Miriam Durante

L-type Ca2+ channel current characteristics are preserved in rat tail artery myocytes after one-day storage

To develop a cheap and simple method of storing for 24‐h vascular tissue and single myocytes while preserving therein the biophysical and pharmacological characteristics of L‐type Ca2+ channels and contractile activity.

Quercetin mitochondriotropic derivatives antagonize nitrate tolerance and endothelial dysfunction of isolated rat aorta rings.

Chronic use of glyceryl trinitrate is limited by serious side effects, inter alia tolerance and endothelial dysfunction of coronary and resistance arteries. The natural flavonoid quercetin has been shown to counteract the development of glyceryl trinitrate tolerance in vitro. Two mitochondriotropic, 4-O-triphenylphosphoniumbutyl derivatives of quercetin (QTA-3BTPI and Q-3BTPI) were compared to quercetin for protection against glyceryl trinitrate-induced tolerance and endothelial dysfunction of isolated rat aorta rings. Both QTA-3BTPI and Q-3BTPI significantly counteracted the reduced vascular responsiveness to both glyceryl trinitrate and acetylcholine caused by prolonged exposure of the vessel to glyceryl trinitrate itself, their potency being much greater than that of quercetin. QTA-3BTPI, however, turned out to cause endothelial dysfunction per se. Since Q-3BTPI antagonized in vitro nitrate tolerance and endothelial dysfunction of vessels, this encourages assessing whether this effect is displayed also in vivo during long-term glyceryl trinitrate treatment.

The vasodilator papaverine stimulates L-type Ca(2+) current in rat tail artery myocytes via a PKA-dependent mechanism.

Papaverine is an opium alkaloid, primarily used as an antispasmodic drug and as a cerebral and coronary vasodilator. Its phosphodiesterase inhibitory activity promotes increase of cAMP levels mainly in the cytosol. As cAMP is known to modulate L-type Ca(2+) channel activity, here we tested the proposition that papaverine could affect vascular channel function. An in-depth analysis of the effect of papaverine on Ba(2+) or Ca(2+) current through L-type Ca(2+) channel [IBa(L) or ICa(L)], performed in rat tail artery myocytes using either the whole-cell or the perforated patch-clamp method, was accompanied by a functional study on rat aorta rings. Papaverine increased current amplitude under both the perforated or whole-cell configuration. Stimulation of the current by papaverine was concentration-, Vh-, frequency-, and charge carrier-dependent, and fully reverted by drug washout. The PKA inhibitor H89, but not the PKG inhibitor Rp-8-Br-cGMPS, antagonised papaverine- as well as IBMX- (another phosphodiesterase inhibitor) induced IBa(L) stimulation. In cells pre-treated with IBMX, application of papaverine failed to increase current amplitude. Papaverine sped up the inactivation kinetics of IBa(L), though only at concentrations ≥ 30 μM, and shifted the voltage dependence of the inactivation curve to more negative potentials. In rings, the vasorelaxing activity of papaverine was enhanced by previous treatment with nifedipine. In conclusion, papaverine stimulates vascular L-type Ca(2+) channel via a PKA-dependent mechanism, thus antagonising its main vasodilating activity.

Murrayafoline A modulation of rat vascular myocyte Cav1.2 channel: functional, electrophysiological and molecular docking analysis

The carbazole alkaloid murrayafoline A (MuA) enhances contractility and the Ca2+ currents carried by the Cav1.2 channels [ICa1.2] of rat cardiomyocytes. As only few drugs stimulate ICa1.2, this study was designed to analyse the effects of MuA on vascular Cav1.2 channels.

Vascular L-Type Ca2+ Channel Blocking Activity of Sulfur-Containing Indole Alkaloids from Glycosmis petelotii

In the search for novel natural compounds endowed with potential antihypertensive activity, a new sulfur-containing indole alkaloid, N-demethylglypetelotine (2), and its known analogue glypetelotine (1), were isolated from the leaves of Glycosmis petelotii. Their structures were established on the basis of spectroscopic evidence. The two alkaloids were assessed for vasorelaxing activity on rat aorta rings and for L-type Ba(2+) current [I(Ba(L))] blocking activity on single myocytes isolated from rat tail artery. Both glypetelotine and N-demethylglypetelotine inhibited phenylephrine-induced contraction with IC50 values of 20 and 50 μM, respectively. The presence of endothelium did not modify their spasmolytic effect. Neither glypetelotine nor N-demethylglypetelotine affected Ca(2+) release from the sarcoplasmic reticulum induced by phenylephrine. The spasmolytic effect of glypetelotine increased with membrane depolarization. In the presence of 60 mM K(+), both compounds inhibited, in a concentration-dependent manner, the contraction induced by cumulative addition of Ca(2+), this inhibition being inversely related to Ca(2+) concentration. Glypetelotine and, less efficiently N-demethylglypetelotine, inhibited I(Ba(L)), the former compound also affecting I(Ba(L)) kinetics. In conclusion, glypetelotine is a novel vasorelaxing agent which antagonizes L-type Ca(2+) channels.

Functional, electrophysiological and molecular docking analysis of the modulation of Cav 1.2 channels in rat vascular myocytes by murrayafoline A.

The carbazole alkaloid murrayafoline A (MuA) enhances contractility and the Ca2+ currents carried by the Cav1.2 channels [ICa1.2] of rat cardiomyocytes. As only few drugs stimulate ICa1.2, this study was designed to analyse the effects of MuA on vascular Cav1.2 channels.

2-Aryl- and 2-amido-benzothiazoles as multifunctional vasodilators on rat artery preparations

The neuroprotective agent riluzole [2-amino-6-(trifluoromethoxy)benzothiazole] has been shown to antagonize neuronal high-voltage activated Ca(2+) currents. In the search for novel scaffolds leading to potential antihypertensive agents, a series of 2-aryl- and 2-amido-benzothiazoles (HUP) were assessed for their vasorelaxing property on rat aorta rings and for their L-type Ba(2+) currents [I(Ba(L))] blocking activity on single myocytes isolated from the rat tail artery. HUP5 and HUP30, the most potent of the series, inhibited phenylephrine-induced contraction with IC₅₀ values in the range 3-6 µM. The presence of endothelium did not modify their spasmolytic activity. Both HUP5 and HUP30 increased tissue levels of cGMP and shifted to the left the concentration-response curve to sodium nitroprusside. In rings precontracted by phenylephrine, tetraethylammonium or 1H-[1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one (ODQ) shifted to the right the concentration-relaxation curves of HUP5 and HUP30. The antispasmodic effect of HUP5 and HUP30 was more marked on rings stimulated with 25/30 mM than with 60 mM K(+). HUP5 and HUP30 antagonized both extracellular Ca(2+) influx and Ca(2+) mobilization from intracellular stores in response to phenylephrine: this effect was not modified by the presence of ODQ. I(Ba(L)) was partly inhibited by HUP5 and blocked by HUP30 in a concentration-dependent as well as ODQ-independent manner. In conclusion, HUP5 and HUP30 are vasorelaxing agents that stimulate soluble guanylyl cyclase, activate K(+) channels, and block extracellular Ca(2+) influx. The present benzothiazole derivatives form a novel class of multifunctional vasodilators which may give rise to effective antihypertensive agents.

In Vitro Vasoactivity of Zerumbone from Zingiber zerumbet

The sesquiterpene zerumbone, isolated from the rhizome of Zingiber zerumbet Sm., besides its widespread use as a food flavouring and appetiser, is also recommended in traditional medicine for the treatment of several ailments. It has attracted great attention recently for its effective chemopreventive and therapeutic effects observed in various models of cancer. To assess the zerumbone safety profile, a pharmacology study designed to flag any potential adverse effect on vasculature was performed. Zerumbone was tested for vasorelaxing activity on rat aorta rings and for L-type Ba(2+) current blocking activity on single myocytes isolated from the rat-tail artery. The spasmolytic effect of zerumbone was more marked on rings stimulated with 60 mM than with 30 mM K(+) (IC50 values of 16 µM and 102 µM, respectively). In the presence of 60 mM K(+), zerumbone concentration-dependently inhibited the contraction induced by the cumulative additions of Ca(2+), this inhibition being inversely related to the Ca(2+) concentration. Phenylephrine-induced contraction was inhibited by the drug, though less efficiently and independently of the presence of an intact endothelium, without affecting Ca(2+) release from the intracellular stores. Zerumbone inhibited the L-type Ba(2+) current (estimated IC50 value of 458.7 µM) and accelerated the kinetics of current decay. In conclusion, zerumbone showed an overall weak in vitro vasodilating activity, partly attributable to the blocking of the L-type Ca(2+) channel, which does not seem to represent, however, a serious threat to its widespread use.

Vascular Toxicity Risk Assessment of MC18 and MC70, Novel Potential Diagnostic Tools for In Vivo PET Studies

The P‐glicoprotein (P‐gp) inhibitor MC18 has been recently proposed as a valuable PET tracer to measure P‐gp expression in vivo. The aim of this study was to evaluate the toxic hazard towards the vasculature of MC18 along with the structurally related and more potent P‐gp inhibitor MC70. Their effects on A7r5 and EA.hy926 cells viability, on the mechanical activity of fresh and cultured rat aorta rings as well as on Cav1.2 channel current (ICa1.2) of A7r5 cells were studied. At concentrations >10 μM, MC18 and MC70 decreased cell viability causing evident morphological changes. In fresh rat aorta rings, both compounds (0.1–100 μM) antagonized phenylephrine‐induced contraction in a concentration‐dependent manner, with IC50 values in the range of 1.67–14.49 μM, whereas only MC18 caused a concentration‐dependent decrease of the 60 mM K+ (K60)‐induced responses. In rings cultured for 7 days with both compounds (1–10 μM), 10 μM MC70 significantly reduced, while 10 μM MC18 completely prevented the contractile response to both phenylephrine and K60. MC18 and MC70 (0.1–100 μM) inhibited ICa1.2 in a concentration‐dependent manner with IC50 values of 16.81 and 32.13 μM, respectively. These findings demonstrate that MC18‐induced vascular effects took place at concentrations that are at least three orders of magnitude higher than those (≤10 nM) allowing in vivo measurement of P‐gp expression. Thus, MC18, and possibly MC70, can be considered promising PET tools for in vivo P‐gp quantification.

MC225, a novel probe for P-glycoprotein PET Imaging at the blood-brain barrier: in vitro cardiovascular safety evaluation

Abstract:The P-glycoprotein (P-gp) substrate MC225, at concentrations ⩽10 nM, is a valuable radiotracer for positron emission tomography imaging of P-gp function in rats and mice. The aim of this study was to evaluate its potential toxic hazard toward the cardiovascular system through an in-depth analysis of its effects on rat aorta rings, on CaV1.2 channel current (ICa1.2) of A7r5 cells and on Langendorff-perfused rat heart. In aortic rings, MC225 relaxed phenylephrine-induced contraction in a concentration-dependent and endothelium-independent manner, with an IC50 value of about 1 &mgr;M. At concentrations ≥3 &mgr;M, it antagonized the response to cumulative concentrations of K+. MC225, 1 and 10 &mgr;M, inhibited ICa1.2 by 15% and 31%, respectively, without affecting either current activation or inactivation kinetics. In Langendorff-perfused rat hearts, only 10 &mgr;M MC225 significantly decreased left ventricular pressure and increased coronary perfusion pressure while reducing heart rate and prolonging the cardiac cycle length as well as the atrioventricular conduction time (PQ interval) on the electrocardiogram. Lower concentrations of the drug were ineffective. These findings demonstrate that MC225-induced cardiovascular effects took place at concentrations that are at least 2 orders of magnitude higher than those allowing in vivo measurement of P-gp function. Therefore, MC225 represents a promising positron emission tomography tool for in vivo straightforward P-gp quantification.