Isolated Rabbit Aortic Rings Research Articles

Royal jelly causes hypotension and vasodilation induced by increasing nitric oxide production.

Among royal jelly’s (RJ) various biological activities, its possible antihypertension and vasorelaxation effects deserve particular attention, but the underlying mechanisms of action remain unclear. Therefore, this study used the spontaneously hypertensive rats (SHR) hypertension model and the isolated rabbit thoracic aorta rings model to explore the mechanisms underlying the hypotension and vasorelaxation effects of RJ. Rats were divided into the following groups (n = 6): WKY‐control group, SHR‐control group, and SHR‐RJ group. SHR‐RJ group was received 1 g/kg of RJ via oral administration daily for 4 weeks. Systolic blood pressure (SBP), diastolic blood pressure (DBP), heart rate (HR), and nitric oxide (NO) level were detected. In addition, the mechanism of vasodilation of RJ was investigated using an isolated rabbit aortic ring technique. RJ significantly reduced SBP and DBP as well as increased NO levels of SHR in vivo. RJ caused vasorelaxation of the isolated aorta rings, and this effect was inhibited by atropine (M3 receptor blocker), L‐NAME (nitric oxide synthase inhibitor), methylene blue (guanylate cyclase inhibitor), and indomethacin (cyclooxygenase inhibitor). Moreover, RJ could markedly suppress the NE‐induced intracellular Ca2+ releases and high K+‐induced extracellular Ca2+ influx in denuded aortic rings. In addition, RJ can also increase cGMP levels and the production of NO in isolated aortic rings. The present study showed that RJ has antihypertensive effects and was associated with increased NO production. In addition, RJ contains muscarinic receptor agonist, possibly an acetylcholine‐like substance, and induces vasodilation through NO/cGMP pathway and calcium channels.

Vascular mechanisms underlying the hypotensive effect of Rumex acetosa

Context: Rumex acetosa L. (Polygonaceae) is well known in traditional medicine for its therapeutic efficacy as an antihypertensive.Objective: The study investigates antihypertensive potential of crude methanol extract (Ra.Cr) and fractions of Rumex acetosa in normotensive and hypertensive rat models and probes the underlying vascular mechanisms.Materials and methods: Ra.Cr and its fractions were tested in vivo on normotensive and hypertensive Sprague-Dawley rats under anaesthesia for blood pressure lowering effect. In vitro experiments on rat and Oryctolagus cuniculus rabbit aortae were employed to probe the underlying vasorelaxant mechanism.Results: In normotensive rats under anaesthesia, Ra.Cr caused fall in MAP (40 mmHg) at 50 mg/kg with % fall of 27.88 ± 4.55. Among the fractions tested, aqueous fraction was more potent at the dose of 50 mg/kg with % fall of 45.63 ± 2.84. In hypertensive rats under similar conditions, extract and fractions showed antihypertensive effect at same doses while aqueous fraction being more potent, exhibited 68.53 ± 4.45% fall in MAP (70 mmHg). In isolated rat aortic rings precontracted with phenylephrine (PE), Ra.Cr and fractions induced endothelium-dependent vasorelaxation, which was partially blocked in presence of l-NAME, indomethacin and atropine. In isolated rabbit aortic rings pre-contracted with PE and K+-(80 mM), Ra.Cr induced vasorelaxation and shifted Ca2+ concentration–response curves to the right and suppressed PE peak formation, similar to verapamil, in Ca2+-free medium.Discussion and conclusions: The data indicate that l-NAME and atropine-sensitive endothelial-derived NO and COX enzyme inhibitors and Ca2+ entry blocking-mediated vasodilator effect of the extract explain its antihypertensive potential.

Blood Pressure Lowering Effect of the Extract of Aerial Parts of Capparis aphylla is Mediated Through Endothelium-Dependent and Independent Mechanisms

This investigation was aimed to provide pharmacological evidences for the medicinal use of Capparis aphylla in hypertension. In normotensive anesthetized rats, intravenous administration of the crude extract of Capparis aphylla (Ca.Cr; 3–100 mg/kg) caused a fall in mean arterial pressure (MAP), which was partially blocked in the presence of atropine (2 mg/kg). In isolated rabbit aortic rings, Ca.Cr inhibited phenylephrine (1 μM) and high K+ (80 mM) precontractions with respective EC50 values of 0.10 (0.07–0.15) and 1.22 mg/mL (1.00–1.50), suggesting calcium channel blocking (CCB) activity with a predominant inhibitory effect on receptor operated Ca2+ channels. Pretreatment of the arotic rings with Ca.Cr (0.1–1 mg/mL) caused a rightward shift in the Ca2+ concentration response curves, similar to verapamil. In isolated rat aorta preparations, Ca.Cr caused a partial endothelium-dependent L-NAME/atropine-sensitive vasodilator effect. In guinea-pig atria, Ca.Cr suppressed both rate and force of spontaneous atrial contractions with respective EC50 values of 1.35 (1.01–1.79) and 1.60 mg/mL (1.18–2.17), which remained unchanged in the presence of atropine (1 μM). These data indicate that the blood pressure (BP) lowering effect of the crude extract of Capparis aphylla is mediated through a vasodilator and cardiac depressant effect. The vasodilator effect is partly mediated by an endothelium-dependent, atropine-sensitive NO pathway, while the CCB effect is partly responsible for endothelium-independent vasodilatation and also for the cardiac depressant effect; thus, this study provides pharmacologic evidence with respect to the medicinal use of the plant in hypertension.

Hydroxyl radical: A possible mediator of nitroglycerin-induced vascular relaxation

The endothelium-derived relaxing factor, nitric oxide (NO·), is involved in acetylcholine (Ach) and nitroglycerin (NTG)-induced relaxation of vascular smooth muscle. It is known that NO· interacts with the superoxide anion (O2·−) at physiological pH and this interaction leads to the generation of hydroxyl radicals (·OH). Hydroxyl radicals are known to induce dilation and act as mediators in acetylcholine (Ach)-induced relaxation of rabbit aortic smooth muscle. Thus, it was hypothesized that ·OH may be involved in NTG-induced relaxation. To test this hypothesis we investigated the effect of NTG on norepinephrine (NE)-precontracted isolated rabbit aortic rings in the absence and/or presence of O2·− [superoxide dismutase (SOD)] and ·OH scavengers [dimethylthiourea (DMTU) or mannitol]. Superoxide dismutase, DMTU, and mannitol markedly reduced NTG-induced relaxation of isolated rabbit aortic rings. Superoxide dismutase produced a 32% reduction in NTG-induced vasodilation. Pretreatment with DMTU and/or mannitol reduced NTG-induced relaxation by 56% and 49%, respectively. These results suggest that ·OH may be a mediator involved in NTG-induced vascular relaxation of rabbit aorta.

Blood pressure lowering, cardiovascular inhibitory and bronchodilatory actions of Achillea millefolium

Achillea millefolium Linn. (Asteraceae) is used in folk medicine for the treatment of overactive cardiovascular and respiratory ailments. This study describes its hypotensive, cardio-depressant, vasodilatory and bronchodilatory activities. The crude extract of Achillea millefolium (Am.Cr) caused a dose-dependent (1-100 mg/kg) fall in arterial blood pressure of rats under anaesthesia. In spontaneously beating guinea-pig atrial tissues, Am.Cr exhibited negative inotropic and chronotropic effects. In isolated rabbit aortic rings, Am.Cr at 0.3-10 mg/mL relaxed phenylephrine (PE, 1 µm) and high K(+) (80 mm)-induced contractions, as well as suppressed the PE (1 µm) control peaks obtained in Ca(++) -free medium, like that caused by verapamil. The vasodilator effect of Am.Cr was partially blocked by N(ω) -nitro-l-arginine methyl ester in endothelium intact preparations. In guinea-pig tracheal strips, Am.Cr inhibited carbachol (CCh, 1 µm) and K(+) -induced contractions. These results indicate that Achillea millefolium exhibits hypotensive, cardiovascular inhibitory and bronchodilatory effects, thus explaining its medicinal use in hyperactive cardiovascular and airway disorders, such as hypertension and asthma.

Pharmacological basis for the medicinal use of Zanthoxylum armatum in gut, airways and cardiovascular disorders

This study describes the gut, airways and cardiovascular modulatory activities of Zanthoxylum armatum DC. (Rutaceae) to rationalize some of its medicinal uses. The crude extract of Zanthoxylum armatum (Za.Cr) caused concentration-dependent relaxation of spontaneous and high K(+) (80 mM)-induced contractions in isolated rabbit jejunum, being more effective against K(+) and suggestive of Ca(++) antagonist effect, which was confirmed when pretreatment of the tissues with Za.Cr shifted Ca(++) concentration-response curves to the right, like that caused by verapamil. Za.Cr inhibited the castor-oil-induced diarrhea in mice at 300-1000 mg/kg. In rabbit tracheal preparations, Za.Cr relaxed the carbachol (1 microM) and high K(+)-induced contractions, in a pattern similar to that of verapamil. In isolated rabbit aortic rings, Za.Cr exhibited vasodilator effect against phenylephrine (1 microM) and K(+)-induced contractions. When tested in guinea pig atria, Za.Cr caused inhibition of both atrial force and rate of spontaneous contractions, like that caused by verapamil. These results indicate that Zanthoxylum armatum exhibits spasmolytic effects, mediated possibly through Ca(++) antagonist mechanism, which provides pharmacological base for its medicinal use in the gastrointestinal, respiratory and cardiovascular disorders.

High‐ but not low‐dose folic acid improves endothelial function in coronary artery disease

While folic acid (FA) reduces plasma homocysteine (Hcy), whether the simultaneous improvement in endothelial function is dependent on Hcy lowering per se is questionable. In the present study the relationship between FA dose, Hcy lowering and endothelial function in patients with coronary artery disease (CAD) was investigated. Eighty-four patients with CAD received either 400 microg FA or 5 mg placebo daily for a 6-week treatment period. A further 44 patients with CAD received either 100 mg kg(-1) day(-1) of betaine or placebo for a 6-week treatment period. Flow-mediated dilatation (FMD), a measure of endothelial function, was assessed before and after the 6-week periods. Isometric tension and Western blotting were used to investigate the effect of FA on endothelial function and endothelial nitric oxide synthase (eNOS) dimerization in isolated rabbit aortic rings and cultured porcine aortic endothelial cells (PAEC), respectively. Both 400 micro g day(-1) and 5 mg day(-1) FA significantly increased plasma folate and decreased plasma Hcy. The FMD improved significantly after 6 weeks' treatment of 5 mg day(-1) FA but did not correlate with the reduction in Hcy. There was no change in FMD in either the 400 micro g FA or placebo group. In a subgroup analysis of 11 patients in the betaine group, despite a reduced Hcy, a significant impairment in FMD was observed. In the in vitro studies FA, but not betaine, reversed methionine-induced endothelial dysfunction. Moreover, the FA promoted eNOS dimerization in cultured PAEC. These data suggest that FA dose-dependently improves endothelial function in CAD via a mechanism independently of Hcy lowering. It may involve promotion of eNOS dimerization.

C-reactive protein-induced in vitro vasorelaxation is an artefact caused by the presence of sodium azide in commercial preparations.

Although C-reactive protein (CRP) is increasingly recognized as an independent risk factor for acute myocardial events, recent evidence suggests that it can directly induce vasorelaxation. This study aimed to investigate the mechanism of this CRP-induced response. Isometric tension recordings were used to measure endothelium-dependent and endothelium-independent vascular smooth muscle relaxation in isolated rabbit aortic rings. CRP generated in-house by genetic engineering and expressed in Chinese hamster ovary cells, CRP purified from ascites, and CRP obtained from commercial sources were assessed for vasorelaxing properties. Only the commercial CRP preparation induced vasorelaxation; more than half maximal relaxation was observed at 0.025 microg/mL and maximum relaxation attained at 0.25 microg/mL. Commercial CRP contains high levels of sodium azide, a well-known vasorelaxant. Removal of this agent by dialysis abolished the vasodilatory effect of commercial CRP. Sodium azide alone at concentrations equivalent to that present in the commercial CRP produced a near-identical relaxation pattern to the undialyzed commercial product. CRP has no vasorelaxant properties per se, and the reported vasorelaxant ability of CRP is an artifact caused by sodium azide present in commercial preparations of this agent.

Enhanced vasoconstriction and reduced vasorelaxation induced by testosterone and nandrolone in hypercholesterolemic rabbits

Androgenic-anabolic steroids (AAS) are widely abused by athletes and this abuse has been associated with many serious circulatory events including sudden cardiac death, myocardial infarction and cardiac hypertrophy. The effect of chronic treatment for 16 weeks with testosterone (25 mg −1 kg −1 week −1) and nandrolone (50 mg −1 kg −1 week −1) on serum lipids of male hypercholesterolemic New Zealand rabbits was investigated. The responses of isolated rabbit aortic rings to some vasoconstrictors (epinephrine, serotonin and endothelin-1) and vasodilators (adenosine and sodium nitroprusside) were also measured after treatment. Testosterone and nandrolone significantly reduced HDL-cholesterol levels, potentiated vasoconstriction responses to epinephrine, serotonin and endothelin-1, and attenuated vasorelaxant responses to sodium nitroprusside in rabbits. Nandrolone also caused a significant increase in LDL-cholesterol levels. No significant changes in adenosine relaxation were found in rabbits. The results of the present study showed that the abuse of AAS in presence of hypercholesterolemia can enhance atherogenicity and vasospasm as well as attenuation of vasorelaxation. Therefore the abuse of AAS is harmful to the vascular system and should be prohibited.

Enhanced vasoconstriction and reduced vasorelaxation induced by testosterone and nandrolone in hypercholesterolemic rabbits

Androgenic-anabolic steroids (AAS) are widely abused by athletes and this abuse has been associated with many serious circulatory events including sudden cardiac death , myocardial infarction and cardiac hypertrophy . The effect of chronic treatment for 16 weeks with testosterone (25 mg −1 kg −1 week −1 ) and nandrolone (50 mg −1 kg −1 week −1 ) on serum lipids of male hypercholesterolemic New Zealand rabbits was investigated. The responses of isolated rabbit aortic rings to some vasoconstrictors (epinephrine, serotonin and endothelin-1) and vasodilators (adenosine and sodium nitroprusside) were also measured after treatment. Testosterone and nandrolone significantly reduced HDL-cholesterol levels, potentiated vasoconstriction responses to epinephrine, serotonin and endothelin-1, and attenuated vasorelaxant responses to sodium nitroprusside in rabbits. Nandrolone also caused a significant increase in LDL-cholesterol levels. No significant changes in adenosine relaxation were found in rabbits. The results of the present study showed that the abuse of AAS in presence of hypercholesterolemia can enhance atherogenicity and vasospasm as well as attenuation of vasorelaxation. Therefore the abuse of AAS is harmful to the vascular system and should be prohibited.

Synthesis and Structure of Novel 2,3,5,6‐Tetrahydro‐1H‐imidazo[2,1‐b][1,3,5]benzotriazepines with Vasocontractile Activity in Rabbit Aortic Rings.

A series of novel 2,3,5,6-tetrahydro-1H-imidazo[2,1-b][1,3,5]benzotriazepines (3a-n) and hydrochlorides (4a-b) were prepared and their structure was determined by IR and NMR spectroscopic data as well as by X-ray analysis of the hydrochloride 4a. The newly synthesized benzotriazepine 4b exhibited concentration-dependent vasocontractile activity in isolated rabbit aortic rings. Rimalkalim was found to induce a relaxation in rabbit aortic rings precontracted by 4b (3x10(-5) mol). Present results indicate that K(ATP)-dependant channels may contribute in the contractile activity of benzotriazepine 4b.

Synthesis and structure of novel 2,3, 5,6-tetrahydro-1 H-imidazo[2,1-b][1,3,5]benzotriazepines with vasocontractile activity in rabbit aortic rings

A series of novel 2,3,5,6-tetrahydro-1 H-imidazo[2,1-b][1,3,5]benzotriazepines ( 3a– n) and hydrochlorides ( 4a– b) were prepared and their structure was determined by IR and NMR spectroscopic data as well as by X-ray analysis of the hydrochloride 4a. The newly synthesized benzotriazepine 4b exhibited concentration-dependent vasocontractile activity in isolated rabbit aortic rings. Rimalkalim was found to induce a relaxation in rabbit aortic rings precontracted by 4b (3×10 −5 mol). Present results indicate that K ATP-dependant channels may contribute in the contractile activity of benzotriazepine 4b.

Hydroxyl radical: A possible mediator of nitroglycerin-induced vascular relaxation

The endothelium-derived relaxing factor, nitric oxide (NO·), is involved in acetylcholine (Ach) and nitroglycerin (NTG)-induced relaxation of vascular smooth muscle. It is known that NO· interacts with the superoxide anion (O2·−) at physiological pH and this interaction leads to the generation of hydroxyl radicals (·OH). Hydroxyl radicals are known to induce dilation and act as mediators in acetylcholine (Ach)-induced relaxation of rabbit aortic smooth muscle. Thus, it was hypothesized that ·OH may be involved in NTG-induced relaxation. To test this hypothesis we investigated the effect of NTG on norepinephrine (NE)-precontracted isolated rabbit aortic rings in the absence and/or presence of O2·− [superoxide dismutase (SOD)] and ·OH scavengers [dimethylthiourea (DMTU) or mannitol]. Superoxide dismutase, DMTU, and mannitol markedly reduced NTG-induced relaxation of isolated rabbit aortic rings. Superoxide dismutase produced a 32% reduction in NTG-induced vasodilation. Pretreatment with DMTU and/or mannitol reduced NTG-induced relaxation by 56% and 49%, respectively. These results suggest that ·OH may be a mediator involved in NTG-induced vascular relaxation of rabbit aorta.

Inhibitory effects of melatonin on vascular reactivity: possible role of vasoactive mediators

Melatonin (MEL), the principal hormone of the vertebral pineal gland, elicits several neurobiological effects. However, the effects of MEL on vascular tissues are still vague. The first goal of this study was to investigate the effect of MEL on isolated rabbit aortic rings and its role in the vascular reactivity to contractile agents, noradrenaline (NA) and phenylephrine (PHE) and relaxant agents (acetylcholine and sodium nitroprusside). In addition, the levels of nitric oxide (NO), cGMP, total calcium, lipid peroxides, superoxide dismutase (SOD) and glutathione (GSH) were also investigated in tissue homogenates of rabbit aortic rings preincubated (20 min) in MEL with and without contractile agents. Our results revealed that MEL has an endothelium-dependent vaso-relaxant effect and potentiated significantly the vaso-relaxant effect of acetylcholine. Moreover, MEL (10 −4 M) had a significant inhibitory effect on the contractile responses of aortic rings to both NA and PHE. In comparison with control tissue rings, the levels of lipid peroxides were significantly increased while the levels of GSH, and SOD activities were significantly decreased in tissue homogenates of aortic rings pre-incubated (20 min) in NA or PHE. In addition, the levels of NO and cGMP were significantly lower in tissue rings pre-treated with NA and PHE, respectively. Also, the levels of total calcium were significantly increased only in tissue rings pre-treated with NA. The levels of lipid peroxides were significantly decreased, while the levels of GSH, NO and cGMP and SOD activities were significantly increased in tissue homogenates of aortic rings incubated (20 min) in MEL (10 −4 M) in comparison to ring tissues incubated in NA or PHE alone. In aortic rings incubated in MEL+PHE, the levels of lipid peroxides were significantly lower while the levels of GSH and cGMP and SOD activities were significantly higher than their levels in ring tissues incubated in PHE. In aortic rings incubated in MEL+NA, the levels of lipid peroxides and total calcium were significantly lower while the levels of NO were significantly higher than their levels in ring tissues incubated in NA alone. We conclude that MEL has an endothelium dependent vasorelaxant effect and potentiates the endothelium dependent vasorelaxation induced by acetylcholine. MEL inhibits the contractile responses of aortic rings to NA and PHE. These effects may be, in part, due to re-balancing the pro-oxidant/antioxidants system, lowered calcium content and elevated NO and cGMP levels in vascular tissue.

Influence of sialic acid content of low-density lipoprotein particles on endothelium-dependent vasorelaxation in rabbit aorta

Low-density lipoprotein (LDL) concentration in plasma is an important predictor for atherosclerosis, and desialylated LDL has been proposed to be particularly atherogenic. Atherosclerosis is also associated with vascular endothelial dysfunction. We therefore wished to test the hypothesis that removal of sialic acid residues from LDL increases its ability to inhibit endothelium-dependent vasorelaxation. We studied vasorelaxant responses to acetylcholine (ACh) in isolated rabbit aortic rings as a model of endothelium-dependent relaxation, in the presence or absence of LDL treated either with saline or with neuraminidase, to cleave sialic acid residues. Vasorelaxant responses to ACh were inhibited by 300 μg protein per ml saline-treated LDL ( E max 77.5±4.5 vs. 89.7±2.2% in the absence of LDL, P<0.05). This inhibitory effect was not altered by neuraminidase treatment of LDL. In contrast, 300 μg protein per ml LDL, either saline- or neuraminidase-treated, did not affect vasorelaxant responses to the endothelium-independent dilator sodium nitroprusside. We found no correlation between sialic acid content of saline-treated LDL and its ability to inhibit endothelium-dependent vasorelaxation, in rabbit aortic rings, at a concentration of 300 μg protein per ml. Our results therefore suggest that sialic acid content is not an important determinant of the effect of LDL on vascular endothelium-dependent relaxation.

Homocysteine-induced inhibition of endothelium-dependent relaxation in rabbit aorta: role for superoxide anions.

Hyperhomocysteinemia is associated with endothelial dysfunction, although its mechanism is unknown. Isometric tension recordings and lucigenin chemiluminescence were used to assess the effects of homocysteine exposure on endothelium-dependent and -independent relaxation in isolated rabbit aortic rings and superoxide anion (O(2)(-)) production by cultured porcine aortic endothelial cells, respectively. Homocysteine (0.1 to 10 mmol/L) produced a significant (P<0.001) concentration- and time-dependent inhibition of endothelium-dependent relaxation in response to both acetylcholine and the calcium ionophore A23187. Only the intracellular O(2)(-) scavenger 4,5-dihydroxy-1,3-benzene disulfonic acid (Tiron, 10 mmol/L) significantly (P<0.001) inhibited the effect of homocysteine on acetylcholine- and A23187-induced relaxation. Incubation of porcine aortic endothelial cells with homocysteine (0.03 to 1 mmol/L for up to 72 hours) caused a significant (P<0.001) time-dependent increase in the O(2)(-) released by these cells on the addition of Triton X-100 (1% [vol/vol]), with levels returning to values comparable to those of control cells at the 72-hour time point. These changes in O(2)(-) levels were associated with a time-dependent increase in endothelial cell superoxide dismutase activity, becoming significant (P<0.001) after 72 hours. Furthermore, the homocysteine-induced increase in endothelial cell O(2)(-) levels was completely inhibited (P<0.001) by the concomitant incubation with either Tiron (10 mmol/L), vitamin C (10 micromol/L), or vitamin E (10 micromol/L). These data suggest that the inhibitory effect of homocysteine on endothelium-dependent relaxation is due to an increase in the endothelial cell intracellular levels of O(2)(-) and provide a possible mechanism for the endothelial dysfunction associated with hyperhomocysteinemia.

Effects of the soluble guanylyl cyclase activator, YC-1, on vascular tone, cyclic GMP levels and phosphodiesterase activity.

The vasomotor and cyclic GMP-elevating activity of YC-1, a novel NO-independent activator of soluble guanylyl cyclase (sGC), was studied in isolated rabbit aortic rings and compared to that of the NO donor compounds sodium nitroprusside (SNP) and NOC 18. Similarly to SNP and NOC 18, YC-1 (0.3-300 microM) caused a concentration-dependent, endothelium-independent relaxation that was greatly reduced by the sGC inhibitor 1-H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ 10 microM; 59% inhibition of dilation induced by 100 microM YC-1) suggesting the activation of sGC as one mechanism of action. Preincubation with YC-1 (3 and 30 microM) significantly increased the maximal dilator responses mediated by endogenous NO in aortic rings that was released upon exposure to acetylcholine, and enhanced the dilator response to the exogenous NO-donors, SNP and NOC 18, by almost two orders of magnitude. Vasoactivity induced by SNP and YC-1 displayed different kinetics as evidenced by a longlasting inhibition by YC-1 (300 microM) on the phenylephrine (PE)-induced contractile response, which was not fully reversible even after extensive washout (150 min) of YC-1, and was accompanied by a long-lasting elevation of intracellular cyclic GMP content. In contrast, SNP (30 microM) had no effect on the vasoconstrictor potency of PE, and increases in intravascular cyclic GMP levels were readily reversed after washout of this NO donor compound. Surprisingly, YC-1 not only activated sGC, but also affected cyclic GMP metabolism, as it inhibited both cyclic GMP break down in aortic extracts and the activity of phosphodiesterase isoforms 1-5 in vitro. In conclusion, YC-1 caused persistent elevation of intravascular cyclic GMP levels in vivo by activating sGC and inhibiting cyclic GMP break down. Thus, YC-1 is a highly effective vasodilator compound with a prolonged duration of action, and mechanisms that are unprecedented for any previously known sGC activator.

Tissue distribution of phosphodiesterase families and the effects of sildenafil on tissue cyclic nucleotides, platelet function, and the contractile responses of trabeculae carneae and aortic rings in vitro

Sildenafil is a selective inhibitor of phosphodiesterase type 5 (PDE5), which has been shown to be a clinically effective treatment for erectile dysfunction. Its action results from increased levels of cyclic guanosine monophosphate (cGMP), which is normally degraded by PDE5. This cyclic nucleotide is a second messenger for nitric oxide, which is involved in the regulation of numerous functions, including vascular smooth muscle tone. In an attempt to better predict the effects of sildenafil on cardiovascular function, the distribution of PDE activity was determined with anti-PDE1 and anti-PDE5 antibodies in the human cardiac ventricle and saphenous vein, and in vitro studies were performed on the isolated human cardiac ventricle, corpus cavernosum, saphenous vein, and mesenteric artery as well as on rabbit aorta, dog coronary artery, dog trabecular tissue, and rabbit and human platelets. The major PDE activity in the human cardiac ventricle was shown to be calcium/calmodulin-dependent PDE1, but there was no detectable level of PDE5. In contrast, the human saphenous vein contained PDEs 1, 4, and 5, and the human mesenteric artery contained PDEs 1, 2, 3, 4, and 5. The distribution of PDE5 in the cardiovascular system is consistent with the observed pharmacodynamic and clinical effects of sildenafil. Sildenafil, unlike milrinone, a selective PDE3 inhibitor, had no effect on the isolated trabeculae carneae; this is consistent with the lack of PDE5 expression in cardiac myocytes. Sildenafil selectively increased cGMP levels in coronary vascular smooth muscle tissue but produced no change in cyclic adenosine monophosphate (cAMP) levels, which is consistent with the drug’s selectivity for PDE5. In phenylephrine-contracted isolated rabbit aortic rings, sildenafil enhanced the relaxation induced by the nitric oxide donor glyceryl trinitrate, suggesting that sildenafil may potentiate the hypotensive effects of nitric oxide donor agents on the vasculature, an effect that has been observed clinically. Human platelets were found to contain PDE5, which was inhibited by 50% (IC 50) by sildenafil at a concentration of 6.3 nM, consistent with the IC 50 value in the corpus cavernosum. Sildenafil alone had no direct effect on platelet function, but it potentiated the in vitro antiaggregatory activity of sodium nitroprusside on rabbit and human platelets. The pharmacodynamic and adverse event profiles observed in clinical trials with sildenafil are consistent with the in vitro profile of the tissue distribution of PDE5 and its known mechanism of action as a selective inhibitor of PDE5.

PROTEOLYTIC ACTIVATION OF A PUTATIVE RECEPTOR LEADING TO VASOCONSTRICTION AND PLATELET AGGREGATION

Submandibular enzymatic vasoconstrictor (SEV), a member of the kallikrein family of enzymes, elicits biological effects by a proteolytically mediated mechanism. We studied 1) whether SEV is able to aggregate platelets and 2) whether SEV may activate a receptor other than the cloned thrombin receptor. SEV (10 −8 M) aggregated platelets, released ATP and increased intracellular Ca 2+. Elastase treatment rendered human platelets unresponsive to SEV and thrombin (TH), but not to cathepsin G. In desensitization experiments performed with γ-TH, after two successive additions of approximately 50 nM γ-TH, a third dose elicited 15.8±3.4% of the initial response ( n = 4), but platelets responded to approximately 20 nM SEV by 33.8 ± 7.2% of control ( p < 0.03 vs last response to γ-TH). After desensitization to SEV ( n=4), the response to a third dose was 4 ± 1.3% of control, but γ-TH still induced 37.7 ± 12.4% aggregation ( p < 0.02 vs last response to SEV). Incubation of washed rabbit platelets with α-TH digested with elastase (10 −10 M TH added to 7 μg/ml elastase for 1 min) rendered them unresponsive to additional challenges with TH, but they still responded to an equipotent dose of SEV (2.7 × 10 −9 M) by 86 ± 48% of control. In isolated rabbit aortic rings contracted with 10 −6 M norepinephrine (NE) to 42 ± 3% of maximum, SEV (2.8 × 10 −8 M) caused further contraction to 87 ± 4%. In contrast, α-TH (1.6 × 10 −7 M) tended to relax both NE- and SEV-contracted rings by 14 ± 2 and 16.2 ± 2%, respectively ( n = 3 each). We concluded that part of the platelet-aggregating effect of SEV may be mediated by activation of a receptor(s) different from that of TH. Copyright © 1997 Elsevier Science Ltd

Cardiovascular protection by ginsenosides and their nitric oxide releasing action.

1. In an animal model in vivo, ginsenosides (GS), saponins from Panax ginseng, were shown to protect against myocardial ischaemia/reperfusion damage with concomitant increased 6-keto-PGF1 alpha and decreased lipid peroxidation. 2. In perfused rabbit lung in situ and isolated rabbit aortic rings, GS protected the pulmonary and aortic endothelium against electrolysis-induced free radical injury. Purified components of GS, Rb1 and especially Rg1, relaxed pulmonary vessels and this effect was eliminated by nitro-L-arginine, an inhibitor of nitric oxide (NO) synthase. 3. In cultured bovine aortic endothelial cells, GS enhanced the conversion of [14C]-L-arginine to [14C]-L-citrulline, indicating an increased release of NO. 4. As the neurotransmitter inducing penile erection, NO release was shown to be enhanced by GS in rabbit corpus cavernosum (CC) in vitro. Ginsenosides enhanced both acetylcholine-induced and transmural nerve stimulation-activated relaxation associated with increased tissue cGMP. The latter effect was eliminated by tetrodotoxin and was associated with decreased tissue cGMP. Ginsenoside-enhanced CC relaxation was attenuated by nitro-L-arginine and oxyhaemoglobin, and enhanced by superoxide dismutase. 5. It is postulated that cardiovascular protection by GS may be partly mediated by the release of NO, a potent antioxidant, and that the GS-enhanced release of NO from endothelial cells, especially from perivascular nitric oxidergic nerves in the CC, may partly account for the aphrodisiac effect of Panax ginseng used in traditional Chinese medicine.