Inotropic response to β‐adrenergic receptor stimulation and anti‐adrenergic effect of ACh in endothelial NO synthase‐deficient mouse hearts

Abstract

It is well established that NO released from the endothelium plays an important role in the regulation of vascular tone. This was most stringently demonstrated by the generation of mice deficient in endothelial NO synthase (eNOS), which develop hypertension (Huang et al. 1995;Shesely et al. 1996;Gödecke et al. 1998). Besides its role in the regulation of organ perfusion, NO might also modulate cardiac function (for review see Kelly et al. 1996). The cytokine-mediated depression of cardiac contractility has been attributed to elevated inducible NO 1. The functional consequences of a lack of endothelial nitric oxide synthase (eNOS) on left ventricular force development and the anti-adrenergic effect of acetylcholine (ACh) were investigated in isolated hearts and cardiomyocytes from wild type (WT) and eNOS knockout (eNOS_/_) mice.2. eNOS expression in cardiac myocytes accounted for 20 % of total cardiac eNOS (Western blot analysis). These results were confirmed by RT-PCR analysis.3. In the unstimulated perfused heart, the left ventricular pressure (LVP) and maximal rate of left ventricular force development (dP/dt max ) of eNOS_/_ hearts were not significantly different from those of WT hearts (LVP: 97 ± 11 mmHg WT vs. 111 ± 11 mmHg eNOS_/_; dP/dt max : 3700 ± 712 mmHg s _1 WT vs. 4493 ± 320 mmHg s _1 eNOS_/_).4. The dobutamine (10-300 nM)-induced increase in LVP was enhanced in eNOS_/_ hearts. In contrast, L-type Ca 2+ currents (I Ca,L ) in isolated cardiomyocytes of WT and eNOS_/_ hearts showed no differences after b-adrenergic stimulation. Dibutyryl-cGMP (50 µM) reduced basal I Ca,L in WT cells to 72 ± 12 % while eNOS_/_ I Ca,L was insensitive to the drug. The prestimulated I Ca,L (30 nM isoproterenol) was attenuated by dibutyryl-cGMP in WT and eNOS_/_ cells to the same extent.5. The Ca 2+ (1.5-4.5 mM)-induced increase in inotropy was not different between the two experimental groups and b-adrenergic receptor density was increased by 50 % in eNOS_/_ hearts.6. The contractile effects of dobutamine could be inhibited almost completely by ACh or adenosine. The extent of the anti-adrenergic effect of both compounds was identical in WT and eNOS_/_ hearts. Measurement of I Ca,L in isolated cardiac myocytes yielded similar results.7. These data demonstrate that in the adult mouse (1) lack of eNOS is associated with increased cardiac contractile force in response to b-adrenergic stimulation and with elevated b-adrenergic receptor density, (2) the unaltered response of I Ca,L in eNOS_/_ cardiac myocytes to b-adrenergic stimulation suggests that endothelium-derived NO is important in mediating the whole-organ effects and (3) eNOS is unimportant for the anti-adrenergic effect of ACh and adenosine.

Cardiac Nitric Oxide Synthase 1 Regulates Basal and β-Adrenergic Contractility in Murine Ventricular Myocytes

“…In particular, Gyurko et al 24 found an enhanced maximal LV dP/dT in response to ␤-adrenergic stimulation in NOS3 Ϫ/Ϫ mice in both in vivo-perfused and Langendorff-perfused hearts, whereas others, using the same model, found no change either in calcium current or papillary muscle contractility. 4 Godecke et al, 25 using a differently generated NOS3 Ϫ/Ϫ mouse, also found a greater maximal LV dP/dT in response to dobutamine in isolated hearts. Differences in experimental conditions and preparations might have been in part responsible for the reported inconsistency in experimental findings (see review 3 ).…”

Endothelial nitric oxide synthase decreases β-adrenergic responsiveness via inhibition of the L-type Ca2+ current

“…The observed effects of NOS3 signaling on the ␤-AR response are the opposite of NOS1 (30), that is, studies have shown that NOS3 signaling depresses the functional response to ␤-AR stimulation. For example, mice with specific knockout of NOS3 (NOS3 Ϫ/Ϫ ) have an increased response to ␤-AR stimulation (4,11,19,20,47). Similarly, transgenic mice with cardiac myocyte-specific NOS3 overexpression have a decreased response to ␤-AR stimulation (9,23).…”

cGMP-Dependent Protein Kinase I Mediates the Negative Inotropic Effect of cGMP in the Murine Myocardium

“…The molecular basis for muscarinic inhibition of cardiac contractility is controversial. 1,2 Activation of NO synthase III leading to an increase of the cGMP level has been reported to contribute to muscarinic inhibition 3-5 as well as the irrelevance of NO, 6 NO synthase III,7,8 and cGMP/cGMPdependent protein kinase I (cGKI) 9 for this signaling pathway. The cGMP receptor potentially mediating the negative inotropic effect has not been identified.…”