Cyclic Guanosine Monophosphate Release Research Articles

Paracrine cardioprotective signaling by red blood cells through hypoxia-mediated activation of soluble guanylate cyclase and release of cyclic guanosine monophosphate

Abstract Background Red blood cells (RBCs) are known to regulate cardiovascular function under hypoxic conditions and to mediate cardioprotection via nitric oxide (NO)-like bioactivity. However, the molecular signalling behind this effect and the identity of any mediator released by the RBCs remain unknown. Previous studies revealed that NO activates soluble guanylate cyclase (sGC) and increases formation of the second messenger cyclic guanosine monophosphate (cGMP) in RBCs. The functional role of this signalling in RBCs during hypoxia/ischemia is still unclear. Aim To determine the functional role of the NO-sGC-cGMP signalling pathway in RBCs during hypoxia and myocardial ischemia. Method RBCs collected from wild-type and sGC knockout mice were exposed to 1% hypoxia or normoxia for 1 h. The RBCs or the extracellular supernatant were then administered to isolated Langendorff-perfused mouse hearts subjected to 40 min global ischemia and 60 min reperfusion. Afterwards, left ventricular developed pressure and infarct size were determined. Additional groups of mice and patients with mild hypertension were given oral nitrate (10mM in their drinking water) before the collection of RBCs to further increase NO signalling. Result Administration of RBCs or the extracellular supernatant collected from wild-type mouse RBCs exposed to hypoxia to isolated hearts subjected to ischemia-reperfusion improved post-ischemic cardiac function and reduced infarct size (Fig. 1A and B). By contrast, supernatant collected from hypoxic RBCs of sGC knockout mice failed to induce cardioprotection (Fig. 1C). The cardioprotection induced by hypoxic RBCs of wild-type mice was blocked by MK571, an inhibitor of cyclic nucleotide transport (Fig. 1D). Hypoxia increased extracellular export of hate cGMP from mouse RBCs (Fig. 1E), and exogenous cGMP resulted in similar cardioprotection induced by the supernatant. The cardioprotective effect of RBCs was blocked by an inhibitor of cardiac cGMP-dependent protein kinase G (PKG) (Fig. 1F) and was associated with increased cardiac PKG-dependent phosphorylation of vasodilator-stimulated phosphoprotein. Oral administration of nitrate to mice for 4 weeks to increase NO bioactivity further enhanced the cardioprotective effect of hypoxic RBCs. RBCs collected from patients randomized to a 5-week nitrate-rich diet induced cardioprotection in the isolated rat heart via an effect dependent on sGC activation in the RBCs (Fig 2). Conclusion RBCs generate and export cGMP as a physiological response to hypoxia mediating cardioprotection via a paracrine effect. This effect can be further augmented by a simple dietary intervention with nitrate suggesting preventive and therapeutic opportunities in ischemic heart disease.Figure 1Figure 2

Transient Receptor Potential Channel Opening Releases Endogenous Acetylcholine, which Contributes to Endothelium-Dependent Relaxation Induced by Mild Hypothermia in Spontaneously Hypertensive Rat but Not Wistar-Kyoto Rat Arteries.

Mild hypothermia causes endothelium-dependent relaxations, which are reduced by the muscarinic receptor antagonist atropine. The present study investigated whether endothelial endogenous acetylcholine contributes to these relaxations. Aortic rings of spontaneously hypertensive rats (SHRs) and normotensive Wistar-Kyoto (WKY) rats were contracted with prostaglandin F2 α and exposed to progressive mild hypothermia (from 37 to 31°C). Hypothermia induced endothelium-dependent, Nω-nitro-l-arginine methyl ester-sensitive relaxations, which were reduced by atropine, but not by mecamylamine, in SHR but not in WKY rat aortae. The responses in SHR aortae were also reduced by acetylcholinesterase (the enzyme responsible for acetylcholine degradation), bromoacetylcholine (inhibitor of acetylcholine synthesis), hemicholinium-3 (inhibitor of choline uptake), and vesamicol (inhibitor of acetylcholine release). The mild hypothermia-induced relaxations in both SHR and WKY rat aortae were inhibited by AMTB [N-(3-aminopropyl)-2-[(3-methylphenyl)methoxy]-N-(2-thienylmethyl)-benzamide; the transient receptor potential (TRP) M8 inhibitor]; only those in SHR aortae were inhibited by HC-067047 [2-methyl-1-[3-(4-morpholinyl)propyl]-5-phenyl-N-[3-(trifluoromethyl)phenyl]-1H-pyrrole-3-carboxamide; TRPV4 antagonist] while those in WKY rat aortae were reduced by HC-030031 [2-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydro-7H-purin-7-yl)-N-(4-isopropylphenyl)acetamide; TRPA1 antagonist]. The endothelial uptake of extracellular choline and release of cyclic guanosine monophosphate was enhanced by mild hypothermia and inhibited by HC-067047 in SHR but not in WKY rat aortae. Compared with WKY rats, the SHR preparations expressed similar levels of acetylcholinesterase and choline acetyltransferase, but a lesser amount of vesicular acetylcholine transporter, located mainly in the endothelium. Thus, mild hypothermia causes nitric oxide-dependent relaxations by opening TRPA1 channels in WKY rat aortae. By contrast, in SHR aortae, TRPV4 channels are opened, resulting in endothelial production of acetylcholine, which, in an autocrine manner, activates muscarinic receptors on neighboring cells to elicit endothelium-dependent relaxations in response to mild hypothermia.

Activation of Guanylate Cyclase-C Attenuates Stretch Responses and Sensitization of Mouse Colorectal Afferents

Irritable bowel syndrome (IBS) is characterized by altered bowel habits, persistent pain and discomfort, and typically colorectal hypersensitivity. Linaclotide, a peripherally restricted 14 aa peptide approved for the treatment of IBS with constipation, relieves constipation and reduces IBS-associated pain in these patients presumably by activation of guanylate cyclase-C (GC-C), which stimulates production and release of cyclic guanosine monophosphate (cGMP) from intestinal epithelial cells. We investigated whether activation of GC-C by the endogenous agonist uroguanylin or the primary downstream effector of that activation, cGMP, directly modulates responses and sensitization of mechanosensitive colorectal primary afferents. The distal 2 cm of mouse colorectum with attached pelvic nerve was harvested and pinned flat mucosal side up for in vitro single-fiber recordings, and the encoding properties of mechanosensitive afferents (serosal, mucosal, muscular, and muscular-mucosal; M/M) to probing and circumferential stretch studied. Both cGMP (10-300 μM) and uroguanylin (1-1000 nM) applied directly to colorectal receptive endings significantly reduced responses of muscular and M/M afferents to stretch; serosal and mucosal afferents were not affected. Sensitized responses (i.e., increased responses to stretch) of muscular and M/M afferents were reversed by cGMP, returning responses to stretch to control. Blocking the transport of cGMP from colorectal epithelia by probenecid, a mechanism validated by studies in cultured intestinal T84 cells, abolished the inhibitory effect of uroguanylin on M/M afferents. These results suggest that GC-C agonists like linaclotide alleviate colorectal pain and hypersensitivity by dampening stretch-sensitive afferent mechanosensitivity and normalizing afferent sensitization.

World Health Organization Pulmonary Hypertension Group 2: Pulmonary hypertension due to left heart disease in the adult—a summary statement from the Pulmonary Hypertension Council of the International Society for Heart and Lung Transplantation

Pulmonary hypertension associated with left heart disease is the most common form of pulmonary hypertension encountered in clinical practice today. Although frequently a target of therapy, its pathophysiology remains poorly understood and its treatment remains undefined. Pulmonary hypertension in the context of left heart disease is a marker of worse prognosis and disease severity, but whether its primary treatment is beneficial or harmful is unknown. An important step to the future study of this important clinical problem will be to standardize definitions across disciplines to facilitate an evidence base that is interpretable and applicable to clinical practice. In this current statement, we provide an extensive review and interpretation of the current available literature to guide current practice and future investigation. At the request of the Pulmonary Hypertension (PH) Council of the International Society for Heart and Lung Transplantation (ISHLT), a writing group was assembled and tasked to put forth this document as described above. The review process was facilitated through the peer review process of the Journal of Heart and Lung Transplantation and ultimately endorsed by the leadership of the ISHLT PH Council.

Atrial natriuretic peptide protects against ischemia-reperfusion injury in the isolated rat heart

Background Atrial natriuretic peptide (ANP), a stimulator of particulate guanylate cyclase, has been found to protect against reoxygenation-induced hypercontracture in isolated cardiomyocytes by increasing cyclic guanosine monophosphate synthesis. The purpose of this study was to investigate the cardioprotective effects of ANP against ischemia-reperfusion injury in isolated rat hearts. Methods Twenty-four hearts were perfused with ANP at 0.01, 0.1, and 1 μmol/L or without ANP (n = 6 each) in normoxic conditions. Because 0.1 μmol/L ANP induced a threefold increase in cyclic guanosine monophosphate release into the coronary effluent without any effect on cardiac function, we used the 0.1 μmol/L ANP dose for ischemia-reperfusion studies. Eighteen hearts were subjected to 15 minutes of normothermic global ischemia followed by 15 minutes of reperfusion. The hearts were divided into three groups (n = 6 each). In group 1, ANP was added before ischemia. In group 2, ANP was added to the reperfusate. Hearts were untreated in the control group. Results In group 1, the postischemic recovery of cardiac output, coronary flow , and cyclic guanosine monophosphate release was similar to the control group. In group 2, the recovery of cardiac output was significantly better than the control group (82.1% ± 9.8% vs 61.8% ± 6.8%, respectively, p < 0.01) with a similar trend to recovery of coronary flow (90.7% ± 8.5% vs 79.3% ± 11.8%, respectively). The improved cardiac function was closely related to a significant increase in postischemic cyclic guanosine monophosphate release. Conclusions Administration of ANP at the time of reperfusion protects the myocardium from ischemia-reperfusion injury. The concentrations of administration must not only increase the release of cyclic guanosine monophosphate release, but also lack negative inotropic effects.

Nitric oxide and thromboxane A2-mediated pulmonary microvascular dysfunction.

To examine whether the lung releases nitric oxide (NO) in response to thromboxane A2 and to examine the local release of NO as a protective compensatory mechanism by which the lung responds to the proinflammatory and vasoactive effects of thromboxane A2. The lungs of anesthetized Sprague-Dawley rats were perfused in vitro with Krebs-Henseleit buffer that contained an inhibitor of NO synthase (nitroglycerinenitro-L-arginine methyl ester [L-NAME]) (10(-4) mol/L), an NO donor (sodium nitroprusside) (10(-8) mol/L), or perfusate alone. Following equilibration, the thromboxane A2 receptor agonist 9,11-dideoxy-11alpha, 9alpha-epoxymethanoprostaglandin F2alpha(U-46619) (7.1 X 10(-8) mol/L) was added to the perfusate. Fifteen minutes later, the capillary filtration coefficient, pulmonary arterial pressure, and vascular resistance were measured. Pulmonary NO release was assessed by quantitating the release of cyclic guanosine monophosphate into the perfusate. The capillary filtration coefficient of lungs exposed to U-46619 was 3.5 times greater than that of lungs perfused with buffer alone (P<.05). The addition of sodium nitroprusside reduced the increase in capillary filtration coefficient associated with U-46619 by 50% (P<.05) whereas L-NAME had no effect. The addition of U-46619 to the perfused lung caused a 3.0+/-0.4 mm Hg increase in pulmonary artery pressure (P<.01) with a corresponding rise in total vascular resistance (P<.05). This effect was exacerbated by L-NAME (P<.05) and inhibited by sodium nitroprusside (P<.05). Exposure of the isolated lungs to U-46619 caused a 4-fold increase in cyclic guanosine monophosphate levels within the perfusate. These data are consistent with the hypothesis that NO release may be an important protective mechanism by which the lung responds to thromboxane A2.

Atrial Natriuretic Peptide-Induced Release of Cyclic Guanosine Monophosphate by Coronary Bypass Grafts

Background. Superior long-term patency rates of the internal mammary artery (IMA) versus saphenous vein (SV) after coronary artery bypass grafting are well documented. Higher production rates of vasodilating and platelet-inhibiting mediators (prostacyclin and nitric oxide) by the IMA seem to have a major impact on its long-term durability and resistance to coronary artery graft disease. For the right gastroepiploic artery (RGEA) marked release of protective mediators is reported as well. The vasodilating effect of cyclic guanosine monophosphate (cGMP) released after stimulation by atrial natriuretic peptide might serve as another graft protective system. The aim of the present study was to determine cGMP release by IMA, RGEA, and SV after atrial natriuretic peptide challenge. Methods. Samples of human IMA (n = 19), RGEA (n = 7), and SV (n = 18) discarded during coronary artery bypass grafting were stimulated with 10 −6 mol/L atrial natriuretic peptide after a resting phase in nutrient me-dium. Release of cGMP was determined by 125-iodide radioimmunoassay. Results. Basal cGMP production rates of the IMA (759.9 ± 277.0 fmol/cm 2) and RGEA (739.9 ± 186.0 fmol/cm 2) were higher than production rates of SV (281.2 ± 64.0 fmol/cm 2). Application of atrial natriuretic peptide led to a statistically significant increase of cGMP release in IMA grafts (1,939.3 ± 778.0 fmol/cm 2), whereas RGEA (618.4 ± 141.3 fmol/cm 2) and SV (221.7 ± 64.5 fmol/cm 2) remained at basal levels ( p < 0.05). Conclusions. From these data we conclude that the IMA in comparison with the RGEA and SV produces more extracellular cGMP when stimulated by atrial natriuretic peptide. This effect might support the cGMP-mediated protective properties of nitric oxide and could underline the extraordinary suitability of the IMA as a bypass conduit.

Impaired Relaxing Response to Isoprenaline in Isolated Thoracic Aorta of Nephrotic Rats: Decrease in Release of EDRF from Endothelial Cells

Isoprenaline-induced relaxation was investigated in aortas from control and daunomycin-induced nephrotic rats. In the endothelium-intact aortas precontracted with phenylephrine, the isoprenaline-induced relaxation and cyclic adenosine monophosphate (AMP) accumulation were significantly less in nephrotic rats than in control animals. Removal of the endothelium, pretreatment with methylene blue (MB), a guanylate cyclase inhibitor, or NW-nitro-L-arginine methyl ester (L-NAME), a nitric oxide (NO) synthase inhibitor, markedly reduced the relaxation induced by isoprenaline in nephrotic and control animals. The increase in cyclic AMP content induced by isoprenaline also was inhibited by these treatments. In addition, the difference in the isoprenaline-induced relaxation and cyclic AMP accumulation between nephrotic and control preparations was abolished by these treatments. The tissue cyclic guanosine monophosphate (GMP) level was not affected by isoprenaline. In the presence of zaprinast (Zap), a cyclic GMP phosphodiesterase inhibitor, the cyclic GMP level in the endothelium-intact tissues was significantly lower in nephrotic rats than in control animals. Removal of endothelium or pretreatment with MB or L-NAME markedly reduced cyclic GMP content in nephrotic and control animals. In the endothelium-denuded tissues, the isoprenaline-induced relaxation and cyclic AMP accumulation were markedly potentiated by a low concentration of nitroprusside (NP). In the endothelium-intact aortas precontracted with phenylephrine, relaxations induced by dobutamine, salbutamol, and forskolin in nephrotic rats were not significantly different from those in control animals. In the endothelium-intact aortas precontracted with KCl, the isoprenaline-induced relaxation also was significantly less in nephrotic rats than in control animals. Pretreatment with prazosin, but not yohimbine, abolished this difference. These results indicate that nephrosis decreases the relaxing response of the endothelium-intact aortas to isoprenaline. In addition, these results suggest that the endothelium-derived relaxing factor (EDRF) released from the endothelial cells markedly enhances isoprenaline-induced increase in the tissue level of cyclic AMP. The decreased relaxing response to isoprenaline in nephrotic rats may be caused by the decrease in the endothelial-dependent cyclic GMP release caused by alpha 1-adrenoceptor activation by isoprenaline.

Renal atrial peptide receptors and natriuresis in two-kidney, one clip hypertension.

It has been suggested that the impaired natriuretic response of the clipped kidney in two-kidney, one clip hypertensive rats is related to downregulation of renal atrial natriuretic peptide receptors. To test this hypothesis, blood volume expansion and atrial peptide binding studies were performed in this model. Infusion of 1% and then 1.5% body weight donor blood (n = 6) caused a progressive increase in plasma immunoreactive atrial natriuretic peptide (107 +/- 26 to 168 +/- 31 to 427 +/- 154 pg/ml, p less than 0.001); the sodium excretion of the nonclipped kidney rose from 230 to 2,200 to 4,000 neq/min (p less than 0.01) but that of the clipped kidney did not rise significantly. There was a highly significant correlation between log cyclic guanosine monophosphate and log sodium excretion by the nonclipped (r2 = 0.749) but not the clipped (r2 = 0.046) kidney. Between clipped and nonclipped kidneys, the association constant (5.26 +/- 0.89 versus 5.17 +/- 0.64 x 10(9)/mol) and apparent binding site density (575 +/- 92 versus 500 +/- 74 fmol/mg protein) for atrial peptide binding in isolated glomeruli did not differ. Assay of atrial peptide-induced cyclic guanosine monophosphate release by isolated glomeruli showed that clipped and nonclipped kidneys were equally responsive. Binding affinity and receptor density did not differ in homogenates prepared from inner medullas of clipped and nonclipped kidneys. These results show that the blunted natriuretic response in clipped kidneys was not associated with any relative decrease in number or function of glomerular or papillary atrial natriuretic peptide receptors.

Release of cyclic guanosine monophosphate evaluated as a diagnostic tool in cardiac diseases

Concentrations of atrial natriuretic peptide (ANP) are increased in plasma of patients with impaired cardiac and renal function. The second messenger of ANP, cyclic guanosine monophosphate (cGMP), is released into the plasma specifically upon stimulation of cells with ANP. Although nitrates can also activate intracellular cGMP synthesis, we detected no increase in plasma cGMP concentrations after infusions of glycerol trinitrate. Because immunoreactive ANP is highly susceptible to degradation and nonspecific influences in blood samples, determinations of ANP require immediate centrifugation and storage of plasma at -20 degrees C. In contrast, we found that cGMP is stable for five days in vitro in blood samples containing EDTA. In 147 healthy blood donors, the upper cutoff value for plasma cGMP was 6.60 nmol/L, not significantly different (P greater than 0.05) from that for 222 patients with disorders other than cardiovascular and renal. In 69 patients with manifest congestive heart failure (NYHA stages II-IV), 65 had increased cGMP values. Using the above cutoff value for cGMP gave diagnostic sensitivity of 94.2% and specificity of 93.7%. Plasma cGMP may thus provide an alternative for routine clinical measurements of ANP in cardiac diseases in the absence of renal disorders.