Vascular Preparations Research Articles

Comparison of the Effects of Levosimendan and Papaverine on Human Internal Mammary Artery and Saphenous Vein

Internal mammary artery (IMA) and saphenous vein (SV) are two most common vessels used in coronary artery bypass grafting (CABG). In the present study, the effect of levosimendan (a novel inotropic/vasodilator compound) and papaverine are compared, using IMA and SV obtained from CABG patients. Unused segments of IMA and SV of each patient were cut into two rings of 3 mm length. Four rings (two from IMA, two from SV) were suspended in an organ bath, contracted with norepinephrine and then the responses to the cumulative concentrations of levosimendan and papaverine were recorded. Levosimendan relaxed IMA but failed to relax SV completely. In addition, its IC50 value (concentration which reduced 50% of initial contraction) to relax SV was about 300 times higher than the IC50 value for IMA. Its arteriodilating concentration was in the range of its clinically effective inotropic concentration. Papaverine relaxed both vascular preparations completely. Its IC50 value was about 2.4 times higher toward SV in comparison to that of IMA. It is concluded that levosimendan is much more potent to relax norepinephrine-induced contraction of human IMA in comparison to its effect on human SV. It may have the potential to be used as a mixed inotropic/arteriodilator compound in several clinical settings including CABG in which it can increase cardiac contractility and prevent IMA vasospasm.

The effect of α-adrenoceptor agonists and L-NMMA on cutaneous postocclusive reactive hyperemia

Postocclusive reactive hyperemia (PRH) is considered to be a locally mediated vascular reaction independent of sympathetic activity. However, experiments on animal vascular preparations have shown that α 2-adrenoceptor activity interferes with the production of endothelium-derived nitric oxide (NO) that has been found to play an important role in the PRH response. We attempted to elucidate the role of endothelium-derived NO in the cutaneous PRH response. Especially, we aimed to clarify the interference of the α-adrenoceptor activity with NO mechanism in cutaneous microcirculation. We studied the effect of intradermal microinjection of the NO synthase inhibitor L-NMMA alone, α 1-agonist phenylephrine alone, α 2-agonist clonidine alone and L-NMMA in combination with each α-agonist. The effect of the saline solution injection was used as a reference value. Laser-Doppler flux was monitored in 11 healthy volunteers before and after a 4-minute and an 8-minute occlusion of digital arteries. The application of L-NMMA significantly reduced resting LD-flux ( p < 0.05) only in combination with α 2-agonist but not α 1-agonist. The application of L-NMMA alone changed the time in which LD flux reached half of the preocclusive value during the PRH response (T/2) only after 4-minute ( p < 0.05) but not after an 8-minute occlusion. Phenylephrine (α 1-agonist) alone shortened ( p < 0.05), while clonidine (α 2-agonist) alone prolonged T/2 ( p < 0.05) of 8-minute PRH. The combined application of L-NMMA and clonidine abolished the effect of clonidine alone on 8-minute PRH. In contrast, the combination of L-NMMA and phenylephrine did not cause any change of the PRH response when compared to the injection of phenylephrine alone. Our finding is consistent with the hypothesis that α 2-adrenoceptor activity (in the condition of maximal agonist stimulation) could interfere with NO mechanisms in cutaneous microcirculation, probably by increasing NO production.

The Endogenous Brain ConstituentN-Arachidonoyl l-Serine Is an Activator of Large Conductance Ca2+-Activated K+Channels

The novel endocannabinoid-like lipid N-arachidonoyl L-serine (ARA-S) causes vasodilation through both endothelium-dependent and -independent mechanisms. We have analyzed the vasorelaxant effect of ARA-S in isolated vascular preparations and its effects on Ca(2+)-activated K(+) currents in human embryonic kidney cells stably transfected with the alpha-subunit of the human, large conductance Ca(+)-activated K(+) (BK(Ca)) channel [human embryonic kidney (HEK) 293hSlo cells]. ARA-S caused relaxation of rat isolated, intact and denuded, small mesenteric arteries preconstricted with (R)-(-)-1-(3-hydroxyphenyl)-2-methylaminoethanol hydrochloride (pEC(50), 5.49 and 5.14, respectively), whereas it caused further contraction of vessels preconstricted with KCl (pEC(50), 5.48 and 4.82, respectively). Vasorelaxation by ARA-S was inhibited by 100 nM iberiotoxin. In human embryonic kidney cells stably transfected with the alpha-subunit of the human BK(Ca) channel cells, ARA-S and its enantiomer, N-arachidonoyl-D-serine, enhanced the whole-cell outward K(+) current with similar potency (pEC(50), 5.63 and 5.32, respectively). The potentiation was not altered by the beta(1) subunit or mediated by ARA-S metabolites, stimulation of known cannabinoid receptors, G proteins, protein kinases, or Ca(2+)-dependent processes; it was lost after patch excision or after membrane cholesterol depletion but was restored after cholesterol reconstitution. BK(Ca) currents were also enhanced by N-arachidonoyl ethanolamide (pEC(50), 5.27) but inhibited by another endocannabinoid, O-arachidonoyl ethanolamine (pIC(50), 6.35), or by the synthetic cannabinoid O-1918 [(-)-1,3-dimethoxy-2-(3-3,4-trans-p-menthadien-(1,8)-yl)-orcinol] (pIC(50), 6.59), which blocks ARA-S-induced vasodilation. We conclude the following. 1) ARA-S directly activates BK(Ca) channels. 2) This interaction does not involve cannabinoid receptors or cytosolic factors but is dependent on the presence of membrane cholesterol. 3) Direct BK(Ca) channel activation probably contributes to the endothelium-independent component of ARA-S-induced mesenteric vasorelaxation. 4) O-1918 is a BK(Ca) channel inhibitor.

Apocynin-induced vasodilation involves Rho kinase inhibition but not NADPH oxidase inhibition

The present study was designed to test the hypothesis that NADPH oxidase inhibition with apocynin would lower blood pressure and improve endothelial function in spontaneously hypertensive rats (SHRs). Although apocyin effectively dilated arterial segments in vitro, it failed to lower blood pressure or improve endothelial function. Further experiments were performed in normotensive rats and in NADPH oxidase subunit knock-out mice to test if apocynin-induced vasodilation depends on NADPH oxidase inhibition at all. SHRs were treated with apocynin orally or i.v. Arterial pressure was recorded directly. Rat and mouse arterial function was investigated in vitro by small vessel wire myography. NADPH oxidase activity was measured in human granulocytes and in rat vascular preparations. Rho kinase activity was determined by Western blot analysis. Apocynin did not reduce arterial pressure acutely in SHR when given at 50, 100, or 150 mg kg(-1) day(-1) orally over 1-week intervals or when given i.v. Apocynin potently inhibited granulocyte NADPH oxidase but not vascular NADPH-oxidase-dependent oxygen radical formation unless exogenous peroxidase was added to vascular preparations. Apocynin dilated rat intrarenal and coronary arteries independently of pharmacological interventions that reduce vascular superoxide radical abundance and actions. Aortic rings from p47phox(-/-) mice were more sensitive to apocynin-induced dilation than wild-type aortic rings. Rho kinase inhibition reduced or prevented the inhibitory effect of apocynin on agonist-induced vasoconstriction and apocynin inhibited the phosphorylation of Rho kinase substrates. Apocynin per se does not inhibit vascular NADPH-oxidase-dependent superoxide formation. Its in vitro vasodilator actions are not due to NADPH oxidase inhibition but may be explained at least in part by inhibition of Rho kinase activity. The discrepancy between apocynin-induced vasodilation in vitro and the failure of apocynin to lower arterial pressure in SHR suggests opposing effects on arterial pressure-regulating systems in vivo. Its use as a pharmacological tool to investigate vascular NADPH oxidase should be discontinued.

Irrigação do timo em aves da linhagem Paraíso Pedrês (Gallus gallus domesticus)

A vascularização arterial do timo (número, origem e ordenação) bem como a distribuição parenquimal dos ramos penetrantes foram estudadas em 40 aves da linhagem Paraíso Pedrês. Trinta aves foram injetadas com látex e dissecadas, enquanto 10 aves tiveram seus sistemas arteriais injetados com resina (metil metacrilato e mercox) para a preparação de moldes vasculares. A principal fonte de irrigação encontrada foi a associação dos ramos oriundos das artérias comuns do nervo vago, tireóideas e ingluviais, sendo que seus ramos penetravam o parênquima dos lobos, principalmente pelas suas extremidades cranial e caudal. A partir da penetração, os ramos tímicos apresentavam distribuição predominante para a periferia do lobo, formando uma trama capilar poligonal, com espaços irregulares, característica de um órgão linforreticular.

Diverse TRPC heteromultimers form store‐operated channels in native vascular smooth muscle preparations

Store‐operated channels (SOCs) are activated following depletion of intracellular Ca2+ stores and the canonical transient receptor potential (TRPC) family of channels are possible molecular candidates of SOCs. Here we describe diverse properties and putative molecular identities of SOCs in different vascular preparations.In rabbit mesenteric (MA) and coronary artery (CA) myocytes SOCs were activated by the store depleting reagents BAPTA‐AM and cyclopiazonic acid and in 1.5mM [Ca2+]o these SOCs had conductances of about 2pS (Saleh et al. 2006) and 3 pS and reversal potentials (EREV) of about +20mV and −10mV respectively. In 0mM [Ca2+]o the conductances were about 7pS in MA and 3pS in CA whilst EREV were both about −10mV respectively, highlighting different biophysical properties of SOCs in these two preparations.MA SOCs were selectively inhibited by anti‐TRPC1/C5 whilst SOCs from CA were specifically inhibited by anti‐TRPC1/C5/C6. SOCs recorded from portal vein myocytes had similar biophysical properties to MA SOCs (Albert &amp; Large, 2002) but were blocked by anti‐TRPC1/C5/C7.These data suggest that TRPC channels can form alternative heteromultimers with a similar SOCE function in different vascular smooth muscle preparations. Moreover, these data show that anti‐TRPC antibodies can be used as ‘immunopharmacological’ reagents with a remarkable degree of selectivity to determine the composition of heteromultimeric ion channel proteins in native tissue.Funded by British Heart Foundation and The Wellcome Trust.

Pioglitazone and Losartan Modify Hemodynamic and Metabolic Parameters and Vascular Prostanoids in Fructose-Overloaded Rats

This study analyzes the effects of losartan (AT1 blocker) and pioglitazone (insulin sensitizer), alone and in combination, in the fructose-overloaded rat, a model of metabolic syndrome. All treatments (nine weeks) reduced blood pressure and triglyceridemia and also restored the diminished release of vasodilator prostaglandins (prostacyclin in aorta and mesenteric vascular bed and prostaglandin E2 in the latter). Pioglitazone, alone and in combination with losartan, reduced the release of the vasoconstrictor thromboxane in controls and fructose rats in both vascular preparations. In conclusion, although combination therapy and single treatments exerted similar effects, there may still be some advantage to the combined treatment.

α2B-adrenoceptor agonist ST-91 antagonizes β2-adrenoceptor-mediated relaxation in rat mesenteric artery rings

We sought an isolated vascular preparation and experimental setting where the function of α2B-adrenoceptors could be demonstrated by non-recombinant technique. ST-91 (2-[2,6-diethylphenylamino]-2-imidazoline), an α2B-adrenoceptor agonist with a mixed α adrenergic receptor type/subtype selection profile antagonized the relaxant effect of isoproterenol in endothelium-denuded rat mesenteric artery rings precontracted with phenylephrine. At 10−7 M of ST-91, the antagonism was characterized by a rightward shift of isoproterenol dose–response curve (A50=6.81±1.40 e−7 (n=4) vs the control 1.29±0.25 e−7 M (n=4)) with no Emax depression. At 10−6 M the Emax depression was prevalent (36.1±7.0% (n=4) vs the control 79.9±5.1% (n=4)); both actions could be antagonized by the α2-adrenoceptor antagonist yohimbine. The not subtype-selective α2-adrenoceptor agonist xylazine (10−7 M) did not affect the relaxant action of isoproterenol. Present findings are discussed in the light of previously reported hemodynamic effects attributed to α2B-adrenoceptors in receptor subtype-knockout animals.

Myogenic reprogramming of retina-derived cells following their spontaneous fusion with myotubes

Satellite cells are recognized as the main source for myoblasts in postnatal muscle. The possible participation of other cell types in myofiber maintenance remains a subject of debate. Here, we investigated the potential of vascular preparations from mouse retina to undergo myogenesis when cultured alone or with differentiated primary myogenic cultures. The choice of retina, an organ richly supplied with capillary network and anatomically separated from skeletal muscles, ensures that the vasculature preparation is devoid of satellite cells. We demonstrate that retina-derived cells spontaneously fuse with preexisting myotubes and contribute additional myonuclei, some of which initiate expression of muscle-specific genes after fusion. Myogenic differentiation of retinal cells prior to their fusion with preexisting myotubes was not detected. Although originating from vasculature preparations, nuclei undergoing myogenic reprogramming were contributed by cells that were neither endothelial nor blood borne. Our results suggest smooth muscle/pericytes as the possible source, and that myogenic reprogramming depends on the muscle specific transcription factor MyoD. Our studies provide insights into a novel avenue for myofiber maintenance, relying on nuclei of non-myogenic origin that undergo fusion and subsequent myogenic conversion within host myofibers. This process may support ongoing myofiber maintenance throughout life.

B-9972 (D-Arg-[Hyp3,Igl5,Oic7,Igl8]-bradykinin) Is an Inactivation-Resistant Agonist of the Bradykinin B2Receptor Derived from the Peptide Antagonist B-9430 (D-Arg-[Hyp3,Igl5,D-Igl7,Oic8]-bradykinin): Pharmacologic Profile and Effective Induction of Receptor Degradation

The bradykinin B(2) receptor is a heptahelical receptor regulated by a cycle of phosphorylation, endocytosis, and extensive recycling at the cell surface following agonist stimulation. B-9430 (d-Arg-[Hyp(3),Igl(5),D-Igl(7),Oic(8)]-bradykinin) is a second generation peptide antagonist found to be competitive at the human B(2) receptor and insurmountable at the rabbit B(2) receptor (contractility assays, isolated human umbilical and rabbit jugular veins). Two isomers of this peptide were prepared: B-10344 (D-Arg-[Hyp(3),Igl(5),Oic(7),D-Igl(8)]-bradykinin; inverted sequence Oic(7), D-Igl(8)) and B-9972 (D-Arg-[Hyp(3),Igl(5),Oic(7),Igl(8)]-bradykinin); they are low- and high-potency agonists, respectively, in vascular preparations. The potency gap between bradykinin and B-9972 is narrow in contractility assays, despite the fact that B-9972 affinity is 7-fold inferior at the rabbit B(2) receptor (radioligand binding competition assay). The effects of agonists on receptors were compared using two chimerical constructions based on rabbit B(2) receptors: conjugate of the B(2) receptor with green fluorescent protein (B(2)R-GFP) and the N-terminally tagged conjugate of the myc epitope with the B(2) receptor. Imaging and immunoblotting showed that B-9972 induced a persistent endocytosis of cell surface B(2) receptors in human embryonic kidney 293 cells with slow receptor degradation (weak after 3 h of treatment, important at 12 h) and B(2)R-GFP desensitization ([(3)H]bradykinin endocytosis and extracellular signal-regulated kinase 1/2 phosphorylation assays). Bradykinin was not active in this respect but when combined with captopril, induced some degradation. B-9430 reduced the endocytosis and degradation of B(2) receptors by the agonists. The results illustrate the agonist-antagonist transition in B(2) receptor peptide ligands with a constrained C-terminal structure, the importance of species in their pharmacological profile, and the possibility of selectively degrading receptors using a peptidase-resistant agonist.

A new class of nitric oxide‐releasing derivatives of cetirizine; pharmacological profile in vascular and airway smooth muscle preparations

The pharmacological properties of compounds NCX 1512 and NCX 1514, synthesized by linking the histamine H1-receptor antagonist cetirizine to NO-releasing spacer groups, are reported. The aim was to establish if the compounds retained the antihistamine action of the parent compound, to assess their efficacy as NO donors and to test if they had broader antiallergic activity than cetirizine in the lung. Antihistamine activity of NCX 1512 and NCX 1514 was investigated in vitro in the guinea pig ileum, in tracheal rings (GPTR) and lung parenchymal strips (GPLP) of the guinea-pig. The NO-releasing capacity was investigated in vascular preparations; the isolated rabbit and guinea-pig aorta and guinea-pig pulmonary artery. Kinetics of NO release were assessed in a rat whole blood assay. Both NCX 1512 and NCX 1514 retained activity as H1-receptor antagonists in the guinea pig ileum and airway preparations. The NO-releasing NCX compounds relaxed the rabbit aorta, an action prevented by the guanylyl cyclase inhibitor ODQ (10 microM). NCX 1512 and NCX 1514 did not relax the antigen (ovalbumin) pre-contracted GPTR, whereas the NO donors NCX 2057 and DEA-NONOate relaxed guinea-pig pre-contracted vascular and tracheal preparations. Cetirizine (1-100 microM) and NCX 1512 (1-100 microM) reduced the cumulative (0.01-100 microg ml(-1)) ovalbumin-induced constriction in GPTR, but had no significant effect in GPLP. NCX 1512 and NCX 1514 act as antihistamines and NO donors. However, there was no improved effect compared to cetirizine on antigen-induced constriction of the central and peripheral lung.

Endothelium-dependent vascular responses induced by leukotriene B 4

Leukotriene B 4 (LTB 4) is an inflammatory mediator derived from the 5-lipoxygenase pathway of arachidonic acid metabolism and has recently implicated in the pathogenesis of atherosclerosis. There are two membrane bound receptors for LTB 4: BLT 1 and BLT 2, which represent the high and low affinity receptors, respectively. BLT receptors are expressed on leukocytes, and LTB 4 is a potent chemoattractant for neutrophils, eosinophils, and T lymphocytes. Recent studies have in addition shown that LTB 4 is an indirectly acting vasoconstrictor of isolated vascular preparations. In the guinea pig aorta, the LTB 4-induced contractions were inhibited by endothelium-denudation. In addition, pre-treatment with the NO synthase inhibitor, L-NOARG, significantly enhanced the contractions induced by LTB 4. The contractile response induced by LTB 4 in the guinea pig aorta was abolished by the selective BLT 1 receptor antagonist U75302 and the expression of BLT 1 receptor mRNA in the guinea pig aorta was established by RT-PCR. Taken together, these results suggest that LTB 4 activates BLT 1 receptors on the endothelium of the guinea pig aorta, associated with the release of both contractile factors and NO.

Vasculoprotective Effects of Anti-Tumor Necrosis Factor-α Treatment in Aging

Vascular aging is associated with dysregulation of tumor necrosis factor (TNF)-alpha expression. TNF-alpha is a master regulator of vascular proatherogenic phenotypic changes, and it has been linked to endothelial dysfunction and apoptosis. To test the hypothesis that anti-TNF-alpha treatment exerts vasculoprotective effects in aging, aged (29 months old) F344 rats were treated with etanercept (1 mg/kg/week for 4 weeks), which binds and inactivates TNF-alpha. In aged carotid arteries, relaxations to acetylcholine were decreased, and endothelial O2* production was increased (as shown by dihydroethidine fluorescence measurements). Etanercept treatment significantly improved responses to acetylcholine and decreased vascular NAD(P)H oxidase activity and expression. In aged carotid and coronary arteries, there were increases in DNA fragmentation rate and caspase 3/7 activity (indicating an increased rate of apoptotic cell death), which were attenuated by etanercept treatment. In aged vessels, there was an up-regulation of inflammatory markers, including inducible nitric-oxide synthase and intercellular adhesion molecule-1, which was decreased by etanercept treatment. In carotid arteries of young animals, recombinant TNF-alpha elicited endothelial dysfunction, oxidative stress, and increased apoptosis and proinflammatory gene expression, mimicking many of the symptoms of vascular aging. Thus, we propose that anti-TNF-alpha treatment exerts anti-aging vasculoprotective effects.

Field stimulation-induced tetrodotoxin-resistant vasorelaxation is mediated by sodium hypochlorite

This study was done to determine the mechanism of field stimulation-induced tetrodotoxin (TTX)- and NG- nitro-l-arginine (LNA)-resistant vasorelaxation. Field stimulation with platinum and carbon, but not with silver, electrodes (30 V, 30 HZ, 2-5 ms pulse width) as well as electrically stimulated salt (0.9% NaCl) solution (ESSS) or Krebs solution caused 100% relaxation of phenylephrine-contracted rat aortic strips, which was TTX and LNA resistant and endothelium independent. ESSS also relaxed other vascular preparations (rabbit aorta and renal artery, dog coronary artery, pig ductus arteriosus, and rat portal vein). The electric current generated hypochlorite (OCl-) and H2O2 from the salt solution; however, vasorelaxation was caused by NaOCl and not by H2O2. ESSS and NaOCl caused contraction failure of spontaneously beating right atria of rats and did not affect uterine contractions, vascular cAMP, cGMP, or the pH of the tissue bath. Field stimulation, ESSS, and NaOCl did not relax aortic preparations contracted by 32 mmol/L potassium and their vasorelaxant effects on phenylephrine-contracted rat aortic strips and rings were completely reversed by tetraethylammonium and partially by glibenclamide and iberiotoxin. We conclude that electric pulses generate the oxidant OCl- from the salt solution, which causes vasorelaxation by increasing K+ conductance.

Differential effects of ouabain on the vasodilator actions of nitric oxide and S-nitrosothiols in vivo: Relevance to the identity of EDRF/EDHF

Objectives This study examined the role of Na +/K +-ATPase in the vasodilator actions of nitric oxide (NO), S-nitrosothiols and the endothelium-dependent agonist, acetylcholine. Methods The vasodilator responses elicited by intravenous injections of (i) the NO-donors, sodium nitroprusside and MAHMA NONOate, (ii) the S-nitrosothiols, l- S-nitrosocysteine and S-nitrosocoenzyme A, and (iii) acetylcholine, in urethane-anesthetized rats. Results The NO-donors, S-nitrosothiols and acetylcholine elicited dose-dependent depressor responses and reductions in hindquarter (HQR) and mesenteric (MR) vascular resistances. The depressor responses and associated reductions in HQR elicited by NO-donors were markedly attenuated after injection of ouabain. In contrast, the depressor responses and reductions in HQR elicited by the S-nitrosothiols and acetylcholine were not affected. The reductions in MR elicited by all vasodilator agents were exaggerated after injection of ouabain. Finally, the decomposition of sodium nitroprusside, MAHMA NONOate, l- S-nitrosocysteine and S-nitrosocoenzyme A to NO upon addition to rat blood or vascular preparations was not affected by ouabain. Conclusion This study demonstrates that ouabain has opposing effects on NO-mediated vasodilation in resistance arteries in the hindquarter and mesenteric beds of the rat. The similarity of effects of ouabain on the vasodilator actions of acetylcholine, l- S-nitrosocysteine and S-nitrosocoenzyme A as opposed to the NO-donors supports the possibility that endothelium-derived relaxing factor released by acetylcholine in resistance arteries is an S-nitrosothiol.

Aldosterone Antagonism in Chronic Kidney Disease

Angiotensin-converting enzyme (ACE) inhibitors are potent members of the arsenal to treat chronic kidney disease (CKD). By reducing blood pressure (BP) and disproportionately decreasing intraglomerular pressure, this class of drugs also reduces proteinuria and slows progression of CKD (1,2). Given the high prevalence of cardiovascular disease in this population, it is noteworthy that ACE inhibitors also decrease the incidence of stroke, myocardial infarction (MI), and cardiovascular mortality in patients who are at high cardiovascular risk (3). More recently, data reporting similar benefits of angiotensin receptor blockers (ARB) support their use in the treatment of CKD as well, especially in individuals with type 2 diabetes (4). Furthermore, one sizable study suggested that the combination of an ACE inhibitor and ARB may be more effective than either agent alone (5). Parving and colleagues (6) called attention to the effects of high-dose ARB therapy, up to three times the recommended dose, as an additional means of effectively interrupting the renin-angiotensin-aldosterone system (RAAS). By logical extension, further blockade of the RAAS by direct antagonism of aldosterone also may prove beneficial. Indeed, aldosterone seems to be a potent effector of renal injury (7–9). In the rare cases of primary aldosteronism and its functional analogue, the even more rare Liddle’s syndrome, the observed renal injury probably is independent of the more proximal elements of the RAAS (10,11). Animal models provide an expeditious tool for assessing pathophysiologic change and the efficacy of intervention. The classic model of primary aldosteronism, the mineralocorticoid–nephrectomy–high-salt model of hypertension, develops systemic and glomerular capillary hypertension and sustains renal damage (12). In the remnant kidney model of CKD, ACE inhibitors and ARB attenuate renal injury (13). However, this protection, which is associated with suppression of aldosterone secretion, is abrogated by exogenous aldosterone infusion with return of …

Molecular characterization of Kv2.1 channels in sheep vasculature: Role of Kv channels in regulating vascular tone

The voltage-gated potassium channel 2.1 (Kv2.1) is regarded as one of the primary oxygen sensitive potassium channels in the pulmonary circulation. The dilator function of Kv2.1 is enhanced in isolated sheep conduit compared to resistance pulmonary arteries, and may be increased during development of the same animal. However, neither the functional role nor distributions of Kv2.1 in other vasculatures have been described in detail. Thus, the goal of this study was to use RT-PCR and tension-recording to compare the transcript expression and dilator influence of Kv2.1 channels between isolated sheep conduit and resistance pulmonary, coronary and renal arteries. Using human Kv2.1 channel primers, we demonstrated the expression of Kv2.1 channel mRNA in all vascular preparations. 4-Aminopyridine (4-AP) (0.001–3 mM), a nonspecific blocker of Kv channels, produced concentrationdependent contractions of conduit pulmonary and renal arteries. On the other hand, 4-AP had no significant effect on the basal tone of isolated sheep resistance artery and coronary arteries. These results suggest that Kv channels may play an important role in regulating the tone of sheep conduit and renal arteries, but they do not appear to contribute to the regulation of basal tone in resistance pulmonary arteries and coronary arteries. The latter observation raises the possibility that Kv2.1 channels are not the primary contributor to hypoxic pulmonary vasoconstriction in sheep. IVRI research support, India.

Characterisation of CGRP receptors in the human isolated middle meningeal artery

Although the understanding of migraine pathophysiology is still incomplete, there seems to be little doubt that dilatation of cranial blood vessels, including meningeal arteries, is involved in the headache phase of migraine. Since calcitonin gene-related peptide (CGRP) has been implicated in this vasodilatation, the present study set out to compare the relaxant effects of the endogenous ligand h-αCGRP, and [ethylamide-Cys 2,7]h-αCGRP ([Cys(Et) 2,7]h-αCGRP), a CGRP 2 receptor agonist, on human isolated middle meningeal artery segments, precontracted with KCl. Classical Schild plot analysis was used to characterise the receptor population in this artery using BIBN4096BS and h-αCGRP 8–37 as antagonists. h-αCGRP relaxed arterial segments more potently than [Cys(Et) 2,7]h-αCGRP (pEC 50: 8.51 ± 0.16 and 7.48 ± 0.24, respectively), while the maximal responses to these agonists were not significantly different. BIBN4096BS equipotently blocked the relaxations induced by both agonists with a p A 2 of ∼ 10 and with a Schild plot slope not significantly different from unity. h-αCGRP 8–37 also antagonised the response to h-αCGRP with a p A 2 of 6.46 ± 0.16 and a Schild plot slope not different from unity. Furthermore, the results obtained from RT-PCR studies confirmed the presence of all the essential components required for a functional CGRP 1 receptor in these arteries. Considering the high antagonist potency of BIBN4096BS, coupled to the lower agonist potency of [Cys (Et) 2,7]h-αCGRP, it is reasonable to suggest a predominant role of CGRP 1 receptors in the human middle meningeal artery. This view is reinforced by Schild plot analysis, which revealed a slope of unity in all experiments, giving further evidence for a homogeneous CGRP receptor population in this vascular preparation.

Methanandamide Hyperpolarizes Gastric Arteries by Stimulation of TRPV1 Receptors on Perivascular CGRP Containing Nerves

Endogenous as well as synthetic cannabinoids have potent vasodilatory actions in a variety of vascular preparations. Their precise mechanism of action is as yet unclear, but several studies point to the activation of type 1 vanilloid (TRPV1) receptors on primary afferent perivascular nerves, stimulating the release of calcitonin gene related peptide (CGRP). Given the documented gastroprotective function of these nerves, and the various gastrointestinal effects reported for cannabinoids, we explored a possible link between these systems in the gastric circulation by comparing responses of small gastric arteries to cannabinoids and to calcitonin gene related peptide using conventional microelectrode techniques. Exposure of small gastric arteries to the stable endocannabinoid analogue methanandamide caused a hyperpolarization of the vascular smooth muscle cells, which was completely abolished by the vanilloid receptor antagonist capsazepine (P < 0.01). Exposure to exogenous calcitonin gene related peptide evoked fully reproducible (P > 0.05) hyperpolarizations with similar time course, unaffected by capsazepine. Preincubation with glibenclamide, an inhibitor of ATP-sensitive potassium (KATP) channels, reversed both responses to methanandamide (P < 0.01) and calcitonin gene related peptide (P < 0.05). Similar results were found in rat mesenteric arteries. These findings show that cannabinoids stimulate TRPV1 receptors, presumably causing the release of calcitonin gene related peptide, which hyperpolarizes the smooth muscle cells by activation of KATP channels. Because membrane hyperpolarization is a powerful mediator of vasorelaxation, this novel pathway might prove to be an important mechanism affording gastroprotection.

Synthesis and smooth muscle-selective relaxant activity of a piperidine analogue: 1-(4′-fluorophenacyl)-4-hydroxy-4-phenyl-piperidinium chloride

The antispasmodic and vasodilator activities of a newly synthesized piperidine derivative (1-(4'-fluorophenacyl)-4-hydroxy-4-phenyl-piperidinium chloride) were studied in vitro. The test compound exhibited a dose-dependent relaxant effect on the spontaneous and K+ (75 mM)-induced contractions of isolated rabbit jejunum with respective EC50 values of 0.01 mM (0.01-0.02, 95% Cl) and 0.30 mM (0.17-0.56). The Ca++ channel blocking (CCB) activity was confirmed when the test compound (0.1-0.2 mM) shifted the Ca++ dose-response curves to the right, similar to that produced by verapamil (0.1-1.0 microM), a standard CCB. In the isolated rabbit aorta, the test compound showed a dose-dependent vasodilator effect on K+ (75 mM)-induced contractions with an EC50 value of 0.08 mM (0.02-0.26) while also suppressed the norepinephrine (1 microM) control peak responses with EC50 value of 0.08 mM (0.05-0.13, n=5). When tested in Langendorff perfused rabbit heart preparation, the test compound exhibited a negligible inhibitory effect on the rate or force of atrial and ventricular contractions when tested up to 5 mM. The results show smooth muscle-selective relaxant effect of the test compound on intestinal and vascular preparations mediated possibly via blockade of voltage and receptor-operated Ca++ channels.