cAMP Signal Transduction, A Potential Compensatory Pathway for Coronary Endothelial NO Production After Heart Failure
Objective— This study investigated whether cAMP signal transduction regulates coronary microvascular NO production after heart failure (HF), a state in which endothelial NO synthase (eNOS) is downregulated.
Methods and Results— Myocardial microvessels were isolated. Nitrite, the hydration product of NO, from these vessels was quantified by using the Griess reaction. Forskolin (10−4 mol/L), 8-bromo-cAMP (10−2 mol/L), isoproterenol (10−4 mol/L), or adrenomedullin (10−6 mol/L) significantly increased nitrite release by 78±8, 84±14, 71±11, and 73±15 pmol/mg, respectively, from isolated microvessels from normal canine hearts (P<0.05 versus control). Bradykinin (10−5 mol/L) and acetylcholine (10−5 mol/L) increased nitrite release by 83±13 and 72±6 pmol/mg, respectively (P<0.05 versus control). However, NO production induced by bradykinin and acetylcholine was markedly reduced after HF (46±7 and 39±7 pmol/mg, respectively; P<0.05 versus normal), reflecting eNOS downregulation (55% in eNOS protein). Surprisingly, NO production in response to forskolin, 8-bromo-cAMP, isoproterenol, and adrenomedullin not only was preserved but also was substantially enhanced in these microvessels after HF (121±14, 124±21, 107±18, and 122±16 pmol/mg, respectively; P<0.05 versus normal group) and was associated with an upregulation of protein kinase B (220% increase in protein kinase B protein). All these responses were in an NO synthase or a protein kinase A inhibitor–blockable manner.
Conclusions— Our data indicate that cAMP signal transduction may be an important potential compensatory pathway to increase myocardial microvascular NO production after HF when eNOS is downregulated.
A number of physiological factors, such as shear stress, and acetylcholine and bradykinin regulate endothelial NO production.1–4⇓⇓⇓ Adenosine and β-adrenergic receptor agonists are widely believed to cause endothelium-independent vasodilation by increasing intracellular cAMP in smooth muscle.3,4⇓ However, growing evidence5–11⇓⇓⇓⇓⇓⇓ indicates that many classic endothelium-independent vasodilators, such as isoproterenol, adenosine, and prostacyclin, cause, at least in part, endothelium-dependent and NO-mediated vasodilation. We have also found that stimulation of cAMP signal transduction (ie, endothelium-independent vasodilation) increases endothelial NO production in isolated canine and porcine coronary blood vessels.12,13⇓ Taken together, these results suggest that there is a cAMP signal transduction system in blood vessels that may also participate in the regulation of endothelial NO production under normal or pathophysiological conditions. Heart failure (HF) is associated with an impaired endothelial NO production,14–20⇓⇓⇓⇓⇓⇓ which has been attributed to downregulation of endothelial NO synthase (eNOS)21 and may be an important mechanism contributing to the progressive dysfunction in clinical and experimental chronic HF. Our previous studies have shown defective muscarinic or B2-kinin receptor–mediated endothelial NO production in blood vessels from dogs with pacing-induced HF20–23⇓⇓⇓ or in explanted failing human hearts.24 Whether cAMP signal transduction still functions in endothelium and whether this pathway could play a role in the regulation of endothelial NO production after HF remains unknown. Therefore, the goal of the present study was to determine whether stimulation of cAMP signal transduction affects coronary endothelial NO production after pacing-induced HF and whether NO production mediated by this pathway is altered after HF.
Please refer to http://atvb.ahajournals.org for online Methods supplement.
Twenty adult mongrel dogs (body weight 21 to 30 kg) in 2 groups, normal (n=11) and pacing-induced HF (n=9), were used in the present study. HF was induced by rapid left ventricular (LV) pacing for 4 weeks. All of these studies were approved by the Institutional Animal Care and Use Committee of New York Medical College and conform to current Guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health and American Physiological Society.
Isolation and Incubation of Myocardial Microvessels
Isolation of coronary microvessels from the LV free wall of the canine heart was performed according to a previously described method,12,13,20–22,24⇓⇓⇓⇓⇓ originally developed by Gerritsen and Printz.25
Preparation of eNOS and PKB Protein From Myocardial Microvessels for Western Blotting
Western Blotting was used to quantify eNOS (135-kDa) and protein kinase B (PKB, 59-kDa) protein from isolated myocardial microvessels in normal and failing canine hearts. Western blotting has been used in our previous studies.21
cAMP Signal Transduction–Mediated NO Production From Myocardial Microvessels From Normal and Failing Canine Heart
Increasing concentrations of forskolin (10−10 to 10−4 mol/L), 8-bromo-cAMP (10−8 to 10−2mol/L), isoproterenol (10−10 to 10−4 mol/L), or adrenomedullin (10−12 to 10−6 mol/L) were incubated with tissue at 37°C for 20 minutes, and nitrite release was measured. To confirm the role of NO synthase, protein kinase A (PKA), adenylyl cyclase, and phosphatidylinositol 3-kinase (PI3-kinase) in the stimulation of NO production, NG-nitro-l-arginine (L-NAME, 10−4 mol/L), Rp-cAMP (10−3 mol/L), dideoxyadenosine (10−4 mol/L), and LY294002 (3×10−5 mol/L) were used. Propranolol (10−4 mol/L) was used to block the effect of isoproterenol.
Effects of Bradykinin and Acetylcholine on NO Production From Myocardial Microvessels From Normal and Failing Canine Heart
Increasing concentrations of bradykinin and acetylcholine (10−10 to 10−5 mol/L), HOE 140 (10−5 mol/L), and atropine (10−5 mol/L) were studied.
Body weight, weight of the total heart, and weight of the LV free wall in these dogs were 25±1 and 25±1 kg, 201±7 and 266±11 g (P<0.05 versus normal), and 75±4 and 97±6 g in normal and HF groups, respectively. The data in the figures are the changes in nitrite production in picomoles per milligram wet weight tissue per 20-minute incubation, whereas the data in the text are percentage of changes in nitrite and absolute values. Changes in hemodynamics in the group with end-stage HF indicate that there were marked decreases in LV systolic pressure (92±3 mm Hg), LV dP/dt (1219±76 mm Hg/s), and mean arterial pressure (77±2 mm Hg) compared with values in the normal group (134±4 mm Hg, 2859±88 mm Hg/s, and 109±3 mm Hg, respectively; all P<0.05) and significant increases in LV end-diastolic pressure (25±1 mm Hg) and heart rate (119±4 bpm) in the HF group compared with the normal group (7±0.9 mm Hg and 83±7 bpm, respectively; all P<0.05). Similar data have been published in our previous studies.20–23⇓⇓⇓
Expression of eNOS and PKB Protein in Myocardial Microvessels From Normal and Failing Canine Heart
Expression of eNOS and PKB protein in microvessels from normal and failing canine hearts are shown in Figure 1. eNOS (135-kDa) protein was markedly reduced in microvessels from failing canine hearts (−55%; Figure 1, top panel). Surprisingly, PKB (59-kDa) protein was substantially elevated in these blood vessels after HF (220%; Figure 1, bottom panel).
Effects of Forskolin and 8-Bromo-cAMP on cAMP Signal Transduction–Mediated NO Production From Myocardial Microvessels From Normal and Failing Canine Heart
Forskolin and 8-bromo-cAMP concentration-dependently increased nitrite production in normal and failing canine hearts. Surprisingly, the change in nitrite release in response to forskolin (Figure 2) or 8-bromo-cAMP was significantly elevated after HF. For instance, forskolin (10−10 to 10−4 mol/L) and 8-bromo-cAMP (10−8 to 10−2 mol/L) increased nitrite release by 12±7% to 95±9% and by 20±4% to 102±12%, respectively (from control value, 82±4 and 82±4 pmol/mg, respectively; P<0.05). After HF, forskolin and 8-bromo-cAMP increased nitrite release by 44±13% to 256±71% and by 51±22% to 233±47%, respectively (from control value, 60±7 and 60±8 pmol/mg, respectively; P<0.05). When the effects of the highest concentration of forskolin and 8-bromo-cAMP were compared with the normal group, the changes in nitrite release increased by 55% and 48%, respectively, in microvessels from failing canine heart. L-NAME, Rp-cAMP, dideoxyadenosine, or LY294002 reduced nitrite release induced by the highest concentration of forskolin (Figure 3) or 8-bromo-cAMP in normal and HF groups. Dideoxyadenosine had no effect on 8-bromo-cAMP–induced NO formation in either group (data not shown).
Effects of Isoproterenol and Adrenomedullin on cAMP Signal Transduction–Mediated NO Production From Myocardial Microvessels From Normal and Failing Canine Hearts
Isoproterenol and adrenomedullin increased nitrite production in a concentration-dependent manner in normal and failing canine hearts. Interestingly, the change in nitrite production in response to isoproterenol or adrenomedullin (Figure 4) was also elevated after HF. For instance, isoproterenol (10−10 to 10−4 mol/L) and adrenomedullin (10−12 to 10−6 mol/L) significantly increased nitrite release by 17±3% to 65±14% and by 19±7% to 86±17%, respectively (from control value, 85±3 and 81±4 pmol/mg, respectively; P<0.05). After HF, isoproterenol and adrenomedullin increased nitrite release by 36±15% to 205±41% and by 39±19% to 198±20%, respectively (from control value, 57±7 and 61±3 pmol/mg, respectively; P<0.05). When the effects of the highest concentration of isoproterenol and adrenomedullin were compared with normal group values, the changes in nitrite release increased 51% and 67%, respectively, in failing canine hearts. L-NAME, Rp-cAMP, dideoxyadenosine, or LY294002 inhibited nitrite release by the highest concentration of isoproterenol and adrenomedullin (Figure 5) in the normal and HF groups. Propranolol entirely eliminated the effects of isoproterenol on NO production in normal and failing canine hearts (data not shown).
Effects of Bradykinin and Acetylcholine on NO Production From Myocardial Microvessels From Normal and Failing Canine Heart
Bradykinin and acetylcholine (10−10 to 10−5 mol/L) increased nitrite release by 35±12% to 128±33% and by 14±3% to 105±9%, respectively (from control value, 76±7 and 73±4 pmol/mg, respectively). After HF, the changes in nitrite release induced by bradykinin and acetylcholine were markedly reduced to 26±13% to 84±12% and 14±7% to 78±17%, respectively (from control value, 57±4 and 56±5 pmol/mg, respectively; P<0.05). When the effects of the highest concentration of bradykinin and acetylcholine were compared with normal group values, the changes in nitrite release were reduced by 45% and 46%, respectively, in failing canine hearts. L-NAME, HOE 140, or atropine blocked the effects of bradykinin and acetylcholine on nitrite release (data not shown).
The most significant findings in the present study are 2-fold. First, stimulation of cAMP signal transduction increased endothelial NO production from isolated canine myocardial microvessels in pacing-induced HF. This effect is mediated by PKA and PI3-kinase. Thus, there is an innate cAMP-NO pathway in these microvessels that may play an important role in the regulation of myocardial microvascular NO production in pathological states. Second, the change in NO production in response to the stimulation of cAMP signal transduction was markedly elevated after HF, despite a low basal level of NO formation in these microvessels, and this was associated with decrease in eNOS and increase in PKB protein expression. In contrast, B2-kinin receptor– or muscarinic receptor–mediated NO production was substantially impaired, reflecting eNOS downregulation in these blood vessels. The present study suggests that cAMP signal transduction may serve as a crucial compensatory pathway for coronary microvascular NO production during HF.
NO derived from vascular endothelium plays an important physiological role in the regulation of many biological functions. A number of clinical and experimental studies14–24⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓ have demonstrated that HF is characterized by depressed systemic and cardiac endothelial NO production. Defective endothelial NO production has been recognized as an important mechanism contributing to the progressive deterioration after HF. Any method that restores endothelial NO production may be beneficial for control of this disease. ACE inhibitors have been shown to have a beneficial effect, at least in part, in improving hemodynamics and protecting the heart via promoting endothelial NO production.26
We20,22,24⇓⇓ have previously found that although bradykinin and acetylcholine have a significant effect on NO production from microvessels, the ability of endothelium to release NO in response to these agonists is reduced after HF. Numerous studies in vivo from our laboratory and others17,20,23⇓⇓ have repeatedly reported a reduction of muscarinic receptor–mediated and NO-dependent vasodilatory responses after HF. This has been thought to be due to the reduction of eNOS protein.21 Stimulation of cAMP signal transduction increases endothelial NO production5–13⇓⇓⇓⇓⇓⇓⇓⇓; however, whether the cAMP-NO pathway is still functional after HF is unknown. Interestingly, in the present study, we found that NO production in response to the stimulation of cAMP signal not only was preserved but also was elevated after HF despite a marked reduction of eNOS in these blood vessels. In contrast, bradykinin- or acetylcholine-induced NO generation was substantially reduced after HF. Our results suggest that there is a selectively impaired bradykinin- or acetylcholine-induced endothelial NO formation in myocardial microvasculature during HF, whereas the elevation of NO production after stimulation of cAMP signal transduction may serve to compensate for the downregulation of eNOS expression.
It is well known that HF is associated with myocardial dysfunction due to β-adrenergic receptor downregulation.27–30⇓⇓⇓ During HF, activation of the sympathetic nervous system constitutes a major component of the neurohumoral compensation.31 However, increased catecholamines, such as norepinephrine, are cardiotoxic and may produce cardiac myocyte injury in concentrations found in the HF.27 Because norepinephrine is a β-adrenergic receptor agonist (60-fold selective for β1- versus β2-receptors and 10-fold selective for β1- versus α1-receptors), a β1-adrenergic receptor–mediated cardiac cytotoxic effect on the myocyte is thought to be a mechanism involved in the development of HF.28 This is the fundamental basis for the use of β-adrenergic antagonists in the treatment of chronic HF.27 However, clinical and experimental studies have demonstrated that myocardial β-receptors are markedly downregulated (in theory, this is due to constant stimulation via chronically elevated circulating catecholamines after HF)27–29,31⇓⇓⇓ and that the extent of reduction in β-adrenergic receptors is directly related to the severity of this disease.30 It has been demonstrated that in canine and human myocytes, there are ≈85% β1-adrenergic receptors and 15% β2-adrenergic receptors, whereas coronary blood vessels, specifically coronary arterioles, contain almost exclusively β2-adrenergic receptors.29,30,32,33⇓⇓⇓ During HF, there is a dramatic selective β1-adrenergic receptor downregulation but little or no change in β2-adrenergic receptors in myocytes,27–33⇓⇓⇓⇓⇓⇓ and there appears to be no change in β-adrenergic receptors in blood vessels.28 Even so, unfortunately, the function of β2-adrenergic receptor–mediated cAMP signal transduction, especially the vascular β2-adrenergic receptor–mediated endothelial cAMP signal transduction on the regulation of cardiovascular function during HF, has never received much attention, despite the fact that β-adrenergic receptors are present on endothelium.34 In the present study, the change in nitrite release from coronary microvessels in response to the highest concentration of isoproterenol increased ≈60% in failing hearts, and this effect was eliminated by propranolol or inhibition of adenylyl cyclase or PKA, indicating an elevated function of the vascular β-adrenergic receptor–cAMP–NO pathway after HF. This finding is consistent with a recent study in which Larosa et al31 found that at an early stage of HF, there was a profound reduction in relaxation of circumflex coronary arteries in response to isoproterenol after endothelium removal, indicating a significant relaxation mediated by endothelial β-adrenergic receptors. These authors speculated that this could be due to an enhanced release of endothelium-derived relaxing factors in these failing hearts. It is likely that after HF, stimulation of β-adrenergic receptors (possibly β2-adrenergic receptors) may have a significant impact on the endothelium, ie, an increase in endothelial NO release, and that cAMP signal transduction in the vascular wall may act differently from the myocardial cAMP-signaling system.
Adrenomedullin, a potent vasodilating peptide, can be synthesized in endothelial and smooth muscle cells.35–37⇓⇓ Although the effect of adrenomedullin-induced vasorelaxation was thought to be due to a subsequent increase in cAMP in vascular smooth muscle cells,35 increasing evidence suggests a major effect of adrenomedullin on NO release from endothelium.35,38–42⇓⇓⇓⇓⇓ However, the intracellular signal transduction pathway in endothelium is unclear. In our previous study12 and the present study, we found that adrenomedullin substantially increased NO production from canine coronary microvessels in normal and pacing-induced failing hearts. Unexpectedly, the change in NO production in response to adrenomedullin was significantly increased in failing hearts, indicating that adrenomedullin has a even greater effect on endothelial NO formation after HF. L-NAME, Rp-cAMP, dideoxyadenosine, and LY294002 all blocked the effect of adrenomedullin, indicating that adrenomedullin shares a common mechanism with forskolin, most likely through the stimulation of a cAMP signal–PKA/PI3-kinase–regulated pathway. Plasma adrenomedullin levels are elevated in a variety of disease states.38–40⇓⇓ Furthermore, plasma levels of adrenomedullin are positively correlated with LV function in HF in humans and predict survival in septic shock.42 Taking our results together with these observations, we propose that adrenomedullin may be an important endogenous hormone that regulates endothelial NO through cAMP signal transduction and that this may be part of the mechanism of ameliorating or modulating cardiovascular dysfunction by adrenomedullin. Indeed, it has been shown that plasma adrenomedullin38 and adenosine43 levels are elevated during HF and that the elevations of adrenomedullin and adenosine are negatively correlated with the severity of HF.42,43⇓ Therefore, we speculate that there may be an increased role for cAMP signal transduction in the endothelium after HF. All the cAMP-increasing agents used in the present study stimulated the production of NO in normal and failing canine hearts, reaching a level that is high enough to cause coronary vasodilation similar to that induced by bradykinin and acetylcholine in normal vessels.44,45⇓ We suggest that cAMP signal transduction may be an important potential compensatory pathway in myocardial microvascular NO production during HF.
Recently, Chen et al46 and Butt et al47 found that AMP-activated protein kinase rapidly activates cardiac eNOS in endothelium by phosphorylation of eNOS at Ser1177 and dephosphorylation of eNOS at Thr495 (perhaps by activation of protein phosphatase 1). Studies by Fulton et al48 and Dimmeler et al49 also found that eNOS is an efficient substrate for PKB, a serine/threonine protein kinase Akt that can be phosphorylated and activated by cAMP.50 This enzyme can phosphorylate eNOS directly through a PI3-kinase–related pathway. Collectively, cAMP signal transduction increases NO release, perhaps via activation of cAMP-dependent PKA and the subsequent phosphorylation of eNOS by PKB through a PI3-kinase–mediated mechanism. We found that although it is difficult to make a definitive conclusion regarding the mechanism by which all these cAMP-increasing agents significantly increased the release of NO in microvessels from pacing-induced failing canine hearts, this increase was associated with a >2-fold increase in PKB protein in these microvessels. This may explain the higher NO production in response to the stimulation of cAMP signal transduction in these microvessels after HF. Thus, it seems that there are 2 different NO-forming pathways in myocardial microvessels: (1) a bradykinin- or acetylcholine-mediated NO-pathway and (2) the cAMP signal transduction–regulated NO pathway. Stimulation of the cAMP-NO pathway may have a potential beneficial effect in the treatment of HF.
There are 2 limitations to the present study. First, these experiments were entirely performed in vitro. We have previously found that during stunning in the conscious pig, there was an enhanced cAMP-induced coronary dilation mediated by NO.13 The compensatory effect of NO induced by cAMP in vivo during HF needs to be confirmed. Although the mechanism for cAMP-mediated NO production and its augmentation in HF have been addressed extensively with the use of pharmacological agents in the present study, more molecular evidence for these mechanisms, such as cAMP signal transduction–mediated phosphorylation of PKB and eNOS, remains to be elucidated in future studies.
Understanding the mechanism of intracellular cAMP signal transduction in the control of endothelial NO production during HF, a state in which eNOS is downregulated, may have major clinical implications. The present study provides new insight into the regulation of endothelial NO production and may suggest novel therapeutic targets for the design of drugs aimed at improving endothelial function in cardiovascular diseases (such as HF) that are associated with dysfunction in the biosynthesis of NO.
This study was supported by grants PO-1 HL-43023, HL-50142, and HL-61290 from the National Heart, Lung, and Blood Institute. Dr Zhang was supported by an intramural grant (B4932-31) from New York Medical College.
Received April 29, 2002; revision accepted May 29, 2002.
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