Class II phosphoinositide 3-kinase α-isoform (PI3K-C2α) is involved in regulating KCl-induced vascular smooth muscle contraction. The current study was to investigate the effects of sevoflurane (SEVO) and isoflurane (ISO) on KCl-elicited PI3KC2α mediated vasoconstriction in rat aortic smooth muscle.

Aldosterone is involved in many cardiovascular diseases with increased oxidative stress. Aldosterone-induced cardiac fibrosis is abolished by blockade of angiotensin II Type-1 (AT1) receptor. Recently, non-genomic vasoconstrictor effects of aldosterone were reported in various vascular beds. We tested the hypothesis that aldosterone stimulates angiotensin AT1 receptor, and causes vasoconstriction by increasing oxidative stress in coronary microcirculation. Coronary arterioles (60-120 microm) were isolated from spontaneously hypertensive rats (SHR) and control Wistar Kyoto (WKY) rats, aged 23-26 weeks. They were cannulated, and pressurized at 60 cm H2O. Effect of aldosterone (10(-15) to 10(-6) M) on coronary arteriolar diameter was examined. Aldosterone rapidly and dose-dependently decreased coronary arteriolar diameter in WKY rats and SHR (diameter changes, 8.4+/-0.7% vs 13.9+/-0.8%, P<0.05). Aldosterone-induced vasoconstriction was enhanced by 1.6-folds in SHR compared to WKY rats (P<0.05). Mineralocorticoid receptor antagonist spironolactone (10(-6) M) did not influence aldosterone-induced vasoconstriction. Selective angiotensin AT1 receptor blocker valsartan (10(-4) M) or candesartan (10(-7) M) abolished aldosterone-induced vasoconstriction. Similarly, superoxide dismutase (SOD, 300 U/ml), and NADPH oxidase inhibitor apocynin (10(-4) M) abolished it. Moreover, the vasoconstrictor effect of aldosterone disappeared in denuded vessels. Real-time quantitative RT-PCR revealed that angiotensin AT1 receptor mRNA level in coronary arterioles of SHR was upregulated by 1.5-folds compared to that in WKY rats (P<0.05). Aldosterone causes vasoconstriction in coronary arterioles, and this vasoconstrictor effect is enhanced by genetically defined hypertension. Aldosterone-induced vasoconstriction is mediated by angiotensin AT1 receptor presumably via oxidative stress.

Oxygen diffusion across the alveolar wall is compromised by low alveolar oxygen but also by pulmonary edema, and leads to hypoxemia and hypoxic pulmonary vasoconstriction (HPV). To test, whether inhibition of alveolar fluid reabsorption results in an increased pulmonary arterial pressure and whether this effect enhances HPV, we established a model, where anesthetized rats were ventilated with normoxic (21% O2) and hypoxic (13.5% O2) gas received aerosolized amiloride and lipopolisaccharide (LPS) to inhibit alveolar fluid reabsorption. Right ventricular systolic pressure (RVsP) was measured as an indicator of pulmonary arterial pressure. Oxygen pressure (PaO2) and saturation (SaO2) in femoral arterial blood served as indicator of oxygen diffusion across the alveolar wall. Aerosolized amiloride and bacterial LPS decreased PaO2 and SaO2 and increased RVsP even when animals were ventilated with normoxic gas. Ventilation with hypoxic gas decreased PaO2 by 35 mmHg and increased RVsP by 10 mmHg. However, combining hypoxia with amiloride and LPS did not aggravate the decrease in PaO2 and SaO2 and had no effect on the increase in RVsP relative to hypoxia alone. There was a direct relation between SaO2 and PaO2 and the RVsP under all experimental conditions. Two hours but not 1 h exposure to aerosolized amiloride and LPS in normoxia as well as hypoxia increased the lung wet-to-dry-weight ratio indicating edema formation. Together these findings indicate that inhibition of alveolar reabsorption causes pulmonary edema, impairs oxygen diffusion across the alveolar wall, and leads to an increased pulmonary arterial pressure.

Propofol is known to cause vasorelaxation of several systemic vascular beds. However, its effect on the pulmonary vasculature remains controversial. In the present study, we investigated the effects of propofol on human pulmonary arteries obtained from patients who had undergone surgery. Arterial rings were mounted in a Multi-Myograph system for measurement of isometric forces. U46619 was used to induce sustained contraction of the intrapulmonary arteries, and propofol was then applied (in increments from 10-300 µM). Arteries denuded of endothelium, preincubated or not with indomethacin, were used to investigate the effects of propofol on isolated arteries. Propofol exhibited a bifunctional effect on isolated human pulmonary arteries contracted by U46619, evoking constriction at low concentrations (10-100 µM) followed by secondary relaxation (at 100-300 µM). The extent of constriction induced by propofol was higher in an endothelium-denuded group than in an endothelium-intact group. Preincubation with indomethacin abolished constriction and potentiated relaxation. The maximal relaxation was greater in the endothelium-intact than the endothelium-denuded group. Propofol also suppressed CaCl2-induced constriction in the 60 mM K+-containing Ca2+-free solution in a dose-dependent manner. Fluorescent imaging of Ca2+ using fluo-4 showed that a 10 min incubation with propofol (10-300 µM) inhibited the Ca2+ influx into human pulmonary arterial smooth muscle cells induced by a 60 mM K+-containing Ca2+-free solution. In conclusion, propofol-induced arterial constriction appears to involve prostaglandin production by cyclooxygenase in pulmonary artery smooth muscle cells and the relaxation depends in part on endothelial function, principally on the inhibition of calcium influx through L-type voltage-operated calcium channels.

Exaggerated vasoconstriction plays a very important role in the hypertension, a major component of metabolic syndrome (MetS). In the current work, the potential protective effect of methanol extract of fruit hulls of Garcinia mangostana L. on the exaggerated vasoconstriction in MetS has been investigated. In addition, the bioactive fraction and compounds as well as the possible mechanism of action have been illustrated.

Contraction-mediated blunting of postjunctional α-adrenergic vasoconstriction (functional sympatholysis) is attenuated in skeletal muscle of ageing males, brought on by altered postjunctional α1 - and α2 -adrenergic receptor sensitivity. The extent to which postjunctional α-adrenergic vasoconstriction occurs in the forearms at rest and during exercise in postmenopausal women remains unknown. The novel findings indicate that contraction-mediated blunting of α1 - (via intra-arterial infusion of phenylephrine) but not α2 -adrenergic (via intra-arterial infusion of dexmedetomidine) vasoconstriction was attenuated in postmenopausal women compared to young women. Additional important findings revealed that postjunctional α-adrenergic vasoconstrictor responsiveness at rest does not appear to be affected by age in women. Collectively, these results contribute to our understanding of local neurovascular control at rest and during exercise with age in women.

Eukaryotic elongation factor 2 (eEF2) kinase (eEF2K) repressively regulates protein translation through phosphorylating eEF2. We previously showed that expression and activity of eEF2K are increased in isolated mesenteric arteries from spontaneously hypertensive rats (SHR) contributing to development of essential hypertension. Furthermore, we have recently shown that 7-Amino-1-cyclopropyl-3-ethyl-1,2,3,4-tetrahydro-2,4-dioxopyrido[2,3-d]pyrimidine-6-carboxamide (A484954), a selective eEF2K inhibitor, induces endothelium-dependent relaxation in isolated mesenteric arteries from SHR inducing an antihypertensive effect. In order to test the hypothesis that inhibition of eEF2K activity induces vasodilatation by suppressing sympathetic nerve activity, we examined the effects of A484954 on perivascular sympathetic nerve stimulation-induced contraction in isolated renal artery from normotensive and hypertensive rats. Electrodes were placed near the isolated renal arteries that were applied with transmural nerve stimulation (TNS). Then, contraction of the arteries was isometrically measured. A484954 inhibited TNS-induced contraction. The A484954-mediated inhibition of TNS-induced contraction was significantly prevented by NG-nitro-L-arginine methyl ester. In SHR isolated renal artery, TNS-induced contraction was enhanced compared with normotensive Wistar rats. Furthermore, A484954-mediated inhibition of TNS-induced contraction in SHR was enhanced compared with Wistar rats. In conclusion, this study demonstrates for the first time that A484954 inhibits perivascular sympathetic nerve stimulation-induced vasoconstriction at least in part perhaps through nitric oxide (NO) release from NO-operating nerve.

ATP, norepinephrine and NPY are co-released by sympathetic nerves innervating arteries. ATP elicits vasoconstriction via activation of smooth muscle P2X receptors. The functional interaction between neuropeptide Y (NPY) and P2X receptors in arteries is not known. In this study we investigate the effect of NPY on P2X1-dependent vasoconstriction in mouse mesenteric arteries. Suramin or P2X1 antagonist NF449 abolished α,β-meATP evoked vasoconstrictions. NPY lacked any direct vasoconstrictor effect but facilitated the vasoconstrictive response to α,β-meATP. Mesenteric arteries expressed Y1 and Y4 receptors, but not Y2 or Y5. Y1 receptor inhibition (BIBO3304) reversed NPY facilitation of the α,β-meATP-evoked vasoconstriction. L-type Ca2+ channel antagonism (nifedipine) had no effect on α,β-meATP-evoked vasoconstrictions, but completely reversed NPY facilitation. Electrical field stimulation evoked sympathetic neurogenic vasoconstriction. Neurogenic responses were dependent upon dual α1-adrenergic (prazosin) and P2X1 (NF449) receptor activation. Y1 receptor antagonism partially reduced neurogenic vasoconstriction. Isolation of the P2X1 component by α1-adrenergic blockade allowed faciliatory effects of Y1 receptor activation to be explored. Y1 receptor antagonism reduced the P2X1 receptor component during neurogenic vasoconstriction. α1-adrenergic and P2X1 receptors are post-junctional receptors during sympathetic neurogenic vasoconstriction in mesenteric arteries. In conclusion, we have identified that NPY lacks a direct vasoconstrictor effect in mesenteric arteries but can facilitate vasoconstriction by enhancing the activity of P2X1, following activation by exogenous agonists or during sympathetic nerve stimulation. The mechanism of P2X1 facilitation by NPY involved activation of the NPY Y1 receptor and the L-type Ca2+ channel.

We investigated the integration of sympathetic vasoconstriction and local vasodilation in the skeletal muscle and skin microvasculature of humans. In 39 healthy volunteers, we simultaneously measured the blood flow index in the flexor carpi radialis muscle using diffuse correlation spectroscopy and the skin using laser-Doppler flowmetry. We examined the effects of acute sympathoexcitation induced by forehead cooling on relatively weak and robust vasodilatory responses during postocclusive reactive hyperemia (PORH) induced by 70-sec and 10-min arterial occlusion in the upper arm. To increase sympathetic tone during PORH, forehead cooling was begun 60 sec before the occlusion release and ended 60 sec after the release. In the 70-sec occlusion trials, acute sympathoexcitation reduced the peak and duration of vasodilation in both skeletal muscle and skin. The inhibition of vasodilation by sympathoexcitation was blunted in both tissues by the robust vasodilatory stimulation produced by the 10-min occlusion, and the degree of blunting was greater in skeletal muscle than in skin, especially the initial and peak responses. Sympathoexcitation reduced the peak vasodilation only in skin, while it accelerated the initial vasodilation only in skeletal muscle. However, the decline in vasodilation after the peak was significantly hastened in skeletal muscle, shortening the duration of the vasodilation. We conclude that, in humans, the integration of sympathetic vasoconstriction and local vasodilation has different effects in skeletal muscle and skin and is likely an important contributor to the selective control of perfusion in the microcirculations of different tissues.

During organ culture of intact vessels, endothelin receptors (ETRs) were upregulated in vascular smooth muscle cells (VSMCs) by various stimuli, but whether inflammation alters ETR expression in vivo remains unclear. We aimed to explore the effects of lipopolysaccharide (LPS) challenge on ETR expression in the VSMC in vivo. Male Sprague-Dawley rats received a single intraperitoneal injection of LPS (5 mg/kg body weight) or normal saline (NS) for 6 hrs. The function and expression of ETR type A (ETA) and type B (ETB) were evaluated in the mesenteric arteries without endothelium, by using myograph system, real-time quantitative PCR, Western blot, and immunohistochemical staining, respectively. Serum tumor necrosis factor-α (TNF-α) level was assessed by using enzyme-linked immunosorbent assay. The results showed that, compared to control (NS) group, LPS treatment potently enhanced the vasoconstriction mediated by ETA or ETB in rat mesenteric artery, with elevated maximum effects. ETA and ETB expressions in the VSMC were increased at both mRNA and protein levels after LPS treatment, paralleled with activation of the NF-κB pathway and augmented serum TNF-α level. Conclusively, in the rat model of immediate systemic inflammation induced by LPS, ETA and ETB expressions were increased in the mesenteric arterial VSMC, paralleled with enhanced receptor-mediated vasoconstriction and activation of the NF-κB pathway. Our data has for the first time demonstrated the upregulation of ETRs in VSMCs by LPS-induced immediate inflammation in vivo.

Patients with type 2 diabetes mellitus (T2DM) have reduced vasodilatory responses during exercise partially attributable to low nitric oxide (NO) levels. Low NO contributes to greater α-adrenergic mediated vasoconstriction in contracting skeletal muscle. We hypothesized boosting NO bioavailability via 8wks of active beetroot juice (BRA, 4.03 mmol nitrate, 0.29 mmol nitrite, n = 19) improves hyperemia, via reduced α-mediated vasoconstriction, during handgrip exercise relative to nitrate/nitrite-depleted beetroot juice (BRP, n = 18) in patients with T2DM.

The potent vasoconstrictor peptides, endothelin 1 (ET-1) and angiotensin II control adaptation of blood vessels to fluctuations of blood pressure. Previously we have shown that the circulating level of ET-1 is regulated through its proteolytic cleavage by secreted serine carboxypeptidase, cathepsin A (CathA). However, genetically-modified mouse expressing catalytically inactive CathA S190A mutant retained about 10-15% of the carboxypeptidase activity against ET-1 in its tissues suggesting a presence of parallel/redundant catabolic pathway(s). In the current work we provide direct evidence that the enzyme, which complements CathA action towards ET-1 is a retinoid-inducible lysosomal serine carboxypeptidase 1 (Scpep1), a CathA homolog with previously unknown biological function. We generated a mouse strain devoid of both CathA and Scpep1 activities (DD mice) and found that in response to high-salt diet and systemic injections of ET-1 these animals showed significantly increased blood pressure as compared to wild type mice or those with single deficiencies of CathA or Scpep1. We also found that the reactivity of mesenteric arteries from DD mice towards ET-1 was significantly higher than that for all other groups of mice. The DD mice had a reduced degradation rate of ET-1 in the blood whereas their cultured arterial vascular smooth muscle cells showed increased ET-1-dependent phosphorylation of myosin light chain 2. Together, our results define the biological role of mammalian serine carboxypeptidase Scpep1 and suggest that Scpep1 and CathA together participate in the control of ET-1 regulation of vascular tone and hemodynamics.

Inositol 1,4,5-trisphosphate receptors (IP(3)Rs) regulate diverse physiological functions, including contraction and proliferation. There are three IP(3)R isoforms, but their functional significance in arterial smooth muscle cells is unclear. Here, we investigated relative expression and physiological functions of IP(3)R isoforms in cerebral artery smooth muscle cells. We show that 2-aminoethoxydiphenyl borate and xestospongin C, membrane-permeant IP(3)R blockers, reduced Ca(2+) wave activation and global intracellular Ca(2+) ([Ca(2+)](i)) elevation stimulated by UTP, a phospholipase C-coupled purinergic receptor agonist. Quantitative PCR, Western blotting, and immunofluorescence indicated that all three IP(3)R isoforms were expressed in acutely isolated cerebral artery smooth muscle cells, with IP(3)R1 being the most abundant isoform at 82% of total IP(3)R message. IP(3)R1 knockdown with short hairpin RNA (shRNA) did not alter baseline Ca(2+) wave frequency and global [Ca(2+)](i) but abolished UTP-induced Ca(2+) wave activation and reduced the UTP-induced global [Ca(2+)](i) elevation by approximately 61%. Antibodies targeting IP(3)R1 and IP(3)R1 knockdown reduced UTP-induced nonselective cation current (I(cat)) activation. IP(3)R1 knockdown also reduced UTP-induced vasoconstriction in pressurized arteries with both intact and depleted sarcoplasmic reticulum (SR) Ca(2+) by approximately 45%. These data indicate that IP(3)R1 is the predominant IP(3)R isoform expressed in rat cerebral artery smooth muscle cells. IP(3)R1 stimulation contributes to UTP-induced I(cat) activation, Ca(2+) wave generation, global [Ca(2+)](i) elevation, and vasoconstriction. In addition, IP(3)R1 activation constricts cerebral arteries in the absence of SR Ca(2+) release by stimulating plasma membrane I(cat).

Chronic intermittent hypoxia (IH) induces activation of the sympathoadrenal system, which plays a pivotal role in attenuating hypoxic pulmonary vasoconstriction (HPV) via central β1-adrenergic receptors (AR) (brain) and peripheral β2AR (pulmonary arteries). Prolonged hypercatecholemia has been shown to upregulate β3AR. However, the relationship between IH and β3AR in the modification of HPV is unknown. It has been observed that chronic stimulation of β3AR upregulates inducible nitric oxide synthase (iNOS) in cardiomyocytes and that IH exposure causes expression of iNOS in RAW264.7 macrophages. iNOS has been shown to have the ability to dilate pulmonary vessels. Hence, we hypothesized that chronic IH activates β3AR/iNOS signaling in pulmonary macrophages, leading to the promotion of NO secretion and attenuated HPV. Sprague-Dawley rats were exposed to IH (3-min periods of 4-21% O2) for 8 h/d for 6 weeks. The urinary catecholamine concentrations of IH rats were high compared with those of controls, indicating activation of the sympathoadrenal system following chronic IH. Interestingly, chronic IH induced the migration of circulating monocytes into the lungs and the predominant increase in the number of pro-inflammatory pulmonary macrophages. In these macrophages, both β3AR and iNOS were upregulated and stimulation of the β3AR/iNOS pathway in vitro caused them to promote NO secretion. Furthermore, in vivo synchrotron radiation microangiography showed that HPV was significantly attenuated in IH rats and the attenuated HPV was fully restored by blockade of β3AR/iNOS pathway or depletion of pulmonary macrophages. These results suggest that circulating monocyte-derived pulmonary macrophages attenuate HPV via activation of β3AR/iNOS signaling in chronic IH.

Signaling via the neuronal NOS (nNOS) splice variant nNOSmu is essential for skeletal muscle health and is commonly reduced in neuromuscular disease. nNOSmu is thought to be the predominant source of NO in skeletal muscle. Here we demonstrate the existence of what we believe to be a novel signaling pathway, mediated by the nNOS splice variant nNOSbeta, localized at the Golgi complex in mouse skeletal muscle cells. In contrast to muscles lacking nNOSmu alone, muscles missing both nNOSmu and nNOSbeta were severely myopathic, exhibiting structural defects in the microtubule cytoskeleton, Golgi complex, and mitochondria. Skeletal muscles lacking both nNOSmu and nNOSbeta were smaller in mass, intrinsically weak, highly susceptible to fatigue, and exhibited marked postexercise weakness. Our data indicate that nNOSbeta is a critical regulator of the structural and functional integrity of skeletal muscle and demonstrate the existence of 2 functionally distinct nNOS microdomains in skeletal muscle, created by the differential targeting of nNOSmu to the sarcolemma and nNOSbeta to the Golgi. We have previously shown that sarcolemmal nNOSmu matches the blood supply to the metabolic demands of active muscle. We now demonstrate that nNOSbeta simultaneously modulates the ability of skeletal muscle to maintain force production during and after exercise. We conclude therefore that nNOS splice variants are critical regulators of skeletal muscle exercise performance.

Sepsis is a complex syndrome characterized by the development of progressive dysfunction in multiple organs. The aim of the present study was to investigate the protective effect of curcumin against lipopolysaccharide (LPS)-induced vasoconstrictive dysfunction, and to investigate the possible underlying mechanism. Male Sprague-Dawley rats were randomly divided into the following groups: Control, sepsis and curcumin. A sepsis model was established by an intraperitoneal (i.p.) injection of 5 mg/kg LPS. Thoracic aortic rings obtained from the rats were mounted in an organ bath and the vasoconstriction of the rings was recorded. In addition, the serum E-selectin levels were determined by an enzyme-linked immunosorbent assay. The expression levels of thrombospondin (TSP)-1 and transforming growth factor (TGF)-β1 in the aortic tissue were detected by immunohistochemistry. Vasoconstriction of the aortic rings was found to significantly decrease in the sepsis rats when compared with the control group. However, curcumin (10 or 20 mg/kg, i.p.) prevented the vasoconstrictive dysfunction induced by LPS. The serum level of E-selectin and the expression levels of TSP-1 and TGF-β1 significantly increased in the sepsis rats when compared with the control group rats; however, the levels decreased significantly following treatment with curcumin (10 or 20 mg/kg). Furthermore, hematoxylin and eosin staining revealed that curcumin alleviated the LPS-induced damage in the aortic tunica intima and tunica media. Therefore, the results indicated that curcumin alleviates LPS-induced vasoconstrictive dysfunction in the thoracic aorta of rats. In addition, the inhibition of TSP-1 and TGF-β1 expression may be involved in the mechanism underlying this protective effect.

Mercury is an environmental pollutant that reduces nitric oxide (NO) bioavailability and increases oxidative stress, having a close link with cardiovascular diseases, as carotid atherosclerosis, myocardial infarction, coronary heart disease and hypertension. One of the main sites affected by oxidative stress, which develops atherosclerosis, is the aorta. Under acute exposure to low mercury concentrations reactive oxygen species (ROS) production were only reported for resistance vessels but if low concentrations of mercury also affect conductance arteries it is still unclear. We investigated the acute effects of 6 nM HgCl(2) on endothelial function of aortic rings measuring the reactivity to phenylephrine in rings incubated, or not, with HgCl(2) for 45 min, the protein expression for cyclooxygenase 2 (COX-2) and the AT1 receptor. HgCl(2) increased Rmax and pD2 to phenylephrine without changing the vasorelaxation induced by acetylcholine and sodium nitroprusside. Endothelial damage abolished the increased reactivity to phenylephrine. The increase of Rmax and pD2 produced by L-NAME was smaller in the presence of HgCl(2). Enalapril, losartan, indomethacin, furegrelate, the selective COX-2 inhibitor NS 398, superoxide dismutase and the NADPH oxidase inhibitor apocynin reverted HgCl(2) effects on the reactivity to phenylephrine, COX-2 protein expression was increased, and AT1 expression reduced. At low concentration, below the reference values, HgCl(2) increased vasoconstrictor activity by reducing NO bioavailability due to increased ROS production by NADPH oxidase activity. Results suggest that this is due to local release of angiotensin II and prostanoid vasoconstrictors. Results also suggest that acute low concentration mercury exposure, occurring time to time could induce vascular injury due to endothelial oxidative stress and contributing to increase peripheral resistance, being a high risk factor for public health.

Cytochrome P450 (CYP) signalling pathway has been shown to play a vital role in the vasoreactivity of wild type mouse ophthalmic artery. In this study, we determined the expression, vascular responses and potential mechanisms of the CYP-derived arachidonic acid metabolites. The expression of murine CYP (Cyp2c44) and soluble epoxide hydrolase (sEH) in the wild type ophthalmic artery was determined with immunofluorescence, which showed predominant expression of Cyp2c44 in the vascular smooth muscle cells (VSMC), while sEH was found mainly in the endothelium of the wild type ophthalmic artery. Artery of Cyp2c44-/- and sEH-/- mice were used as negative controls. Targeted mass spectrometry-based lipidomics analysis of endogenous epoxide and diols of the wild type artery detected only 14, 15-EET. Vasorelaxant responses of isolated vessels in response to selective pharmacological blockers and agonist were analysed ex vivo. Direct antagonism of epoxyeicosatrienoic acids (EETs) with a selective inhibitor caused partial vasodilation, suggesting that EETs may behave as vasoconstrictors. Exogenous administration of synthetic EET regioisomers significantly constricted the vessels in a concentration-dependent manner, with the strongest responses elicited by 11, 12- and 14, 15-EETs. Our results provide the first experimental evidence that Cyp2c44-derived EETs in the VSMC mediate vasoconstriction of the ophthalmic artery.

Development of combination therapies has received significant interest in recent years. Previously, a two-receptor one-transducer (2R-1T) model was proposed to characterize drug interactions with two receptors that lead to the same phenotypic response through a common transducer pathway. We applied, for the first time, the 2R-1T model to characterize the interaction of noradrenaline and arginine-vasopressin on vasoconstriction and performed inter-species scaling to humans using this mechanism-based model.

Cleistanthus collinus leaf extracts are consumed for suicidal purposes in southern India. The boiled decoction is known to be more toxic than the fresh leaf juice. Although several compounds have been isolated and their toxicity tested, controversy remains as to which compounds are responsible for the high level of toxicity of C. collinus. We report herein that cleistanthoside A is the major toxin in the boiled aqueous extract of fresh leaves and causes death in rats in small doses. The toxicity of the boiled extract prepared in the manner described can be attributed entirely to cleistanthoside A. Cleistanthin A could also be isolated from the boiled extract, albeit in trace amounts. As hypotension not responding to vasoconstrictors is the cause of death in patients who have consumed the boiled extract, effects of cleistanthoside A on the determinants of blood pressure, namely, force of cardiac contraction and vascular resistance, were tested in isolated organ experiments. Cleistanthoside A has a direct vasoconstrictor effect; however, it inhibits ventricular contractility. Therefore, the notion that the shock in C. collinus poisoning is of vascular origin must be considered carefully, and the possibility of cardiogenic shock must be studied. We present the crystal structure of cleistanthin A and show the potency of fast NMR methods (NOAH4-BSCN-NUS) in the full spectral assignment of cleistanthoside A as a real-world sample of a natural product. We also compare the results of the NOAH4-BSCN-NUS NMR experiments with conventional NMR methods.