Atrial natriuretic factor and sodium nitroprusside increase cyclic GMP in cultured rat lung fibroblasts by activating different forms of guanylate cyclase.

We used cultured rat lung fibroblasts to evaluate the role of particulate and soluble guanylate cyclase in the atrial natriuretic factor (ANF)-induced stimulation of cyclic GMP. ANF receptors were identified by binding of 125I-ANF to confluent cells at 37 degrees C. Specific ANF binding was rapid and saturable with increasing concentrations of ANF. The equilibrium dissociation constant (KD) was 0.66 +/- 0.077 nM and the Bmax. was 216 +/- 33 fmol bound/10(6) cells, which corresponds to 130,000 +/- 20,000 sites/cell. The molecular characteristics of ANF binding sites were examined by affinity cross-linking of 125I-ANF to intact cells with disuccinimidyl suberate. ANF specifically labelled two sites with molecular sizes of 66 and 130 kDa, which we have identified in other cultured cells. ANF and sodium nitroprusside produced a time- and concentration-dependent increase in intracellular cyclic GMP. An increase in cyclic GMP by ANF was detected at 1 nM, and at 100 nM an approx. 100-fold increase in cyclic GMP was observed. Nitroprusside stimulated cyclic GMP at 10 nM and at 1 mM a 500-600-fold increase in cyclic GMP occurred. The simultaneous addition of 100 nM-ANF and 10 microM-nitroprusside to cells resulted in cyclic GMP levels that were additive. ANF increased the activity of particulate guanylate cyclase by about 10-fold, but had no effect on soluble guanylate cyclase. In contrast, nitroprusside did not alter the activity of particulate guanylate cyclase, but increased the activity of soluble guanylate cyclase by 17-fold. These results demonstrate that rat lung fibroblasts contain ANF receptors and suggest that the ANF-induced stimulation of cyclic GMP is mediated entirely by particulate guanylate cyclase.     

Growth control in chick embryo fibroblasts; no evidence for a specific role for cyclic purine nucleotides

Cyclic AMP and cyclic GMP concentrations were measured in cultures of normal chick embryo fibroblasts and those transformed by Rous sarcoma virus under different growth conditions. No significant and reproducible correlation between the nucleotide levels and the rate of proliferation was observed. Neither release of normal cells from density dependent inhibition of growth nor transformation of the cultures by different strains of Rous sarcoma virus affected the concentrations of cyclic AMP or cyclic GMP. Activities of cellular cyclic nucleotide phosphodiesterases, enzymes involved in regulating the level of the nucleotides, were not directly affected by growth-stimulating concentrations of insulin or neuraminidase. Growth stimulation by insulin did not alter the activities of cellular cAMP-dependent protein kinase. These results do not support the hypothesis that cyclic AMP or cyclic GMP has a specific role in the growth control of chick embryo fibroblasts.     

Peptide binding to intestinal epithelium: distinct sites for insulin, EGF and VIP

Several peptides, including insulin, epidermal growth factor and vasoactive intestinal polypeptide bind to intestinal epithelial cells. However, it is unclear whether one binding site binds several peptides or whether separate sites exist for each peptide. These studies were designed to examine the specificity of peptide binding sites on intestinal epithelial cells. Peptide binding was measured directly with [125I]radiolabelled peptides to isolated enterocytes prepared from rabbit ileum. The characteristics of insulin and epidermal growth factor binding were similar. Both insulin and epidermal growth factor specific binding was saturable, directly correlated to cell concentration and temperature and pH dependent. The total number of insulin binding sites per cell was 4500, that for epidermal growth factor was 2280. Scatchard analysis for both peptides produced curvilinear plots. Dissociation of both peptides from the binding site was increased in the presence of their respective unlabelled peptide. However, insulin specific binding was not altered by epidermal growth factor, and epidermal growth factor specific binding was unaffected by insulin. Further, both insulin and epidermal growth factor failed to inhibit the specific binding of vasoactive intestinal polypeptide to ileal enterocytes, and vasoactive intestinal polypeptide did not inhibit insulin or epidermal growth factor specific binding. These studies demonstrate that insulin, epidermal growth factor and vasoactive intestinal polypeptide interact with three distinct membrane binding sites on the enterocyte.     

Growth factor concentrations and growth-promoting activity in human milk following premature birth

We have measured growth factor concentrations in human milk from mothers of term and premature infants to identify any adaptive responses to premature delivery. Measurements included concentrations of epidermal growth factor and insulin and the growth-promoting activity of milk in vitro, estimated by the stimulation of rats of protein accumulation in cultured human fibroblasts. Compared with women delivering at full-term, mothers of premature infants produced milk containing higher concentrations of epidermal growth factor and increased growth-promoting activity in vitro, changes which were probably maintained throughout lactation. The anabolic effect of human milk in cultured human fibroblasts could be attributed partially but not entirely to epidermal growth factor, suggesting that the concentrations of additional growth factors were also increased following premature delivery. Insulin did not contribute to the extra growth-promoting activity; premature delivery depressed the insulin concentration significantly on the first two days of lactation and, thereafter, milk from mothers of term or premature infants contained similar amounts of insulin. Growth factor concentrations were also measured in cow's milk-based formulae. These formulae contained low concentrations of epidermal growth factor and insulin and reduced growth-promoting activity compared with human milk. Changes in milk growth factor concentrations may occur as a compensatory mechanism to accelerate growth and development in pre-term infants, and if so, it follows that premature infants could benefit more from their own mother's milk than from pooled human milk or from cow's milk-based formulae.     

Regulation of Neuronal Nitric Oxide and Cyclic GMP Formation by Ca2+

Nitric oxide (NO) acts as a messenger molecule in the CNS by activating soluble guanylyl cyclase. Rat brain synaptosomal NO synthase was stimulated by Ca2+ in a concentration-dependent manner with half-maximal effects observed at 0.3 microM and 0.2 microM when its activity was assayed as formation of NO and L-citrulline, respectively. Cyclic GMP formation was apparently inhibited, however, at Ca2+ concentrations required for the activation of NO synthase, indicating a down-regulation of the signal in NO-producing cells. Purified synaptosomal guanylyl cyclase was not inhibited directly by Ca2+, and the effect was not mediated by a protein binding to guanylyl cyclase at low or high Ca2+ concentrations. In cytosolic fractions, the breakdown of cyclic GMP, but not that of cyclic AMP, was highly stimulated by Ca2+, and 3-isobutyl-1-methylxanthine did not block this reaction effectively. The effects of Ca2+ on cyclic GMP hydrolysis and on apparent guanylyl cyclase activities were abolished almost completely in the presence of the calmodulin antagonist calmidazolium, whose effect was attenuated by added calmodulin. Thus, a Ca2+/calmodulin-dependent cyclic GMP phosphodiesterase is highly active in synaptic areas of the brain and may prevent elevations of intracellular cyclic GMP levels in activated, NO-producing neurons.     

Co-purification of an atrial natriuretic factor receptor and particulate guanylate cyclase from rat lung

An atrial natriuretic factor (ANF) receptor from rat lung was solubilized with Lubrol-PX and purified by sequential chromatographic steps on GTP-agarose, DEAE-Sephacel, phenyl-agarose, and wheat germ agglutinin-agarose. The ANF receptor was enriched 19,000-fold. The purified receptor has a binding profile and properties that correspond to the affinity and specificity found in membranes and crude detergent extracts. Polyacrylamide gel electrophoresis of the purified preparation in the presence of sodium dodecyl sulfate and dithiothreitol showed the presence of one major protein band with a molecular mass of 120,000 daltons. When purified preparations were incubated with 125I-ANF, then cross-linked with disuccinimidyl suberate, the 120,000-dalton protein was specifically radiolabeled. This high affinity binding site for ANF co-purified with particulate guanylate cyclase. Particulate guanylate cyclase was purified to a specific activity of 19 mumol cyclic GMP produced/min/mg of protein utilizing Mn-GTP as substrate. This represented a 15,000-fold purification compared to the initial lung membrane preparation with Lubrol-PX. Gel permeation high performance liquid chromatography and glycerol density gradient sedimentation studies of the purified preparation also resulted in co-migration of specific ANF binding and guanylate cyclase activities. The co-purification of these activities suggests that both ANF binding and guanylate cyclase activities reside in the same macromolecular complex. Presumably ANF binding occurs at the external membrane surface and cyclic GMP synthesis at the internal membrane surface of this transmembrane glycoprotein.     

Characteristics of a new binding protein distinct from the kinase for guanosine 3':5'-monophosphate in rat platelets

A new type of cyclic GMP binding protein was recently identified in our laboratory (Hamet, P. and Coquil, J.-F. (1978) J. Cyclic Nucleotide Res. 4, 281--290). The binding, recovered in the supernatant fractions, is highly specific for cyclic GMP and is clearly distinct from the binding to cyclic GMP-dependent protein kinase. Chromatography on DEAE-Sepharose separated the cyclic GMP binding protein from cyclic AMP binding, cyclic AMP-dependent kinase activities, and from guanylate cyclase. The optimal binding occurs at high pH and in the presence of thiol reagents. Several phosphodiesterase inhibitors increase the affinity of binding (Kd was 353 +/- 60 nM in the absence and 13.4 +/- 1.5 nM in the presence of 1-methyl-3-isobutyl-xanthine). The molecular weight of the binding protein was determined to be about 176,000 and the sedimentation coefficient was 6.4 S. While the binding and phosphodiesterase activities co-migrated on DEAE-Sepharose, gel filtration and sucrose gradients, certain treatments (such as increasing the concentrations of salt and heating) were able to influence one activity while having no effect on the other. Hence, the binding activity may be involved in the regulation of the activity of cyclic GMP phosphodiesterase. Since the binding protein appears to be the only 'receptor' for cyclic GMP detectable in platelets, this protein and/or its relation to cyclic GMP phosphodiesterase may play a role in the mechanism of action of cyclic GMP in platelets.     

Insulin-mediated antilipolysis in permeabilized rat adipocytes

Elucidating the mechanism by which insulin inhibits lipolysis has been hampered by the unavailability of a broken cell preparation in which the intact cell responses to the hormone could be duplicated. Here we report, using digitonin-permeabilized rat adipocytes, that physiological concentrations of insulin inhibit cyclic AMP-activated lipolysis despite the absence of cytosolic and plasma membrane integrity. Cyclic AMP (1.0 mM) maximally activates lipolysis in permeabilized adipocytes greater than 10-fold. Insulin inhibits this activation in a biphasic manner with maximum inhibition of 59 +/- 8% (N = 7) at 10(-9) M. At the submaximal concentrations of cyclic AMP (1.0 to 10 microM), insulin (10(-9) M) inhibits lipolysis 80 to 90%. Additionally, the antilipolytic effect of insulin is rapid (3 min) and it is specific, with the relatively inactive desoctapeptide analogue of insulin being three orders of magnitude less inhibitory than native insulin. In contrast to permeabilized cells, intact cells demonstrate only a small lipolytic response to cyclic AMP which is insensitive to insulin. These findings suggest the following about insulin's antilipolytic effects: 1) an intact cell is not required; 2) the intracellular mechanism of action does not require physiological concentrations of the freely diffusible cytosolic components; and 3) a site of insulin action independent of adenylate cyclase may play a major role.     

Characteristics of a new binding protein distinct from the kinase for guanosine 3′:5′-monophosphate in rat platelets

A new type of cyclic GMP binding protein was recently identified in our laboratory (Hamet, P. and Coquil, J.-F. (1978) J. Cyclic Nucleotide Res. 4, 281–290). The binding, recovered in the supernatant fractions, is highly specific for cyclic GMP and is clearly distinct from the binding to cyclic GMP-dependent protein kinase. Chromatography on DEAE-Sepharose separated the cyclic GMP binding protein from cyclic AMP binding, cyclic AMP-dependent kinase activities, and from guanylate cyclase. The optimal binding occurs at high pH and in the presence of thiol reagents. Several phosphodiesterase inhibitors increase the affinity of binding (K_d was 353 ± 60 nM in the absence and 13.4 ± 1.5 nM in the presence of 1-methyl-3-isobutyl-xanthine). The molecular weight of the binding protein was determined to be about 176 000 and the sedimentation coefficient was 6.4 S. While the binding and phosphodiesterase activities co-migrated on DEAE-Sepharose, gel filtration and sucrose gradients, certain treatments (such as increasing the concentrations of salt and heating) were able to influence one activity while having no effect on the other. Hence, the binding activity may be involved in the regulation of the activity of cyclic GMP phosphodiesterase. Since the binding protein appears to be the only ‘receptor’ for cyclic GMP detectable in platelets, this protein and/or its relation to cyclic GMP phosphodiesterase may play a role in the mechanism of action of cyclic GMP in platelets.     

Reduced mRNA and a nonsense mutation in the insulin-receptor gene produce heritable severe insulin resistance.

Leprechaunism is an autosomal recessive syndrome of severe insulin resistance and is characterized by intrauterine growth restriction, acanthosis nigricans, hirsutism, and loss of glucose homeostasis. Here we report a new female patient of Hispanic and Afro-American descent whose fibroblasts and lymphoblasts had markedly impaired insulin binding (less than 10% of that in controls). Insulin binding to lymphoblasts established from both unrelated parents was partially impaired. Insulin-like growth factor-I (IGF-I) and epidermal growth factor (EGF) binding to the patient's fibroblasts were within the normal range. Insulin stimulation of receptor autophosphorylation and kinase activity was markedly reduced in the patient's fibroblasts. The patient's fibroblasts had both a reduced number of immunoreactive insulin receptor (6% of those in controls) and concomitantly reduced amounts of insulin-receptor mRNA, suggesting that both mutations inherited by the patient reduced insulin-receptor mRNA. Sequencing of the insulin-receptor gene and cDNA indicated that the patient was heterozygous for a paternally derived mutation at bp 1333, converting Arg372 to a STOP codon. This nonsense mutation was observed in the insulin-receptor gene, but not in cDNA, indicating reduced amounts of mRNA for the allele containing this mutation. The coding sequence of the maternally inherited insulin-receptor allele was normal. Both the marked reduction in insulin-receptor mRNA in the compound heterozygous fibroblasts of the proband and the partially reduced insulin binding in maternal cells suggest that the maternally derived mutation is located in an insulin-receptor gene sequence that controls cellular mRNA content.     

An examination of possible mechanisms of angiotensin II-stimulated steroidogenesis.

Dog and rat adrenal glomerulosa cells and subcellular fractions have been utilized to evaluate the mechanism of angiotensin II- and angiotensin III-induced aldosterone production. The effects of angiotensin, ACTH, and potassium have been compared on cyclic AMP and cyclic GMP in isolated glomerulosa cells and adenylate cyclase activity in subcellular fractions. The effect of angiotensin II has also been assessed on Na+-K+-activated ATPase of plasma membrane enriched fractions of dog and rat adrenals. We have demonstrated no effect of angiotensin II or angiotensin III on either adenylate cyclase, cyclic AMP, cyclic GMP, or Na+-K+-dependent ATPase activity over a wide range of concentrations. Potassium ion in concentrations that stimulate significant aldosterone production was also without effect. The negative effects of angiotensin and potassium were contrasted against a positive correlation between an ACTH-induced effect on aldosterone production, adenylate cyclase, and cyclic AMP accumulation. These studies have served to demonstrate that neither adenylate cyclase, cyclic AMP, cyclic GMP, or Na+-K+-activated ATPase seem to be directly involved in the mechanism of action of angiotensins on aldosterone production in the rat and dog adrenal glomerulosa.     

Insulin-like growth factor I and insulin rapidly increase casein kinase II activity in BALB/c 3T3 fibroblasts

We have tested whether growth factors added to serum-deprived BALB/c 3T3 fibroblasts alter the casein kinase II activity measured in cell extracts. A rapid phosphocellulose chromatography method was developed that provides a 40-fold partial purification of casein kinase II activity assayed with the specific substrate peptide Arg-Arg-Glu-Glu-Glu-Thr-Glu-Glu-Glu. Using this technique, kinase activity is stimulated 1.6-2.5-fold when isolated from fibroblasts treated with insulin or insulin-like growth factor I (IGF-I). The activated kinase activity exhibits the specific properties of casein kinase II such as the ability to utilize [gamma-32P]GTP as phosphate donor and marked inhibition by low concentrations of heparin. Activation of casein kinase II appears specific for these hormones because epidermal growth factor and platelet-derived growth factor have no effect on the kinase activity when added to fibroblasts under conditions where they markedly stimulate [3H]thymidine incorporation into DNA. Increases of casein kinase II activity by insulin and IGF-I were detected within 1 min of their addition to cell cultures. IGF-I is more potent in stimulating casein kinase II than insulin in mouse fibroblasts. These results demonstrate that casein kinase II is a selective target for insulin and IGF-I action in BALB/c fibroblasts, consistent with the hypothesis that this kinase plays a role in cellular signaling by these hormones.     

Induction of DNA synthesis in dog thyrocytes in primary culture: synergistic effects of thyrotropin and cyclic AMP with epidermal growth factor and insulin

We have investigated the growth effects of thyrotropin (TSH) (mimicked by forskolin and acting through cyclic AMP), epidermal growth factor (EGF), serum (10%) and insulin on quiescent dog thyroid epithelial cells in primary culture in a serum-free defined medium. These cells were previously shown to retain the capacity to express major thyroid differentiation markers. In the presence of insulin and after a similar prereplicative phase of 18 +/- 2h, TSH, EGF, and serum promoted DNA synthesis in such quiescent cells only a minority of which had proliferated in vitro before stimulation. The combination of these factors induced more than 90% of the cells to enter S phase within 48 h and near exponetial proliferation. Analysis of the cell cycle parameters of the stimulated cells revealed that the G1 period duration was similar to the length of the prereplicative phase of quiescent thyroid cells; this might indicate that they were in fact in an early G1 stage rather than in G0 prior to stimulation. TSH and EGF action depended on or was potentiated by insulin. Strikingly, nanomolar concentrations of insulin were sufficient to support stimulation of DNA synthesis by TSH, while micromolar concentrations of insulin were required for the action of EGF. This suggests that insulin supported the action of TSH by acting on its own high affinity receptors, whereas its effect on EGF action would be related to its somatomedinlike effects at high supraphysiological concentrations. Insulin stimulated the progression in the prereplicative phase initiated by TSH or forskolin. In addition, in some primary cultures TSH must act together with insulin to stimulate early events of the prereplicative phase. In the presence of insulin, EGF, and forskolin, an adenylate cyclase activator, markedly synergized to induce DNA synthesis. Addition of forskolin 24 h after EGF or EGF 24 h after forskolin also resulted in amplification of the growth response but with a lag equal to the prereplicative period observed with the single compound. This indicates that events induced by the second factor can no longer be integrated during the prereplicative phase set by the first factor. These findings demonstrate the importance of synergistic cooperation between hormones and growth factors for the induction of DNA synthesis in epithelial thyroid cells and support the proposal that essentially different mitogenic pathways--cyclic AMP-dependent or independent--may coexist in one cell.     

Independence of the insulin effect from the guanosine-3',5'-monophosphate level in muscle tissue

The role of cyclic GMP in the insulin effect was investigated using isolated frog sartorii. A study was made of the effect of exogenous cyclic GMP, dibutyryl cyclic GMP, 8-bromo-cyclic GMP on xylose transport, glycogen synthesis and muscle respiration. Only dibutyryl cyclic GMP (1.10(-6) - 10(-4) M) alone was observed to have a stimulating effect on glycogen synthesis and respiration. The xylose transport was but slightly accelerated only following a 20 hours incubation of muscles in the cyclic GMP solution. Cyclic GMP was shown to penetrate the muscle fibres. The cyclic GMP content in muscles was equal to 22.7 +/- 2.0 pM per gram of wet weight. Insulin exerted no effect on cyclic GMP concentration in muscles. The data obtained do not allow to conclude that cyclic GMP may serve as a mediator in realization of the insulin effect on membrane and intracellular processes.     

Involvement of the adenylyl cyclase signaling system in the action of insulin and mollusk insulin-like peptide

Involvement of the adenylyl cyclase signaling system in the mechanism of action of the mammalian insulin and epidermal growth factor as well as of insulin-like peptide isolated from the bivalve mollusk Anodonta cygnea has been studied. It was shown for the first time that insulin and insulin-like peptide exert in vitro the GTP-dependent stimulating action on the adenylyl cyclase activity. Epidermal growth factor has an analogous effect. Effectiveness of the peptides decreased in the order insulin-like peptide > epidermal growth factor > insulin in the foot smooth muscles of A. cygnea and insulin > epidermal growth factor > insulin-like peptide in the skeletal muscles of rat.     

Production and characterization of antibody blocking epidermal growth factor:receptor interactions

Membranes were prepared from the human epithelioid carcinoma cell line A-431 which has approx. 2 . 10(6) epidermal growth factor receptors per cell. This membrane preparation which retained a high epidermal growth factor binding specific activity was used as an antigen to produce antisera in rabbits. Double-immunodiffusion experiments demonstrated that the immune serum contained precipitating antibodies to several components of detergent solubilized A-431 membranes. The immunoglobulin G fraction of this immune sera inhibited 125I-labeled epidermal growth factor binding to receptors in: (1) intact human and mouse cells; (2) membrane preparations from A-431 cells and human placenta, and (3) solubilized A-431 membranes. Inhibition of 125I-labeled epidermal growth factor binding was observed with divalent and monovalent fragments of immunoglobulin G prepared from the immunoglobulin G fraction. Also, the immunoglobulin G fraction blocked growth factor binding to membranes at low temperature (5 degrees C). Anti-A-431 antibody blocked the induction of DNA synthesis in quiescent fibroblasts by epidermal growth factor in a manner similar to that of anti-epidermal growth factor antibody. Addition of either anti-A431 or anti-epidermal growth factor antibodies to fibroblasts at times up to 5 h after the addition of epidermal growth factor completely reversed the hormone's mitogenic potential. At later times (after 12 h) addition of either antibody was without effect on the stimulation of DNA synthesis by epidermal growth factor. Anti-A-431 antibody did not block the induction of DNA synthesis in fibroblasts by fibroblast growth factor or serum.     

Regulation by insulin of gluconeogenesis in isolated rat hepatocytes.

Insulin (10nM) completely suppressed the stimulation of gluconeogenesis from 2 mM lactate by low concentrations of glucagon (less than or equal to 0.1 nM) or cyclic AMP (less than or equal to 10 muM), but it had no effect on the basal rate of gluconeogenesis in hepatocyctes from fed rats. The effectiveness of insulin diminished as the concentration of these agonists increased, but insulin was able to suppress by 40% the stimulation by a maximally effective concentration of epinephrine (1 muM). The response to glucagon, epinephrine, or insulin was not dependent upon protein synthesis as cycloheximide did not alter their effects. Insulin also suppressed the stimulation by isoproterenol of cyclic GMP. These data are the first demonstration of insulin antagonism to the stimulation of gluconeogenesis by catecholamines. Insulin reduced cyclic AMP levels which had been elevated by low concentrations of glucagon or by 1 muM epinephrine. This supports the hypothesis that the action of insulin to inhibit gluconeogenesis is mediated by the lowering of cyclic AMP levels. However, evidence is presented which indicates that insulin is able to suppress the stimulation of gluconeogenesis by glucagon or epinephrine under conditions where either the agonists or insulin had no measurable effect on cyclic AMP levels. Insulin reduced the glucagon stimulation of gluconeogenesis whether or not extracellular Ca2+ were present, even though insulin only lowered cyclic AMP levels in their presence. Insulin also reduced the stimulation by epinephrine plus propranolol where no significant changes in cyclic AMP were observed without or with insulin. In addition, insulin suppressed gluconeogenesis in cells that had been preincubated with epinephrine for 20 min, even though the cyclic AMP levels had returned to near basal values and were unaffected by insulin. Thus insulin may not need to lower cyclic AMP levels in order to suppress gluconeogenesis.     

Production and characterization of antibody blocking epidermal growth factor: Receptor interactions

Membranes were prepared from the human epithelioid carcinoma cell line A-431 which has approx. 2 · 10⁶ epidermal growth factor receptors per cell. This membrane preparation which retained a high epidermal growth factor binding specific activity was used as an antigen to produce antisera in rabbits. Double-immunodiffusion experiments demonstrated that the immune serum contained precipitating antibodies to several components of detergent solubilized A-431 membranes.The immonoglobulin G fraction of this immune sera inhibited ¹²⁵I-labeled epidermal growth factor binding to receptors in: (1) intact human and mouse cells; (2) membrane preparations from A-431 cells and human placenta, and (3) solubilized A-431 membranes. Inhibition of ¹²⁵I-labeled epidermal growth factor binding was observed with divalent and monovalent fragments of immunoglobulin G prepared from the immunoglobulin G fraction. Also, the immunoglobulin G fraction blocked growth factor binding to membranes at low temperature (5°C).Anti-A-431 antibody blocked the induction of DNA synthesis in quiescent fibroblasts by epidermal growth factor in a manner similar to that of anti-epidermal growth factor antibody. Addition of either anti-A-431 or anti-epidermal growth factor antibodies to fibroblasts at times up to 5 h after the addition of epidermal growth factor completely reversed the hormone's mitogenic potential. At later times (after 12 h) addition of either antibody was without effect on the stimulation of DNA synthesis by epidermal growth factor. Anti-A-431 antibody did not block the induction of DNA synthesis in fibroblasts by fibroblast growth factor or serum.     

An insulin mediator preparation serves to stimulate the cyclic GMP activated cyclic AMP phosphodiesterase rather than other purified insulin-activated cyclic AMP phosphodiesterases

An insulin mediator preparation was obtained from rat hepatocytes which had been treated with insulin. This preparation inhibited adenylate cyclase activity. It stimulated the activity of homogeneous preparations of both the cytosolic and membrane-bound forms of rat liver cyclic GMP-activated cyclic AMP phosphodiesterase. It failed to activate homogeneous preparations of both the peripheral plasma membrane and 'dense-vesicle' cyclic AMP phosphodiesterases. The insulin mediator preparation stimulated cyclic GMP-activated cyclic AMP phosphodiesterase activity in a dose-dependent fashion with a hill coefficient of 0.46. Insulin caused the dose-dependent production of mediator activity in intact hepatocytes with a Ka of 9 pM, although concentrations of insulin greater than 10 nM progressively reduced stimulatory activity.     

Decreased tissue guanylate cyclase activity in glycosuric Djungarian hamsters (Phodopus sungorus) that is correctable with insulin

Twelve hyperglycemic, glycosuric, and ketonuric Djungarian hamsters with average blood glucose concentrations of 295+-32 mg/dl were compared to twelve non-glycosuric, but ketonuric Djungarian hamsters with average blood glucose concentrations of 88+-11 mg/dl with regards to their cyclic nucleotide metabolism. The glycosuric Djungarian hamsters had decreased guanylate cyclase (E.C.4.6.1.2.) activity in vitro and cyclic GMP levels in vivo in liver, lung, kidney, colon, heart, spleen, and pancreas that was approximately 50% of the guanylate cyclase activity in these same tissues of non-glycosuric Djungarian hamsters. The decreased tissue guanylate cyclase activity and cyclic GMP levels in the glycosuric animals could be restored to the level of non-glycosuric Djungarian hamsters with 100 U regular insulin, but not with 50 or 10 U of regular insulin. Fifty and 100 U of regular insulin also increased the level of guanylate cyclase activity in the non-glycosuric (control) animals. There was no change in adenylate cyclase (E.C.4.6.1.1.) activity but there were increased cyclic AMP levels in the glycosuric when compared to the non-glycosuric Djungarian hamsters that were correctable with 100 U of insulin. We conclude that guanylate cyclase activity is decreased in the peripheral tissues of glycosuric Djungarian hamsters as compared to non-glycosuric Djungarian hamsters and that insulin modulates this enzyme.