cGMP Release Research Articles

Abstract 13027: Differential Direct Effects of Equal Hypotensive Natriuretic Peptides (NPs) of ANP, BNP and CNP on Left Ventricular Contractile Performance in Heart Failure: Assessment by Left Ventricular Pressure-Volume Analysis

Background: Natriuretic peptides (NPs) play a crucial role in maintaining cardiovascular homeostasis. NPs stimulate the production and release of cGMP, leading to the vasodilating and natriuretic actions. In heart failure (HF), circulating and cardiac ANP, BNP, and CNP are increased and exhibit a range of actions. However, although they serve as therapeutic agents, their direct cardiac effects in HF are uncertain due to the confounding influence of NPs-produced changes in loading condition on conventional measures of LV function. We test the hypothesis that equal hypotensive 3 NPs may have different inotropic effects on LV contractility and relaxation in HF. Methods: We assessed the cardiac effects of intravenous infusion (20 min) of ANP (2 μg/kg plus 0.5 μg/kg/min), BNP (2 μg/kg plus 0.04 μg/kg/min) and CNP (2 μg/kg plus 0.4 μg/kg/min) on different days in 6 instrumented conscious dogs with pacing-induced HF by using pressure (P)-volume (V) analysis, a load-independent measure of LV contractility. Results: Versus baselines, 3 NPs produced arterial vasodilation with similar and significant decreases in LV end-systolic pressure (10 to 12 mmHg) with relatively unchanged heart rate. ANP caused significant reductions (13%) of E ES (4.2 vs 4.8 mmHg/ml) and M SW (54.6 vs 62.8 mmHg). The time constant of LV relaxation (τ, 45.1 vs 37.6 ms) was lengthened. The LV-arterial coupling, E ES / E A (arterial elastance) (0.57 vs 0.58) was unaltered. The peak mitral flow, dV/dt max was only increased by 7% (178 vs 166 ml/s). With BNP, there were no significant changes in E ES (5.1 vs 4.9 mmHg/ml) and M SW, but E ES /E A was improved 30% (0.74 vs 0.57) due to decreased E A . τ (33.4 vs 37.9 ms) was significantly shortened and dV/dt max increased 15% (189 vs 165 ml/s). In contrast, CNP produced significant increases (~30%) in E ES (6.3 vs 4.8 mmHg/ml) and M SW (80.5 vs 62.4 mmHg) with enhanced increase in E ES /E A (50%, 0.87vs 0.58), but decrease in τ (25%, 28.4 vs 38.1 ms) and significantly greater augmented dV/dt max (25%, 205 vs 164 ml/s). Similar observations of NPs were made at constant heart rate, or after autonomic blockade. Conclusion: In conscious dogs with HF, equal hypotensive ANP, BNP and CNP have negative, no effect, and positive inotropic effects on LV contractility and relaxation, respectively.

Vasorelaxant Effect of Novel Nitric Oxide-Hydrogen Sulfide Donor Chalcone in Isolated Rat Aorta: Involvement of cGMP Mediated sGC and Potassium Channel Activation.

Recently, nitric oxide (NO) and hydrogen sulfide (H2S) donating moieties were extensively studied for their role in the vasculature as they are responsible for many cellular and pathophysiological functioning. The objective of the present study is to evaluate novel NO and H2S donating chalcone moieties on isolated rat aorta for vasorelaxation, and to investigate the probable mechanism of action. To extend our knowledge of vasorelaxation by NO and H2S donor drugs, here we investigated the vasorelaxing activity of novel NO and H2S donating chalcone moieties on isolated rat aorta. The mechanism of vasorelaxation by these molecules was investigated by performing in vitro cGMP mediated sGC activation assay and using Tetraethylammonium chloride (TEA) as a potassium channel blocker and Methylene blue as NO blocker. Both NO and H2S donating chalcone moieties were found to be potent vasorelaxant. The compound G4 and G5 produce the highest vasorelaxation with 3.716 and 3.789 M of pEC50, respectively. After the addition of TEA, G4 and G5 showed 2.772 and 2.796 M of pEC50, respectively. The compounds Ca1, Ca2, and D7 produced significant activation and release of cGMP mediated sGC which was 1.677, 1.769 and 1.768 M of pEC50, respectively. The vasorelaxation by NO-donating chalcones was blocked by Methylene blue but it did not show any effect on H2S donating chalcones. The vasorelaxing potency of NO-donating molecules was observed to be less affected by the addition of TEA but H2S donors showed a decrease in both efficacy and potency. The cGMP release was more in the case of NO-donating molecules. The tested compounds were found potent for relaxing vasculature of rat aorta.

Abstract 027: ProANP 31-67 and Renal Function in Heart Failure

The physiologic interplay between heart and kidney is best understood in the context of heart failure (HF). Two-thirds of HF patients suffer from cardiorenal syndrome (CRS) - heart failure with renal insufficiency wherein both organs contribute to their reciprocal decline. Four peptides are released from an atrial natriuretic prohormone in response to atrial-ventricular wall stress in HF. One in particular, a well characterized 28 a.a cyclic peptide from the C-terminus termed ANP elicits systemic vasodilation via cGMP release and was developed as Natrecor® to treat acute HF. Limited preclinical data exists regarding the remaining three N-terminal peptides although all three have been explored for clinical utility in a limited series of Phase 1 and 2a clinical trials in the U.S. and in Australia. We have examined a 37 a.a. linear peptide, mid-sequence in the prohormone termed proANP 31-67 (MP3167) to better understand its role in CRS. MP3167 is unique among the four as it has the longest half-life of all in humans (1-1/2 hours vs. minutes), is highly acidic (pI=3.2; -5 net charge), appears resistant to degradation by neprilysin and has been reported to act through PGE2 although the tissue source in humans is unknown. We show for the first time that MP3167 elicits a 3-fold increase in PGE2 release over basal levels predominantly in human renal medullary tissue (from 2401 ± 97.2 to 8437 ± 62.9 pg/ml, N=3, p<0.001). The positive control, beta hydroxyl butyric acid (BOH) also increased PGe2 (from 2401 ± 97.2 to 7749 ±120 pg/ml, N=3, p<0.01). In 3D cultured cells isolated from digested tissue , we also observed an increase in MP3167 stimulated PGE2 (From 41.1 ± 1.3 to 62.9 ± 3.19 pg/ml, N=3, p<0.05). The PGE2 signal co-localizes with cells stained by L1-CAM antibody implicating collecting tubules/ducts at the cortical-medullary interface. As the receptor and second messenger pathway for MP3167 is unknown, we have isolated L1-CAM positive cells and created immortalized cell lines of human origin. Initial findings reiterate MP3167 stimulated PGE2 release, reaching a maximum by ~10 min. These human studies will enable better understanding of MP3167 as a regulator of renal function and the paracrine actions of PGE2 in rebalancing heart and renal function in CRS associated HF.

Establishment and validation of a microfluidic capillary gel electrophoresis platform method for purity analysis of therapeutic monoclonal antibodies.

Capillary and microfluidic chip electrophoresis technologies are heavily utilized for development, characterization, release, and stability testing of biopharmaceuticals. Within the biopharmaceutical industry, CE-SDS and M-CGE are commonly used for purity determination by separation and quantitation of size-based variants. M-CGE is used primarily as an R&D tool for product and process development, while cGMP release and stability testing applications are commonly reserved for CE-SDS. This paper describes the establishment of an M-CGE platform method to be used for R&D and cGMP applications, including release and stability testing, for monoclonal antibodies. The M-CGE platform method enables testing for product development support and cGMP release and stability using the same method, and utilization of one CE technology for the entire lifecycle of a biopharmaceutical product. Critical method parameters were identified, and the analytical design space of those critical parameters was defined using design of experiments (DOE) studies. Once defined through DOE studies, the method design space was validated according to ICH Q2 (R1) guidelines. Additional molecules of the same validated class were verified for use in the method by experimental confirmation of accuracy, specificity, and stability indicating capabilities. The platform method model facilitates rapid utilization of the method in development and GMP testing environments, and eliminates the need for individual validations for assets of the same class entering early stage development.

Counter-flow suggests transport of dantrolene and 5-OH dantrolene by the organic anion transporters 2 (OAT2) and 3 (OAT3).

Dantrolene is the only available drug for the treatment of malignant hyperthermia, a life-threatening inborn sensitivity of the ryanodine receptor (RyR1) in skeletal muscles to volatile anesthetics. Dantrolene is metabolized in the liver to 5-OH dantrolene. Both compounds are zwitterions or net negatively charged. Here, we investigated interactions of dantrolene and 5-OH dantrolene with solute carrier (SLC) family members occurring in skeletal muscle cells, hepatocytes, and renal proximal tubule cells. SLC22A8 (organic anion transporter 3, OAT3) was very sensitive to both compounds exhibiting IC50 values of 0.35±0.03 and 1.84±0.34μM, respectively. These IC50 concentrations are well below the plasma concentration in patients treated with dantrolene (3-28μM). SLC22A7 (OAT2) was less sensitive to dantrolene and 5-OH dantrolene with IC50 values of 15.6±2.1 and 15.8±3.2μM, respectively. SLCO1B1 (OATP1B1), SLCO1B3 (OATP1B3), and SLCO2B1 (OATP2B1) mainly interacted with 5-OH dantrolene albeit with higher IC50 values than those observed for OAT2 and OAT3. Dantrolene and 5-OH dantrolene failed to inhibit uptake of 1-methyl-4-phenylpyrimidinium (MPP) by OCT1 and of carnitine by OCTN2. In counter-flow experiments on OAT3, dantrolene and 5-OH dantrolene decreased pre-equilibrated cellular [3H]estrone-3-sulfate (ES) content as did the transported substrates glutarate, furosemide, and bumetanide. With OAT2, dantrolene and 5-OH dantrolene slightly decreased the pre-equilibrated [3H]cGMP content. If no other transporter markedly contributes to uptake or release of ES or cGMP, respectively, these data suggest that OAT3 and OAT2 may be involved in absorption, metabolism, and excretion of dantrolene and its metabolite 5-OH dantrolene.

Effects of Immediate Stimulation on Single Nucleotide and Catecholamine Level in Chinese Tree Shrews

The present study examined the effects of the immediate stimuli on the levels of catecholamine and single nucleotide of Chinese tree shrew by using RIA approach. When tree shrews were under immediate stimulation, compared to the control animals, the levels of the epinephrine (E), norepinephrine (NE), adenosine monophosphate (cAMP), and cyclic guanosine phosphate (cGMP) in blood were significantly increased. Also, the acute stimulation could significantly enhance the cAMP and cGMP levels of the midbrain ventral tegmental area (VTA), prefrontal cortex (PFC), hippocampus (Hipp), and testis (Testicle), and the dopamine (DA) levels in PFC, Hipp, striatum, and nucleus accumbens (NAc). However, when tree shrews were treated with reserpine, both the E and NE levels were significantly reduced (P< 0.01), indicating that the reserpine interventions could reduce or prevent those stress-related damages. Also, the mitochondrial index (the volume density, surface density and density) in hippocampus cells were significantly increased by 55.21%, 55.21% and 55.21% respectively (P<0.01), compared to those of the control animals housed. Our results demonstrated that the immediate stimulus can significantly affect the synthesis and release of epinephrine, norepinephrine, DA, cMAP, and cGMP in Chinese tree shrews. This study was supported by the following Chinese funding agencies: the National Natural Science Foundation of China (31060283), the National Social Science Fund (07BSH054), the Lanzhou Social Science Fund (07-1-64), and the Academic Innovation Team Building Program Management of Gansu Institute of Political Science and Law (GZF2013CXTD003).

Linaclotide Inhibits Colonic Nociceptors and Relieves Abdominal Pain via Guanylate Cyclase-C and Extracellular Cyclic Guanosine 3′,5′-Monophosphate

Linaclotide is a minimally absorbed agonist of guanylate cyclase-C (GUCY2C or GC-C) that reduces symptoms associated with irritable bowel syndrome with constipation (IBS-C). Little is known about the mechanism by which linaclotide reduces abdominal pain in patients with IBS-C. We determined the effects of linaclotide on colonic sensory afferents in healthy mice and those with chronic visceral hypersensitivity. We assessed pain transmission by measuring activation of dorsal horn neurons in the spinal cord in response to noxious colorectal distention. Levels of Gucy2c messenger RNA were measured in tissues from mice using quantitative reverse transcription polymerase chain reaction and in situ hybridization. We used human intestinal cell lines to measure release of cyclic guanosine-3',5'-monophosphate (cGMP) by linaclotide. We performed a post-hoc analysis of data from a phase III, double-blind, parallel-group study in which 805 patients with IBS-C were randomly assigned to groups given an oral placebo or 290 μg linaclotide once daily for 26 weeks. We quantified changes in IBS-C symptoms, including abdominal pain. In mice, linaclotide inhibited colonic nociceptors with greater efficacy during chronic visceral hypersensitivity. Intra-colonic administration of linaclotide reduced signaling of noxious colorectal distention to the spinal cord. The colonic mucosa, but not neurons, was found to express linaclotide's target, GC-C. The downstream effector of GC-C, cGMP, was released after administration of linaclotide and also inhibited nociceptors. The effects of linaclotide were lost in Gucy2c(-/-) mice and prevented by inhibiting cGMP transporters or removing the mucosa. During 26 weeks of linaclotide administration, a significantly greater percentage of patients (70%) had at least a 30% reduction in abdominal pain compared with patients given placebo (50%). We have identified an analgesic mechanism of linaclotide: it activates GC-C expressed on mucosal epithelial cells, resulting in the production and release of cGMP. This extracellular cGMP acts on and inhibits nociceptors, thereby reducing nociception. We also found that linaclotide reduces chronic abdominal pain in patients with IBS-C.

Linaclotide induces secretion of cGMP from mouse colonic epithelium

Linaclotide is a novel receptor guanylyl cyclase C (GC-C) agonist approved for treatment of abdominal pain and constipation in patients with irritable bowel syndrome with constipation (IBS-C). Linaclotide effects on bowel movements are mediated by the intracellular cGMP that is produced upon activation of GC-C. It is hypothesized that the effects of linaclotide on abdominal pain are mediated by extracellular cGMP, which was shown to decrease the activity of pain-sensing nerves [1]. Here we used an ex vivo Ussing chamber assay to measure the secretion of cGMP from the mouse colonic mucosa in response to linaclotide treatment. Ion transport and epithelial barrier function were monitored by measuring short-circuit current (Isc) and trans-epithelial electrical resistance (TEER). Stimulation with linaclotide (1 µM) elicited a robust short-circuit current across mouse colonic epithelium. Isc reached a maximum within ten minutes following stimulation with linaclotide and remained steady during the duration of the study (60 min). Treatment of colonic mucosa with linaclotide induced release of cGMP from the apical, as well as, the basolateral side of the epithelium. The time course of cGMP accumulation in the basolateral bath of the Ussing chamber was linear with an estimated cGMP secretion rate equal to 23 fmol/min×cm2. The trans-epithelial electrical resistance of the colonic mucosa remained high over the course of the study indicating that the barrier to diffusion of cGMP between apical and basolateral sides remained intact throughout the study.. In summary, these data demonstrate that linaclotide-stimulated mouse colonic epithelium secretes cGMP from both the apical and basolateral sides and that cGMP is available in the submucosal interstitial space to inhibit colonic nociceptors.

269 Acute Psychological Stress Increases Small Intestinal Permeability in Healthy Volunteers via CRH-Mediated Activation of Mast Cells

G A A b st ra ct s region, MRP4 was significantly down-regulated compared with expression in biopsies from healthy subjects (P,0.001, n=7-11). Conclusions: Key components of the GC-C/cGMP signalling pathway are expressed within human colonic mucosa. However, expression of the cGMP transporter MRP4 was significantly reduced in rectosigmoid biopsies from patients with IBS-C compared to biopsies from healthy subjects. Reduced MRP4 expression may result in reduced basal release of cGMP from intestinal epithelial cells in these patients and it may underlie some aspects of the pathophysiology of IBS. 1.Gastroenterology,2011,140(5),S1,S-538. Supported by NHMRC Australia, Ironwood Pharmaceuticals Inc.

Atrionatriuretic Peptide Improves Left Ventricular Function After Myocardial Global Ischemia-Reperfusion in Hypoxic Hearts

Atrionatriuretic peptide (ANP) is reported to be useful for attenuating myocardial ischemia-reperfusion injury and improving left ventricular function after reperfusion. However, ANP may be either ineffectual or harmful in cases where the myocardium has been chronically hypoxic since birth. This can be a result of the concomitant high levels of cyclic guanosine monophosphate (cGMP) produced within the myocardium. This study aimed to verify the validity of using ANP to improve left ventricular function after myocardial ischemia-reperfusion injury. For this purpose, a cyanotic congenital disease model that was developed using isolated rat hearts was used. Hearts were obtained from Sprague-Dawley rats that were housed from birth until 6 weeks of age either in a hypoxic environment with 13-14% FiO(2) (hypoxic group) or in ambient air (normoxic group). These hearts were subjected to 30min of normothermic global ischemia followed by 30min of reperfusion using the Langendorff technique. Left ventricular functional recovery in hearts administered ANP (0.1µM) into the reperfusion solution was compared with those hearts that were not administered ANP in both hypoxic (without ANP: n=6, with ANP: n=6, with ANP and HS-142-1[an antagonist of ANP]: n=6) and normoxic hearts (without ANP: n =6, with ANP: n=6). In the hypoxic hearts, ANP administration improved the percent recovery of the left ventricular developed pressure (76.3±9.2% without ANP vs. 86.9±6.7% with ANP), maximum first derivative of the left ventricular pressure (82.4±1.1% without ANP vs. 95.8±6.5% with ANP), and heart rate (85.6±4.7% without ANP vs. 96.1±5.2% with ANP) after reperfusion. The improvement and recovery of these cardiac functions were closely related to significantly increased levels of postischemic cGMP release after ANP administration. The effect of ANP was blocked by HS-142-1. The improvements observed in the hypoxic group were similar to those found in the normoxic group. ANP administration during reperfusion improved left ventricular function after myocardial acute global ischemia-reperfusion equally in both the chronically hypoxic and age-matched normoxic groups.

Binding of cGMP to the transducin-activated cGMP phosphodiesterase, PDE6, initiates a large conformational change involved in its deactivation

Retinal photoreceptor phosphodiesterase (PDE6), a key enzyme for phototransduction, consists of a catalytic subunit complex (Pαβ) and two inhibitory subunits (Pγs). Pαβ has two noncatalytic cGMP-binding sites. Here, using bovine PDE preparations, we show the role of these cGMP-binding sites in PDE regulation. Pαβγγ and its transducin-activated form, Pαβγ, contain two and one cGMP, respectively. Only Pαβγ shows [(3)H]cGMP binding with a K(d) ∼ 50 nM and Pγ inhibits the [(3)H]cGMP binding. Binding of cGMP to Pαβγ is suppressed during its formation, implying that cGMP binding is not involved in Pαβγγ activation. Once bound to Pαβγ, [(3)H]cGMP is not dissociated even in the presence of a 1000-fold excess of unlabeled cGMP, binding of cGMP changes the apparent Stokes' radius of Pαβγ, and the amount of [(3)H]cGMP-bound Pαβγ trapped by a filter is spontaneously increased during its incubation. These results suggest that Pαβγ slowly changes its conformation after cGMP binding, i.e. after formation of Pαβγ containing two cGMPs. Binding of Pγ greatly shortens the time to detect the increase in the filter-trapped level of [(3)H]cGMP-bound Pαβγ, but alters neither the level nor its Stokes' radius. These results suggest that Pγ accelerates the conformational change, but does not add another change. These observations are consistent with the view that Pαβγ changes its conformation during its deactivation and that the binding of cGMP and Pγ is crucial for this change. These observations also imply that Pαβγγ changes its conformation during its activation and that release of Pγ and cGMP is essential for this change.

Cirrhosis decreases vasoconstrictor response to electrical field stimulation in rat mesenteric artery: role of calcitonin gene-related peptide

Our study determines alterations in the vasoconstrictor response elicited by electric field stimulation (EFS) in mesenteric arteries from cirrhotic rats treated with CCl(4), and how calcitonin gene-related peptide (CGRP) participates in this response. Vasoconstriction induced by EFS was analysed in the absence and presence of the CGRP receptor antagonist CGRP(8-37) in arterial segments from control and cirrhotic rats. The vasodilator response to exogenous CGRP was tested in both groups of rats, and the interference of the guanylate cyclase inhibitor ODQ or the K(ATP) channel blocker glibenclamide was analysed only in segments from cirrhotic rats. The vasodilator response to the K(ATP) channel opener pinacidil and to 8-bromo-cyclic GMP was tested. The K(ATP) currents were recorded using the patch-clamp technique. Expression of receptor activity-modifying protein 1 (RAMP1), calcitonin receptor-like receptor, Kir 6.1 and sulfonylurea receptor 2B (SUR2B) was also analysed. Release of CGRP and cGMP was measured. The EFS-elicited vasoconstriction was less in segments from cirrhotic rats. The presence of CGRP(8-37) increased the EFS-induced response only in segments from cirrhotic rats. The CGRP-induced vasodilatation was greater in segments from cirrhotic rats, and was inhibited by ODQ or glibenclamide. Both pinacidil and 8-bromo-cyclic GMP induced a stronger vasodilator response in segments from cirrhotic rats. Pinacidil induced greater K(ATP) currents in cirrhotic myocytes. Expression of RAMP1, calcitonin receptor-like receptor, Kir 6.1 and SUR2B was not modified by liver cirrhosis. Liver cirrhosis increased CGRP release, but did not modify cGMP formation. The decreased vasoconstrictor response to EFS in cirrhosis is mediated by increased vasodilator response to CGRP, as well as increased K(ATP) channel gating. This effect of CGRP may play a role in the splanchnic vasodilatation present in liver cirrhosis.

Transpulmonary B-Type Natriuretic Peptide Uptake and Cyclic Guanosine Monophosphate Release in Heart Failure and Pulmonary Hypertension: The Effects of Sildenafil

We sought to identify factors that discriminate heart failure (HF) patients with normal and elevated pulmonary vascular resistance (PVR) and to elucidate the role of cyclic guanosine monophosphate (cGMP)-dependent vasodilation. Mechanisms of PVR increase in patients with chronic HF are incompletely understood. Twenty-two HF patients with high pulmonary vascular resistance (H-PVR) (>200 dyn.s.cm(-5)) were compared with 24 matched low pulmonary vascular resistance (L-PVR) patients of similar age, sex, body size, HF severity, and volume status who were undergoing invasive hemodynamic study. Pulmonary arterial (PA) and venous blood samples from a wedged PA catheter were used to calculate transpulmonary B-type natriuretic peptide (BNP) uptake and cGMP release. The H-PVR patients were re-examined 1 h after a 40-mg oral dose of sildenafil. Although transpulmonary BNP uptake was similar (p = 0.2), cGMP release was diminished in the H-PVR patients (-1.9 vs. 27.8 nmol.min(-1); p = 0.005). Transpulmonary BNP uptake and cGMP release correlated in the L-PVR patients (R = 0.6, p = 0.004) but not in the H-PVR. The H-PVR patients also had lower PA compliance, systemic arterial compliance (by 47% and 20%, p < 0.001 and p < 0.03), and cardiac index. Sildenafil reduced PVR (-47%), systemic resistance (-24%) and heart rate (-8%), increased cardiac index (+24%), and PA compliance (+87%, all p < 0.001), with a parallel increase of cGMP release (from -5.6 to 16.5 nmol.min(-1), p = 0.047), without affecting BNP uptake or norepinephrine(PA). The PVR response was not dependent on PA wedge pressure or pulmonary hypertension reversibility with prostaglandin E(1). The H-PVR patients have stiffening of both pulmonary and systemic arteries, preserved transpulmonary BNP uptake, but diminished cGMP release, which is reversible by the administration of sildenafil. This study provides in vivo evidence that phosphodiesterase 5A inhibition restores sensitivity of pulmonary vasculature to endogenous cGMP-dependent vasodilators.

Responses of Nitric Oxide–cGMP Release in Acupuncture Point to Electroacupuncture in Human Skin In Vivo Using Dermal Microdialysis

The aim of this study was to examine the release of nitric oxide (NO) and cGMP in response to electroacupuncture (EA) stimulation in the acupuncture point (acupoint), compared to the non-meridian control area. Thirty samples of dermal microdialysis data were collected from 24 volunteers at pericardium (PC) 4 and control area. EA was applied to PC 3 by using a 5-V pulse with a duration of 1.0 milliseconds at 10 Hz for 15 minutes. Dialysate samples were continuously collected 20 minutes each before, during, and after EA for two hours. Total nitrite and nitrate (NO(x)(-)) and cGMP in the dialysate were quantified in a blinded fashion. Dialysate NO(x)(-) concentrations were decreased during a 120-minute dialysis in all groups, but reduced NO(x)(-) levels were attenuated predominantly in PC 4 acupoint at 20-40 minutes after EA PC 3. cGMP concentrations were significantly enhanced in acupoint PC 4 by EA PC 3, but not in the non-meridian area. We suggest that the attenuation of NO(x)(-) reduction during dialysis reflects an increase in NO release induced by EA stimulation in acupoint and that cGMP mediates the signaling functions of NO to improve local microcirculation, which, at least in part, contributes to the effects of acupuncture.

Vascular smooth muscle cell dysfunction in patients with migraine

Migraine is associated with increased risk of cardiovascular disease, but the mechanisms are unclear. To investigate the activity of endothelial and vascular smooth muscle cells (VSMCs) in patients with migraine. Case-control study of 12 patients with migraine without aura and 12 matched healthy control subjects. Endothelial and VSMC components of vascular reactivity were explored by plethysmography measurement of forearm blood flow (FBF) during infusions of vasoactive agents into the brachial artery. Forearm production of nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) was also quantified. In patients with migraine, the vasodilating effect of acetylcholine (ACh), an endothelium-dependent vasodilator, was markedly reduced (p < 0.001 by analysis of variance). In response to the highest dose of ACh, FBF rose to 8.6 +/- 2.2 in patients with migraine and to 22.7 +/- 3.0 mL x dL(-1) x min(-1) in controls (p = 0.001). The dose-response curve to nitroprusside, a vasodilator directly acting on VSMCs, was depressed in patients with migraine (p < 0.001 by analysis of variance). The maximal response of FBF to nitroprusside was 12.1 +/- 2.0 in patients with migraine and 24.1 +/- 1.8 mL x dl(-1) x min(-1) in controls (p < 0.001). During ACh infusion, NO release from the endothelium was similar in patients and controls. In contrast, there was a marked release of cGMP from VSMCs in controls, but not in patients with migraine (-1.9 +/- 2.2 in patients with migraine and -19.1 +/- 5.4 nmol x dL(-1) x min(-1) in controls; p = 0.03). Patients with migraine are characterized by a distinct vascular smooth muscle cell dysfunction, revealed by impaired cyclic guanosine monophosphate and hemodynamic response to nitric oxide.

Impaired glycocalyx barrier properties and increased capillary tube haematocrit

Impaired glycocalyx barrier properties and increased capillary tube haematocrit

Cardioprotective and Vasodilatory Actions of Glucagon-Like Peptide 1 Receptor Are Mediated Through Both Glucagon-Like Peptide 1 Receptor–Dependent and –Independent Pathways

The glucagon-like peptide 1 receptor (GLP-1R) is believed to mediate glucoregulatory and cardiovascular effects of the incretin hormone GLP-1(7-36) (GLP-1), which is rapidly degraded by dipeptidyl peptidase-4 (DPP-4) to GLP-1(9-36), a truncated metabolite generally thought to be inactive. Novel drugs for the treatment of diabetes include analogues of GLP-1 and inhibitors of DPP-4; however, the cardiovascular effects of distinct GLP-1 peptides have received limited attention. Here, we show that endothelium and cardiac and vascular myocytes express a functional GLP-1R as GLP-1 administration increased glucose uptake, cAMP and cGMP release, left ventricular developed pressure, and coronary flow in isolated mouse hearts. GLP-1 also increased functional recovery and cardiomyocyte viability after ischemia-reperfusion injury of isolated hearts and dilated preconstricted arteries from wild-type mice. Unexpectedly, many of these actions of GLP-1 were preserved in Glp1r(-/-) mice. Furthermore, GLP-1(9-36) administration during reperfusion reduced ischemic damage after ischemia-reperfusion and increased cGMP release, vasodilatation, and coronary flow in wild-type and Glp1r(-/-) mice, with modest effects on glucose uptake. Studies using a DPP-4-resistant GLP-1R agonist and inhibitors of DPP-4 and nitric oxide synthase showed that the effects of GLP-1(7-36) were partly mediated by GLP-1(9-36) through a nitric oxide synthase-requiring mechanism that is independent of the known GLP-1R. These data describe cardioprotective actions of GLP-1(7-36) mediated through the known GLP-1R and novel cardiac and vascular actions of GLP-1(7-36) and its metabolite GLP-1(9-36) independent of the known GLP-1R. Our data suggest that the extent to which GLP-1 is metabolized to GLP-1(9-36) may have functional implications in the cardiovascular system.

Inhaled Agonists of Soluble Guanylate Cyclase Induce Selective Pulmonary Vasodilation

Nitric oxide-independent agonists of soluble guanylate cyclase (sGC) have been developed. We tested whether inhalation of novel dry-powder microparticle formulations containing sGC stimulators (BAY 41-2272, BAY 41-8543) or an sGC activator (BAY 58-2667) would produce selective pulmonary vasodilation in lambs with acute pulmonary hypertension. We also evaluated the combined administration of BAY 41-8543 microparticles and inhaled nitric oxide (iNO). Finally, we examined whether inhaling BAY 58-2667 microparticles would produce pulmonary vasodilation when the response to iNO is impaired. In awake, spontaneously breathing lambs instrumented with vascular catheters and a tracheostomy tube, U-46619 was infused intravenously to increase mean pulmonary arterial pressure to 35 mm Hg. Inhalation of microparticles composed of either BAY 41-2272, BAY 41-8543, or BAY 58-2667 and excipients (dipalmitoylphosphatidylcholine, albumin, lactose) produced dose-dependent pulmonary vasodilation and increased transpulmonary cGMP release without significant effect on mean arterial pressure. Inhalation of microparticles containing BAY 41-8543 or BAY 58-2667 increased systemic arterial oxygenation. The magnitude and duration of pulmonary vasodilation induced by iNO were augmented after inhaling BAY 41-8543 microparticles. Intravenous administration of 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), which oxidizes the prosthetic heme group of sGC, markedly reduced the pulmonary vasodilator effect of iNO. In contrast, pulmonary vasodilation and transpulmonary cGMP release induced by inhaling BAY 58-2667 microparticles were greatly enhanced after treatment with ODQ. Inhalation of microparticles containing agonists of sGC may provide an effective novel treatment for patients with pulmonary hypertension, particularly when responsiveness to iNO is impaired by oxidation of sGC.

Increased insulin‐stimulated endothelin‐1 release is a distinct vascular phenotype distinguishing Cushing's disease from metabolic syndrome

Although much is known about the anti-inflammatory effects of an acute corticosteroid therapy, little is known about the effects on chronic hypercortisolism on endothelial dysfunction and proinflammatory alterations in patients with Cushing's disease (CD). We studied 9 patients with CD, 10 patients with metabolic syndrome and 27 normal controls. The tests consisted of an intravenous bolus of 0.1 U/kg insulin combined with a euglycaemic clamp technique with an arterialized forearm and assessment of the training parameters deep-venous balance of forearm glucose uptake (as an index of insulin sensitivity); NO(x) (nitric oxide end-products), c-GMP (second messenger of nitric oxide) and endothelin-1 release, as indices of endothelial function and proinflammatory systemic markers. Forearm glucose uptake incremental area was significantly lower in Cushing's disease and in the metabolic syndrome than in controls, suggesting a state of severe insulin resistance. Compared to controls and to the metabolic syndrome, basal and insulin-stimulated NO(x) release incremental areas were significantly reduced in Cushing's disease, while forearm c-GMP release was similarly decreased in CD and metabolic syndrome. By contrast, endothelin-1 incremental areas after insulin bolus were significantly higher in CD than in controls and the metabolic syndrome, in the presence of increased TNF-alpha, IL-6 and CRP levels. Forearm glucose uptake incremental area significantly correlated with NO(x) incremental area, forearm c-GMP release incremental area, TNF-alpha levels and ET-1 incremental area. In patients with CD, supraphysiological insulin levels are not able to overcome the insulin resistance due to chronic hypercortisolism. Furthermore, an increased proatherogenic risk profile is characterized by decreased nitric oxide synthesis and activity, enhanced endothelin-1 levels and increased proinflammatory markers.

Vardenafil increases coronary flow response to hypercapnic acidosis in isolated guinea pig heart

The hypothesis was tested that vardenafil, a PDE5 inhibitor, specifically enhances coronary vasodilation during acidosis. In isolated constant pressure perfused guinea pig hearts, infusion of vardenafil (<or= 1 microM) increased coronary flow concentration-dependently 34 % above baseline. In parallel, cGMP release increased (0.44 +/- 0.094 vs. 0.14 +/- 0.017 pmol/min x g at 0.5 microM vardenafil vs. baseline). Flow increases occurred in the absence of changes in heart function (LVP, heart rate, dP/dt(max), heart rate - pressure product). Infusion of the NO synthase blocker L-NMMA (100 microM) caused a rightward shift of the dose-response curve of vardenafil. To test whether vardenafil treatment may enhance metabolic coronary vasodilation, arterial pCO(2) was raised from 38 to 61 mmHg, which resulted in a steady state flow increase of 18.8 +/- 4.5%. Infusion of vardenafil, given at a threshold flow enhancing concentration, doubled the coronary flow response during hypercapnic acidosis to 38.4 +/- 4.2 % (p=0.004). This flow amplification during acidosis was not shared by the K(ATP) channel opener cromakalim, indicating a specific effect of vardenafil on flow control during myocardial acidosis. We conclude that vardenafil specifically relaxes coronary resistance vessels through NO/cGMP-dependent pathways and increases the coronary flow response toward hypercapnic acidosis. This finding further supports the importance of the NO-cGMP axis in mediation of this flow response.