RESUMEN

Calcium homeostasis is intimately regulated by protein kinase phosphorylation cascades that are also involved in the induction and maintenance of cardiac hypertrophy. In addition, the development of cardiac hypertrophy has been associated with alteration in the activation of the adrenergic system. Therefore, we investigated the specific role of protein kinase A (PKA) and C (PKC) on cardiac muscle contractile activity in the presence and absence of adrenergic stimulation. Isolated left atrial preparations from sham- and volume overload-induced cardiac hypertrophied rats were superfused with Tyrode and electrically stimulated at 0.75 Hz. Contraction was assessed in the basal and pre-stimulated (norepinephrine, 10(-9)M) states. Specific inhibitors, KT 5720 for PKA and Ro-32-0432 for PKC, were used. Peak tension development in left atria from sham-operated rats was more sensitive to PKC- than PKA-inhibition, whereas this differential sensitivity was abolished in the hypertrophied hearts. This difference was mainly due to an increase in the role of PKA in the contractile response. Developed peak tension by left atria from shunt rats was higher than that from sham rats, but when expressed to relative tissue mass, hypertrophied muscle showed weaker contraction than that from the sham group. In addition, the left atrial velocity of contraction in the sham is PKA-sensitive, while that of the shunt is PKC-sensitive. Furthermore, the velocity of relaxation shows dependency on both protein kinases, with PKC having a greater effect than PKA in the hypertrophied group. NE increased the PTD and the velocity of contraction (+dT/dt) through PKA and PKC dependent mechanisms, without affecting the velocity of relaxation (-dT/dt) in atrial muscle from sham rats. In contrast, during eccentric hypertrophy NE effectively reduced PTD as well as the -dT/dt through a PKC-dependent mechanism. The present study demonstrates that during early development of moderate eccentric cardiac hypertrophy there is: (1) a reduced specific peak tension developed due to an imbalance in the PKA and PKC activation; (2) a change in the protein kinase dependence of the velocity of contraction and relaxation from PKA to PKC with atrial hypertrophy; and (3) a negative inotropic response to adrenergic receptor stimulation. These functional responses may play a critical role in the cardiac performance during the progression of eccentric cardiac hypertrophy into the decompensated phase and heart failure.

Asunto(s)

RESUMEN

Angiotensin II (Ang II) mediates its effects through its non-tyrosine-kinase G protein coupled Ang-II type 1 receptor (AT1). Growing evidence indicates that a functional insulin-like growth factor-1 (IGF-1) tyrosine kinase receptor is required for Ang-II-induced mitogenesis. Along with Ang II, we have previously shown that changes in IGF-1 receptor binding at myofibers are causative agents for cardiac eccentric hypertrophy. This study investigated the interaction of the renin-angiotensin system with the IGF-1 receptor during the development and regression of cardiac hypertrophy. Alterations in IGF-1 binding were evaluated in the CHAPS-pretreated perfused heart. Four weeks of aortocaval shunt increased relative heart mass by 76% without a major change in body mass or systolic blood pressure. Binding studies showed that IGF-1 has a higher affinity for the cardiac myofibers of shunt than sham rats. Two weeks of treatment with the angiotensin-converting enzyme (ACE) inhibitor captopril (0.5 g/L in drinking water) or the AT1-antagonist losartan (10 mg/(kg x day)) reduced cardiac hypertrophy by 54 and 42%, respectively. However, while both ACE inhibition and AT1-antagonist treatments produced equivalent regression in ventricular hypertrophy, captopril was more efficacious than losartan in the regression of atrial hypertrophy. Regression of cardiac hypertrophy in the shunt by either captopril or losartan was accompanied with a reduction or normalization of the elevated IGF-1 affinity. Thus, the induction and regression of cardiac eccentric hypertrophy seems to be largely dependent on cross talk between the renin-angiotensin system and the IGF-1 axis at the receptor level.

Asunto(s)

RESUMEN

Insulin-resistance is associated with a number of disease states such as diabetes, syndrome X, and hypertension. These situations may be coupled to insulin-resistance through the insulin signaling system as a common pathway. The purpose of this study was to investigate the receptor binding alterations in streptozotocin-induced diabetic rats, spontaneously hypertensive rats and aortocaval shunted rats (eccentric cardiac hypertrophy). A physical model describing a 1:1 stoichiometry of ligand binding with its receptor is proposed describing reversible binding of [(125)I]insulin or [(125)I]IGF-1 at the microvascular endothelial as well as with the cardiac myocytes after CHAPS-treatment. Analysis of the collected effluents are curve-fitted with a conservation equation and a first-order Bessel function which allowed the calculation of the forward binding constants (k(n)), the reversible constants (k(-n)), the dissociation constants (k(d)) and the residency time constants (tau). The results showed that streptozotocin-induced diabetic rats showed insulin-resistance through alterations in the kinetics of insulin receptor binding. The normotensive controls of the spontaneously hypertension rats (SHR) carry themselves insulin-resistant receptors whose binding to insulin worsens in the hypertensive SHR. Negative cooperativity between insulin-like growth factor IGF-1 and insulin receptors could be a causative factor predisposing for insulin-resistance in the aortocaval shunted rats to insulin resistance. The defects may be occurring at the receptor level in insulin-dependent diabetes mellitus, Wistar-Kyoto rats and spontaneously hypertensive rats. In conclusion, alterations in the kinetics of insulin binding to its receptor seem to play a central role for the initiation of insulin-resistance during the various pathophysiological states.

Asunto(s)

RESUMEN

The purpose of this study is to assess the effect of an apparent alteration in intracellular pH and the effect of amiloride on the activity of the Na+/H+ antiporter in perfused rat kidney. Rat kidney-Na+ retention was determined using tracer 22Na in perfusate composed of HCl-glycine buffer (pH 3.80 to pH 5.92) or NH4OH-glycine buffer (pH 6.22-7.95) containing Na+ to match physiologic concentrations. Plotting renal Na+ retention for 10 min versus pH in absence of amiloride showed two classical uncompetitive activator curves for H+, one curve from pH 4.19 to 5.10 and another from pH 6.22 to 7.95. H+ acts as an uncompetitive reversible binding substrate with the receptor triggering activation of the exchanger already sequestered with Na+, thus yielding two Ka values for the exchanger suggesting non-first order kinetics. Using an equation derived for uncompetitive-activation binding of Nao+ and Hi+, plotting [mM Na+ mg protein-1 10 min-1]-1 versus [H+], two linear plots are observed on Cartesian coordinates with abscissa intersecting at 47 +/- 1 microM, pKa = 4.32 +/- 0.02 (pH 4.19-5.10) and 4.21 +/- 0.02 microM, pKa = 5.38 +/- 0.01 (pH 6.22-7.95), respectively. Perfusing buffer containing 2 mM amiloride, completely inactivated the antiporter showing stronger inhibition between pH 3.80 and 5.92. Results suggest the presence of two uncompetitive binding sites for H+ with the Na+/H+ exchanger. One is a high affinity binding site at physiological intracellular apparent pH, and another is a low affinity binding site at ischaemic apparent pH, implying the existence of two titration sites for intracellular pH regulation.

Asunto(s)

Asunto(s)

RESUMEN

The effects of metabolic inhibition on K+ background currents and action potential duration were investigated in neonatal rat ventricle cells during early development. Action potentials and ionic currents were measured with the patch clamp technique in current and voltage clamp mode in cells isolated with collagenase from 1 day and 7 day old rats. During the first postnatal week, the cell surface increased from 1700 to 2210 microm2 and the membrane hyperpolarized from -66.1 to -72.0 mV. Concomitantly the action potential shortened and the plateau became more negative. Inhibition of oxidative phosphorylation (50 microM 2,4 DNP) or of glycolysis in 1 day old rats (5 mM 2-deoxyglucose, 2-DG) also shortened the action potential by about 50% after 5 min exposure. The background current measured in the absence of INa, ICa,L, and Ito included: (1) an inward rectifying component whose I/V curves crossed over when measured in 6, 15, or 30 mM [K]o and showed an increase in slope conductance when [K]o was raised. Inward rectification was abolished by 2.4 mM Ba2+ in 1 day old cells and by 0.2 mM one week after birth; (2) a glibenclamide (100 microM) sensitive component that developed with time after membrane rupture (5-10 min) showing a higher current density in 7 than in 1 day old animals (1.4 vs 0.2 microA x cm-2 at -50 mV); and (3) a small and almost linear leak component of comparable amplitude in both age groups. Inhibition of oxidative phosphorylation with 2.5 microM carbonylcyanide m-chlorophenylhydrazone induced the development of background currents with different properties in both age groups: An inwardly rectifying Ba2+ sensitive current in 1 day old cells and a glibenclamide sensitive outwardly rectifying current in the 7 day old group. In contrast, exposure to 5 mM 2-DG provoked in all cells the development of an outwardly rectifying current that was blocked by glibenclamide. We conclude that the electrophysiologic response to metabolic inhibition is determined by the relative importance of the metabolic pathways present which in turn depends on the developmental state of the cells.

Asunto(s)

RESUMEN

In order to assess the interaction between the cAMP-dependent and the cGMP-dependent phosphorylation pathways on the slow Ca2+ current (ICa(L)), whole-cell voltage-clamp experiments were conducted on embryonic chick heart cells. Addition of 8Br-cGMP to the bath solution reduced the basal (unstimulated) ICa(L). Intracellular application of the catalytic subunit of PK-A (PK-A(cat); 1.5 microM) via the patch pipette rapidly potentiated ICa(L) by 215 +/- 16%) (n = 4); subsequent addition of 1 mM 8Br-cGMP to the bath reduced the amplitude of ICa(L) towards the initial control values (123 +/- 29%). Intracellular application of PK-G (25 nM pre-activated by 10(-7) M cGMP), rapidly inhibited the basal ICa(L) by 64 +/- 6% (n = 8). Heat-denatured PK-G was ineffective. Subsequent additions of relatively high concentrations of 8Br-cAMP (1 mM) or isoproterenol (ISO, 1-10 microM) did not significantly remove the PK-G blockade of ICa(L). The results of the present study suggest that: (a) 8Br-cGMP can inhibit the basal or stimulated (by PK-A(cat)) ICa(L) in embryonic chick myocardial cells. (b) PK-G applied intracellularly inhibits the basal ICa(L).

Asunto(s)