Stimulation of uridine, leucine, and sulfate incorporation into rat cartilage macromolecules by adenosine 3',5'-monophosphate.

Conflicting findings on the ability of cAMP analogs or phophodiesterase inhibitors to stimulate precursor incorporation into macromolecules of rat cartilage have been reported. Therefore, the effects of these compounds on the incorporation of uridine into RNA, leucine into proteins, and sulfate into proteoglycans have been reexamined in cartilage from normal and hypophysectomized rats. When cartilage was incubated for 24 h in a medium with the test agents and then pulsed for 2 h in the basal medium containing labeled precursors, both monobutyryl cAMP (BucAMP) and methylisobutylxanthine (MIX) enhanced the ability of the tissue to incorporate precursors into macromolecules. The effect of BucAMP was significant in most cases at a concentration of 30 microM, optimal at concentrations of 100-300 microM, and diminished at a concentration of 1000 microM. Similar stimulation was produced by dibutyrul cAMP [(Bu)2cAMP] or 8-dimethylamino cAMP, but monobutyryl cGMP was ineffective. MIX in a concentration of 20 microM increased precursor incorporation in most cases, and a concentration of 100 microM was optimal; at a concentration of 500 microM, MIX had no significant effect on leucine or sulfate incorporation. When cartilage from hypophysectomized rats was incubated in a medium with the test agents for 4-6 h and the labeled precursors were added for the last 2 h, BucAMP did not increase incorporation of any of the precursors. MIX was also ineffective, even though tissue cAMP levels were increased. However, precursor incorporation was increased by exposure to partially purified rat somatomedin for the same periods. The degree of stimulation of sulfate incorporation induced by either BucAMP or MIX was proportional to the time of exposure to these agents. Preincubation of cartilage in basal medium alone for 22 h or longer increased basal sulfate incorporation, but caused only a slight enhancement of the action of BucAMP. The addition of synthetic bovine PTH-(1-34) (1 microM) to the incubation medium increased sulfate incorporation into hypophysectomized rat cartilage by 24 h, and this effect was potentiated by MIX (10 microM). No stimulation was detectable by 6 h, even with MIX in the medium. PTH-(1-34) increased the cartilage cAMP level, and this effect was also potentiated by MIX. In the presence of MIX, PTH-(1-34) increased the level of cAMP within 30 min, while the rat somatomedin preparation had no effect on the cAMP level during 60 min of incubation. The level of cartilage cGMP was not raised by either PTH or somatomedin.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of endotoxemia on cyclic nucleotides in the unanesthetized sheep.

The effects of endotoxemia on hemodynamics, pulmonary fluid, and solute exchange and cGMP and cAMP concentrations in lung lymph and pulmonary artery and left atrial blood were studied in six unanesthetized sheep. Cyclic AMP levels increased early in the endotoxin reaction, reaching peak concentration 1 hr after endotoxemia (during the period of pulmonary hypertension). Cyclic GMP levels increased gradually during the endotoxin reaction, reaching peak concentrations 5 hr after endotoxemia (during the period of "increased pulmonary vascular permeability"). The changes observed in cyclic nucleotide levels in lung lymph and pulmonary artery and left atrial blood suggested pulmonary production of cGMP but not cAMP. Cyclic AMP concentrations correlated with PPA, Qlymph, and a drop in the L/P ratio, whereas lung lymph cGMP correlated with an increased clearance of protein in the lymph. These results further characterize the sheep endotoxin reaction and suggest a possible role for cyclic nucleotides in the pathogenesis of the changes in lung vascular function that follow endotoxin infusion in sheep.

Similarity of somatomedin inhibitor in sera from starved, hypophysectomized, and diabetic rats: distinction from a heat-stable inhibitor of rat cartilage metabolism.

The inhibitory effects of sera from starved, hypophysectomized, and alloxan-diabetic rats on basal and somatomedin-stimulated sulfate incorporation into cartilage from hypophysectomized rats were compared. The somatomedin inhibitory activity in serum from diabetic rats behaved like that in serum from starved rats on heating at 60 C. Both were labile in the native sera (pH 8.3-8.4), but activity was conserved to a large extent by lowering the pH to 7.4 and diluting the sera before heating. In all of these sera the peak of the somatomedin inhibitory activity was eluted from a column of Sephacryl S-200 at pH 7.4 just after albumin, and lesser amounts were eluted with albumin and higher molecular weight components. Activity of this type was undetectable in fractions prepared from sera that had been heated at 60 C. These results indicate the similarity of this inhibitor in starved, hypophysectomized, and diabetic rat sera. Certain fractions of both starved and diabetic rat sera, which were eluted from a column of Sephacryl S-200 beyond the total bed volume, contained heat-stable inhibitory activity. In contrast to the effects of the heat-labile inhibitor, these fractions only inhibited basal sulfate incorporation into hypophysectomized rat cartilage under the assay conditions employed. This heat-stable inhibitor was not detected in fractions of hypophysectomized rat serum, and inhibitory concentrations of corticosterone were present in fractions of starved and diabetic rat sera containing the material. The findings suggest that the heat-stable inhibitor is corticosterone.

Effects of cyclic nucleotides on deoxyribonucleic acid synthesis in hypophysectomized rat cartilage: stimulation of thymidine incorporation and potentiation of the action of somatomedin by analogs of adenosine 3',5'-monophosphate or a cyclic nucleotide phosphodiesterase inhibitor.

The actions of cyclic nucleotides on basal and somatomedin-stimulated thymidine incorporation into DNA by costal cartilage from hypophysectomized rats were investigated. Three analogs of cAMP (dibutyryl, 8-bromo, and 8-dimethylamino derivatives, which are alternate activators of cAMP-dependent protein kinase and resistant to degradation by cAMP phosphodiesterase but represent a wide difference in potency as phosphodiesterase inhibitors) in range of concentrations from about 10(-5) to 3 X 10(-4) M enhanced basal and somatomedin-stimulated thymidine incorporation. Each cAMP analog at optimal concentration produced combined effects with a suboptimal concentration of somatomedin which were additive or greater. cAMP itself, 5'-AMP, adenosine, 8-Br-5'-AMP, 8-Br-AMPT, and cGMP at concentrations from 10(-7)--10(-3) M or dibutyryl cGMP at concentrations from 10(-10)--10(-3) M did not reproduce the effects of the cAMP analogs. A phosphodiesterase inhibitor (1-methyl-3-isobutylxanthine) at concentrations of 100 or 500 microM also potentiated the effects of somatomedin. At 100- or 500-microM concentrations, the phosphodiesterase inhibitor increased cartilage levels of cAMP and cGMP. These results suggest a role for cAMP in DNA synthesis in rat cartilage. However, they fail to support the hypothesis that all effects of somatomedin on that process are mediated by cAMP, since stimulation of thymidine incorporation by the hormone can be demonstrated in cartilage maximally stimulated by analogs of cAMP.

Stimulation of cyclic 3':5'-guanosine monophosphate levels in rat spleen cells by lipopolysaccharide preparations.

LPS greatly increases cGMP in rat fetal liver cells without affecting cAMP. The present experiments were undertaken to determine whether this effect occurs in an adult tissue, which might be exposed to LPS in vivo. Therefore cyclic nucleotides were measured in adult male rat spleen cells incubated in vitro in the presence or absence of LPS. A clear cGMP elevation was found in cultures treated with LPS. This was first evident at 2 hr and persisted for 4 hr. In contrast, cAMP was unaffected by LPS even in the presence of the phosphodiesterase inhibitor, MIX. CGMP rose progressively with LPS doses ranging from 0.8 to 20 ng, and addition of MIX potentiated the cGMP stimulatory effect. Several LPS concentrates prepared by different techniques and LPS exposed to vigorous heat treatment exhibited cGMP activity, whereas absorption of LPS with Limulus lysate abolished the cGMP response. Thus LPS increases cGMP in rat spleen cells in a dose-and time-dependent manner without affecting cAMP. Although the significance of this cGMP increase is unknown, its occurrence in spleen cells (a tissue likely to come in contact with LPS in vivo), its production by very small quantities of LPS, and the delayed but persistent nature of the effect (analogous to the aciton of cholera toxin on cAMP) are all consistent with an important role for cGMP in the action of LPS on cells.

Somatomedin and analogues of cyclic AMP increase the number of cells synthesizing DNA in cartilage from hypophysectomized rats.

The effects of somatomedin and certain nucleotides on nuclear labelling of cartilage cells with [3H]thymidine were determined by autoradiography. Segments of costal cartilage from hypophysectomized rats were incubated for 24 h in a basal medium with or without additions and then pulsed for 2 h with [3H]thymidine in the basal medium. Both somatomedin (0.1 U/ml) and Bt2cAMP (10(-4)M) increased the number of labelled nuclei, and the combined effects were more than additive. A parallelism between the effects of these agents on nuclear labelling and their effects on total thymidine incorporation into DNA was demonstrated. The 8-bromated derivative of cAMP (10(-4)M) also enhanced chondrocyte nuclear labelling, but neither 8-Br-5'-AMP (10(-4)7) nor 8-Br-cGMP (10(-4)M) exhibited actions of the cAMP analogues. It is concluded that in cartilage obtained from hypophysectomized rats and incubated under the specified conditions (1) both somatomedin and cAMP analogues increase the number of cells synthesizing DNA as well as total thymidine incorporation into DNA, (2) the effects of the hormone and cyclic nucleotide in combination are synergistic, and (3) the increased incorporation of labelled thymidine into DNA reflects increased DNA synthesis and not merely an alteration of the specific activity of the intracellular thymidine nucleotide pool.

Evidence that endotoxin is the cyclic 3':5'-GMP--promoting factor in erythropoietin preparations.

Since Ep preparations are contaminated with endotoxin, the possibility that the latter might be the factor in crude Ep which increases cGMP levels in rat fetal liver cells was examined. Endotoxin produced a striking elevation of cGMP in rat fetal liver cells without affecting cAMP levels or heme synthesis. Absorption with Limulus lysate of more than 99% of the endotoxin in a crude Ep preparation caused a parallel decrease in the cGMP-promoting activity without reduction of heme synthetic potency. It is concluded that endotoxin is the component of crude Ep which increases cGMP levels in rat fetal liver. The precise role of elevated cGMP in the action of endotoxin on cells and the universality of this effect remain to be determined.

Insulin-glucagon interaction in controlling splanchnic glucose production in normal man.

The interaction of glucagon and insulin in controlling hepatic glucose production in man has been inferred from studies of immunoreactive glucagon and insulin. This study directly examines the interaction of glucagon and insulin in controlling net splanching glucose production (NSGP) in eight normal men. Glucagon was infused iv at 5 ng/kg/min for 15 min and resultant arterial glucagon levels (1.3 X 10(-10) M) did not exceed the physiologic portal range. In four normal men NSGP increased 2.3-fold by 5 min and remained elevated for 15 min. There was no change in arterial insulin concentration. To study the effect of exogenous insulin on this glucagon-induced increase in NSGP, insulin was infused at 10 mU/kg/min in four normal men to achieve arterial immunoreactive insulin concentrations of 1500 muU/ml (1 X 10(-8) M). Blood glucose was stabilized by glucose infusions. During insulin and glucose administration, NSGP was suppressed and net splanchnic glucose uptake occurred. After 40 min of insulin and glucose pretreatment, a 5 ng/kg/min glucagon infusion resulted in no increase in NSGP (arterial insulin: glucagon molar ratio of approximately 100). In two subjects the glucagon infusion rate was then increased to 15 ng/kg/min (arterial insulin: glucagon molar ratio of approximately 33), resulting in stimulation of NSGP. These studies provide evidence that insulin in high concentration can suppress glucagon-stimulated NSGP in normal man.

Transient stimulatory effect of sustained hyperglucagonemia on splanchnic glucose production in normal and diabetic man.

Insulin can modulate glucagon-stimulated hepatic glucose production and is considered to be the major factor acting in vivo to exert a couterregulatory action to glucagon. The insulin-dependent diabetic, therefore, might be especially vulnerable to enhanced hepatic glucose production promoted by glucagon. To investigate this hypothesis, low-dose glucagon infusions were administered to normal and diabetic men to compare the effects of glucagon on net splanchnic glucose production (NSGP). Four normal and three insulin-dependent, ketosis-prone, hyperglycemic diabetic men (insulin withheld for 24 hours) underwent brachial-artery-hepatic-vein catheterization. Each received a 90-minute glucagon infusion at 5 ng/kg./min. Glucagon levels rose four-to-fivefold in both groups, plateauing at 300-600 pg./ml. In the normals, NSGP rose from 92+/-12 to 211+/-31 mg./min. at 15 minutes and returned to basal levels by 45 minutes. Insulin measured in the hepatic vein rose from 19+/-6 to 33+/-11 muU/.ml., while plasma glucose rose 17 mg./dl. In the insulin-dependent diabetics, NSGP rose from 78+/-24 to a peak of 221+/-33 mg./min. at 30 minutes and then fell sharply to 113+/-15 mg./min. at 60 minutes despite continuing hyperglucagonemia. Plasma glucose in the diabetics rose 21 mg./dl. These data suggest a mechanism that acts to rapidly diminish glucagon-induced hepatic glucose production in diabetic man but does not appear to be mediated by increased insulin secretion.

Somatomedin.

Somatomedin is a peptide component of serum which has been postulated to mediate the action of growth hormone on skeletal tissue. Direct effects on cartilage include stimulation of the synthesis of mucopolysacharide, protein, and nucleic acids. Insulin-like effects on non-skeletal tissues and cells have been described, and a relationship to NSILA-S and MSA has been suggested. The liver may be an important source. Non-specificity of bioassays is a problem. Growth hormone deficiency, malnutrition, therapy with corticosteroids or estrogens, and a type of dwarfism characterized by high serum growth hormone are associated with decreased somatomedin. An unexplained phenomenon is the normal somatomedin with low or undetectable growth hormone in certain cases of craniopharyngioma or other tumors involving the hypothalamus. Somatomedin is increased in acromegaly.

Effects of glucagon on lipolysis and ketogenesis in normal and diabetic men.

The effect of glucagon (50 ng/kg/min) on arterial glycerol concentration and net splanchnic production of total ketones and glucose was studied after an overnight fast in four normal and five insulin-dependent diabetic men. Brachial artery and hepatic vein catheters were inserted and splanchnic blood flow determined using indocyanine green. The glucagon infusion resulted in a mean circulating plasma level of 4,420 pg/ml. In the normal subjects, the glucagon infusion resulted in stimulation of insulin secretion indicated by rising levels of immunoreactive insulin and C-peptide immunoreactivity. Arterial glycerol concentration (an index of lipolysis) declined markedly and net splanchnic total ketone production was virtually abolished. In contrast, the diabetic subjects secreted no insulin (no rise in C-peptide immunoreactivity) in response to glucagon. Arterial glycerol and net splanchnic total ketone production in these subjects rose significantly (P=<0.05) when compared with the results in four diabetics who received a saline infusion after undergoing the same catheterization procedure.Net splanchnic glucose production rose markedly during glucagon stimulation in the normals and diabetics despite the marked rise in insulin in the normals. Thus, the same level of circulating insulin which markedly suppressed lipolysis and ketogenesis in the normals failed to inhibit the glucagon-mediated increase in net splanchnic glucose production. It is concluded (a) that glucagon at high concentration is capable of stimulating lipolysis and ketogenesis in insulin-deficient diabetic man; (b) that insulin, mole for mole, has more antilipolytic activity in man than glucagon has lipolytic activity; and (c) that glucagon, on a molar basis, has greater stimulatory activity than insulin has inhibitory activity on hepatic glucose release.

Effect of glucagon on net splanchnic cyclic AMP production in normal and diabetic men.

Glucagon activates hepatic adenylate cyclase, thereby increasing acutely the liver content of cyclic AMP (cAMP) as well as the release of cAMP into the hepatic vein. Insulin, on the other hand, antagonizes this glucagon-mediated cAMP production, thus providing a hypothetical mechanism through which insulin might correct some of the metabolic abnormalities of diabetes. To study this hormonal interaction in man, net splanchnic cAMP production (NScAMPP) was investigated in normal and insulin-dependent diabetic men under basal conditions and in response to intravenous glucagon, 50 ng/kg/min for 2 h. In normals (n=19), basal hepatic vein cAMP concentration was 23.6+/-1.1 nM and NScAMPP was 1.7+/-0.6 nmol/min. Glucagon stimulated NScAMPP in four normal subjects to a peak of 99.6+/-43 nmol/min at 25 min with a subsequent fall to 12.4+/-5.1 nmol/min by 90 min despite continuing glucagon infusion. Endogenous insulin secretion was stimulated as indicated by rising levels of immunoreactive insulin and C-peptide (connecting peptide) immunoreactivity, raising the possibility that endogenous insulin might be responsible for the fall in NScAMPP that followed the initial spike. In the diabetics (n=8), basal hepatic vein cAMP concentration was 24.7+/-1.2 nM and NScAMPP was undetectable. Glucagon stimulated NScAMPP in five diabetics to a peak of 169.9+/-42.6 with a subsequent fall to 17.4+/-3.9 nmol/min by 90 min even though endogenous insulin secretion was not stimulated (no rise in C-peptide immunoreactivity). Although the mean increase in NScAMPP was greater in the diabetics, the two groups did not differ significantly.Conclusions. In normal resting man the liver is a significant source of circulating cAMP. Diabetics do not release abnormally large amounts of hepatic cAMP under basal conditions. Glucagon markedly enhances hepatic cAMP release with a spike-decline pattern in both normal and diabetic men. The decline in hepatic cAMP release despite continuing glucagon stimulation is due to factors other than a stimulation of insulin secretion.