Natural Vasodilators: Mechanisms and Therapeutic Potential in Cardiovascular Diseases

Nonvascular smooth muscle, such as the iris sphincter, receives double reciprocal innervation: stimulation of the parasympathetic nervous system (cholinergic muscarinic), which functions through the polyphosphoinositide (PPI) signaling pathway, contracts it, while activation of the sympathetic nervous system (beta-adrenergic), which functions through the cAMP system, relaxes it. Interactions between the two second messenger systems are important in regulation of smooth muscle tone and represent an important focal point for pharmacological manipulation. Here, I have summarized the experimental evidence in support of the hypothesis that the cross talk between cAMP and the PPI cascade could constitute a biochemical correlate for this functional antagonism. Recent studies suggest that cAMP inhibition is on Ca2+ mobilization rather than myosin light chain phosphorylation. Thus, cAMP-elevating agents, which inhibit agonist-induced PPI hydrolysis, are effective relaxants. Furthermore, inositol 1,4,5-trisphosphate (IP3) appears to be involved in both Ca2+ release from the sarcoplasmic reticulum and in Ca2+ influx through the plasma membrane, and since a reduction in intracellular Ca2+ ([Ca2+]i) is the underlying mechanism for cAMP-mediated relaxation, an important target for cAMP inhibition would be either to inhibit IP3 production or to stimulate IP3 inactivation. In the iris sphincter and other nonvascular smooth muscle there is reasonable experimental evidence that shows that cAMP inhibits phospholipase C activation and stimulates IP3 3-kinase activity, both of which can result in: [i) reduction in IP3 concentrations and (ii) reduction in IP3-dependent Ca2+ mobilization, which may lead to muscle relaxation. In addition to IP3-induced Ca2+ mobilization, changes in [Ca2+]i are the result of the interplay of many processes which may also serve as potential sites for cAMP inhibition. A great deal of progress has been made on the cross talk between cAMP and the PPI signaling cascade in the past decade, and there will be more on the regulation of the second messenger systems and their involvement in smooth muscle tone in the coming years. Clearly, an understanding of the physiological and pathophysiological regulation of smooth muscle tone is central to the development of novel therapeutic agents for the treatment of diseases such as asthma and glaucoma, where cAMP-elevating drugs are currently employed.

The Journal of PhysiologyVolume 523, Issue suppl p. 47S-53S Symposium ProceedingFree Access Regulation of Smooth Muscle Tone First published: 01 February 2000 https://doi.org/10.1111/j.1469-7793.2000.tb00236.xAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Volume523, IssuesupplFebruary 2000Pages 47S-53S RelatedInformation

Capacitative Ca2+ entry and the regulation of smooth muscle tone.

Interaction between spontaneous and neurally mediated regulation of tone in the corpus cavernosum smooth muscle (CCSM) of the rabbit was investigated. Changes in isometric muscle tension, intracellular Ca2+ concentration ([Ca2+]i) and membrane potential were recorded. CCSM developed spontaneous contractions, transient increases in [Ca2+]i (Ca2+ transients) and depolarizations. This spontaneous activity was abolished by blocking L-type Ca2+ channels (nicardipine, 1 mum), sarcoplasmic reticulum Ca2+ pump activity (cyclopiazonic acid, 10 microm), Ca2(+)-activated Cl- channels (niflumic acid, 10 mum) or cyclooxygenase-2 (COX-2; NS-398, 1 microm). Transmural nerve stimulation initiated either alpha-adrenergic contractions or nitrergic relaxations of CCSM depending on the level of muscle tone. NS-398 suppressed nerve-evoked contractions by about 70% but caused only a 40% reduction in the corresponding Ca2+ transient. Blocking nitric oxide synthase with N(omega)-nitro-l-arginine (LNA, 100 microm) reinforced nerve-evoked Ca2+ transients by about 150%, whilst increasing the corresponding Ca2+ transients by only 20%. In CCSM preparations that had been pre-contracted with either noradrenaline (0.3 microm) or prostaglandin F(2alpha) (0.1 microm), nerve stimulation inhibited about 70% of the contraction and caused only a 20% decrease in [Ca2+]i. Fluorescent immunohistochemistry with COX-2 antibodies and the reverse transcriptase-polymerase chain reaction (RT-PCR) method showed that the enzyme and its mRNA were highly expressed in the CCSM. These results suggest that spontaneously produced prostaglandins (PGs) not only contribute to the generation of spontaneous contractions but also facilitate nerve-evoked contractions. Conversely, spontaneously released nitric oxide (NO) suppresses excitation. Thus, interaction between spontaneous and neurally mediated regulation of CCSM tone may be fundamental to maintaining the muscle contractility. In addition, both PGs and NO appear to alter CCSM tone with only small changes in [Ca2+]i.

MP19-09 NSC23766, A RAC GTPASE INHIBITOR, INHIBITS SMOOTH MUSCLE CONTRACTION IN THE HYPERPLASTIC HUMAN PROSTATE

Circulating factors access cell bodies of vagal afferents in nodose ganglia (NG) via the occipital artery (OA). Constrictor responses of OA segments closer in origin from the external carotid artery (ECA) differ from segments closer to NG. Our objective was to determine the role of endothelium in this differential vasoreactivity in rat OA segments. Vasoreactivity of OA segments (proximal segments closer to ECA, distal segments closer to NG) was examined in wire myographs. We evaluated 1) vasoconstrictor effects of 5-hydroxytryptamine (5-HT) in intact and endothelium-denuded OA segments in absence/presence of soluble guanylate cyclase (SGC) inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), 2) vasodilator responses elicited by the endothelium dependent vasodilator, acetylcholine (ACh), in intact or endothelium-denuded OA segments in absence/presence of ODQ, and 3) vasodilator responses elicited by NO-donor MAHMA NONOate, in intact OA segments in absence/presence of ODQ. Intact distal OA responded more to 5-HT than intact proximal OA. Endothelium denudation increased 5-HT potency in both OA segments, especially proximal OA. ODQ increased maximal responses of 5-HT in both segments, particularly proximal OA. ACh similarly relaxed both OA segments, effects abolished by endothelial denudation and attenuated by ODQ. MAHMA NONOate elicited transient vasodilation in both segments. Effects of ODQ against ACh were segment dependent whereas those against MAHMA NONOate were not. The endothelium regulates OA responsiveness in a segment-dependent fashion. Endothelial cells at the OA-ECA junction more strongly influence vascular tone than those closer to NG. Differential endothelial regulation of OA tone may play a role in controlling blood flow and access of circulating factors to NG.NEW & NOTEWORTHY This study demonstrates that the endothelium-dependent regulation of smooth muscle tone of occipital arteries is segment-dependent. Endothelial cells at the occipital artery-external carotid artery junction (entryway of blood flow to the nodose ganglia) more strongly influence vascular tone than those closer to the nodose ganglia. This differential endothelial regulation of occipital artery tone may control blood flow and access of circulating factors to the nodose ganglia.

Physical inactivity and sedentary lifestyle are risk factors for obesity which may lower quality of life and increase risk for cardiovascular disease (CVD) morbidity and mortality. Obesity detrimentally alters vascular structure and function. Individuals who are obese and sedentary tend to exhibit lower capillary density. This is important because after ingesting food or exercising, obese individuals may show altered rates of skeletal muscle vasodilatation. Thus, vascular dysfunction inhibits the ability of skeletal muscles to meet metabolic demands. Metabolic supply-demand imbalances can lead to microvascular abnormalities and insulin resistance. An impaired vasodilatory response is reported in obesity and is marked by decreased nitric oxide (NO) bioavailability; there is an altered balance between NO producing enzymes (eNOS) and NO quenching enzymes ((NAD(P)Hoxidase). Imbalance between NO formation and NO quenching leads to the increase of superoxide anions that act as free radicals in the body to induce oxidative stress. Oxidative stress propagates further reduction in NO availability. While exercise has many health benefits such as improving cardiorespiratory fitness (CRF) and reducing metabolic risk factors, many individuals do not engage in a regular exercise regimen, citing ‘lack of time’ as the underlying culprit. High intensity training (HIT) is a time-effective and aerobically beneficial mode of exercise, alternative to moderate-intensity continuous training (MICT), for cardiometabolic disease risk management. However, low-volume protocols at a high-intensity have been reported as strenuous, presenting complex barriers to obese individuals. Without supervision, adherence can be low. Laboratory-based HIT protocols may not accurately mimic the applicability and feasibility of home-based HIT protocols. In an article recently published in The Journal of Physiology, Scott and colleagues evaluated the effect of a virtually supervised at-home HIT programme on muscle capillarization and muscle microvascular eNOS/NAD(P)Hoxidase ratio in obese individuals with elevated CVD risk (Scott et al. 2019). Flow-mediated dilatation (FMD) was used to assess brachial artery NO-mediated endothelial function. FMD significantly increased after HIT training, with no difference between HIT groups. Pulse wave velocity (PWV) significantly decreased after the 12-week mark in all three HIT groups, suggesting a reduction in aortic stiffness. Blood pressure (BP) remained unchanged. These data suggest that HIT training may enhance endothelial function and reduce aortic stiffness in obese adults. Obesity and lack of physical activity affect the balance between eNOS and eNOS serine1177. Immunofluorescence microscopy using cryosectioned muscle biopsy samples was used to analyse NO-producing enzymes (eNOS content and ser1177 phosphorylation) and NO quenching enzymes (NOX2 and p47phox) that critically suppresses the effect of NO. When normalized to eNOS content (eNOS ser1177/eNOs ratio), Ser1177 content levels decreased in the arterioles and capillaries with training. Terminal arteriole NOX2 content and skeletal capillary NOX2 content was significantly lower in all groups following HIT training. Increased eNOS content and reduced NOX2 content suggest an improved balance between NO synthesis and NO quenching by superoxide anion, resulting in enhanced microvascular NO bioavailability. From the image analysis using immunofluorescence microscopy, capillarization measures were quantified specific to fibres, containing at least 50 completed fibres per assessment. Capillary-to-fibre ratio, capillary-fibre perimeter exchange index and capillary contacts (number of capillaries around a fibre) were all increased by training and were all higher in type I fibres than type II fibres, with no group differences. Augmented capillarization measures may allow for an increased O2 delivery and extraction, probably contributing to the higher seen with HIT training reported by Scott et al. (2019) and throughout the literature in obese adults. GLUT4 and intramuscular triglyceride content (IMTG) were chosen as classical markers for myocyte adaptations to training. Total muscle fibre GLUT4 content was augmented following training, with a similar increase in GLUT4 content in type II fibres across the HIT groups. HIT training increased total IMGT content in all three groups, governed by an increase in lipid droplet density. Although insulin resistance is coupled with high IMGT, these results suggest that there is a greater capacity to oxidize IMGT. There is a greater efficiency of the muscle to consume fatty acids from the IMGT pool for oxidation. Increased GLUT4 coupled with increased IMGT oxidation may lead to an overall improvement in insulin sensitivity. Scott et al. (2019) eloquently demonstrated that a home-based HIT programme can effectively improve vascular function, endothelial eNOS regulation, capillarization and myocyte adaptations in obese individuals. Another noteworthy finding, less touched on by the authors, is the improvement of cardiovascular risk markers after home-based HIT intervention. Elevated BMI is associated with increased aortic stiffness (Croymans et al. 2014), a strong predictor of heart failure, kidney disease, stroke, myocardial infarction and cognitive decline; all important co-morbidities of obesity. Scott & colleagues (2019) determined that PWV was reduced after 12 weeks of HIT in obese individuals. Central aortic stiffness is mediated by many factors including arterial wall distending pressure and smooth muscle tone. Thus, reductions in aortic stiffness with exercise training may be related to improvements in endothelial function, as NO contributes to arterial compliance (Sugawara et al. 2009). Improvements in PWV may also be related to reductions in sympathetic nervous system (SNS) activity, a mechanism not explored by Scott et al. SNS activity is an important regulator of smooth muscle tone and has been shown to be elevated in the obese population (Figueroa et al. 2012). An increase in SNS activity results in vasoconstriction and increased vascular resistance, which can lead to increased aortic stiffness. Therefore, it is plausible to conclude that 12 weeks of progressive HIT training may effectively reduce SNS activity and relax smooth muscle tone, resulting in reduced PWV in obese individuals (Sugawara et al. 2009). Understanding potential mechanisms of improved vascular function with exercise is important for designing targeted interventions to improve cardiometabolic health in obesity. Scott et al. (2019) demonstrated that adherence to an at-home HIT exercise programme is achievable for obese adults. This is noteworthy, as many may think that the difficulty of exercises used in a home HIT programme such as that described by Scott et al. (2019) may deter participation. Indeed, excess body fat may limit range of motion and prevent participants from performing exercises appropriately. Moreover, jumping tasks and high impact plyometric-type movements are often thought to increase stress on joints, which could lead to orthopedic complications and musculoskeletal injury in overweight and obese adults. However, this was not case. HIT training was demonstrated to be a safe and effective exercise modality for obese adults. In summary, Scott et al. (2019) demonstrated that a home-based HIT programme aids in removing barriers to exercise and improved cardiovascular health markers in obese adults. Obese individuals can take comfort in ‘NO-ing’ that improved metabolic and vascular health is only a few weeks of clapping jumping jacks away! None declared. All authors have approved the final version of the manuscript and agree to be accountable for all aspects of the work. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed. None declared. We'd like to acknowledge Dr Kevin Heffernan for his helpful review of this manuscript.

Nitric oxide (NO) and atrial natriuretic peptide (ANP) signalling via cGMP controls smooth muscle tone. One important signalling pathway of cGMP-dependent protein kinase type I (cGKI) is mediated by IRAG (IP(3) receptor associated cGKI substrate) which is highly expressed in smooth muscle tissues. To elucidate the role of IRAG for NO- and ANP-mediated smooth muscle tone regulation, cGKI localization, and for its possible function in blood pressure adjustment, we generated IRAG-knockout mice by targeted deletion of exon 3. IRAG deletion prevented stable interaction of IP(3) receptor type I (IP(3)RI) with cGKIbeta determined by cGMP affinity chromatography. Confocal microscopy in vascular smooth muscle cells (VSMCs) showed that localization of cGKIbeta and cGKIalpha did not change in absence of IRAG. NO-, ANP-, and cGMP-dependent relaxation of hormone-contracted aortic vessels and colon was significantly affected in IRAG-knockout mice. The suppression of cGMP-induced relaxation was not rescued by selective expression of cGKIbeta in smooth muscle from cGKIbeta-transgenic mice. NO-, ANP-, and cGMP-mediated inhibition of the hormone-induced increase in intracellular calcium concentration measured by Fura2 was suppressed in IRAG-deficient VSMC. Telemetric measurements revealed that IRAG-deficient animals exhibited normal basal tone, but were resistant to blood pressure reduction induced by lipopolysaccharide-treatment. These findings indicate that signalling of cGKIbeta via IRAG is an essential functional part for regulation of smooth muscle tone and of intracellular calcium by NO (exogenously applicated or endogenously synthesized) and by ANP. IRAG signalling does not modulate basal tone but might be important for blood pressure regulation under pathophysiological conditions.

Functional role of TRPC proteins in vivo: lessons from TRPC-deficient mouse models

The nitric oxide (NO)-cGMP signal transduction pathway plays an important role in the regulation of pulmonary vascular tone and resistance in pulmonary hypertension. A number of studies have demonstrated that endothelial (e) and inducible nitric oxide synthases (NOS) are upregulated in hypoxia-exposed rat lung. These changes in NOS expression have been found to correlate with the process of pulmonary vascular remodeling in hypoxia-induced pulmonary hypertension, and remodeling is increased in the absence of eNOS. In this study, we examined the expression and localization of soluble guanylate cyclase (sGC), the primary receptor for NO, in hypoxia- and normoxia-treated rat lungs. Male Sprague-Dawley rats were exposed to hypoxia (10% O(2), normobaric) or normoxia for 1, 3, 5, and 21 days. The lungs were used for Western analysis of sGC protein, sGC enzyme activity, immunohistochemistry using antiserum against sGC alpha(1)- and beta(1)-subunits, and nonradioactive in situ hybridization (NRISH) using a digoxigenin-labeled sGC alpha(1)-subunit cRNA probe. Western blot analysis revealed a more than twofold increase of sGC protein alpha(1)-subunit in rat lungs exposed to 3, 5, and 21 days of hypoxia, correlating well with sGC enzyme activity. Immunohistochemistry and NRISH demonstrated increased expression of sGC in the smooth muscle cells of the pulmonary arteries and arterioles in the hypoxic rat lungs when compared with normoxic controls. Based on our results, the upregulation of sGC may play an important role in the regulation of smooth muscle tone and pressure in the pulmonary circulation during chronic hypoxia.

The small GTP-binding protein, RhoA, and its downstream effector protein, rho-kinase, have been implicated in the pathogenesis of a number of cardiovascular diseases. The activation of rho-kinase is involved in the development of increased vascular tone, endothelial dysfunction, inflammation, and restenosis; and that the inhibition of rho-kinase has been shown to have a beneficial effect in a variety of cardiovascular disorders. It is our hypothesis that rho-kinase inhibitors promote vasodilation independent of the mechanism that increases vasoconstrictor tone and moreover, the RhoA/rho-kinase pathway has a role in the regulation of smooth muscle tone under physiological conditions. The objective of this review is to improve our current understanding of the role of RhoA/rho-kinase pathway in the regulation of vasoconstrictor tone and the use of rho-kinase inhibitors in the treatment of cardiovascular disorders with an emphasis on pulmonary hypertension.

Basal tone in the internal anal sphincter (IAS) is primarily myogenic. Neurohumoral substances like angiotensin II may partially provide external signal for the basal tone in the IAS. The sphincteric relaxation on the contrary is neurogenic by activation of non-adrenergic non-cholinergic (NANC) nerves that release nitric oxide (NO), vasoactive intestinal polypeptide (VIP) and perhaps carbon monoxide. Because of the presence of spontaneous tone, the IAS offers an excellent model to investigate the nature of the inhibitory neurotransmission for NANC relaxation. Work from different laboratories in different species concludes that NO is the major contributor in the NANC relaxation. This may invoke the role of other inhibitory neurotransmitters such as VIP, working partly via NO. An understanding of the basic regulation of basal tone in the IAS and nature of inhibitory neurotransmission are critical in the pathophysiology and therapeutic potentials in the anorectal motility disorders.

Chapter 26 - Cyclic GMP-Mediated Signaling Mechanisms in Smooth Muscle

Background Resistive breathing (RB) due to airflow limitation is the pathophysiologic hallmark of chronic obstructive pulmonary disease (COPD). Nitric oxide (NO) is a physiological regulator of smooth muscle tone that acts through activation of soluble guanylyl cyclase (sGC). We hypothesized that increased smooth muscle tone limiting airflow in COPD could result from reduced sGC. Herein, we investigated the expression and downstream signalling of sGC in RB.

The NO/cGMP signal transduction is involved in the regulation of a variety of physiological processes e.g. smooth muscle relaxation and platelet aggregation. As signalling molecule, NO has diverse effects with NO-sensitive guanylyl cyclase (NO-GC) being accepted as the most important NO receptor. To differentiate between cGMP-dependent and -independent effects of NO inhibitors for the NO-GC are broadly used. The commonly used inhibitor for NO-GC is ODQ (1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one). The precise mechanism of NO-GC inhibition by ODQ remains unclear. Recently, we have generated mice deficient in NO-GC (GC-KO). GC-KO mice show a pronounced increase in blood pressure, underlining the importance of NO in the regulation of smooth muscle tone in vivo. We showed a total lack of NO affecting smooth muscle tone and platelet aggregation which confirms NO-GC as the only NO target regulating these two functions in mice. Using these KO mice we can evaluate the specificity of ODQ as inhibitor of NO-GC. In fact, ODQ used at low μM concentrations is a good inhibitor of cGMP signalling, allowing its use to investigate the cGMP dependence of NO effects. However, high NO concentrations elicited relaxation responses in ODQ-treated WT smooth muscle via NO-GC as they were absent in GC-KO tissue. This shows that NO-induced effects in the presence of ODQ do not necessarily indicate cGMP independence.