Mechanisms of L-type Ca(2+) current downregulation in rat atrial myocytes during heart failure.

Downregulation of the L-type Ca(2+) current (I(Ca)) is an important determinant of the electrical remodeling of diseased atria. Using a rat model of heart failure (HF) due to ischemic cardiopathy, we studied I(Ca) in isolated left atrial myocytes with the whole-cell patch-clamp technique and biochemical assays. I(Ca) density was markedly reduced (1.7+/-0.1 pA/pF) compared with sham-operated rats (S) (4.1+/-0.2 pA/pF), but its gating properties were unchanged. Calcium channel alpha(1C)-subunit quantities were not significantly different between S and HF. The beta-adrenergic agonist isoproterenol (1 micromol/L) had far greater stimulatory effects on I(Ca) in HF than in S (2.5- versus 1-fold), thereby suppressing the difference in current density. Dialyzing cells with 100 micromol/L cAMP or pretreating them with the phosphatase inhibitor okadaic acid also increased I(Ca) and suppressed the difference in density between S and HF. Intracellular cAMP content was reduced more in HF than in S. The phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine had a greater effect on I(Ca) in HF than in S (76.0+/-11.2% versus 15.8+/-21.2%), whereas the inhibitory effect of atrial natriuretic peptide on I(Ca) was more important in S than in HF (54.1+/-4.8% versus 24.3+/-8.8%). Cyclic GMP extruded from HF myocytes was enhanced compared with S (55.8+/-8.0 versus 6.2+/-4.0 pmol. mL(-1)). Thus, I(Ca) downregulation in atrial myocytes from rats with heart failure is caused by changes in basal cAMP-dependent regulation of the current and is associated with increased response to catecholamines.

Nebivolol induces calcium-independent signaling in endothelial cells by a possible beta-adrenergic pathway.

Nebivolol is a highly selective beta1-adrenoreceptor-blocking agent with a peculiar pharmacodynamic profile. It has peripheral acute vasodilating properties that are mediated by modulation of the endogenous production of nitric oxide. In this study we analyzed the different signaling pathways implicated in the response of human umbilical vein endothelial cells to nebivolol. Its effect on endothelial transduction pathways was determined by assaying phospholipase C and A2 activities and cyclic adenosine monophosphate (AMP) production. Variations in intracellular calcium concentration were also measured. Our results showed that nebivolol activates a calcium-independent transduction pathway that implicates an increase in adenylate cyclase and phospholipase A2 activity. Beta1- or beta2-Adrenoreceptor antagonists do not inhibit the action of nebivolol. However, its action on cyclic AMP production is inhibited by bupranolol, a beta1-3-adrenoreceptor antagonist, and S-(-)-cyanopindolol, a selective beta3-adrenoreceptor antagonist. Nebivolol also dose-dependently increased nitrite production. This effect was inhibited by bupranolol, suggesting that the possible action of nebivolol on beta3-adrenoreceptor is involved in its vasodilating properties. This study suggests that nebivolol could behave as a beta3-adrenoreceptor agonist and induce some calcium-independent pathways implicating phospholipase A2 and adenylate cyclase. This agonistic activity of nebivolol seems to be responsible for its endothelium-dependent vasodilating activity.

Characterization of effects of endothelin-1 on the L-type Ca2+ current in human atrial myocytes.

The effects of endothelin-1 (ET-1) on the L-type Ca2+ current (I(Ca)) were examined in whole cell patch-clamped human atrial myocytes. Depending on the initial current density, ET-1 (10 nM) increased the amplitude of I(Ca) by 99 +/- 7% or decreased it by 33 +/- 2%. The stimulatory effect predominated on current of low density (2.3 +/- 0.2 pA/pF), whereas I(Ca) of higher density (5.8 +/- 0.3 pA/pF) was inhibited by ET-1. After I(Ca) stimulation by 1 microM isoproterenol, ET-1 always inhibited the current by 32 +/- 7% (P < 0.05), an effect that was suppressed by pretreating myocytes with pertussis toxin. Atrial natriuretic peptide (ANP) inhibited I(Ca) (41 +/- 3%) by reducing intracellular cAMP concentration. In ANP-treated myocytes, the stimulatory effect of ET-1 on I(Ca) predominated (52 +/- 7%). The inhibitory effect of ET-1 on I(Ca) was blocked by the ET(A) antagonist BQ-123, whereas the stimulatory effect was suppressed by the ET(B) agonist BQ-788. We conclude that ET-1 has opposite effects on I(Ca) depending on the baseline amplitude of current, and both subtype ET receptors are implicated in the signal transduction pathways.

Endothelium-independent conversion of angiotensin I by vascular smooth muscle cells.

The conversion of angiotensin I (AT-I) to angiotensin II (AT-II) by angiotensin I-converting enzyme (ACE) is a key step in the action of angiotensins. ACE is constitutively expressed in endothelial cells, but can also be detected at low levels in smooth muscle cells (SMC). Furthermore, in rats the ACE activity can be induced in SMC in vivo by experimental hypertension or vascular injury and in vivo by corticoid treatment. This study was therefore undertaken to evaluate the conversion of AT-I and its subsequent effects in SMC in basal conditions and after stimulation by dexamethasone. Using rat and human SMC, showed that dexamethasone induced ACE expression and that this enzyme was functional, leading to AT-II-dependent intracellular signaling. A fourfold increase in phospholipase C activity in response to AT-I was observed in dexamethasone-activated SMC compared with quiescent SMC. This effect of dexamethasone on signal transduction is dependent on ACE activity, whereas AT-II receptor parameters remain unchanged. The action of AT-I was blocked by an AT1 receptor antagonist, suggesting that it was mediated by AT-II. Similarly, dexamethasone-induced ACE expression was present in human SMC, and calcium signaling was mobilized in response to AT-I in activated human cells. Experiments performed with cocultures of endothelial cells and SMC in a Transwell system showed that the response to AT-I was limited to the compartment where AT-I was localized, suggesting that AT-I does not pass through the endothelial cell barrier to interact with underlying SMC. Our data suggest that in rat, as in human SMC, the conversion of AT-I into AT-II and the signal transduction in response to AT-I are ACE expression-dependent. In addition, the present findings show that this SMC response to AT-I is endothelium-independent, supporting the idea of a local generation of AT-II in the vascular wall.

Tyrosine kinase and protein kinase C regulate L-type Ca(2+) current cooperatively in human atrial myocytes.

The effects of tyrosine protein kinases (TK) on the L-type Ca(2+) current (I(Ca)) were examined in whole cell patch-clamped human atrial myocytes. The TK inhibitors genistein (50 microM), lavendustin A (50 microM), and tyrphostin 23 (50 microM) stimulated I(Ca) by 132 +/- 18% (P < 0.001), 116 +/- 18% (P < 0.05), and 60 +/- 6% (P < 0.001), respectively. After I(Ca) stimulation by genistein, external application of isoproterenol (1 microM) caused an additional increase in I(Ca). Dialyzing the cells with a protein kinase A inhibitor suppressed the effect of isoproterenol on I(Ca) but not that of genistein. Inhibition of protein kinase C (PKC) by pretreatment of cells with 100 nM staurosporine or 100 nM calphostin C prevented the effects of genistein on I(Ca). The PKC activator phorbol 12-myristate 13-acetate (PMA), after an initial stimulation (75 +/- 17%, P < 0.05), decreased I(Ca) (-36 +/- 5%, P < 0.001). Once the inhibitory effect of PMA on I(Ca) had stabilized, genistein strongly stimulated the current (323 +/- 25%, P < 0.05). Pretreating myocytes with genistein reduced the inhibitory effect of PMA on I(Ca). We conclude that, in human atrial myocytes, TK inhibit I(Ca) via a mechanism that involves PKC.