A rapid and strong apoptotic process is triggered by hyperosmotic stress in cultured rat cardiac myocytes.

In all cell types, the maintenance of normal cell volume is an essential homeostatic function. Relatively little is known about the induction of apoptosis by hyperosmotic stress and its molecular mechanism in terminally differentiated cardiac myocytes. We compared the apoptotic response of cultured neonatal rat cardiomyoctes to hyperosmotic stress by sorbitol (SOR) with those induced by doxorubicin (Doxo) or angiotensin II (Ang II). We also examined the apoptotic-signaling pathway stimulated by the hyperosmotic stress. Apoptosis was assessed by the observation of: (1) cell viability, (2) DNA fragmentation detected by the TUNEL method and by agarose gel electrophoresis, and (3) poly(ADP-ribose)polymerase (PARP) degradation, and Bcl-XS and Bcl-XL levels by Western blot analysis. Exposure of cardiomyocytes to 0.3 M SOR for 24 h resulted in decreased cell viability and increased generation of oligosomal DNA fragments (2.5-fold of controls). At this time, 83 +/- 5% of SOR-treated myocytes were TUNEL-positive (vs 23.7 +/- 6.8% in controls; P<0.01). PARP levels also decreased by approximately 42% when cardiac myocytes were exposed to SOR. Hyperosmotic stress induced a more rapid and stronger apoptotic response in cardiomyocytes than Doxo or Ang II. In addition, SOR increased 3.2-fold Bcl-XS proapoptotic protein without changes in Bcl-XL antiapoptotic protein levels and in the p53-transactivating activity. Taken together, these results strongly suggest that hyperosmotic stress triggers cardiac myocyte apoptosis in a p53-independent manner, being earlier and stronger than apoptosis induced by Doxo and Ang II.

Extracellular regulated kinase, but not protein kinase C, is an antiapoptotic signal of insulin-like growth factor-1 on cultured cardiac myocytes.

This study aims to elucidate the signaling pathway for insulin-like growth factor-1 (IGF-1) in cultured neonatal rat cardiomyocytes and particularly the role of IGF-1 in cardiac apoptosis. IGF-1 stimulated polyphosphoinositide turnover, translocation of protein kinase C (PKC) isoforms (alpha, epsilon, and delta) from the soluble to the particulate fraction, activation of phospholipid-dependent and Ca(2+)-, phospholipid-dependent PKC, and activation of the extracellular-regulated kinase (ERK). IGF-1 attenuated sorbitol-induced cardiomyocyte viability and nuclear DNA fragmentation. These antiapoptotic effects of IGF-1 were blocked by PD-098059 (an MEK inhibitor) but not by bisindolylmaleimide I (BIM, a specific PKC inhibitor). The ERK pathway may therefore be an important component in the mechanism whereby IGF-1 exerts its antiapoptotic effect on the cardiomyocyte.

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.

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 beta-casein and alpha-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.

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 beta-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.

Selective increase in cardiac IGF-1 in a rat model of ventricular hypertrophy.

There is evidence that insulin-like growth factor-1 (IGF-1) plays a role in the development of left ventricular hypertrophy, but it is uncertain whether cardiac IGF-1 changes before or after hypertension is established, and whether circulating IGF-1 are involved in cardiac hypertrophy. We have investigated changes in circulating and left ventricular IGF-1 and in the expression of the IGF-1 gene in the left ventricles of rats during the development of hypertensive left ventricular hypertrophy (Goldblatt model; 2 kidney-1 clamped). Our results show that the left ventricular contents of IGF-1 and its mRNA were increased at one and four weeks of hypertension and hypertrophy, and that both returned to control values after nine weeks. These changes were unrelated to the seric concentration of IGF-1 in the blood. These results show that local rather than circulating IGF-1 levels contributed to the development of renovascular hypertensive left ventricular hypertrophy.

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 beta-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-gamma1, 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 EC50 values 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.

Cyclic AMP-dependent protein kinase and mechanical heart function in ventricular hypertrophy induced by pressure overload or secondary to myocardial infarction.

The role of cyclic AMP-dependent protein kinase (PKA) and systolic function during the development of left ventricular hypertrophy (LVH) still remain uncertain. The aim of this work is to study PkA activity and mechanical heart function in two experimental heart hypertrophy models: specifically, one induced by pressure overload (Goldblatt model: two kidneys, one clamped, Gb); and another secondary to myocardial infarction (MI) generated by ligation of the left coronary artery. Hypertension in the Gb group becomes evident by the third and fourth week after surgery without any significant change in the corresponding sham group. The myocardial infarction group did not show any change in systolic pressure. Different degrees of LVH for the two experimental models were observed. Relative cardiac mass (RCM) and relative ventricular mass (RVM) increased 23 and 16%, respectively, above the sham-operated rats in MI group (P < 0.05). For the pressure overload model, the increase values were 42 and 44%, respectively (P < 0.05). Left ventricular hypertrophy was also evaluated through quantitative changes in cardiac beta-myosin heavy chain which agreed with morphometric studies in Goldblatt rats. Ventricular PKA activity did not show any significant difference with respect to the sham-operated group after induction of pressure overload. For the MI model, ventricular PKA activity changed only at day 7 post-infarction with a 289% increase above the sham-operated group (P < 0.05). The absence of activation of ventricular PKA after constriction of renal artery or myocardial infarction was also corroborated by the patterns of PKA-dependent phosphorylated proteins. While force-generating capacity was increased, there was no change in ventricular PKA activity, indicating that there is no relation between this enzyme and systolic stress-strain regression lines in either pressure overload or myocardial infarction conditions. Cyclic AMP-dependent protein kinase activity had no relation with development of cardiac hypertrophy in the two experimental models of LVH. These findings contribute to the hypothesis for a multifactorial interaction of different intracellular biochemical and molecular mechanisms in the genesis of cardiac hypertrophy.

Changes in protein kinase C activity, subcellular distribution and protein phosphorylation during the lactogenic cycle in the rat mammary tissue.

Direct and indirect evidence emphasizes the participation of classical protein kinase C (cPKC) in the development and function of the mammary gland. This work shows that there are changes not only in total cPKC activity during the lactogenic cycle, but also in the relative amounts of the soluble and particulate cPKC activities and that the time-course of these two events are not the same. The time-course of translocation from the cytoplasm to the plasma membrane suggests that the soluble and particulate forms of the enzyme may be associated with growth and differentiation of the tissue, respectively. Phosphorylation patterns also show characteristic and significant differences throughout the development of the gland. These results suggest that both total mammary cPKC activity and its subcellular forms change in accordance with the proliferative and differentiative stages of the mammary gland, and that the enzyme translocation occurs during the transition from pregnancy to lactation.

Changes in cyclic AMP dependent protein kinase and active stiffness in the rat volume overload model of heart hypertrophy.

OBJECTIVE: The aim was to clarify the role of cyclic AMP dependent protein kinase (PKA) and changes in mechanical heart function during development of cardiac hypertrophy induced by volume overload. METHODS: Protein and DNA contents, PKA activity, and peak systolic stress-strain relationships in hearts from animals submitted to aortocaval shunt were assessed as a function of time. Sham operated (control) rats were used as controls. RESULTS: Heart weight to body weight ratio and cardiac protein content per heart increased from d 7 (p < 0.005 and p < 0.01, respectively) reaching their highest values by d 56; the same occurred with cardiac DNA content. PKA activity.g-1 tissue in soluble extracts of hearts from rats with aortocaval shunt increased by 2.7-fold on d 2 (p < 0.005), reached a ninefold peak increase by d 7 (p < 0.0001) and declined to fourfold by d 56 with respect to control values. The end peak systolic stress-strain relation slopes were: control, 368(SEM 14) g.cm-2 (n = 16); aortocaval shunt values: 2 d, 514(28) g.cm-2 (n = 6); 7 d, 579(10) g.cm-2 (n = 7); and 56 d, 554(28) g.cm-2 (n = 7). The force generating capacity at 0% strain was also significantly higher in the shunt groups as compared to sham operated controls (p < 0.01). Early activation of PKA was also confirmed through endogenous cardiac protein phosphorylation. SDS-PAGE gel electrophoretogram and autoradiography showed more heavily phosphorylated bands in aortocaval shunt hearts than in the control group. CONCLUSIONS: PKA activity and the slope of systolic stress-strain regression line followed a similar trend throughout the study, with an early increase in both variables by d 2 in the shunt group, reaching a peak at d 7, and decreasing thereafter but remaining higher than in controls. PKA activity appears to be related to increased force generating capacity rather than to hypertrophy or increased cardiac protein content. Thus PKA activation is an early biochemical event after aortocaval shunt, followed later by cardiac hypertrophy. Changes in PKA activity showed a similar trend to mechanical heart function over time. These findings help to explain the changes in the mechanical properties of the heart preceding the development of cardiac hypertrophy in the rat model of volume overload.

Changes in beta-adrenergic receptors of rat heart and adipocytes during volume-overload induced cardiac hypertrophy.

Modification of cardiac beta-adrenergic receptors (beta-AR), resulting from the stimulation of the sympathetic nervous system, is one of the most important factors in the generation of cardiac hypertrophy and heart failure. In this research, we propose the utilization of adipocytes as an alternative to the use of predominantly beta 2-AR subtype containing circulating lymphocytes for the convenient assessment of cardiac failure in the experimentally, volume-overload induced heart hypertrophy in rats. Using this model, we measured beta-AR both in the heart and adipocytes of male rats 2, 7, 21 and 56 days after creating an aorta-cava fistula. Whereas an increase (58%) in cardiac beta-AR density from day 7 to 21 was followed by a decrease in this measurement (30%) on day 56 [changes expressed as percentage of controls; no significant changes in beta-AR affinity (Kd) were recorded at any of the time interval studied], adipocytes beta-AR density showed a progressive increase starting on day 21 (87%) which continued until the end (131%) of the study period. This lack of correlation of the beta-AR population in both tissues supports the need for a specific evaluation of the beta 1-AR subtype in the heart and adipocyte in order to evaluate the usefulness of adipocyte cells as an alternative to assess cardiac failure.

Neurochemical evidence for the presence of sympathetic nerve terminals in the rat mammary gland: Changes during the lactogenic cycle.

Experiments were undertaken to obtain neurochemical evidence of the presence of sympathetic nerve terminals in the rat mammary gland and the changes occurring in them during the lactogenic cycle. The norepinephrine (NE) content of the gland changed during the lactogenic cycle. Higher levels of NE were found during virginity and involution, whereas a lower level was found at 14 days of lactation. Surgical and chemical (with 6-hydroxydopamine) denervation reduced the norepinephrine content of the gland by 61 and 90%, respectively. Uptake of [(3)H]norepinephrine by the mammary gland was saturable and specifically blocked by cocaine. No changes in the maximal capacity of incorporation during the lactogenic cycle were found, but the affinity of NE for the transmembrane carrier was low during lactation, as was the NE content, suggesting a decrease in the sympathetic nerve activity during this stage of the lactogenic cycle. In support of this, we found a decrease in total NE released after stimulation with 80 mM KCI. The neurochemical evidence obtained during this research strongly suggests that rat mammary gland is innervated by sympathetic nerves and that their activity changes during the lactogenic cycle.

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.

Epidermal growth factor increases cyclic AMP levels in pregnant rat mammary gland explants.

Addition of epidermal growth factor (EGF) to a rat mammary organ culture results in increase levels of intracellular cyclic AMP. This dose dependent EGF effect occurs at physiological concentrations of the hormone and was maximal in early and middle pregnancy.

[Beta adrenergic receptors in circulating lymphocytes of patients with chronic heart failure]. / Receptores beta adrenérgicos en linfocitos circulantes de pacientes con insuficiencia cardiaca crónica.

Severe decompensated chronic heart failure is associated to increased levels of circulating catecholamines and decreased density of myocardial beta-adrenergic receptors. In 14 patients with stable, class II-III heart failure we studied circulating lymphocytes to determine the number of beta adrenergic receptors, the dissociation constant of 3H dihydroalprenolol (kd) and the intracellular content of cyclic AMP (AMPc). Results (mean +/- SEM) were compared to those obtained in 10 healthy controls. The number of beta receptors was significantly decreased (105 +/- 16 vs 185 +/- 24, fmol/mg of membrane protein, p less than 0.01). No differences were found in Kd (1.65 +/- 0.2 vs. 1.36 +/- 0.28 nm) nor the level of AMPc (7.9 +/- 2.1 vs 7.1 +/- 2.9 pmol/mg protein), respectively. The decreased number of beta adrenergic receptors in the circulating lymphocytes may be related to the increased level of circulating catecholamines that have been shown to be present during exercise in these patients.

Hormonal modulation of the rat mammary gland gamma-glutamyltranspeptidase.

1. The in vivo effect of estradiol and domperidone (a hypophysis stimulator of prolactin secretion) in immature ovariectomized-adrenalectomized and hypophysectomized rat mammary gland was studied. 2. gamma-Glutamyltranspeptidase activity was used to evaluate the role of estradiol in the specific response of the gland to prolactin. 3. Our results suggest that the activity of gamma-glutamyltranspeptidase, like casein and lactose synthetase, is part of the specific response of the gland to prolactin.

The role of thyroid hormones in the control of beta-adrenergic receptors in rat mammary gland.

We have studied the effect of hyperthyroidism and hypothyroidism on the lactating rat mammary gland number of beta-adrenergic receptors and basal intracellular cyclic AMP levels. We also describe the effects of isoproterenol, cholera toxin and forskolin challenge on these parameters. Using cyclic AMP levels as an indication of receptor functionality our results indicate that both thyroid states are accompanied by a decrease in the response to beta-adrenergic receptor stimulation.