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.

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.

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.

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.

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.

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.

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.

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.

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.

Interleukin-6/interleukin-21 signaling axis is critical in the pathogenesis of pulmonary arterial hypertension.

IL-6 is a multifunctional proinflammatory cytokine that is elevated in the serum of patients with pulmonary arterial hypertension (PAH) and can predict the survival of patients with idiopathic PAH (IPAH). Previous animal experiments and clinical human studies indicate that IL-6 is important in PAH; however, the molecular mechanisms of IL-6-mediated pathogenesis of PAH have been elusive. Here we identified IL-21 as a downstream target of IL-6 signaling in PAH. First, we found that IL-6 blockade by the monoclonal anti-IL-6 receptor antibody, MR16-1, ameliorated hypoxia-induced pulmonary hypertension (HPH) and prevented the hypoxia-induced accumulation of Th17 cells and M2 macrophages in the lungs. Consistently, the expression levels of IL-17 and IL-21 genes, one of the signature genes for Th17 cells, were significantly up-regulated after hypoxia exposure in the lungs of mice treated with control antibody but not in the lungs of mice treated with MR16-1. Although IL-17 blockade with an anti-IL-17A neutralizing antibody had no effect on HPH, IL-21 receptor-deficient mice were resistant to HPH and exhibited no significant accumulation of M2 macrophages in the lungs. In accordance with these findings, IL-21 promoted the polarization of primary alveolar macrophages toward the M2 phenotype. Of note, significantly enhanced expressions of IL-21 and M2 macrophage markers were detected in the lungs of IPAH patients who underwent lung transplantation. Collectively, these findings suggest that IL-21 promotes PAH in association with M2 macrophage polarization, downstream of IL-6-signaling. The IL-6/IL-21-signaling axis may be a potential target for treating PAH.

H2S concentrations in the heart after acute H2S administration: methodological and physiological considerations.

In this study, we have tried to characterize the limits of the approach typically used to determine H2S concentrations in the heart based on the amount of H2S evaporating from heart homogenates-spontaneously, after reaction with a strong reducing agent, or in a very acidic solution. Heart homogenates were prepared from male rats in control conditions or after H2S infusion induced a transient cardiogenic shock (CS) or cardiac asystole (CA). Using a method of determination of gaseous H2S with a detection limit of 0.2 nmol, we found that the process of homogenization could lead to a total disappearance of free H2S unless performed in alkaline conditions. Yet, after restoration of neutral pH, free H2S concentration from samples processed in alkaline and nonalkaline milieus were similar and averaged ∼0.2-0.4 nmol/g in both control and CS homogenate hearts and up to 100 nmol/g in the CA group. No additional H2S was released from control, CS, or CA hearts by using the reducing agent tris(2-carboxyethyl)phosphine or a strong acidic solution (pH < 2) to "free" H2S from combined pools. Of note, the reducing agent DTT produced a significant sulfide artifact and was not used. These data suggest that 1) free H2S found in heart homogenates is not a reflection of H2S present in a "living" heart and 2) the pool of combined sulfides, released in a strong reducing or acidic milieu, does not increase in the heart in a measurable manner even after toxic exposure to sulfide.

Methylene blue counteracts H2S toxicity-induced cardiac depression by restoring L-type Ca channel activity.

We have previously reported that methylene blue (MB) can counteract hydrogen sulfide (H2S) intoxication-induced circulatory failure. Because of the multifarious effects of high concentrations of H2S on cardiac function, as well as the numerous properties of MB, the nature of this interaction, if any, remains uncertain. The aim of this study was to clarify 1) the effects of MB on H2S-induced cardiac toxicity and 2) whether L-type Ca(2+) channels, one of the targets of H2S, could transduce some of the counteracting effects of MB. In sedated rats, H2S infused at a rate that would be lethal within 5 min (24 µM·kg(-1)·min(-1)), produced a rapid fall in left ventricle ejection fraction, determined by echocardiography, leading to a pulseless electrical activity. Blood concentrations of gaseous H2S reached 7.09 ± 3.53 µM when cardiac contractility started to decrease. Two to three injections of MB (4 mg/kg) transiently restored cardiac contractility, blood pressure, and V̇o2, allowing the animals to stay alive until the end of H2S infusion. MB also delayed PEA by several minutes following H2S-induced coma and shock in unsedated rats. Applying a solution containing lethal levels of H2S (100 µM) on isolated mouse cardiomyocytes significantly reduced cell contractility, intracellular calcium concentration ([Ca(2+)]i) transient amplitudes, and L-type Ca(2+) currents (ICa) within 3 min of exposure. MB (20 mg/l) restored the cardiomyocyte function, ([Ca(2+)]i) transient, and ICa The present results offer a new approach for counteracting H2S toxicity and potentially other conditions associated with acute inhibition of L-type Ca(2+) channels.

Cell-sheet therapy with omentopexy promotes arteriogenesis and improves coronary circulation physiology in failing heart.

Cell-sheet transplantation induces angiogenesis for chronic myocardial infarction (MI), though insufficient capillary maturation and paucity of arteriogenesis may limit its therapeutic effects. Omentum has been used clinically to promote revascularization and healing of ischemic tissues. We hypothesized that cell-sheet transplantation covered with an omentum-flap would effectively establish mature blood vessels and improve coronary microcirculation physiology, enhancing the therapeutic effects of cell-sheet therapy. Rats were divided into four groups after coronary ligation; skeletal myoblast cell-sheet plus omentum-flap (combined), cell-sheet only, omentum-flap only, and sham operation. At 4 weeks after the treatment, the combined group showed attenuated cardiac hypertrophy and fibrosis, and a greater amount of functionally (CD31(+)/lectin(+)) and structurally (CD31(+)/α-SMA(+)) mature blood vessels, along with myocardial upregulation of relevant genes. Synchrotron-based microangiography revealed that the combined procedure increased vascularization in resistance arterial vessels with better dilatory responses to endothelium-dependent agents. Serial (13)N-ammonia PET showed better global coronary flow reserve in the combined group, mainly attributed to improvement in the basal left ventricle. Consequently, the combined group had sustained improvements in cardiac function parameters and better functional capacity. Cell-sheet transplantation with an omentum-flap better promoted arteriogenesis and improved coronary microcirculation physiology in ischemic myocardium, leading to potent functional recovery in the failing heart.

Treadmill running improves hindlimb arteriolar endothelial function in type 1 diabetic mice as visualized by X-ray microangiography.

BACKGROUND: Vascular function is impaired in patients with diabetes, however diabetic vascular dysfunction is ameliorated by exercise training. We aimed to clarify which hindlimb arterial segments are affected by treadmill running in the hindlimbs of streptozocin-induced type 1 diabetic mice in vivo. METHODS: Mice were divided into 3 groups; healthy control, diabetic control, and diabetic-running groups. The exercise regimen was performed by treadmill level running mice for 60 min/day, for 4 weeks. Thereafter, we examined the vascular response to systemic acetylcholine administration in the left hindlimb of anesthetized-ventilated mice using either 1) X-ray microangiography to visualize the arteries or 2) ultrasonic flowmetry to record the femoral arterial blood flow. RESULTS: X-ray imaging clearly visualized the hindlimb arterial network (~70-250 µm diameter). The vasodilator response to acetylcholine was significantly attenuated locally in the arterioles <100 µm diameter in the diabetic group of mice compared to the control group of mice. Post-acetylcholine administration, all groups showed an increase in hindlimb vascular conductance, but the diabetic mice showed the smallest increase. Overall, compared to the diabetic mice, the treadmill-running mice exhibited a significant enhancement of the vasodilator response within the arterioles with diabetes-induced vasodilator dysfunction. CONCLUSIONS: Diabetes impaired acetylcholine-induced vasodilator function locally in the arteries <100 µm diameter and decreased hindlimb vascular conductance responded to acetylcholine, while regular treadmill running significantly ameliorated the impaired vasodilator function, and enhanced the decreased conductance in the diabetic mice.

Pulmonary vascular tone is dependent on the central modulation of sympathetic nerve activity following chronic intermittent hypoxia.

Chronic intermittent hypoxia (IH) provokes a centrally mediated increase in sympathetic nerve activity (SNA). Although this sympathetic hyperexcitation has been linked to systemic hypertension, its effect on the pulmonary vasculature is unclear. This study aimed to assess IH-mediated sympathetic excitation in modulating pulmonary vasculature tone, particularly acute hypoxia vasoconstrictor response (HPV), and the central ß-adrenergic signaling pathway for facilitating the increase in SNA. Sprague-Dawley rats were exposed to IH (cycle of 4% O2 for 90 s/air for 90 s) for 8 h/day for 6 weeks. Subsequently, rats were anesthetized and either pulmonary SNA was recorded (electrophysiology), or the pulmonary vasculature was visualized using microangiography. Pulmonary sympathetic and vascular responses to acute hypoxia were assessed before and after central ß1-adrenergic receptor blockade (Metoprolol, 200 nmol i.c.v.). Chronic IH increased baseline SNA (110% increase), and exacerbated the sympathetic response to acute hypoxia. Moreover, the magnitude of HPV in IH rats was blunted compared to control rats (e.g., 10 and 20% vasoconstriction, respectively). In only the IH rats, ß1-receptor blockade with metoprolol attenuated the hypoxia-induced increase in pSNA and exacerbated the magnitude of acute HPV, so that both sympathetic and HPV responses were similar to that of control rats. Interestingly, the expression of ß1-receptors within the brainstem was similar between both control and IH rats. These results suggest that the centrally mediated increase in SNA following IH acts to blunt the local vasoconstrictor effect of acute hypoxia, which reflects an inherent difference between vasodilator and vasoconstrictor actions of SNA in pulmonary and systemic circulations.

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.

Myosin heads are displaced from actin filaments in the in situ beating rat heart in early diabetes.

Diabetes is independently associated with a specific cardiomyopathy, characterized by impaired cardiac muscle relaxation and force development. Using synchrotron radiation small-angle x-ray scattering, this study investigated in the in situ heart and in real-time whether changes in cross-bridge disposition and myosin interfilament spacing underlie the early development of diabetic cardiomyopathy. Experiments were conducted using anesthetized Sprague-Dawley rats 3 weeks after treatment with either vehicle (control) or streptozotocin (diabetic). Diffraction patterns were recorded during baseline and dobutamine infusions simultaneous with ventricular pressure-volumetry. From these diffraction patterns myosin mass transfer to actin filaments was assessed as the change in intensity ratio (I(1,0)/I(1,1)). In diabetic hearts cross-bridge disposition was most notably abnormal in the diastolic phase (p < 0.05) and to a lesser extent the systolic phase (p < 0.05). In diabetic rats only, there was a transmural gradient of contractile depression. Elevated diabetic end-diastolic intensity ratios were correlated with the suppression of diastolic function (p < 0.05). Furthermore, the expected increase in myosin head transfer by dobutamine was significantly blunted in diabetic animals (p < 0.05). Interfilament spacing did not differ between groups. We reveal that impaired cross-bridge disposition and radial transfer may thus underlie the early decline in ventricular function observed in diabetic cardiomyopathy.

Oxidative stress augments pulmonary hypertension in chronically hypoxic mice overexpressing the oxidized LDL receptor.

Chronic hypoxia is one of the main causes of pulmonary hypertension (PH) associated with ROS production. Lectin-like oxidized low-density lipoprotein receptor (LOX)-1 is known to be an endothelial receptor of oxidized low-density lipoprotein, which is assumed to play a role in the initiation of ROS generation. We investigated the role of LOX-1 and ROS generation in PH and vascular remodeling in LOX-1 transgenic (TG) mice. We maintained 8- to 10-wk-old male LOX-1 TG mice and wild-type (WT) mice in normoxia (room air) or hypoxia (10% O2 chambers) for 3 wk. Right ventricular (RV) systolic pressure (RVSP) was comparable between the two groups under normoxic conditions; however, chronic hypoxia significantly increased RVSP and RV hypertrophy in LOX-1 TG mice compared with WT mice. Medial wall thickness of the pulmonary arteries was significantly greater in LOX-1 TG mice than in WT mice. Furthermore, hypoxia enhanced ROS production and nitrotyrosine expression in LOX-1 TG mice, supporting the observed pathological changes. Administration of the NADPH oxidase inhibitor apocynin caused a significant reduction in PH and vascular remodeling in LOX-1 TG mice. Our results suggest that LOX-1-ROS generation induces the development and progression of PH.

Assessment of the serotonin pathway as a therapeutic target for pulmonary hypertension.

Blockade of the serotonin reuptake transporter (5-HTT), using fluoxetine, has been identified as a potential therapeutic target for preventing and, importantly, reversing pulmonary hypertension (PH). This study utilized synchrotron radiation microangiography to determine whether fluoxetine could prevent or reverse endothelial dysfunction and vessel rarefaction, which underpin PH. PH was induced by a single injection of monocrotaline (MCT; 60 mg kg(-1)). Following MCT administration, rats received daily injections of either saline or fluoxetine (MCT+Fluox; 10 mg kg(-1)) for three weeks. A third group of rats also received the fluoxetine regime, but only three weeks after MCT (MCT+FluoxDelay). Control rats received daily injections of saline. Pulmonary microangiography was performed to assess vessel branching density and visualize dynamic changes in vessel diameter following (i) acute fluoxetine or (ii) acetylcholine, sodium nitroprusside, BQ-123 (ET-1A receptor blocker) and L-NAME (NOS inhibitor). Monocrotaline induced PH that was inevitably terminal. `Delayed' treatment of fluoxetine (MCT+FluoxDelay) was unable to reverse the progression of PH. Early fluoxetine treatment pre-PH (i.e. MCT+Fluox) attenuated but did not completely prevent vascular remodeling, vessel rarefaction and an increase in pulmonary pressure, and it did not prevent pulmonary endothelial dysfunction. Interestingly, fluoxetine treatment did counter-intuitively prevent the onset of right ventricular hypertrophy. Using synchrotron radiation microangiography, selective blockade of the serotonin reuptake transporter alone is highlighted as not being sufficient to prevent pulmonary endothelial dysfunction, which is the primary instigator for the inevitable onset of vascular remodeling and vessel rarefaction. Accordingly, potential therapeutic strategies should aim to target multiple pathways to ensure an optimal outcome.

Impaired pulmonary blood flow distribution in congestive heart failure assessed using synchrotron radiation microangiography.

Synchrotron radiation microangiography is a powerful tool for assessing adverse changes in pulmonary vessel density associated with primary pulmonary hypertension (PH). Congestive heart failure (CHF) leads to a `secondary' onset of PH, yet it is unknown whether secondary PH is also associated with reduced vessel density. This study utilized synchrotron radiation to assess both pulmonary vessel density and endothelial function in a Dahl rat model of CHF with secondary PH. High salt-fed Dahl salt-sensitive (Dahl-S) and salt-resistant (Dahl-R) rats were anesthetized and microangiography was performed to assess the pulmonary vessel density and vascular responses to (i) sodium nitroprusside (5.0 µg kg(-1) min(-1)), (ii) acetylcholine (3.0 µg kg(-1) min(-1)) and (iii) ET-1A receptor blockade, BQ-123 (1 mg kg(-1)). Dahl-S rats developed CHF and secondary PH as evident by endothelial dysfunction, impaired vasodilatory responses to acetylcholine, enhanced vasodilatory responses to BQ-123 and extensive pulmonary vascular remodeling. Consequently, the pulmonary vessel density was adversely reduced. Interestingly, the etiology of secondary PH manifests with structural and functional changes that are comparable with that previously reported for primary PH. One important discrepancy, however, is that ET-1 modulation of pulmonary vessels is most striking in vessels with a diameter range of 100-200 µm in secondary PH, in contrast to a range of 200-300 µm in primary PH. Such discrepancies should be considered in future studies investigating primary and secondary forms of PH.