Impaired Nitric Oxide Release Research Articles

Apelin‐Induced Relaxation is Impaired in Coronary Arteries from Spontaneously Hypertensive Rats

Hypertension is a major modifiable risk factor for coronary artery disease. The pathophysiological mechanisms underlying hypertension as a risk factor for coronary artery disease are not fully understood. Apelin is a vasoactive peptide that binds to APJ receptors, which are highly expressed throughout the cardiovascular system, including coronary arteries. APJ receptors signal via G-protein-dependent and -independent pathways, including activation of G-protein-coupled-receptor kinase 2 (GRK2), which can potentially lead to inhibition of eNOS and nitric oxide (NO) production. We have previously shown that apelin causes endothelium-dependent, NO-mediated relaxation of coronary arteries from normotensive animals (JPET 366:265, 2018), but its effects in hypertensive coronary arteries are unknown. Here, we tested the hypothesis that apelin-induced relaxation is impaired in coronary arteries from spontaneously hypertensive rats (SHR). Western blot analysis demonstrated increased expression of GRK2 in cultured coronary endothelial cells from SHR in comparison with normotensive WKY (control) rats. Isolated coronary arterial rings were mounted in wire myographs for isometric tension recording and contracted with 5-HT (10-7 M). Apelin (10-9-10-6 M) caused endothelium-dependent relaxation of WKY coronary arteries (pD2 = 7.00 ± 0.11; Emax = 54 ± 4% relaxation; n=6), but had no effect in rings from SHR. Conversely, endothelium-dependent relaxations to an APJ receptor biased agonist, CMF-019 (10-9-10-5 M), which preferentially activates the G-protein-dependent pathway with minimal effect on GRK2, did not differ between SHR and WKY. In the presence of apelin (10-7 M), endothelium-dependent relaxation to acetylcholine (ACh) (10-9-10-5 M) was impaired in SHR coronary arteries (pD2 = 6.82 ± 0.10 vs 6.52 ± 0.18, and Emax = 81 ± 8 vs 60 ± 10% relaxation, in the absence and presence of apelin, respectively; n=5; p<0.05); however, apelin had no effect on ACh-induced relaxation of WKY coronary arteries. By contrast, ACh-induced relaxation of SHR and WKY coronary arterial rings was unaffected by CMF-019 (10-7 M). Moreover, the GRK2 inhibitor, CMPD 101 (3 × 10-5 M), partially restored the relaxation response to apelin in SHR coronary arteries (pD2 = 6.24 ± 0.30; Emax = 42 ± 13% relaxation; n=3). Relaxations in response to the NO-donor, DEA NONOate (10-9-10-4 M), were not altered by apelin (10-7 M) treatment in either WKY or SHR coronary arteries. Taken together, the data indicate that apelin failed to cause relaxation in hypertensive coronary arteries, likely due to impaired production or release of NO from endothelial cells rather than interfering with the action of NO on coronary smooth muscle cells. In SHR coronary arteries, apelin instead actively inhibited relaxation to another endothelium-dependent vasodilator; i.e. ACh. That the effects of the G-protein biased agonist, CMF-019, did not differ between WKY and SHR coronary arteries is consistent with a role for GRK2 activation in the altered response to apelin in hypertensive arteries. APJ receptor signaling via the GRK2 pathway may contribute to both the loss of relaxation to apelin itself, as well as the ability of apelin to inhibit endothelium-dependent relaxation to ACh in coronary arteries from SHR.

Microcirculation as a goal of correction in macroangiopathy (atherogenesis)

Background. The microcirculatory system (MCS) is a network of blood vessels that includes arterioles, capillaries, venules, and terminal lymphatic vessels. Microcirculation is characterized by the constant variability. Factors of atherogenesis development due to MCS dysfunction include shear stress, hyperglycemia, dyslipidemia, systemic and local inflammation, hypoxia and endothelial dysfunction mediated by oxidative stress. Laser Doppler flowmetry (LDF) is used to study microcirculation in the clinical settings. The advantages of LDF include simplicity, accessibility and non-invasiveness.&#x0D; Objective. To describe the features of microcirculation disorders and their elimination.&#x0D; Materials and methods. Analysis of literature data on this topic; own study. The study involved 98 patients (59 females; 39 males) with a mean age of 52.0 years. The first group consisted of patients with coronary heart disease (CHD) and chronic heart failure of I-IIA grades, the second – of relatively healthy individuals. All patients underwent LDF, ultrasound examination of the carotid arteries, and determination of anthropometric parameters.&#x0D; Results and discussion. MCS dysfunction is not only a risk factor for atherogenesis, but also a trigger for its acute complications (myocardial infarction, stroke, sudden death). Nitric oxide (NO) deficiency plays an important role in this. A potential target of therapeutic influence in the treatment of coronary heart disease is not only macrovascular system, but also vasa vasorum. The condition of the latter determines the course of atherosclerosis. According to the results of our own study, patients with CHD demonstrated a muscle mass decrease, an increase in waist and hip circumference, and in body mass index. In addition, the groups differed in thickness of the intima-media complex of both common carotid arteries (right common carotid artery: CHD group – 0.79±0.18 mm; group of relatively healthy individuals – 0.69±0.13 mm, p&lt;0,05; left common carotid artery: CHD group – 0.81±0.19 mm, group of relatively healthy individuals – 0.70±0.14 mm, p&lt;0,05). When assessing the indicators of wavelet analysis of LDF, a significant decrease in the rate of microcirculation and capillary blood flow reserve is revealed in the CHD group, as well as an increase in peripheral vascular resistance. According to previous own studies, sorbitol (Reosorbilact, “Yuria-Pharm”) and pentoxifylline (Latren, “Yuria-Pharm”) can be used to correct microcirculation disorders. The use of these drugs leads to vasodilation of precapillary sphincters and improvement of regional microperfusion.&#x0D; Conclusions. 1. Disorders of MCS are the pathogenetic factors of the atherogenesis. 2. Laser Doppler flowmetry is used to study microcirculation in the clinical settings. 3. In patients with CHD there is an increase in neuro- and myotonus of the MCS, which is associated with the impaired release of nitric oxide. 4. Changes in microcirculation contribute to the development of atherosclerosis, which should be taken into account when choosing treatment for such patients. 5. Sorbitol (Reosorbilact) and pentoxifylline (Latren) can be used to correct microcirculation disorders.

Contribution of peptidyl prolyl isomerase (Pin1) to development of pulmonary hypertension via pulmonary vascular endothelial cell dysfunction

Abstract Background Endothelial dysfunction is thought to be a major contributor to overall pathogenesis of vasculopathy seen in pulmonary hypertension (PH), which is manifested by the impaired release of nitric oxide (NO) generated through endothelial nitric oxide synthase (eNOS) in endothelial cells. Activation of human eNOS is regulated by phosphorylation at multiple sites including Thr33 and Ser114, which residues are followed by Pro. The peptidyl isomerase Pin1 specifically isomerizes the phospho-protein having Ser/Thr-Pro bond and regulates their activity. Pin1 is involved in proliferation, cell cycle, and apoptosis in cancer, by isomerizing some functional molecules such as JNK, JUN, cyclin D, BAX, etc. However, it is controversial whether direct interaction of Pin1 with eNOS and how eNOS activity is altered by Pin1, especially in PH. Purpose We aimed to clarify whether Pin1 contributes to the PH development using Pin1 knockout mice and Pin1 affects the expression of phosphorylated eNOS (p-eNOS) molecule and pulmonary arterial endothelial cell (PAEC) apoptosis. Methods and results Wild (WT) and Pin1-deficient mice (KO) were exposed to hypoxia (10% O2) or normoxia for 3 weeks to generate hypoxia-induced PH. Hypoxia-inducible factor (HIF1α) expression in lungs was significantly enhanced in WT-hypoxia (WH, n=6) and KO-hypoxia (KH, n=6), suggesting that hypoxic response was certainly occurred in these mice. Pulmonary arterial pressure did not elevate in KH compared with KO-normoxia (KN, n=6) and WT-normoxia (WN, n=6), it was significantly increased only in WH (P&amp;lt;0.01), indicating that KO did not develop PH by hypoxia. The gain of RV weight was parallel to the increase of pulmonary arterial pressure. Western blot showed that p-eNOS expression in lungs was significantly decreased in WH compared to WN, however, the expression was not different between KH and KN. It suggests that Pin1 plays a regulatory role in p-eNOS expression in hypoxic response. In cultured PAECs, the expression of p-eNOS and eNOS was markedly increased by siRNA-mediated Pin1 knockdown. Immunoprecipitation study showed the possibility of Pin1 binding to p-eNOS molecule. Apoptosis evaluated by caspase-3/7 activity by fluorescent assay and cleaved caspase-3 expression by Western blot was significantly increased by Pin1 overexpression in PAECs; however, it was significantly decreased by Pin1 knockdown. Moreover, the exaggeration of apoptosis induced by doxorubicin was markedly increased by Pin1 overexpression compared with control in PAECs; however, it was clearly suppressed by Pin1 knockdown. Conclusion This study suggests that endogenous Pin1 contributes to the development of PH partly via the dysfunction of PAECs, that is, by the interference with p-eNOS expression and by the increase of apoptosis inducibility to external stimuli. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): JSPS KAKENHI

The renal and blood pressure response to low sodium diet in P2X4 receptor knockout mice.

In the kidney, purinergic (P2) receptor‐mediated ATP signaling has been shown to be an important local regulator of epithelial sodium transport. Appropriate sodium regulation is crucial for blood pressure (BP) control and disturbances in sodium balance can lead to hypo‐ or hypertension. Links have already been established between P2 receptor signaling and the development of hypertension, attributed mainly to vascular and/or inflammatory effects. A transgenic mouse model with deletion of the P2X4 receptor (P2X4−/−) is known to have hypertension, which is thought to reflect endothelial dysfunction and impaired nitric oxide (NO) release. However, renal function in this model has not been characterized; moreover, studies in vitro have shown that the P2X4 receptor can regulate renal epithelial Na+ channel (ENaC) activity. Therefore, in the present study we investigated renal function and sodium handling in P2X4−/− mice, focusing on ENaC‐mediated Na+ reabsorption. We confirmed an elevated BP in P2X4−/− mice compared with wild‐type mice, but found that ENaC‐mediated Na+ reabsorption is no different from wild‐type and does not contribute to the raised BP observed in the knockout. However, when P2X4−/− mice were placed on a low sodium diet, BP normalized. Plasma aldosterone concentration tended to increase according to sodium restriction status in both genotypes; in contrast to wild‐types, P2X4−/− mice did not show an increase in functional ENaC activity. Thus, although the increased BP in P2X4−/− mice has been attributed to endothelial dysfunction and impaired NO release, there is also a sodium‐sensitive component.

New Insights into Possible Role of NOS-NO-ADMA Pathway Dysfunction in the Development of Cognitive Decline and Dementia: Exploring the Vascular Features of Alzheimer’s Disease

In recent years significant overlap between cardio-metabolic risk factors and cognitive decline has been reported. Cardio-metabolic and vascular factors shown to be associated with Alzheimer’s disease (AD) and other forms of dementia include midlife diabetes and hypertension, cerebrovascular lesions, diminished vascular function, dyslipidemia, obesity, and cigarette smoking. Accordingly, it has been recently proposed that amyloid is not the cause of AD but merely a marker and a later consequence of upstream changes that lead to neuronal and synaptic losses. However, although the idea that features of vascular dysfunction and injury are present in cognitive decline and AD patients was suggested over 25 years ago, the role of cardio-metabolic and cerebrovascular mechanisms in the pathogenesis of AD is far from being fully elucidated. Based on newly proposed vascular hypothesis, there is an impaired structure and function of cerebral blood vessels and cells in patients with cognitive decline and AD which is mediated by vascular oxidative stress. Consistent with these observations, the importance of endothelial dysfunction in the development of AD has been highlighted. One of the most prominent features of endothelial dysfunction is decreased production and bioavailability of Nitric Oxide (NO). Based on versatile properties in physiological as well as in pathophysiological conditions NO is regarded as a key molecule for longevity and cardiovascular health. NO is produced by three different isoforms of the enzyme NO synthase (NOS), namely: endothelial (eNOS), neuronal (nNOS), and inducible (iNOS). Reduced NO bioavailability and altered vascular expression and activity of NOS enzymes are implicated as major molecular players in the process of vascular aging. In the process of aging, the mechanisms by which NOS enzymes promote vascular dysfunction are specific for each NOS isoform. Normal activity of eNOS is required for the balanced production/bioavailability of NO, which is the main prerequisite for optimal vascular function. Conversely, excessive amount of NO produced by iNOS contributes to vascular dysfunction. Furthermore, although the possible role of nNOS-derived NO in aging-associated vascular dysfunction is far from being fully elucidated, experimental findings indicate that impaired perivascular NO release from nNOS increases vasoconstriction in aged arterioles. Oxidative stress and inhibitors of NO synthase are regarded as most potent initiators of disturbed production and bioavailability of NO. Important endogenous inhibitor of all three isoforms of NOS is asymmetrical dimethylarginine (ADMA). It has been recently proposed that ADMA may be a possible link between vascular disease and dementia. Based on this assumption ADMA might represent a unifying pathophysiological pathway linking the presence of vascular risk factors with the onset and progression of cognitive decline and dementia. These observations were made based on good evidence from literature that higher plasma ADMA concentrations favor atherosclerosis and independently predict adverse cardiovascular and cerebrovascular outcomes in several patient groups. However, possible role of ADMA in pathophysiology of cognitive decline and AD is still not fully understood. Prospective studies are warranted aiming at further clarification of ADMA involvement in development and progression of dementia. Moreover, use of ADMA in CSF and /or plasma as prospective biomarker of AD should be explored. According to newly introduced hypothesis disturbed NOS-NO-ADMA pathway is one of the most important attributes of cerebrovascular dysfunction that plays a pathogenic role in the development of cognitive decline and dementia. Experimental data from cell cultures and animal models have demonstrated that dysfunction of the NO-NOS- ADMA pathway results in cell death, blood-brain barrier (BBB) disruption, and brain edema via different pathological mechanisms. However, exact underlining mechanisms through which disturbed NOS-NO-ADMA pathway contributes to cognitive decline and dementia remain to be elucidated. Clarification of these mechanisms may be helpful in the identification of new therapeutic targets for aging and neurodegenerative diseases. Future research should also explain whether endothelial dysfunction precede or follow neurological changes which characterize cognitive decline and dementia. Moreover, clinical and epidemiological studies have shown that healthy nutrition and physical exercise are important non-pharmacological, lifestyle-related interventions that could help in maintaining appropriate vascular tone, adequate cerebral blood flow and normal cognition during aging. It is reasonable to suggest that prevention of cardio-metabolic diseases via endorsement of healthy aging determinants such as control of blood pressure, lipids and glucose levels as well as maintenance of optimal body weight achieved by regular physical activity and nutrition with high anti-oxidant capacity might be effective strategy in the preservation of cognitive function.

Working Memory and Aphasia

In recent years significant overlap between cardio-metabolic risk factors and cognitive decline has been reported. Cardio-metabolic and vascular factors shown to be associated with Alzheimer’s disease (AD) and other forms of dementia include midlife diabetes and hypertension, cerebrovascular lesions, diminished vascular function, dyslipidemia, obesity, and cigarette smoking. Accordingly, it has been recently proposed that amyloid is not the cause of AD but merely a marker and a later consequence of upstream changes that lead to neuronal and synaptic losses. However, although the idea that features of vascular dysfunction and injury are present in cognitive decline and AD patients was suggested over 25 years ago, the role of cardio-metabolic and cerebrovascular mechanisms in the pathogenesis of AD is far from being fully elucidated. Based on newly proposed vascular hypothesis, there is an impaired structure and function of cerebral blood vessels and cells in patients with cognitive decline and AD which is mediated by vascular oxidative stress. Consistent with these observations, the importance of endothelial dysfunction in the development of AD has been highlighted. One of the most prominent features of endothelial dysfunction is decreased production and bioavailability of Nitric Oxide (NO). Based on versatile properties in physiological as well as in pathophysiological conditions NO is regarded as a key molecule for longevity and cardiovascular health. NO is produced by three different isoforms of the enzyme NO synthase (NOS), namely: endothelial (eNOS), neuronal (nNOS), and inducible (iNOS). Reduced NO bioavailability and altered vascular expression and activity of NOS enzymes are implicated as major molecular players in the process of vascular aging. In the process of aging, the mechanisms by which NOS enzymes promote vascular dysfunction are specific for each NOS isoform. Normal activity of eNOS is required for the balanced production/bioavailability of NO, which is the main prerequisite for optimal vascular function. Conversely, excessive amount of NO produced by iNOS contributes to vascular dysfunction. Furthermore, although the possible role of nNOS-derived NO in aging-associated vascular dysfunction is far from being fully elucidated, experimental findings indicate that impaired perivascular NO release from nNOS increases vasoconstriction in aged arterioles. Oxidative stress and inhibitors of NO synthase are regarded as most potent initiators of disturbed production and bioavailability of NO. Important endogenous inhibitor of all three isoforms of NOS is asymmetrical dimethylarginine (ADMA). It has been recently proposed that ADMA may be a possible link between vascular disease and dementia. Based on this assumption ADMA might represent a unifying pathophysiological pathway linking the presence of vascular risk factors with the onset and progression of cognitive decline and dementia. These observations were made based on good evidence from literature that higher plasma ADMA concentrations favor atherosclerosis and independently predict adverse cardiovascular and cerebrovascular outcomes in several patient groups. However, possible role of ADMA in pathophysiology of cognitive decline and AD is still not fully understood. Prospective studies are warranted aiming at further clarification of ADMA involvement in development and progression of dementia. Moreover, use of ADMA in CSF and /or plasma as prospective biomarker of AD should be explored. According to newly introduced hypothesis disturbed NOS-NO-ADMA pathway is one of the most important attributes of cerebrovascular dysfunction that plays a pathogenic role in the development of cognitive decline and dementia. Experimental data from cell cultures and animal models have demonstrated that dysfunction of the NO-NOS- ADMA pathway results in cell death, blood-brain barrier (BBB) disruption, and brain edema via different pathological mechanisms. However, exact underlining mechanisms through which disturbed NOS-NO-ADMA pathway contributes to cognitive decline and dementia remain to be elucidated. Clarification of these mechanisms may be helpful in the identification of new therapeutic targets for aging and neurodegenerative diseases. Future research should also explain whether endothelial dysfunction precede or follow neurological changes which characterize cognitive decline and dementia. Moreover, clinical and epidemiological studies have shown that healthy nutrition and physical exercise are important non-pharmacological, lifestyle-related interventions that could help in maintaining appropriate vascular tone, adequate cerebral blood flow and normal cognition during aging. It is reasonable to suggest that prevention of cardio-metabolic diseases via endorsement of healthy aging determinants such as control of blood pressure, lipids and glucose levels as well as maintenance of optimal body weight achieved by regular physical activity and nutrition with high anti-oxidant capacity might be effective strategy in the preservation of cognitive function.

Endothelial dysfunction: the link between homocysteine and hydrogen sulfide.

High level of homocysteine (hyperhomocysteinemia, HHcy) is associated with increased risk for vascular disease. Evidence for this emerges from epidemiological studies which show that HHcy is associated with premature peripheral, coronary artery and cerebrovascular disease independent of other risk factors. Possible mechanisms by which homocysteine causes vascular injury include endothelial injury, DNA dysfunction, proliferation of smooth muscle cells, increased oxidative stress, reduced activity of glutathione peroxidase and promoting inflammation. HHcy has been shown to cause direct damage to endothelial cells both in vitro and in vivo. Clinically, this manifests as impaired flow-mediated vasodilation and is mainly due to a reduction in nitric oxide synthesis and bioavailability. The effect of impaired nitric oxide release can in turn trigger and potentiate atherothrombogenesis and oxidative stress. Endothelial damage is a crucial aspect of atherosclerosis and precedes overt manifestation of disease. In addition, endothelial dysfunction is also associated with hypertension, diabetes, ischemia reperfusion injury and neurodegenerative diseases. Homocysteine is a precursor of hydrogen sulfide (H2S) which is formed by transulfuration process catalyzed by the enzymes, cystathionine β-synthase and cystathionine γ-lyase. H2S is a gasotransmitter that has emerged recently as a novel mediator in cardiovascular homeostasis. As a potent vasodilator, it plays several roles which include regulation of vessel diameter, protection of endothelium from redox stress, ischemia reperfusion injury and chronic inflammation. However, the precise mechanism by which it mediates these beneficial effects is complex and still remains unclear. Current evidence indicates H2S modulates cellular functions by a variety of intracellular signaling processes. In this review, we summarize the mechanisms of HHcy-induced endothelial dysfunction and the metabolism and physiological functions of H2S as a protective agent.

Nitric Oxide and Atherosclerotic Lesion Progression: An Overview

There is a growing interest regarding the complex pathophysiological relationship between nitric oxide (NO) and the development of atherosclerosis. The endothelial damage induced by atherogenesis may lead to the reduction in concentration or activity both of inducible and endothelial NO synthase with subsequent impaired release of NO. Moreover, impaired NO diffusion from endothelium to vascular smooth muscle cells is followed by decreased sensitivity to its vasodilator action. Finally, an important mechanism would be a local enhanced degradation of NO by increased generation of reactive oxygen species and other free radicals with subsequent cascade of oxidation-sensitive mechanisms in the arterial wall. Therefore, one target for new drugs should be the restoration of NO-mediated signaling pathways in atherosclerotic arteries. Such novel therapeutic strategies may include administration of L-arginine, the precursor of NO, as well as antioxidants, NO donors, and tissue-specific gene-therapy approaches.

Role of Oxidative Stress in the Pathophysiological Mechanism of Erectile Dysfunction

Center for Advanced Research in HumanReproduction, Infertility and Sexual Function, GlickmanUrological Institute, Cleveland Clinic Foundation, Glickman Urological Institute, Marymount Hospital,and the `Department of Internal Medicine and Pediatrics,Case Western Reserve University, MetroHealth MedicalCenter, Cleveland, Ohio.Erectile dysfunction (ED) is defined as the inability toachieve or maintain erections sufficient for satisfactorysexual intercourse (NIH Consensus Conference, 1993).It is estimated that the prevalence of ED will double inthe next 25 years. Normal erectile function depends ona precise balance between psychological, hormonal,neurological, vascular, and cavernosal factors. There-fore, an alteration in any one or combination of thesefactors may lead to ED. For many years, the complexinteraction between the origin of impulse and thenormal erectile response was not clearly understood. Itwas not until the early 1990s when the roles of centraland peripheral phenomena in the normal erectileresponse was proposed. The identification of the rolesof various mediators, as well as their interactions, innormal erectile function is a major development in thestudy of ED.Production of nitric oxide (NO) plays a centralphysiological role in erections. The endothelium is theprimary source of NO (Burnett, 1997). The pathophys-iological mechanism of endothelial dysfunction ismultifactorial, and the major outcome is impairedrelease of NO, which leads to ED. Although NO-mediated relaxation plays a central role in erections,other mediators, such as prostaglandins, endothelin, andbradykinins, are also important in maintaining peniletone. Researchers have made great strides in the pasttwo decades in identifying the pathophysiologicalmechanism in ED. However, the precise pathophysio-logical mechanism is still unclear.There is a growing interest among researchers re-garding the role of oxidative stress in the pathophysi-ological mechanism of ED. Oxidative stress occurs whenthere is an imbalance between pro-oxidants and theability of the antioxidants to scavenge excess reactiveoxygen species. The role of oxidative stress and reactiveoxygen species has been extensively evaluated in thepathophysiological mechanisms of male and femaleinfertility. However, its role in ED has not beeninvestigated comprehensively. Initial reports from bothin vitro and in vivo studies have shown a significantassociation between the production of reactive oxygenspecies and erectile dysfunction, especially in diabeticanimal models.In this article, we discuss the role of oxidative stress inthe pathophysiological mechanism of ED and thetherapeutic interventions and preventive strategies thatmay be beneficial in restoring endothelial function andnormal erectile function.

Vasculoprotective properties of the endothelial glycocalyx: effects of fluid shear stress

The endothelial glycocalyx exerts a wide array of vasculoprotective effects via inhibition of coagulation and leucocyte adhesion, by contributing to the vascular permeability barrier and by mediating shear stress-induced NO release. In this review, we will focus on the relationship between fluid shear stress and the endothelial glycocalyx. We will address the hypothesis that modulation of glycocalyx synthesis by fluid shear stress may contribute to thinner glycocalyces, and therefore more vulnerable endothelium, at lesion-prone sites of arterial bifurcations. Finally, we will discuss the effects of known atherogenic stimuli such as hyperglycaemia on whole body glycocalyx volume in humans and its effect on endothelial function.

L-Arginine administration recovers sarcoplasmic reticulum function in ischemic reperfused hearts by preventing calpain activation

Earlier studies have shown that impaired cardiac contractility in ischemia reperfusion (IR) is associated with alterations in sarcoplasmic reticulum (SR) function. Impaired release of nitric oxide (NO) has been reported during IR, while administration of NO donors, such as L-arginine (LA), has been shown to improve cardiac performance in IR hearts. We therefore investigated the mechanisms underlying the recovery of contractile function in IR hearts treated with LA. Isolated rat hearts subjected to 30 min of global ischemia were reperfused for 60 min. The effects of LA on cardiac performance, SR function and its regulation were examined. IR-induced impairment in cardiac performance was associated with a reduction in SR function and its regulation. IR caused an increase in calpain activity and a decrease in the sarcolemmal and SR nitric oxide synthase (NOS) isoform protein content as well as cytosolic NO levels. Administration of LA prevented contractile dysfunction in IR hearts, which was associated with a recovery of SR function and SR regulation by protein phosphorylation. This was consistent with a recovery in protein levels of major SR Ca2+-cycling and Ca2+-regulatory proteins. LA treatment attenuated an increase in calpain activity, possibly by nitrosylation of calpain, and increased cytosolic NO levels and SR NOS protein content in IR hearts. These results suggest that LA administration improved cardiac contractility by preventing alterations in SR Ca2+ handling and calpain activation in IR hearts.

Novel features of nitric oxide, endothelial nitric oxide synthase, and atherosclerosis

There is a complex pathophysiologic scenario involving nitric oxide (NO), endothelial nitric oxide synthase (eNOS), and the development of atherosclerosis and unstable atheroma. Endothelial damage induced by atherosclerosis leads to the reduction in bioactivity of eNOS with subsequent impaired release of NO. An important mechanism is local enhanced degradation of NO by increased generation of reactive oxygen species and other free radicals, with subsequent cascade of oxidation-sensitive mechanisms in the arterial wall. Novel molecular approaches have resulted in the development of new strains of mice lacking eNOS. These experimental models will help to understand how to implement NO-based therapies against atherosclerosis. L-arginine, the precursor of NO, has demonstrated beneficial effects in atherosclerosis and disturbed shear stress. The target or goal for new drugs should be the complete restoration of NO-mediated signaling pathways in atherosclerotic arteries.

Novel features of nitric oxide, endothelial nitric oxide synthase, and atherosclerosis.

There is a complex pathophysiologic scenario involving nitric oxide (NO), endothelial nitric oxide synthase (eNOS), and the development of atherosclerosis and unstable atheroma. Endothelial damage induced by atherosclerosis leads to the reduction in bioactivity of ENOS with subsequent impaired release of NO. An important mechanism is local enhanced degradation of NO by increased generation of reactive oxygen species and other free radicals, with subsequent cascade of oxidation-sensitive mechanisms in the arterial wall. Novel molecular approaches have resulted in the development of new strains of mice lacking eNOS. These experimental models will help to understand how to implement NO-based therapies against atherosclerosis. L-arginine, the precursor of NO, has demonstrated beneficial effects in atherosclerosis and disturbed shear stress. The target or goal for new drugs should be the complete restoration of NO-mediated signaling pathways in atherosclerotic arteries.

Nitric oxide pathways in Alzheimer's disease and other neurodegenerative dementias

Nitric oxide (NO) is an enzymatic product of nitric oxide synthase (NOS). NO has significant physiological functions and an increasing body of evidence suggests that NO pathways are implicated in a number of neurological disorders, including Alzheimer's disease (AD) and other neurodegenerative dementias. NO is continuously released by endothelial cells in the vascular system, whereas advanced age in the presence of vascular risk factor causes a decrease in cerebral blood flow, involving microvasculopathy with impaired NO release, which in turn results in regional metabolic dysfunction. This finding suggests that vascular pathology plays a crucial role in the pathogenesis of so-called neurodegenerative dementias. Inflammatory responses are commonly found in the brain under a variety of neurodegenerative dementias, including AD and dementia with Lewy bodies, in which up-regulation of NOS expression, suggesting overproduction of NO, is found in neurons and glia. NO is thought to be involved in such neuroinflammation due to its free radical properties, which compromise cellular integrity and viability via mitochondrial damage. Further studies to elucidate NO pathways in neurodegenerative dementias could lead to a better understanding of their pathogenesis and improved therapeutic strategies, and therefore are certainly warranted.

Therapeutic strategies for optimizing PDE-5 inhibitor therapy in patients with erectile dysfunction considered difficult or challenging to treat.

Phosphodiesterase 5 (PDE5) inhibitors prevent the normal hydrolysis of cGMP. As the resulting cGMP accumulation facilitates penile smooth muscle relaxation, PDE5 inhibitors can partially reverse deficiencies in the nitric oxide (NO)/cGMP pathway to treat erectile dysfunction (ED). However, approximately 30-40% of men with ED do not respond to drug therapy. Patients with severe neurologic damage, diabetes mellitus, or severe vascular disease may be resistant to PDE5 inhibitors. Decreased expression or activity of neuronal or endothelial NO synthase (NOS), impaired NO release, or NO destruction will preclude sufficient cGMP formation to permit PDE5 inhibitor efficacy. This article discusses the possible reasons for unresponsiveness and strategies to overcome it. Therapeutic approaches proposed to increase available NO in penile tissue include facilitating NO release by using alpha-2 antagonists, enhancing NO synthesis by providing more substrate for the reaction, and using antioxidants to inhibit NO breakdown by reactive oxygen species.

Association of insulin resistance, hyperleptinemia, and impaired nitric oxide release with in-stent restenosis in patients undergoing coronary stenting

Association of insulin resistance, hyperleptinemia, and impaired nitric oxide release with in-stent restenosis in patients undergoing coronary stenting

G972R IRS-1 variant impairs insulin regulation of endothelial nitric oxide synthase in cultured human endothelial cells.

Impaired insulin-mediated vasodilation might contribute to vascular damage in insulin-resistant states. Little is known about insulin regulation of nitric oxide (NO) synthesis in insulin-resistant cells. The aim of this work was to investigate insulin regulation of NO synthesis in human umbilical vein endothelial cells (HUVECs) carrying the IRS-1 gene G972R variant, known to be associated with impaired insulin activation of the PI3-kinase (PI3-K) pathway in transfected cells. HUVECs were screened for the presence of the G972R-IRS-1 (HUVEC-G972R) variant by restriction fragment length polymorphisms. After 24-hour exposure to 10(-7) mol/L insulin, endothelial NO synthase (eNOS) mRNA (reverse transcription-polymerase chain reaction), eNOS protein levels (Western blotting), and NOS activity (conversion of [(3)H]arginine into [(3)H]citrulline) were increased in wild-type HUVECs (HUVEC-WT), whereas they did not change from baseline in HUVEC-G972R. Compared with HUVEC-WT, in HUVEC-G972R after 2 and 10 minutes of insulin stimulation, IRS-1-associated PI3-K activity was reduced by 47% and 32%, respectively; Akt phosphorylation was decreased by 40% at both time points; and eNOS-Ser1177 phosphorylation was reduced by 38% and 51%, respectively. In HUVEC-WT, eNOS-Thr495 phosphorylation decreased after insulin stimulation. In contrast, in HUVEC-G972R, eNOS-Thr495 phosphorylation increased after insulin stimulation and was 40% greater than in HUVEC-WT. Our data demonstrate that genetic impairment of the (IRS)-1/PI3-K/PDK-1/Akt insulin signaling cascade determines impaired insulin-stimulated NO release and suggest that the G972R-IRS-1 polymorphism, through a direct impairment of Akt/eNOS activation in endothelial cells, may contribute to the genetic predisposition to develop endothelial dysfunction and cardiovascular disease.

Commentary on Selected Contributions

HIGHLIGHTED TOPICSPhysiology of AgingCommentary on Selected ContributionsGary C. Sieck, Gary C. SieckJournal of Applied Physiology, EditorPublished Online:01 Nov 2003https://doi.org/10.1152/japplphysiol.00885.2003MoreSectionsPDF (17 KB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInEmailWeChat In the first Highlighted Topics article featured in this issue of the Journal of Applied Physiology, “Aging impairs nitric oxide and protacyclin mediation of endothelium-dependent dilation in soleus feed arteries,” Woodman et al. assessed the hypothesis that aging impairs endothelium-dependent vasodilation of feed arteries in rat soleus muscle. Such feed arteries play an integral role in the control of blood flow to this muscle during exercise. With age, the ability to increase soleus muscle blood flow during exercise declines. The primary finding of this study was that endothelium-dependent vasodilation, stimulated by increases in intraluminal flow and acetylcholine, was impaired in aged rats. In addition, these results indicated that the age-related decrement in endothelium-dependent vasodilation was due to impaired release of nitric oxide and prostacyclin by the endothelium in senescent arteries. An improved understanding of the cellular mechanisms that account for age-induced endothelial dysfunction may offer therapeutic approaches to attenuate or reverse the detrimental effects of age on skeletal muscle blood flow.In the second article featured in this issue, “Identification of differentially expressed genes between young and old rat soleus muscle during recovery from immobilization-induced atrophy,” Pattison et al. immobilized hindlimbs of young and old rats to cause atrophy and then assessed age-related variations in gene expression. The investigators found that, whereas skeletal muscle from older rats exhibited little to no regrowth after 10 days of hindlimb immobilization, muscles of young rats returned to the precontrol size by 30 days postimmobilization. To assess candidate genes potentially responsible for the defective regrowth following atrophy in the aged rats, the investigators employed oligonucleotide microarrays, assaying 24,000 transcripts. They found that, during recovery from immobilization, young and old rats expressed 64 mRNAs differently. Real-time PCR confirmed 3 of these 64 candidates: Elfin, amphilregulin, and clusterin. Determination of the many genes playing roles in sarcopenia is essential to the development of future therapies that will offer hope to those confined to nursing homes because of physical frailty.This article has no references to display. Download PDF Previous Back to Top Next FiguresReferencesRelatedInformation More from this issue > Volume 95Issue 5November 2003Pages 2163-2163 Copyright & PermissionsCopyright © 2003 the American Physiological Societyhttps://doi.org/10.1152/japplphysiol.00885.2003History Published online 1 November 2003 Published in print 1 November 2003 Metrics

Effect of a beta 2-adrenoceptor stimulation on hyperglycemia-induced endothelial dysfunction.

To investigate whether beta(2)-adrenoceptors exist on endothelial cells and whether a beta(2)-adrenoceptor stimulation might prevent the development of hyperglycemia-induced endothelial dysfunction, porcine aortic endothelial cells (PAECs) were cultured and chronically exposed to either 5 mM D-glucose ("normoglycemia") or 20 mM D-glucose ("hyperglycemia"), with or without 100 nM salbutamol in absence or presence of beta(2)-adrenoceptor antagonist ICI 118,551 [1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxyl]-3-[(1-methylethyl)-amino]-2-butanol] or beta(1)-antagonist metoprolol. For osmotic control, PAECs were exposed to 15 mM L-glucose. We measured nitric oxide release using the met-hemoglobin assay and assessed beta-adrenoceptor density and subtypes by radioligand binding. Furthermore, we determined intracellular NADH and NADPH using high-performance liquid chromatography. High D-glucose concentrations but not L-glucose led to significantly reduced basal and stimulated nitric oxide release. Chronic salbutamol treatment significantly antagonized the impairment of the nitric oxide response, which was inhibited by ICI 118,551 but not by metoprolol. The number of giant cells was significantly increased in hyperglycemia, which could be prevented by salbutamol. Binding of the radioligand (-)-[(125)I]iodocyanopindolol revealed a total beta-adrenoceptor density of 29.8 +/- 3.7 (normoglycemic) and 30.3 +/- 3.6 (hyperglycemic) fmol/mg protein. Displacement by ICI 118,551 revealed beta-adrenoceptor subtype distribution with 30.3 +/- 4.4 (normoglycemic) and 29.1 +/- 3.8% beta(2)-adrenoceptors. NADH production increased in hyperglycemia, which was completely prevented by salbutamol. We conclude that hyperglycemia in PAEC induces endothelial dysfunction with impaired nitric oxide release and that this can be prevented by beta(2)-adrenoceptor stimulation.

Role of endothelial dysfunction in insulin resistance

The endothelium regulates vascular tone through the release of vasodilating and vasoconstricting substances. The most important of these vasodilating substances is nitric oxide (NO), which is also vascular protective and inhibits inflammation, oxidation, vascular smooth muscle cell proliferation, and migration. Damage to the endothelium causes endothelial dysfunction with impaired release of NO and loss of its antiatherogenic protection. Traditional risk factors for coronary artery disease, including diabetes, hypercholesterolemia, hypertension, and low levels of high-density lipoprotein cholesterol, are associated with endothelial dysfunction and thus promote the atherogenic process. More recently, insulin resistance in the absence of overt diabetes or the metabolic syndrome has been associated with endothelial dysfunction. This association provides evidence that the atherosclerotic process may actually begin earlier in the spectrum of insulin resistance, ultimately resulting in a progression of the metabolic syndrome to prediabetes and then to type 2 diabetes. Aggressive treatment of dyslipidemia and hypertension, even before the onset of type 2 diabetes, would appear prudent in decreasing the progression of the atherosclerotic process. The thiazolidinediones are peroxisome proliferator-activated receptor–γ agonists that improve glucose and lipid metabolism. These agents have recently been shown to improve endothelial function in the early stages of insulin resistance. Results from ongoing trials with thiazolidinediones will reveal whether they will also reduce cardiovascular end points.