Non-neuronal cell-derived acetylcholine, a key modulator of the vascular endothelial function in health and disease.

Vascular endothelial cells play an important role in regulating peripheral circulation by modulating arterial tone in the microvasculature. Elevated intracellular Ca2+ levels are required in endothelial cells to induce smooth muscle relaxation via endothelium-dependent mechanisms such as nitric oxide production, prostacyclin, and endothelial cell hyperpolarization. It is well established that exogenous administration of acetylcholine can increase intracellular Ca2+ concentrations, followed by endothelium-dependent vasodilation. Although endogenous acetylcholine's regulation of vascular tone remains debatable, recent studies have reported that endogenously derived acetylcholine, but not neuronal cell-derived acetylcholine, is a key modulator of endothelial cell function. In this minireview, we summarize the current knowledge of the non-neuronal cholinergic system (NNCS) in vascular function, particularly vascular endothelial cell function, which contributes to blood pressure regulation. We also discuss the possible pathophysiological impact of endothelial NNCS, which may induce the development of vascular diseases due to endothelial dysfunction, and the potential of endothelial NNCS as a novel therapeutic target for endothelial dysfunction in the early stages of metabolic syndrome, diabetes, and hypertension.

Transporter-dependent uptake and metabolism of myocardial interstitial serotonin in the rat heart.

To investigate the roles of the serotonin (5-HT) transporter (SERT) and plasma membrane monoamine transporter (PMAT) in 5-HT uptake and its metabolism in the heart, we monitored myocardial interstitial levels of 5-HT and 5-HIAA, a metabolite of 5-HT by monoamine oxidase (MAO), in anesthetized rats using a microdialysis technique. Fluoxetine (SERT inhibitor), decynium-22 (PMAT inhibitor), or their mixture was locally administered by reverse-microdialysis for 60 min. Subsequently, pargyline (MAO inhibitor) was co-administered. Fluoxetine rapidly increased dialysate 5-HT concentration, while decynium-22 gradually increased it. The mixture induced a larger increase in dialysate 5-HT concentration compared to fluoxetine or decynium-22 alone. Fluoxetine increased dialysate 5-HIAA concentration, and this increase was abolished by pargyline. Decynium-22 and the mixture did not change dialysate 5-HIAA concentration, which were not affected by pargyline. Both SERT and PMAT regulate myocardial interstitial 5-HT levels by its uptake; however, 5-HT uptake via PMAT leads to 5-HT metabolism by MAO.

Endothelial Natriuretic Peptide Receptor 1 Play Crucial Role for Acute and Chronic Blood Pressure Regulation by Atrial Natriuretic Peptide.

BACKGROUND: ANP (atrial natriuretic peptide), acting through NPR1 (natriuretic peptide receptor 1), provokes hypotension. Such hypotension is thought to be due to ANP inducing vasodilation via NPR1 in the vasculature; however, the underlying mechanism remains unclear. Here, we investigated the mechanisms of acute and chronic blood pressure regulation by ANP. METHODS AND RESULTS: Immunohistochemical analysis of rat tissues revealed that NPR1 was abundantly expressed in endothelial cells and smooth muscle cells of small arteries and arterioles. Intravenous infusion of ANP significantly lowered systolic blood pressure in wild-type mice. ANP also significantly lowered systolic blood pressure in smooth muscle cell-specific Npr1-knockout mice but not in endothelial cell-specific Npr1-knockout mice. Moreover, ANP significantly lowered systolic blood pressure in Nos3-knockout mice. In human umbilical vein endothelial cells, treatment with ANP did not influence nitric oxide production or intracellular Ca2+ concentration, but it did hyperpolarize the cells. ANP-induced hyperpolarization of human umbilical vein endothelial cells was inhibited by several potassium channel blockers and was also abolished under knockdown of RGS2 (regulator of G-protein signaling 2), an GTPase activating protein in G-protein α-subunit. ANP increased Rgs2 mRNA expression in human umbilical vein endothelial cells but failed to lower systolic blood pressure in Rgs2-knockout mice. Endothelial cell-specific Npr1-overexpressing mice exhibited lower blood pressure than did wild-type mice independent of RGS2, and showed dilation of arterial vessels on synchrotron radiation microangiography. CONCLUSIONS: Together, these results indicate that vascular endothelial NPR1 plays a crucial role in ANP-mediated blood pressure regulation, presumably by a mechanism that is RGS2-dependent in the acute phase and RGS2-independent in the chronic phase.

Increased contribution of KCa channels to muscle contraction induced vascular and blood flow responses in sedentary and exercise trained ZFDM rats.

In resistance arteries, endothelium-dependent hyperpolarization (EDH)-mediated vasodilatation is depressed in diabetes. We hypothesized that downregulation of KCa channel derived EDH reduces exercise-induced vasodilatation and blood flow redistribution in diabetes. To test this hypothesis, we evaluated vascular function in response to hindlimb muscle contraction, and the contribution of KCa channels in anaesthetised ZFDM, metabolic disease rats with type 2 diabetes. We also tested whether exercise training ameliorated the vascular response. Using in vivo microangiography, the hindlimb vasculature was visualized before and after rhythmic muscle contraction (0.5 s tetanus every 3 s, 20 times) evoked by sciatic nerve stimulation (40 Hz). Femoral blood flow of the contracting hindlimb was simultaneously measured by an ultrasonic flowmeter. The contribution of KCa channels was investigated in the presence and absence of apamin and charybdotoxin. We found that vascular and blood flow responses to muscle contraction were significantly impaired at the level of small artery segments in ZFDM fa/fa rats compared to its lean control fa/+ rats. The contribution of KCa channels was also smaller in fa/fa than in fa/+ rats. Low-intensity exercise training for 12 weeks in fa/fa rats demonstrated minor changes in the vascular and blood flow response to muscle contraction. However, the KCa-derived component in the response to muscle contraction was much greater in exercise trained than in sedentary fa/fa rats. These data suggest that exercise training increases the contribution of KCa channels among endothelium-dependent vasodilatory mechanisms to maintain vascular and blood flow responses to muscle contraction in this metabolic disease rat model. KEY POINTS: Microvascular dysfunction in type 2 diabetes impairs blood flow redistribution during exercise and limits the performance of skeletal muscle and may cause early fatigability. Endothelium-dependent hyperpolarization (EDH), which mediates vasodilatation in resistance arteries, is known to be depressed in animals with diabetes. Here, we report that low-intensity exercise training in ZFDM rats increased the KCa channel-derived component in the vasodilator responses to muscle contraction compared to that in sedentary rats, partly as a result of the increase in KCNN3 expression. These results suggest that low-intensity exercise training improves blood flow redistribution in contracting skeletal muscle in metabolic disease with diabetes via upregulation of EDH.

Carrier-mediated serotonin efflux induced by pharmacological anoxia in the rat heart in vivo.

Serotonin (5-HT) accumulates in the heart during myocardial ischaemia and induces deleterious effects on the cardiomyocytes. We aimed to investigate whether carrier-mediated 5-HT efflux contributed to the increase in interstitial 5-HT level during ischaemia. Using microdialysis technique applied to the heart of anaesthetised Wistar rats, myocardial interstitial concentration of 5-HT was measured by electro-chemical detection coupled with high-performance liquid chromatography (HPLC-ECD) while simultaneously various pharmacological agents, which create a similar condition to ischaemia, were locally administered by reverse-microdialysis. Sodium cyanide-induced chemical anoxia increased dialysate 5-HT concentration. A similar increase in dialysate 5-HT concentration was induced by ouabain, an inhibitor of sodium-potassium ATPase and reserpine, an inhibitor of vesicular monoamine transporter. Fluoxetine, a selective serotonin reuptake inhibitor raised the baseline level of 5-HT, and neither sodium cyanide nor the combination of ouabain and reserpine induced further increase in 5-HT in the presence of fluoxetine. The results indicate that reverse transport of 5-HT via SERT, which is caused by an impaired ion gradient, contributes to the rise in interstitial level of 5-HT during ischaemia suggesting carrier-mediated 5-HT efflux occurs in the heart in vivo.

ß-blockade prevents coronary macro- and microvascular dysfunction induced by a high salt diet and insulin resistance in the Goto-Kakizaki rat.

A high salt intake exacerbates insulin resistance, evoking hypertension due to systemic perivascular inflammation, oxidative-nitrosative stress and endothelial dysfunction. Angiotensin-converting enzyme inhibitor (ACEi) and angiotensin receptor blockers (ARBs) have been shown to abolish inflammation and redox stress but only partially restore endothelial function in mesenteric vessels. We investigated whether sympatho-adrenal overactivation evokes coronary vascular dysfunction when a high salt intake is combined with insulin resistance in male Goto-Kakizaki (GK) and Wistar rats treated with two different classes of ß-blocker or vehicle, utilising synchrotron-based microangiography in vivo. Further, we examined if chronic carvedilol (CAR) treatment preserves nitric oxide (NO)-mediated coronary dilation more than metoprolol (MET). A high salt diet (6% NaCl w/w) exacerbated coronary microvessel endothelial dysfunction and NO-resistance in vehicle-treated GK rats while Wistar rats showed modest impairment. Microvascular dysfunction was associated with elevated expression of myocardial endothelin, inducible NO synthase (NOS) protein and 3-nitrotyrosine (3-NT). Both CAR and MET reduced basal coronary perfusion but restored microvessel endothelium-dependent and -independent dilation indicating a role for sympatho-adrenal overactivation in vehicle-treated rats. While MET treatment reduced myocardial nitrates, only MET treatment completely restored microvessel dilation to dobutamine (DOB) stimulation in the absence of NO and prostanoids (combined inhibition), indicating that MET restored the coronary flow reserve attributable to endothelium-derived hyperpolarisation (EDH). In conclusion, sympatho-adrenal overactivation caused by high salt intake and insulin resistance evoked coronary microvessel endothelial dysfunction and diminished NO sensitivity, which were restored by MET and CAR treatment in spite of ongoing inflammation and oxidative-nitrosative stress presumably caused by uninhibited renin-angiotensin-aldosterone system (RAAS) overactivation.

Using Synchrotron Radiation Imaging Techniques to Elucidate the Actions of Hexarelin in the Heart of Small Animal Models.

The majority of the conventional techniques that are utilized for investigating the pathogenesis of cardiovascular disease in preclinical animal models do not permit microlevel assessment of in situ cardiomyocyte and microvascular functions. Therefore, it has been difficult to establish whether cardiac dysfunction in complex multiorgan disease states, such as heart failure with preserved ejection fraction and pulmonary hypertension, have their origins in microvascular dysfunction or rather in the cardiomyocyte. Herein, we describe our approach of utilizing synchrotron radiation microangiography to, first, ascertain whether the growth hormone secretagogue (GHS) hexarelin is a vasodilator in the coronary circulation of normal and anesthetized Sprague-Dawley rats, and next investigate if hexarelin is able to prevent the pathogenesis of right ventricle (RV) dysfunction in pulmonary hypertension in the sugen chronic hypoxia model rat. We show that acute hexarelin administration evokes coronary microvascular dilation through GHS-receptor 1a and nitric oxide, and through endothelium-derived hyperpolarization. Previous work indicated that chronic exogenous administration of ghrelin largely prevented the pathogenesis of pulmonary hypertension in chronic hypoxia and in monocrotaline models. Unexpectedly, chronic hexarelin administration prior to sugen chronic hypoxia did not prevent RV hypertrophy or RV cardiomyocyte relaxation impairment. Small-angle X-ray scattering revealed that super relaxed myosin filaments contributed to diastolic dysfunction, and that length-dependent activation might contribute to sustained contractility of the RV. Thus, synchrotron-based imaging approaches can reveal novel insights into cardiac and coronary functions in vivo.

Contribution of afferent pathway to vagal nerve stimulation-induced myocardial interstitial acetylcholine release in rats.

Vagal nerve stimulation (VNS) has been explored as a potential therapy for chronic heart failure. The contribution of the afferent pathway to myocardial interstitial acetylcholine (ACh) release during VNS has yet to be clarified. In seven anesthetized Wistar-Kyoto rats, we implanted microdialysis probes in the left ventricular free wall and measured the myocardial interstitial ACh release during right VNS with the following combinations of stimulation frequency (F in Hz) and voltage readout (V in volts): F0V0 (no stimulation), F5V3, F20V3, F5V10, and F20V10. F5V3 did not affect the ACh level. F20V3, F5V10, and F20V10 increased the ACh level to 2.83 ± 0.47 (P < 0.01), 4.31 ± 1.09 (P < 0.001), and 4.33 ± 0.82 (P < 0.001) nM, respectively, compared with F0V0 (1.76 ± 0.22 nM). After right vagal afferent transection (rVAX), F20V3 and F20V10 increased the ACh level to 2.90 ± 0.53 (P < 0.001) and 3.48 ± 0.63 (P < 0.001) nM, respectively, compared with F0V0 (1.61 ± 0.19 nM), but F5V10 did not (2.11 ± 0.24 nM). The ratio of the ACh levels after rVAX relative to before was significantly <100% in F5V10 (59.4 ± 8.7%) but not in F20V3 (102.0 ± 8.7%). These results suggest that high-frequency and low-voltage stimulation (F20V3) evoked the ACh release mainly via direct activation of the vagal efferent pathway. By contrast, low-frequency and high-voltage stimulation (F5V10) evoked the ACh release in a manner dependent on the vagal afferent pathway.

Severe Hypoxemia Prevents Spontaneous and Naloxone-induced Breathing Recovery after Fentanyl Overdose in Awake and Sedated Rats.

BACKGROUND: As severe acute hypoxemia produces a rapid inhibition of the respiratory neuronal activity through a nonopioid mechanism, we have investigated in adult rats the effects of hypoxemia after fentanyl overdose-induced apnea on (1) autoresuscitation and (2) the antidotal effects of naloxone. METHODS: In nonsedated rats, the breath-by-breath ventilatory and pulmonary gas exchange response to fentanyl overdose (300 µg · kg · min iv in 1 min) was determined in an open flow plethysmograph. The effects of inhaling air (nine rats) or a hypoxic mixture (fractional inspired oxygen tension between 7.3 and 11.3%, eight rats) on the ability to recover a spontaneous breathing rhythm and on the effects of naloxone (2 mg · kg) were investigated. In addition, arterial blood gases, arterial blood pressure, ventilation, and pulmonary gas exchange were determined in spontaneously breathing tracheostomized urethane-anesthetized rats in response to (1) fentanyl-induced hypoventilation (7 rats), (2) fentanyl-induced apnea (10 rats) in air and hyperoxia, and (3) isolated anoxic exposure (4 rats). Data are expressed as median and range. RESULTS: In air-breathing nonsedated rats, fentanyl produced an apnea within 14 s (12 to 29 s). A spontaneous rhythmic activity always resumed after 85.4 s (33 to 141 s) consisting of a persistent low tidal volume and slow frequency rhythmic activity that rescued all animals. Naloxone, 10 min later, immediately restored the baseline level of ventilation. At fractional inspired oxygen tension less than 10%, fentanyl-induced apnea was irreversible despite a transient gasping pattern; the administration of naloxone had no effects. In sedated rats, when PaO2 reached 16 mmHg during fentanyl-induced apnea, no spontaneous recovery of breathing occurred and naloxone had no rescuing effect, despite circulation being maintained. CONCLUSIONS: Hypoxia-induced ventilatory depression during fentanyl induced apnea (1) opposes the spontaneous emergence of a respiratory rhythm, which would have rescued the animals otherwise, and (2) prevents the effects of high dose naloxone.

Liraglutide treatment improves the coronary microcirculation in insulin resistant Zucker obese rats on a high salt diet.

BACKGROUND: Obesity, hypertension and prediabetes contribute greatly to coronary artery disease, heart failure and vascular events, and are the leading cause of mortality and morbidity in developed societies. Salt sensitivity exacerbates endothelial dysfunction. Herein, we investigated the effect of chronic glucagon like peptide-1 (GLP-1) receptor activation on the coronary microcirculation and cardiac remodeling in Zucker rats on a high-salt diet (6% NaCl). METHODS: Eight-week old Zucker lean (+/+) and obese (fa/fa) rats were treated with vehicle or liraglutide (LIRA) (0.1 mg/kg/day, s.c.) for 8 weeks. Systolic blood pressure (SBP) was measured using tail-cuff method in conscious rats. Myocardial function was assessed by echocardiography. Synchrotron contrast microangiography was then used to investigate coronary arterial vessel function (vessels 50-350 µm internal diameter) in vivo in anesthetized rats. Myocardial gene and protein expression levels of vasoactive factors, inflammatory, oxidative stress and remodeling markers were determined by real-time PCR and Western blotting. RESULTS: We found that in comparison to the vehicle-treated fa/fa rats, rats treated with LIRA showed significant improvement in acetylcholine-mediated vasodilation in the small arteries and arterioles (< 150 µm diameter). Neither soluble guanylyl cyclase or endothelial NO synthase (eNOS) mRNA levels or total eNOS protein expression in the myocardium were significantly altered by LIRA. However, LIRA downregulated Nox-1 mRNA (p = 0.030) and reduced ET-1 protein (p = 0.044) expression. LIRA significantly attenuated the expressions of proinflammatory and profibrotic associated biomarkers (NF-κB, CD68, IL-1ß, TGF-ß1, osteopontin) and nitrotyrosine in comparison to fa/fa-Veh rats, but did not attenuate perivascular fibrosis appreciably. CONCLUSIONS: In a rat model of metabolic syndrome, chronic LIRA treatment improved the capacity for NO-mediated dilation throughout the coronary macro and microcirculations and partially normalized myocardial remodeling independent of changes in body mass or blood glucose.

Threshold and saturation pressures of baroreflex-mediated myocardial interstitial acetylcholine release in rats.

Cardiac microdialysis allows the assessment of cardiac efferent vagal nerve activity from myocardial interstitial acetylcholine (ACh) levels with minimal influence on the neural control of the heart; however, a total picture of the baroreflex-mediated myocardial interstitial ACh release including the threshold and saturation pressures has yet to be quantified. In eight anesthetized Wistar-Kyoto rats, we implanted microdialysis probes in the left ventricular free wall and measured the myocardial interstitial ACh release simultaneously with efferent sympathetic nerve activity (SNA) during a carotid sinus baroreceptor pressure input between 60 and 180 mm Hg. The baroreflex-mediated ACh release approximated a positive sigmoid curve, and its threshold and saturation pressures were not significantly different from those of an inverse sigmoid curve associated with the baroreflex-mediated SNA response (threshold: 94.3 ± 8.6 vs. 99.3 ± 6.0 mm Hg; saturation: 150.0 ± 10.3 vs. 158.8 ± 5.8 mm Hg). The sympathetic and vagal systems have certain levels of activities across most of the normal pressure range.

Hydrogen sulfide intoxication induced brain injury and methylene blue.

Hydrogen sulfide (H2S) remains a chemical hazard in the gas and farming industry. It is easy to manufacture from common chemicals and thus represents a potential threat for the civilian population. It is also employed as a method of suicide, for which incidence has recently increased in the US. H2S is a mitochondrial poison and exerts its toxicity through mechanisms that are thought to result from its high affinity to various metallo-proteins (such as - but not exclusively- the mitochondrial cytochrome c oxidase) and interactions with cysteine residues of proteins. Ion channels with critical implications for the cardiac and the brain functions appear to be affected very early during and following H2S exposure, an effect which is rapidly reversible during a light intoxication. However, during severe H2S intoxication, a coma, associated with a reduction in cardiac contractility, develops within minutes or even seconds leading to death by complete electro-mechanical dissociation of the heart. If the level of intoxication is milder, a rapid and spontaneous recovery of the coma occurs as soon as the exposure stops. The risk, although probably very small, of developing long-term debilitating motor or cognitive deficits is present. One of the major challenges impeding our effort to offer an effective treatment against H2S intoxication after exposure is that the pool of free/soluble H2S almost immediately disappears from the body preventing agents trapping free H2S (cobalt or ferric compounds) to play their protective role. This paper (1) presents and discusses the neurological symptoms and lesions observed in various animals models and in humans following an acute exposure to sub-lethal or lethal levels of H2S, (2) reviews the potential interest of methylene blue (MB), a potent cyclic redox dye - currently used for the treatment of methemoglobinemia - which has potential rescuing effects on the mitochondrial activity, as an antidote against sulfide intoxication.

Diastolic dysfunction is initiated by cardiomyocyte impairment ahead of endothelial dysfunction due to increased oxidative stress and inflammation in an experimental prediabetes model.

Coronary microvessel endothelial dysfunction and nitric oxide (NO) depletion contribute to elevated passive tension of cardiomyocytes, diastolic dysfunction and predispose the heart to heart failure with preserved ejection fraction. We examined if diastolic dysfunction at the level of the cardiomyocytes precedes coronary endothelial dysfunction in prediabetes. Further, we determined if myofilaments other than titin contribute to impairment. Utilizing synchrotron microangiography we found young prediabetic male rats showed preserved dilator responses to acetylcholine in microvessels. Utilizing synchrotron X-ray diffraction we show that cardiac relaxation and cross-bridge dynamics are impaired by myosin head displacement from actin filaments particularly in the inner myocardium. We reveal that increased PKC activity and mitochondrial oxidative stress in cardiomyocytes contributes to rho-kinase mediated impairment of myosin head extension to actin filaments, depression of soluble guanylyl cyclase/PKG activity and consequently stiffening of titin in prediabetes ahead of coronary endothelial dysfunction.

Serotonin uptake via plasma membrane monoamine transporter during myocardial ischemia-reperfusion in the rat heart in vivo.

Serotonin (5-HT) accumulates in the heart during myocardial ischemia and induces deleterious effects on the cardiomyocytes through receptor-dependent and monoamine oxidase-dependent pathways. We aimed to clarify the involvement of extra-neuronal monoamine transporters in the clearance of 5-HT during ischemia and reperfusion in the heart. Using a microdialysis technique in the anesthetized Wistar rat heart, we monitored myocardial interstitial 5-HT and 5-hydroxyindole acetic acid (5-HIAA) concentration by means of electro-chemical detection coupled with high-performance liquid chromatography (HPLC-ECD). Effects of inhibitors of the plasma membrane monoamine transporter (PMAT) and the organic cation transporter 3 (OCT3) (decynium-22 and corticosterone) on the 5-HT and 5-HIAA concentrations during baseline, coronary occlusion, and reperfusion were investigated. Basal dialysate 5-HT concentration were increased by local administration of decynium-22, but not by corticosterone. Addition of fluoxetine, a serotonin transporter (SERT) inhibitor further increased the 5-HT concentration upon during administration of decynium-22. Decynium-22 elevated the background level of 5-HT during coronary occlusion and maintained 5-HT concentration at a high level during reperfusion. Production of 5-HIAA in the early reperfusion was significantly suppressed by decynium-22. These results indicate that PMAT and SERT independently regulate basal level of interstitial 5-HT, and PMAT plays a more important role in the clearance of 5-HT during reperfusion. These data suggest the involvement of PMAT in the monoamine oxidase-dependent deleterious pathway in the heart.

Liraglutide Improves Renal Endothelial Function in Obese Zucker Rats on a High-Salt Diet.

Metabolic syndrome is a common risk factor in chronic kidney disease. We investigated whether liraglutide [(LIRA), a glucagon-like peptide-1 receptor (GLP-1R) agonist] treatment improved renal vascular function and renal remodeling in male Zucker rats on a high-salt diet (6% NaCl). Zucker lean (+/+) and obese (fa/fa) rats (8 weeks old) were treated with vehicle or LIRA (0.1 mg/kg per day) for 8 weeks on a high-salt diet. The glomerular filtration rate (GFR) was measured at 0 and 8 weeks using the fluorescein isothiocyanate/sinistrin method in conscious rats. We used X-ray microangiography to measure renal arterial vessel diameter (70-350 µm) and vessel number in vivo in anesthetized rats. Renal protein expression levels of nitrotyrosine, CD-68, endothelial nitric oxide synthase (eNOS), vascular endothelial growth factor (VEGF), transforming growth factor-ß1, cyclooxygenase-2, and GLP-1R were assessed by western blotting. Renal gene expressions were determined by real-time polymerase chain reaction. In contrast to vehicle-treated rats, fa/fa-LIRA rats improved GFR, nitric oxide (NO)-mediated vasodilation in response to acetylcholine and sodium nitroprusside in small arterial vessels (<200 µm diameter). LIRA treatment increased vessel responsivity to NO donors in comparison with vehicle treatment. Increases in the expressions of proinflammatory, profibrotic, and oxidative stress related genes in fa/fa rats relative to +/+ were unaltered by LIRA, other than a trend toward attenuation of VCAM-1 gene expression. However, LIRA treatment increased protein expressions of eNOS (P = 0.014) and VEGF (P = 0.063), while reducing glomerular macrophage infiltration in comparison with vehicle-treated fa/fa rats. Low-dose LIRA treatment improved renal vascular function through amelioration of vascular dysfunction and improved NO-mediated dilation of small intrarenal arteries and arterioles and a reduction in renal inflammation.

Accentuated antagonism of vagal heart rate control and less potent prejunctional inhibition of vagal acetylcholine release during sympathetic nerve stimulation in the rat.

Complex interactions are known to occur between the sympathetic and parasympathetic controls of the heart. Although sympathetic nerve stimulation (SNS) usually augments the heart rate (HR) response to vagal nerve stimulation (VNS), exogenously administered norepinephrine (NE) can attenuate the HR response as well as the myocardial interstitial acetylcholine (ACh) release during VNS. To provide a basis for an integrative knowledge about the opposing adrenergic effects on the vagal control of the heart, we examined whether SNS significantly attenuates VNS-induced myocardial interstitial ACh release in the in vivo beating heart. In nine anesthetized rats, changes in HR and myocardial interstitial ACh release in response to 5- and 20-Hz VNS were examined in both the absence and presence of a 5-Hz background SNS. The SNS significantly enhanced the VNS-induced HR reduction during 20-Hz VNS (-101.2 ±â€¯33.1 vs. -163.0 ±â€¯34.9 beats/min, P < 0.001, a 60% augmentation). By contrast, the SNS significantly attenuated the ACh release during 20-Hz VNS (4.30 ±â€¯0.72 vs. 3.80 ±â€¯0.75 nM, P < 0.01, a 12% attenuation). In conclusion, SNS exerted only a moderate inhibitory effect on the VNS-induced myocardial interstitial ACh release in the in vivo beating heart.

Modulation of Sympathetic Activity and Innervation With Chronic Ivabradine and ß-Blocker Therapies: Analysis of Hypertensive Rats With Heart Failure.

BACKGROUND: Whether the reduction of heart rate with ivabradine (IVA) could affect sympathetic activation and cardiac innervation in heart failure (HF) remains unknown. PURPOSE: The present study assessed the chronic effects of IVA and ß-blocker on the systemic and local sympathetic nervous systems of hypertensive animals with HF. METHODS AND RESULTS: The Dahl salt-sensitive rats received chronic IVA, bisoprolol (BIS), or placebo (CTL) therapy. The survival of the animal models with IVA and BIS significantly improved (median; 19.7 in IVA and 19.7 in BIS vs 17.0 weeks in CTL, P < .001). A similar decrease in 24-hour heart rate (mean; 305 in IVA and 329 in BIS vs 388 beats/min in CTL, P < .001) without effect on blood pressure, and an improvement in the left ventricular dysfunction (mean fractional shortening; 56.7% in IVA and 47.8% in BIS vs 39.0% in CTL, P < .001) were observed in the animals with IVA and BIS. However, a negative inotropic effect was only observed in the animals with BIS. Excessive urinary noradrenaline excretion in animals with CTL was only suppressed with the use of IVA (mean; 1.35 µg/d in IVA and 1.95 µg/d in BIS vs 2.27 µg/d in CTL, P = .002). In contrast, atrial noradrenaline and acetylcholine depletion in the animals with CTL improved and the tyrosine hydroxylase expression in the both atria were restored with the use of both IVA and BIS. CONCLUSIONS: IVA therapy improved the survival of hypertensive animals with HF. Furthermore, it was associated with the amelioration of systemic sympathetic activation and cardiac sympathetic and parasympathetic nerve innervations. Chronic ß-blocker therapy with negative inotropic effects had beneficial effects only on cardiac innervations.

Central activation of cardiac vagal nerve by α2-adrenergic stimulation is impaired in streptozotocin-induced type 1 diabetic rats.

To elucidate the abnormality of cardiac vagal control in streptozotocin-induced type 1 diabetic rats, we measured left ventricular myocardial interstitial acetylcholine (ACh) release in response to α2-adrenergic stimulation as an index of in vivo cardiac vagal nerve activity. A cardiac microdialysis technique was applied to the rat left ventricle, and the effect of α2-adrenergic stimulation by intravenous medetomidine (100 µg/kg) on myocardial interstitial ACh levels was examined in anesthetized diabetic rats (4-6 weeks after intraperitoneal streptozotocin) and age-matched control rats (protocol 1). The effect of electrical vagal nerve stimulation on ACh levels was also examined in separate rats (protocol 2). In protocol 1, medetomidine increased the ACh levels in control (from 1.76 ±â€¯0.65 to 3.13 ±â€¯1.41 nM, P < 0.05, n = 7) but not in diabetic rats (from 2.01 ±â€¯0.47 to 1.62 ±â€¯0.34 nM, not significant, n = 7). In protocol 2, electrical vagal nerve stimulation at 20 Hz significantly increased the ACh levels in both control (from 1.49 ±â€¯0.26 to 6.39 ±â€¯1.81 nM, P < 0.001, n = 6) and diabetic rats (from 1.77 ±â€¯0.54 to 6.98 ±â€¯1.38 nM, P < 0.001, n = 6). In conclusion, medetomidine-induced central vagal activation was impaired in diabetic rats, whereas peripheral cardiac vagal control of ACh release was preserved. The impairment of central vagal activation may lead to relative sympathetic predominance and promote cardiovascular complications in diabetes.

Ghrelin and vascular protection.

Ghrelin is a small peptide with important roles in the regulation of appetite, gut motility, glucose homeostasis as well as cardiovascular protection. This review highlights the role that acyl ghrelin plays in maintaining normal endothelial function by maintaining the balance of vasodilator-vasoconstrictor factors, inhibiting inflammatory cytokine production and immune cell recruitment to sites of vascular injury and by promoting angiogenesis.

On the Efficacy of Cardio-Pulmonary Resuscitation and Epinephrine Following Cyanide- and H2S Intoxication-Induced Cardiac Asystole.

This study was aimed at determining the efficacy of epinephrine, followed by chest compressions, in producing a return of spontaneous circulation (ROSC) during cyanide (CN)- or hydrogen sulfide (H2S)-induced toxic cardiac pulseless electrical activity (PEA) in the rat. Thirty-nine anesthetized rats were exposed to either intravenous KCN (n = 27) or H2S solutions (n = 12), at a rate that led to a PEA within less than 10 min. In the group intoxicated by CN, 20 rats were mechanically ventilated and received either epinephrine (0.1 mg/kg i.v. n = 10) followed by chest compressions or saline (n = 10, "control CN") when in PEA. PEA was defined as a systolic pressure below 20 mmHg and a pulse pressure of less than 5 mmHg for 1 min. In addition, seven spontaneously breathing rats were also exposed to the same CN protocol, but infusion was stopped when a central apnea occurred; then, as soon as a PEA occurred, epinephrine (0.1 mg/kg IV) was administered while providing manual chest compressions and mechanical ventilation (CPR). Finally, 12 rats were intoxicated with H2S, while mechanically ventilated, and received either saline (n = 6, "control H2S") or epinephrine (n = 6) with CPR when in PEA. None of the control-intoxicated animals resuscitated (10 rats in the control CN group and 6 in the control H2S group). In contrast, all the animals intoxicated with CN or H2S that received epinephrine followed by chest compressions, returned to effective circulation. In addition, half of the spontaneously breathing CN-intoxicated animals that achieved ROSC after epinephrine resumed spontaneous breathing. In all the animals achieving ROSC, blood pressure, cardiac output, peripheral blood flow and [Formula: see text]O2 returned toward baseline, but remained lower than the pre-intoxication levels (p < 0.01) with a persistent lactic acidosis. Epinephrine, along with CPR maneuvers, was highly effective in resuscitating rodents intoxicated with CN or H2S. Since epinephrine is readily available in any ambulance, its place as an important countermeasure against mitochondrial poisons should be advocated. It remains critical to determine whether the systematic administration of epinephrine to any victims found hypotensive following CN or H2S intoxication could prevent PEA, decrease post-ischemic brain injury and increase the efficacy of current antidotes by improving the circulatory status.