from adult mouse hearts, was not activated by glucagon. Finally, the injection of epinephrine (1.6 mg/kg), but not of glucagon (1.0 mg/kg), increased the per cent of cardiac phosphorylase a activity and depleted cardiac glucogen. Since increased levels of cyclic AMP are associated with an increase in the per cent of the cardiac phosphorylase a activity, these experiments provide evidence for the hypothesis that cyclic AMP is not essential for the rate effects of glucagon in the adult mouse.
1. The decreased ability of polysomes isolated from the gastrocnemius of rats bearing the Walker 256 carcinoma to incorporate L-[14C]leucine into protein persisted in the presence of 5-10-5 M aurin tricarboxylic acid and 1-10-2 M NaF. 2. Poly(U)-directed phenylalanine incorporation by such polysome preparations was less than that of similar preparations from normal rats. 3. The ability of gastrocnemius polysomes from tumour-bearing rats to react with puromycin was markedly decreased. Cycloheximide inhibited peptidyl puromycin formation by polysome preparations from both normal and tumour-bearing rats. 4. The ability of recombined 60 S and 40 S subunits prepared from polysomes of tumour-bearing rats to carry out poly(U)-directed polyphenylalanine synthesis was much reduced when assayed over a wide range of magnesium concentrations. Cross-over experiments with subunits from normal and tumour-bearing animals suggested that this was due to a defect in the smaller ribosomal subunit.