Mario Sapag-Hagar

Insulin-like Growth Factor-I Rapidly Activates Multiple Signal Transduction Pathways in Cultured Rat Cardiac Myocytes

In response to insulin-like growth factor-I (IGF-I), neonatal rat cardiac myocytes exhibit a hypertrophic response. The elucidation of the IGF-I signal transduction system in these cells remains unknown. We show here that cardiac myocytes present a single class of high affinity receptors (12,446 ± 3,669 binding sites/cell) with a dissociation constant of 0.36 ± 0.10 nm. Two different β-subunits of IGF-I receptor were detected, and their autophosphorylation was followed by increases in the phosphotyrosine content of extracellular signal-regulated kinases (ERKs), insulin receptor substrate 1, phospholipase C-γ1, and phosphatidylinositol 3-kinase. IGF-I transiently activates c-Raf in cultured neonatal cardiac myocytes, whereas A-raf is activated much less than c-Raf. Two peaks of ERK activity (ERK1 and ERK2) were resolved in cardiac myocytes treated with IGF-I by fast protein liquid chromatography, both being stimulated by IGF-I (with EC50values for the stimulation of ERK1 and ERK2 by IGF-I of 0.10 and 0.12 nm, respectively). Maximal activation of ERK2 (12-fold) and ERK1 (8.3-fold) activities was attained after a 5-min exposure to IGF-I. Maximal activation of p90 S6 kinase by IGF-I was achieved after 10 min, and then the activity decreased slowly. Interestingly, IGF-I stimulates incorporation of [3H]phenylalanine (1.6-fold) without any effect on [3H]thymidine incorporation. These data suggest that IGF-I activates multiple signal transduction pathways in cardiac myocytes some of which may be relevant to the hypertrophic response of the heart.

Effect of inhibitors of signal transduction on IGF-1-induced protein synthesis associated with hypertrophy in cultured neonatal rat ventricular myocytes

IGF‐1 increased 2‐fold protein synthesis in cardiac myocytes. Genistein, whether added during preincubation or with IGF‐1 at the start of incubation, significantly inhibited the IGF‐1‐induced stimulation of protein synthesis, autophosphorylation of the β‐subunit of IGF‐1 receptor and inhibition of ERK. When added 1 or 6 h after IGF‐1, however, genistein was without effect. IGF‐1‐stimulated protein synthesis was also significantly inhibited by PD‐098059, staurosporine, and rapamycin, but not by wortmannin, in cardiac myocytes. Some inhibitors produced a reduction in cell size. Activation of the ERK cascade by IGF‐1 may be responsible for some of the features associated with cardiac myocyte hypertrophy.

γ-glutamyltranspeptidase activity and cyclic AMP levels in rat liver and mammary gland during the lactogenic cycle and in the oestradiol-progesterone pseudo-induced pregnancy

The mammary gland is a tissue subject to close hormonal control over the whole course of the lactation cycle with the most striking changes in hormonal balance occurring at parturition and the onset of lactation [ 11. Studies of enzyme changes in the tissue at this tra@tion have shown dramatic increases in the activity of those enzymes involved in the biosynthesis of the milk constituents and, in some cases, a hormonal regulation of their activity has been implied [2,3]. Although the membrane-bound enzyme 7-glutamyltranspeptidase has been found in most secretory tissues its activity has not yet been fully studied in mammary gland where, as the enzyme initiating the r-glutamyl cycle [4], it may be expected to fill an important role in regulating the entry of amino acids into the cell. The enzyme has been reported to be regulated by cyclic AMP in liver [5] while, in rat seminal vesicles, its activity appears to be testosteronedependent [6]. This investigation is a study of 7-glutamyltranspeptidase in the liver and mammary glands taken from pregnant and lactating rats and from rats in which mammary involution (caused by weaning of the pups) had been allowed to proceed for 5 days. Parallel studies were also carried out on the same tissues from hormonally induced pseudopregnant animals. The results show that the enzyme increases slowly in the pregnant mammary gland and then sharply in

In vivo and in vitro evidence of basic fibroblast growth factor action in mouse mammary gland development

Basic fibroblast growth factor (bFGF) stimulated [3H]thymidine incorporation at all stages of development, although the magnitude of this effect was the greatest in cells derived from pregnant mice. Cells primed with insulin and bFGF synthesized more casein than cells not exposed to either hormone. bFGF inhibited casein synthesis and decreased the amounts of β‐casein and α‐lactalbumin transcripts in cells from pregnant animals simultaneously incubated with insulin, hydrocortisone and prolactin. bFGF content in mammary gland increased with puberty and pregnancy, but decreased markedly in lactation; the number of bFGF receptors in epithelial cells changed in parallel. These data suggest that bFGF may have a physiological role both in stimulating growth and in inhibiting functional differentiation of normal mouse mammary epithelial cells.

Calmodulin and cyclic nucleotide-phosphodiesterase activities in rat mammary gland during the lactogenic cycle

Numerous studies [l-4] have documented that cyclic AMP plays a central role in the hormonal regulation of cell growth and function and that its content in the mammary gland increases progressively during the growth stage of pregnancy and then, at parturition, decreases rapidly to the low levels seen during the lactational period. These findings suggest that cyclic AMP is a negative controlling factor for lactogenesis. It has also been demonstrated that 3’,5’nucleotide phosphodiesterase, the enzyme which hydrolyses cyclic nucleotides, is activated by calmodulin, a thermostable low M, protein already studied in several tissues, but not in mammary gland [S]. Calmodulin is an intracellular calcium receptor protein that binds to Ca2+ when their concentration increases in response to stimulus. This binding induces a distinct change in the shape of the cahnodulin molecule which in turn is capable of binding to any of several enzymes, activating them and so setting in motion the biochemical changes that produce directly the response to the stimulus. Calmodulin can also indirectly modify cell activation by affecting the concentration of calcium itself and that of other important cellular regulators, including cyclic AMP. The finding that Ca*+, in cooperation with calmodulin, can alter cyclic nucleotides concentration indicates one way by which the actions of the 2 regulatory agents may be integrated in regulating the lactogenic cycle in mammary gland. and its activating capacity for the enzyme, both from bovine brain and mammary gland, and analyse its kinetic properties. Our results support the view that calmodulin plays a regulatory role during lactogenesis.

Binding and production of insulin-like growth factor-I in rat mammary gland.

1. Mammary tissue from pregnant rat presents low and high affinity IGF-I functional receptors. 2. Mammary explants from pregnant and lactating rats secrete IGF-I and its production was related to the developmental stage of the gland. 3. An inverse relationship between IGF-I production and tissue binding capacity was observed.

Effects of antihypertensive treatment on cardiac IGF-1 during prevention of ventricular hypertrophy in the rat

There is some evidence that cardiac rather than circulating insulin-like growth factor-1 (IGF-1) levels contribute to the development of renovascular hypertensive left ventricular hypertrophy (LVH), remaining unknown the effects of antihypertensive drugs on IGF-1 levels. We have assessed here the preventive effects of enalapril, losartan, propanolol and alpha-methyldopa on left ventricle (LV) and circulating IGF-1 levels in a rat model of hypertension and LVH (Goldblatt, GB). Our results show that relative LV mass and the LV content of IGF-1 were significantly lower with all antihypertensive drugs in GB rats (p<0.001). Serum concentrations of IGF-1 were lower in GB rats treated with enalapril, alpha-methyldopa and propanolol (p<0.01), but not in those treated with losartan. These results support the hypothesis that local rather than seric IGF-1 contributes to the development of left ventricular hypertrophy induced by pressure overload in the rat.

Glutathione andγ -glutamyl cycle enzymes in rat mammary gland

The main enzymes of theγ-glutamyl cycle during the lactogenic cycle in rat mammary gland were studied. A significant increase was found in all of them with the onset of lactogenesis. The effect of methionine sulfoximine on reduced glutathione concentration was studied in tissue slices of lactating mammary gland. The findings suggest that this compound affects glutathione synthesis by inhibitingγ-glutamylcysteine synthetase.

A role for adrenaline and calmodulin in modulating cyclic AMP levels during the lactogenic cycle.

The effect on lactose production of several external modulators of intracellular cyclic AMP was studied in rat mammary gland tissue slices and explants. Adrenaline, a β‐adrenergic receptor effector, forskolin, a direct adenylate cyclase activator and fluphenazine, a calmodulin inhibitor, all produced an increase in the intracellular level of cyclic AMP and a concomitant inhibition of lactose production. These results suggest a role for adrenaline and calmodulin in modulating cyclic AMP levels in mammary tissue during the lactogenic cycle.

Changes in electrophoretic migration due to impregnation of the paper strips with various buffers

Abstract Paper strips were impregnated with different buffers before serum protein electrophoresis, without altering the rest of the usual experimental conditions : buffer in the vessels, duration of the experiment, voltage, etc. The electropherograms obtained when the composition or the ionic strength of the buffer was modified were different from each other, but characteristic for the buffer solution tested as impregnating solution. It was concluded that protein migration in paper electrophoresis is a special effect that depends on the migration media, on which other usually interfering factors have no influence.