Opposing roles of endothelial and smooth muscle phosphatidylinositol 3-kinase in vasoconstriction: effects of rho-kinase and hypertension

Phosphatidylinositol 3-kinase (PI3K) can activate endothelial nitric oxide synthase (eNOS), leading to production of the vasodilator NO. In contrast, vascular smooth muscle (VSM) PI3K may partially mediate vascular contraction, particularly during hypertension. We tested whether endothelial and VSM PI3K may have opposing functional roles in regulating vascular contraction. Secondly, we tested whether the procontractile protein rho-kinase can suppress endothelial PI3K/eNOS activity in intact arteries, thus contributing to vasoconstriction by G protein-coupled receptor (GPCR) agonists. We studied contractile responses to the GPCR agonist phenylephrine, and the receptor-independent vasoconstrictor KCl, in aortic rings from Sprague-Dawley rats. In endothelium-intact rings, the PI3K inhibitor wortmannin (0.1 microM) markedly augmented responses to phenylephrine (P < 0.05) by approximately 50% but not to KCl. However, in endothelium-denuded or N(G)-nitro-L-arginine methyl ester (L-NAME) (100 microM)-treated rings, wortmannin reduced responses to phenylephrine and KCl (P < 0.05). Furthermore, the rhokinase inhibitor Y-27632 (R-[+]-trans-N-[4-pyridyl]-4-[1-aminoethyl]-cycloheaxanecarboxamide; 1 microM) abolished responses to phenylephrine, and this effect was partially reversed by wortmannin or L-NAME. The ability of wortmannin to oppose the effect of rho-kinase inhibition on contractions to phenylephrine was L-NAME-sensitive. In aortas from angiotensin II-induced hypertensive rats, relaxation to acetylcholine (10 microM) was impaired (P < 0.05), and vasoconstriction by phenylephrine was markedly enhanced and not further augmented by wortmannin. These data suggest that endothelial PI3K-induced NO production can modulate GPCR agonist-induced vascular contraction and that this effect is impaired in hypertension in association with endothelial dysfunction. In addition, endothelial rho-kinase may act to suppress PI3K activity and, hence, attenuate NO-mediated relaxation and augment GPCR-dependent contraction.     

Antidiabetic omarigliptin dilates rabbit aorta by activating voltage-dependent K+ channels and the sarco/endoplasmic reticulum Ca2+-ATPase pump

We investigated the vasodilatory effect of omarigliptin, an oral antidiabetic drug in the dipeptidyl peptidase-4 inhibitor class, and its related mechanisms using phenylephrine (Phe)-induced pre-contracted aortic rings. Omarigliptin dilated aortic rings pre-constricted with Phe in a dose-dependent manner. Pretreatment with the voltage-dependent K⁺ channel inhibitor 4-aminopyridine significantly attenuated the vasodilatory effect of omarigliptin, whereas pretreatment with the inwardly rectifying K⁺ channel inhibitor Ba²⁺, ATP-sensitive K⁺ channel inhibitor glibenclamide, and large-conductance Ca²⁺-activated K⁺ channel inhibitor paxilline did not alter its vasodilation. Pretreatment with the sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) pump inhibitors thapsigargin and cyclopiazonic acid significantly reduced the vasodilatory effect of omarigliptin. Neither cAMP/PKA-related signaling pathway inhibitors nor cGMP/PKG-related signaling pathway inhibitors modulated the vasodilatory effect of omarigliptin. Removal of endothelium did not diminish the vasodilatory effect of omarigliptin. Furthermore, pretreatment with the nitric oxide synthase inhibitor L-NAME or small-conductance Ca²⁺-activated K⁺ channel inhibitor apamin, together with the intermediate-conductance Ca²⁺-activated K⁺ channel inhibitor TRAM-34, did not influence the vasodilatory effect of omarigliptin. In conclusion, omarigliptin induced vasodilation in rabbit aortic smooth muscle by activating voltage-dependent K⁺ channels and the SERCA pump independently of other K⁺ channels, cAMP/PKA- and cGMP/PKG-related signaling pathways, and the endothelium.     

Influence of the endothelium on Ca2+ dependency of angiotensin II-induced contractions of rat aortic rings

Summary— Endothelium-dependent relaxation has been demonstrated to be involved in the regulation of vascular tone and extracellular Ca²⁺ was found to play a prominent role in this process. Since the dependency on extracellular Ca²⁺ appeared to differ considerably within the arterial tree, possibly as a consequence of vessel-related endothelium-dependent mechanisms, we investigated the effects of different compounds affecting Ca²⁺ entry (nifedipine, CoCl₂) on angiotensin II-induced contractions of rat aortic rings with and without endothelium as well as the responses in a Ca²⁺–“free” solution. For this purpose, rat aortic rings were either undone from their endothelial layer by gentle mechanical rubbing or care was taken to keep the intima intact in case rings where endothelium were required. The presence of an intact endothelium was confirmed by acetylcholine-induced relaxation. A stronger responsiveness towards angiotensin I, both after a complete concentration-response curve and after a single maximal concentration of angiotensin II was observed in arterial segments without endothelium. The maximal contraction to a single concentration of angiotensin II (0.1 μM) in the rings without endothelium amounted to 75.8 ± 3.8% of the preceding response to a supramaximal concentration of noradrenaline (= E_max). In rings without the endothelial layer, the contraction was 34.8 ± 3.7% of E_max. This indicates an endothelium-induced relaxation in aortic rings with endothelium. After incubation with the Ca²⁺ entry blocker nifedipine (1 μM) both rings with and without endothelium were inhibited to the same extent, contractions amounted to 30.7 ± 1.8% and 19.6 ± 1.3% of E_max, respectively. However, incubation in a Ca²⁺-“free” medium for 5 min resulted in similar contractions for rings without endothelium (16.4 ± 1.4% of E_max) as for rings with endothelium (15.0 ± 1.6% of E_max). Moreover, CoCl₂ in a concentration of 300 μM hardly inhibited the contraction of rings with an intact endothelium, a contractile response of 30.5 ± 2.8% of E_max was observed. The results of the study suggest that the influx of Ca²⁺ions is indeed responsible for the endothelium-mediated relaxation. However, this influx, which cannot be antagonized by nifedipine, but has shown to be affected by CoCl₂, suggests that channels intensitive to organic Ca²⁺ entry blockers may be involved.     

New insights into the mechanisms of the vasorelaxant effects of apocynin in rat thoracic aorta

Apocynin is a naturally occurring acetophenone widely used as an inhibitor of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Recent data suggested that apocynin might exert NADPH oxidase‐independent pharmacological properties. Among them, vasorelaxant properties have been described, but the mechanisms still give rise to debates. The present study investigated the mechanisms involved in the vasorelaxant effect of apocynin on the in vitro model of rat isolated thoracic aortic rings. Apocynin (30 μm to 10 mm ) induced a dose‐dependent relaxation in both endothelium‐intact and endothelium‐denuded aortic rings with respective EC₅₀ values of 0.78 ± 0.08 and 1.91 ± 0.21 mm . Endothelium removal or inhibition of nitric oxide (NO) synthase with N^ω‐nitro‐l ‐arginine‐methyl ester (l ‐NAME) significantly decreased but did not abolish the effect of apocynin. By contrast, apocynin‐induced relaxation was unchanged after incubation with indomethacin or charybdotoxin plus apamin. In endothelium‐denuded aortas, the vasorelaxant effect of apocynin was significantly reduced by glibenclamide but not by 4‐aminopyridine nor by iberiotoxin. Apocynin significantly decreased Ca²⁺‐induced contraction and inhibited intracellular Ca²⁺mobilization after contraction with phenylephrine. Finally, the acute intravenous injection of apocynin led to an immediate and transient hypotensive effect in spontaneously hypertensive rats (SHR). In conclusion, our data demonstrated that apocynin induces both endothelium‐independent relaxant effects involving inhibition of Ca²⁺mobilization and activation of K_ATP channels in vascular smooth muscle cells and endothelium‐dependent effects mediated by NO. These results should provide a basis for caution when interpreting results on the vascular effects of apocynin.     

Vasodilatory activity and antihypertensive profile mediated by inhibition of phosphodiesterase type 1 induced by a novel sulfonamide compound

LASSBio‐985 is a sulfonamide compound designed as a simplified structure of a nonselective phosphodiesterase type 4 (PDE‐4) inhibitor that promotes vasodilatory activity in vitro. PDE are enzymes responsible for the hydrolysis of cyclic adenosine 3′,5′‐ monophosphate and cyclic guanosine 3′,5′‐monophosphate. Five different isozymes of PDE are found in vascular smooth muscle (PDE1–PDE5). Aortic rings, with or without endothelium, from male normotensive and spontaneously hypertensive rats (SHR) were prepared for isometric tension recording. Blood pressure was measured in Wistar Kyoto (WKY) rats and SHR during intravenous infusion of LASSBio‐985 (10 mg/kg/min) during 15 min. LASSBio‐985 induced a concentration‐dependent vasodilation in aortic rings from normotensive and SHR, which was almost completely inhibited in endothelium‐denuded vessels. Vasodilatory activity was also reduced in endothelium‐intact aortic rings that had been pretreated with N_ω‐nitro‐l ‐arginine methyl ester hydrochloride (l ‐NAME), a nitric oxide synthase inhibitor and 1H‐[1,2,4]oxadiazolod[4,3‐a]quinoxalin‐1‐one (ODQ), a guanylate cyclase inhibitor. LASSBio‐985‐induced vasodilation was also inhibited by sildenafil (100 μm ) and SQ 22536, a PDE5 inhibitor and adenylate cyclase inhibitor, respectively. To evaluate the involvement of some endothelial receptors, atropine, diphenhydramine, HOE 140, naloxone, propranolol, indomethacin, and wortmannin were tested, but none inhibited the effects of LASSBio‐985. The residual effect observed on endothelium‐denuded aortic rings was abolished by nicardipine, a voltage‐sensitive‐Ca²⁺‐channel blocker. Intravenous infusion of LASSBio‐985 (10 mg/kg/min) significantly reduced systolic and diastolic pressures in both WKY and SHR. LASSBio‐985 is a compound with vasodilatory activity, which could be consequent to PDE1 inhibition and voltage‐sensitive‐Ca²⁺‐channel blockade.     

The role of endothelium in phenylephrine- and potassium-induced contractions of the rat aorta during pregnancy

The involvement of endothelium in the contractile responses of rat aortic rings to phenylephrine and potassium chloride in pregnancy was examined. Contractions in response to both agents were significantly greater in rings from non-pregnant rats than in rings from pregnant rats, and they were unaltered by treatment of the rings with indomethacin. De-endothelialization potentiated the contractions of rings from pregnant rats in response to phenylephrine, but had no significant effect on similar rings contracted with potassium chloride. Whereas de-endothelialization had no significant effect on the contractions to phenylephrine in rings from non-pregnant rats, it decreased those of rings from the same type of rats, contracted with potassium chloride. Pregnancy significantly inhibited contractions in response to calcium chloride of rings treated with phenylephrine or potassium chloride. The effect of endothelium removal on contractions to calcium chloride in rings from pregnant and non-pregnant rats treated with phenylephrine or potassium chloride was similar to that observed for phenylephrine-induced and potassium chloride-induced contractions, respectively. Contractions of intact aortic rings from pregnant and non-pregnant rats to phenylephrine in calcium-free medium were similar. Results of the study suggest that the effect of pregnancy on the contractions of the rat aorta in response to phenylephrine and potassium chloride is at least partly mediated by the endothelium and is independent of prostaglandin synthesis. The endothelial factor involved in this effect appears to modulate contractions by interfering with calcium influx through the receptor-operated calcium channels and the voltage-operated calcium channels.     

Effect of inhibition of the electrogenic Na+/K+ pump on the mechanical activity in the rat uterus

Summary— The effects of ouabain and K⁺-free solution were studied in estrogen-primed rat uterine strips under resting tone or repeatedly stimulated with KCl, acetylcholine or oxytocin applied for 20 minutes at 60 minute intervals. These effects were compared with those of the K⁺ channel opener cromakalim. In preparations under resting tone, ouabain (0.1 mM and 0.3 mM) induced rhythmic contractions which disappeared after 20–30 minutes whereas at a higher concentration (1 mM) it evoked a rapid, phasic response followed by a small tonic contraction. Exposure of the strip to a K⁺-free solution induced either rhythmic waves, which ceased after 8–10 minutes, or a single phasic contraction which was followed by a small and slow increase in the resting tone (54 ± 10 mg after 180 min exposure). Nifedipine (0.3 μM) abolished the rhythmic or phasic component of these responses but failed to modify the late small tonic contraction induced by ouabain 1 mM or by K⁺-free solution. Ouabain (0.1–1 mM) or K⁺-free-evoked responses disappeared after short (4 min) or prolonged (60 min) exposure to a Ca²⁺-free, 3 mM EGTA-containing solution. Cromakalim (10 nM ?0.1 mM) did not induce any variation in the resting tone either in the presence or in the absence of Ca²⁺ in the medium. In strips repeatedly stimulated with acetylcholine (0.1 mM) or oxytocin (1 μM), ouabain (0.3 mM), K⁺-free-solution and cromakalim (10 μM) reduced the amplitude of the initial, phasic response and progressively decreased the oscillatory component of the response to these agonists. Conversely, the successive responses evoked by KCl 60 mM in similar experimental conditions were not affected by ouabain or cromakalim. Ouabain (0.3 mM), K⁺-free solution and cromakalim (10 μM) decreased the Ca²⁺-independent, maintained contractions induced by acetylcholine or oxytocin after prolonged exposure to a Ca²⁺-free, EGTA-containing medium. These inhibitory effects were partially or completely reversed in the presence of the non-selective potassium channel blocker tetraethylammonium (10 mM) or in a Ca²⁺-free solution containing 60 mM K⁺. In conclusion, these results suggest that the response induced by ouabain or K⁺-free solution in estrogen-primed rat myometrium involves Ca²⁺ influx through potential-operated calcium channels but not Ca²⁺ release from intracellular stores. In addition, our results show that prolonged exposure to ouabain or K⁺-free medium decreases membrane receptor-mediated responses in rat uterus. This inhibitory effect seems to be the result, at least in part, of a decrease in the cytosolic level of K⁺, due to the inhibition of the electrogenic Na⁺ pump.     

Vasorelaxant effects of the monoterpenic phenol isomers,carvacrol and thymol,on rat isolated aorta

Various essential oils are rich in carvacrol, a monoterpenic phenol isomeric with thymol. This study was undertaken to assess the vasorelaxant effects of thymol and carvacrol in rat isolated aorta and the putative mechanisms underlying these effects. Thymol and carvacrol produced a concentration‐dependent relaxation on the aortic ring preparations pre‐contracted using KCl (IC₅₀ value of 64.40 ± 4.41 and 78.80 ± 11.91 μm , respectively) or using phenylephrine (PHE, 0.1 μm ) (IC₅₀ value of 106.40 ± 11.37 and 145.40 ± 6.07 μm , respectively) and inhibited the concentration‐response curves of aortic rings to PHE or KCl. In Ca²⁺‐free medium with ethylene glycol‐bis(2‐aminoethylether)‐N,N,N′,N′‐tetraacetic acid (2 mm ), thymol and carvacrol both at 1000 μm completely abolished the phasic component of PHE‐induced endothelium‐containing ring contractions. At 400 μm , thymol and carvacrol significantly reduced the CaCl₂‐induced contractions in Ca²⁺‐free medium. Furthermore, both thymol and carvacrol (300 and 1000 μm ) significantly reduced the contraction evoked by phorbol dibutyrate (1 μm ), an activator of protein kinase C. Magnitude of this inhibitory effect was enhanced in the presence of the Ca²⁺ pump inhibitor, thapsigargin (1 μm ). At 1000 μm , neither thymol nor carvacrol altered the resting potential of vascular smooth muscle cells. In conclusion, thymol and carvacrol induced an endothelium‐independent relaxation in rat isolated aorta, an effect that seems mediated through some mechanisms probably involving a transduction pathway between Ca²⁺ release from sarcoplasmic reticulum and/or regulation of the Ca²⁺ sensitivity of the contractile system. Moreover, it’s conceivable that thymol and carvacrol, at low concentrations, block the Ca²⁺ influx through the membrane.     

Inhibitors of calmodulin impair the constitutive but not the inducible nitric oxide synthase activity in the rat aorta.

The possibility that calmodulin inhibitors impair the constitutive but not the inducible nitric oxide synthase(s)-mediated inhibitions of tone was investigated in the rat aorta. The endothelium-dependent relaxations evoked by acetylcholine, ATP and the calcium ionophore A23187 (which are mediated by the constitutive nitric oxide synthase) were inhibited by calmodulin inhibitors [calmidazolium, W-7 and (N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide, hydrochloride, fendiline] and by an inhibitor of nitric oxide synthase, nitro L-arginine. Nitro L-arginine but not calmidazolium reduced the inhibitory influence of the endothelium on the concentration-contraction curves evoked by phenylephrine. Treatment of aortic rings without endothelium with interleukin-1 beta inhibited the contractions to phenylephrine by inducing nitric oxide synthase activity. Nitro L-arginine but not calmidazolium restored the contractility of the aortic rings. The relaxations evoked by a donor of nitric oxide, 3-morpholino-sydnonimine, were minimally affected by calmidazolium and nitro L-arginine. The basal tissue content in, and the production of, guanosine 3',5' cyclic monophosphate evoked by acetylcholine in rings with endothelium were inhibited by calmidazolium and nitro L-arginine. The production of cyclic GMP evoked by interleukin-1 beta in rings without endothelium was inhibited by nitro L-arginine but not by calmidazolium. These observations indicate that calmodulin inhibitors inhibit the constitutive but not the inducible nitric oxide synthase(s) in the rat aorta.     

Amorphous nanosilica particles evoke vascular relaxation through PI3K/Akt/eNOS signaling

There have been several reported studies on the distribution and/or toxicity of nanosilica particles. However, the influence of these particles on blood vessels through which they are distributed is poorly understood. Hence, we investigated the effects of nano‐ and micromaterials on blood vessel shrinkage and relaxation. Nanosilica particles with diameters of 70 nm (nSP70) were used as the nanomaterial, and particles of 300 and 1000 nm (nSP300 and mSP1000, respectively) were used as micromaterials. A rat thoracic aorta was used as the test blood vessel. The nano‐ and micromaterials had no effect on vessel shrinkage. Of the nano‐ and micromaterials tested, only nSP70 strongly evoked vascular relaxation. Vascular relaxation evoked by nSP70 was almost completely inhibited by the phosphoinositide 3‐kinase (PI3K) inhibitor wortmannin. In addition, the selective nitric oxide synthesis inhibitor NG‐nitro‐l ‐arginine methyl ester, which inhibits endothelial nitric oxide synthase (eNOS) downstream of PI3K signaling, inhibited vascular relaxation evoked by nSP70. In an analysis using bovine aortic endothelial cells (bAECs), nSP70 phosphorylated protein kinase B (AKT) and eNOS acted downstream of PI3K signaling. PI3K inhibition by wortmannin reduced AKT and eNOS phosphorylation. These results demonstrated that 70‐nm amorphous nanosilica particles evoked vascular relaxation through PI3K/Akt/eNOS signaling. Moreover, it was suggested that nanomaterials, in general, control or disrupt vascular function by activating a known signal cascade.     

Pharmacological characterization of the vascular muscarinic receptors mediating relaxation and contraction in rabbit aorta.

Studies were performed in the rabbit aortic rings, precontracted with norepinephrine, to determine the subtype(s) of muscarinic receptors involved in endothelium-dependent relaxation and contraction in the absence of endothelium elicited by cholinergic stimuli. Acetylcholine (ACh) and arecaidine propargyl ester (APE), a M2 and M3 agonist, produced a dose-dependent relaxation and contraction in endothelium-intact and endothelium-denuded rabbit aortic rings, respectively. Both of these responses were blocked by the muscarinic receptor antagonist atropine. M1 selective agonist McN-A-343 [4-[N-(3-chlorophenyl)carbamoyloxy]-2-butinyltrimethylammonium+ ++ chloride] did not produce any effect on the tone of precontracted aortic rings. ACh- and APE-induced relaxation in aortic rings with intact endothelium was selectively blocked by M3 receptor antagonists hexahydrosila-difenidol and p-fluoro-hexahydro-sila-difenidol (pA2 of 7.84 and 7.18) but not by M1 antagonist pirenzepine or M2 receptor antagonists AF-DX 116 [11-(2-[(diethylamino)methyl]- 1-piperidinyl]acetyl)-5, 11-dihydro-6H-pyrido-[2,3-b][1,4]-benzo-diazepin-6-one] and methoctramine. ACh- and APE-induced contraction was inhibited by M2 receptor antagonists AF-DX 116 and methoctramine (pA2 of 7.11 and 6.71) but not by pirenzepine, hexahydro-sila-difenidol or p-fluoro-hexahydro-sila-difenidol. ACh- and APE-induced relaxation or contraction were not altered by nicotinic receptor antagonist hexamethonium or cyclooxygenase inhibitor indomethacin. These data suggest that relaxation elicited by cholinergic stimulin in endothelium-intact aortic rings is mediated via release of endothelium-derived relaxing factor consequent to activation of M3 receptors located on endothelial cells, whereas the contraction in aortic rings denuded of their endothelium is mediated via stimulation of M2 receptors located on smooth muscle cells.     

α-Methylnoradrenaline-induced contractions in rat aorta are mediated via α1D-adrenoceptors

Summary— The subtype(s) of α-adrenoceptor-mediating contractions to α-methynoradrenaline in the rat aorta has been investigated by using α-adrenoceptor-selective competitive antagonists and the α₁-adrenoceptor selective agonist, phenylephrine, for comparison. α-Methylnoradrenaline and phenylephrine elicited concentration-dependent contractions in the endothelium-denuded and endothelium-intact aortic rings with similar potencies and maximal effects. α-Methylnoradrenaline- and phenylephrine-induced contractions in endothelium-denuded aortic rings were competitively antagonized by prazosin (pA₂ = 9.38 and 9.13; respectively) and rauwolscine (pA₂ = 7.19 and 6.60, respectively). This confirms that there is an α₁- and a non α₂-adrenoceptor response to α-methylnoradrenaline in the rat aorta. The subtype selective α_1D-adrenoceptor antagonist, BMY 7378, was found to antagonize contractions to α-methylnoradrenaline and phenylephrine competitively in endothelium-denuded and endothelium-intact aortic rings. The pA₂ values of BMY 7378 against α-methylnoradrenaline (8.39 and 8.41; endothelium-intact and endothelium-denuded, respectively) and phenylephrine (8.64 and 8.76; endothelium-intact and endothelium-denuded, respectively), are consistent with its published functional potency and clonal α_1d-adrenoceptor binding affinity. In addition, contractions to α-methylnoradrenaline and phenylephrine in endothelium-denuded aortic rings, were potently inhibited by WB 4101 with pA₂ values of 9.75 and 9.25, respectively. The high pA₂ values for WB 4101 indicate that the α_1B-adrenoceptor subtype does not seem to participate in α-methylnoradrenaline (and phenylephrine) induced contractions in this artery. These results suggest that the α_1D-subtype plays a determining role in rat aorta contractions induced by α-methylnoradrenaline.     

Angiotensin 1‐7 modulates electrophysiological characteristics and calcium homoeostasis in pulmonary veins cardiomyocytes via MAS/PI3K/eNOS signalling pathway

Background

Atrial fibrillation (AF) is the most common sustained arrhythmia, and pulmonary veins (PVs) play a critical role in triggering AF. Angiotensin (Ang)‐(1‐7) regulates calcium (Ca²⁺) homoeostasis and also plays a critical role in cardiovascular pathophysiology. However, the role of Ang‐(1‐7) in PV arrhythmogenesis remains unclear.

Materials and methods

Conventional microelectrodes, whole‐cell patch‐clamp and the fluo‐3 fluorimetric ratio technique were used to record ionic currents and intracellular Ca²⁺ in isolated rabbit PV preparations and in single isolated PV cardiomyocytes, before and after administration of Ang‐(1‐7).

Results

Ang (1‐7) concentration dependently (0.1, 1, 10 and 100 nmol/L) decreased PV spontaneous electrical activity. Ang‐(1‐7) (100 nmol/L) decreased the late sodium (Na⁺), L‐type Ca²⁺ and Na⁺‐Ca²⁺ exchanger currents, but did not affect the voltage‐dependent Na⁺ current in PV cardiomyocytes. In addition, Ang‐(1‐7) decreased intracellular Ca²⁺ transient and sarcoplasmic reticulum Ca²⁺ content in PV cardiomyocytes. A779 (a Mas receptor blocker, 3 μmol/L), L‐NAME (a NO synthesis inhibitor, 100 μmol/L) or wortmannin (a specific PI3K inhibitor, 10 nmol/L) attenuated the effects of Ang‐(1‐7) (100 nmol/L) on PV spontaneous electric activity.

Conclusion

Ang‐(1‐7) regulates PV electrophysiological characteristics and Ca²⁺ homoeostasis via Mas/PI3K/eNOS signalling pathway.     

Mechanism of the endothelium-dependent relaxation evoked by a grape seed extract

GSEs (grape seed extracts) which contain polyphenolic compounds cause an endothelium-dependent relaxation of blood vessels. The aim of the present study was to examine the mechanisms involved in this response. A well-characterized GSE was applied to rabbit aortic rings suspended in organ baths containing Krebs-Henseleit buffer maintained at 37 degrees C. In aortic rings pre-contacted with noradrenaline (norepinephrine), the extract produced a dose-dependent relaxation. The maximum relaxations elicited by the extract (71.9+/-1.0%) were similar to those elicited by acetylcholine (64.2+/-1.5%) (n=12 for each). As expected, the relaxations were abolished by removal of the endothelium and by prior incubation with L-NAME (N(G)-nitro-L-arginine methyl ester), confirming the essential role of eNOS (endothelial NO synthase) in the response. The responses to the GSE were also abolished by incubation with wortmannin and LY294002, which are inhibitors of PI3K (phosphoinositide 3-kinase). These compounds had no effect on the responses to acetylcholine. Using immunoblotting, we also demonstrated that the GSE induced the phosphorylation of both Akt and eNOS in HUVECs (human umbilical vein endothelial cells). Finally, the extract was modified by methylation of the hydroxy groups in the polyphenolic groups and was applied to the aortic rings. The modified extract failed to cause a relaxation. Taken together, these findings suggest that the endothelium-dependent relaxation induced by the GSE was mediated by activation of the PI3K/Akt signalling pathway through a redox-sensitive mechanism, resulting in phosphorylation of eNOS.     

Endothelium-dependent relaxation of rabbit aorta. I. Relaxation stimulated by arachidonic acid

Vascular responses to the addition of arachidonic acid (AA) were characterized in rings of rabbit thoracic aorta. In preparations which were precontracted to a stable plateau by 10(-7) M phenylephrine, AA (10-100 microM) elicited transient relaxation if the endothelium was intact. The same concentrations of AA only contracted tissues in which the endothelium was purposely disrupted. Indomethacin pretreatment (14 microM, 30 min) potentiated the relaxation response in intact rings. Under these conditions, relaxation in response to 100 microM AA was 32(+/- 3)% of the tension stimulated by 10(-7) M phenylephrine. Preincubation with 5,8,11,14-eicosatetraynoic acid (50 microM) or nordihydroguaiaretic acid (25 microM) completely prevented AA-induced relaxation. Contractile responses to AA were examined in rings lacking pre-existing active tension. AA (3-100 microM) elicited rapidly developing and somewhat transient contractions in rings with endothelium. Slow developing contractions which were significantly smaller in magnitude were observed in rings lacking endothelium. Indomethacin or 5,8,11,14-eicosatetraynoic acid pretreatment inhibited AA-stimulated contractions both in intact and de-endothelialized tissues. Nordihydroguaiaretic acid pretreatment had no effect on contractile responses in rings lacking endothelium and slightly potentiated responses in intact rings. We conclude that endothelium-dependent relaxation of rabbit aorta by AA is mediated by a noncyclooxygenase metabolite(s). Contractile responses are mediated by cyclooxygenase metabolites, which in intact rings are derived largely from the vascular endothelium.     

Temperature-dependent basal tone in isolated human saphenous veins: implication of TP-receptors

Abstract— Cutaneous blood vessels are very sensitive to changes in environmental temperature. The influence of variations in local temperature on the mechanisms involved in the basal tone, present in isolated human saphenous veins has not yet been studied. In the present study, segments with and without endothelium of human saphenous veins obtained from coronary bypass surgery patients were mounted for isometric tension recording in oxygenated physiological salt solution (PSS). After stabilisation of the basal tone, the local temperature was rapidly either decreased from 37 °C to 24 °C (cooling) or increased from 37 °C to 42°C (warming). When antagonists or inhibitors were used the preparations were incubated for 30 min with the drugs. During basal conditions, cooling caused relaxations of the saphenous vein segments with endothelium and warming caused contractions; the absence of the endothelium did not modify these responses. In veins without endothelium, the warming‐induced contractions were significantly inhibited by verapamil (10 μM) and by the antagonist of TP‐receptors (receptors for thromboxane A₂) Bay u 3405 (1 μM). The warming induced contractions were not affected by cyclooxygenase or lipoxygenase inhibition. At 37°C, the isoprostanes (8‐iso‐PGE₂ and 8‐iso‐PGF_2α) induced potent contractions that were significantly inhibited by Bay u 3405 (1 μM). The data show that a basal tone is present in isolated resting human saphenous vein segments at 37°C. This basal tone is decreased by local cooling and enhanced by local wanning and is not dependent on the presence of the endothelium. The warming‐induced contraction of the veins is mediated by a non‐cyclooxygenase, non‐lipoxygenase metabolite (iso‐prostanc?) that interacts with TP‐receptors and via an extracellular calcium‐dependent pathway.     

Atorvastatin acutely reduces the reactivity to spasmogens in rat aorta: implication of the inhibition of geranylgeranylation and MYPT‐1 phosphorylation

Statins are known to display benefits in various diseases independently from their cholesterol lowering properties. In this study, we investigated the acute effects of atorvastatin on vascular reactivity to various spasmogens in isolated rat aorta. The responses to noradrenaline (NA, 10^?8–10^?4 m ), endothelin‐1 (ET‐1, 10^?10–10^?7 m ), and potassium chloride (KCl, 10–100 mm ) were evaluated in aortic rings pretreated with atorvastatin (10^?7–10^?4 m , 30 min). To verify the mechanism of action, the effects of atorvastatin were studied in the presence of cholesterol precursor, mevalonate (10^?2 m , 45 min), mevalonate‐derived isoprenoids, namely geranylgeranyl pyrophosphate (GGPP, 5 × 10^?6 m , 30 min) and farnesyl pyrophosphate (FPP, 5 × 10^?6 m , 30 min), and in the absence of endothelium. In parallel, aortic rings were pretreated with the specific inhibitor of Rho kinase, Y‐27632 (10^?7–10^?6 m ). Atorvastatin significantly and concentration‐dependently reduced the contractions to spasmogens in rat aorta. This acute inhibitory effect was also evident in endothelium‐denuded rings. Pretreatment with mevalonate and GGPP, but not with FPP, reversed the inhibitory effect of atorvastatin (10^?4 m ) on NA and ET‐1 induced contractions. Similar to atorvastatin, pretreatment with Y‐27632 inhibited the contractions to NA and KCl in a concentration‐dependent manner. Western blot analysis revealed that both atorvastatin (10^?4 m ) and Y‐27632 (10^?6 m ) pretreatment inhibited the phosphorylation of myosin phosphatase target subunit‐1 (MYPT‐1) triggered by NA, indicating an inhibitory influence on myosin phosphatase. In conclusion, atorvastatin displayed an acute inhibitory effect on vascular contractility evoked by various spasmogens and the inhibitory effect was possibly mediated by the inhibition of mevalonate and GGPP synthesis as well as the prevention of MYPT‐1 phosphorylation induced by Rho/Rho kinase.     

Influence of the endothelium on tone and the response of isolated pig coronary artery to norepinephrine

The influence of the endothelium on smooth muscle tone and the response of the pig right coronary artery to norepinephrine (NE) was studied. Isolated rings of artery with and without endothelium were stretched in the presence of nitroprusside to a tension previously determined to be optimal for contraction. During wash out of the nitroprusside, rings without endothelium spontaneously generated tone representing 24% of the contraction caused by potassium (120 mM); in rings with endothelium no significant spontaneous tone was observed. Relaxations were caused by NE in rings with endothelium contracted with prostaglandin F2 alpha (PGF2 alpha). In rings without endothelium, NE relaxed spontaneously generated tone as well as that produced by PGF2 alpha. Independent of the mode or degree of contraction, rings with endothelium were more sensitive to NE than rings without endothelium. The difference in sensitivity to NE between rings with and without endothelium was likely due to endothelial cell alpha-2 adrenoceptors, inasmuch as the difference was abolished by rauwolscine. In the presence of propranolol and prazosin, endothelium-dependent relaxations were observed which were also inhibited by rauwolscine. Nevertheless, beta adrenoceptors are the predominant mediator of the relaxation to NE of pig coronary smooth muscle, because propranolol caused a greater shift to the right of the relaxation induced by NE compared to that caused by endothelium removal. Accordingly, under resting conditions, NE caused contractions only in the presence of propranolol. These contractions were attenuated by prazosin or rauwolscine, but blocked only by a combination of both alpha adrenoceptor antagonists.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of endothelium-derived relaxing factor involvement in the potentiating effect of parathyroidectomy on norepinephrine-induced rat aortic contraction

Summary— Previous results have shown that the contractile response to norepinephrine (NE) was enhanced in isolated aortae from SHR and normotensive Wistar parathyroidectomized rats. In this work we sought to characterize the contribution of endothelium-derived relaxing factor (EDRF) release to this effect which is not linked to hypertension. Parathyroidectomy (PTX) was performed by surgery on 5 week-old male Wistar rats. Five weeks later intact (E+) and rubbed (E–) aortic rings were mounted in an organ chamber for isometric tension recording. KCl-induced contractions were potentiated in PTX E+ aortae compared to sham operated (SO), (P < 0.05), but not in denuded E- aortae. Similarly NE (1.nM- 10 μM) induced a potentiated contractile response in PTX E+ (P < 0.01), but not in PTX E- rings; nevertheless the sensitivity did not change. After removal of endothelium, the expected enhanced contraction and sensitivity observed in SO rats was not present in PTX. The NO synthase inhibitor L-NAME (20 μM), enhanced sensitivity to NE in SO but not in PTX E+ aortic rings. In addition, hemoglobin (Hb, 10 μM) enhanced NE contraction in SO (P < 0.01) aortic rings, but to a lesser extent in PTX rat aortae. Moreover, in the presence of L-NAME or Hb, SO and PTX aortae displayed a similar contraction. Superoxide dismutase (SOD, 150 U/ml) diminished the NE contraction since NO was protected from degradation but the difference was still present between SO and PTX rat aortae, ruling out the possible implication of superoxide anions in the hyperreactivity of PTX aortae. On the other hand, A23187, which induces EDRF release, reduced the level of NE contraction as expected, but suppressed the PTX enhancing effect and in calcium-free solution the enhancement of contraction after PTX was not observed. These experiments extend to the rat the observations previously obtained in rabbit aorta: extracellular calcium is a major determining factor in NO production. Acetylcholine and A23187 (cumulative doses) produced an endothelium-dependent relaxation which was not significantly modified in NE-pre-contracted PTX aortae compared to SO aortae. L-arginine (100 μM), reversed the L-NAME inhibitory effect and induced an attenuated endothelium-dependent relaxation in PTX vessels (P < 0.01). In conclusion, in rat isolated aortae the enhancing effect of parathyroidectomy on norepinephrine and KCl contractions is due to a diminished endothelial nitric oxide production. This might arise via a decrease of the constitutive NO synthase activity in an extracellular calcium-dependent manner.