Presence Of Functional Endothelium Research Articles

Vaso‐relaxant Effect of Zingiber officinale Extract Involves Release of Nitric Oxide from the Endothelium

IntroductionZingiber officinale (ZO) is an important plant with several medicinal, nutritional and ethnomedical values. The blood pressure lowering effect of ZO has been reported in experimental animals and human and some mechanisms have been postulated, including; blockage of voltage dependent calcium channel, stimulation of muscarinic receptors and serotonergic antagonistic property. The aim of this study is to investigate the vaso‐relaxant effect of the extract of ZO and the mechanism by which it is produced.MethodRhizomes of ZO were extracted in 80% methanol. Thoracic aorta isolated from male rabbits were cut into rings of 3–5 mm, mounted in organ baths containing 25 ml Krebs physiological solution maintained at 37 °C and bubbled continuously with a mixture of 95% O2 and 5% CO2. The aortic rings were equilibrated for 30 min at a resting tension of 1 g. Responses were recorded isometrically via a force displacement transducer connected to a recording system. The presence of functional endothelium was checked by the ability of acetylcholine (1μM) to induce relaxation on phenylepherine (PE, 1μM) contracted rings. The relaxant responses to ZO was recorded by adding cumulative concentrations (10, 20, 40, 80, 100, 500, 1000 μg/ml) of ZO. To investigate the involvement of endothelium in the vaso‐relaxant action, endothelium was removed by gently rubbing the luminal surface of the ring with a polyethylene tube. To investigate which endothelium‐derived vasorelaxant factors were involved, the rings were pre‐incubated with Nitro‐L‐arginine methyl ester (L‐NAME, 10 μM) or indomethacin (10 μM) for 30 min before addition of the spasmogen and ZO. Relaxant responses were expressed as percentage relaxation from PE‐precontraction levels. The EC50 (the concentration required to cause half‐maximal relaxation) was calculated from the concentration‐response curve. Results were expressed as means ± s.e.m. Student t‐test was used to assess the significant differences at (p < 0.05)ResultsZO produced a vaso‐relaxation effect on rings pre‐contracted with PE (1μM) at concentrations of 20–100 μg/ml in endothelium intact rings with an EC50 of 14.8±1.2 μg/ml (Figure 1). This relaxation was not however seen at 500–1000 μg/ml but rather a vaso‐contraction was observed. In endothelium denuded rings, the vaso‐relaxation effect of ZO at 20–100 μg/ml was abolished while vaso‐relaxation was observed with 500–1000 μg/ml, the concentrations that otherwise produced contraction in endothelium intact aortic rings. Pre‐incubation of the tissues with L‐NAME (10μM) abolished the relaxant effect of ZO at 20–100μg/ml but caused relaxation effect with 500–1000 μg/ml similar to that observed with endothelium denuded rings. Pretreatment with indomethacin abolished the relaxant effect of ZO at 20μg/ml but not at 40 μg/ml and above (Figure 2).ConclusionVaso‐relaxant effect of ZO is seen at lower concentrations, endothelium dependent and may involve the release of nitric oxide from the endothelium. There may be interplay between contractile and relaxant compounds in crude extract of ZO at concentrations above 500 μg/ml.Support or Funding InformationThe PhD study grant awarded to I.M Adebisi by Usmanu Danfodiyo University, Sokoto, Nigeria is acknowledgedRelaxation effect of methanol extract of Zingiber officinale (ZO) on phenylephrine‐precontracted aortic rings with intact endothelium. Data represent the mean ± s.e.m. of four individual experiments. * is p<0.05Figure 1Relaxation effect of methanol extract of Zingiber officinale (ZO) on phenylephrine‐precontracted aortic rings with endothelium, without endothelium and effect of L‐NAME (10 μM), and Indomethacin (10 μM) on ZO induced vasorelaxation on phenylephrine‐precontracted aortic rings with endothelium. Data represent the mean ± s.e.m. of four individual experiments. * is p<0.05Figure 2

Membrane Stabilizing Effects of Calcium in Salt-induced Hypertensive Pregnancy

Though Calcium is involved in the mechanisms of increase contractility, it has been shown that at high concentration, calcium ions may cause membrane stabilization in which case the smooth muscle responsiveness decreases. Theoretically, a decrease in membrane permeability to calcium ions caused by increased extracellular calcium concentration stabilizes membranes. Apparently, administration of high level of calcium ion may become useful either by way of preventing or diminishing pregnancy induced hypertension (preeclampsia). The goal of this study was to examine the effect of calcium ion on the sensitivity of blood vessels during pregnancy, especially in salt induced hypertensive pregnancy. The isolated aorta of Forty adult Sprague Dawley rats [four groups of ten rats each; with Group 1=non pregnant fed with normal rat chow, Group 2=normal rat chow + 5% CaCl2 prior to and during 6 weeks feeding on 1.6% NaCl, Group 3=normal rat chow + 8% NaCl for 6 weeks, and Group 4=pregnant rats (350-380) fed on normal rat chow] was cut into 2 mm ring segments and each segment was suspended between two L-shaped holders. The lower holder was fixed to the base of 20 ml organ baths containing physiological salt solution, while the upper holder was connected to isometric transducer coupled to Ugo Bassile recorder. The presence of functional endothelium was ascertained before the start of the experiment by the observation of at least, 42% relaxation to 10-7 M acetylcholine in blood vessels contracted with 10-7 M noradrenaline. Concentration response tests to phenylephrine, KCl, and CaCl2 were done and result compared. Results show that the maximum contraction to phenylephrine of rings from pregnant rats fed on calcium chloride diets were insignificantly different from those of rats fed only with sodium chloride diet. Their sensitivities were however significantly (p<0.05) different. This observation suggests that the effect of Ca2+ feeding may be limited to the sensitivity, rather than the maximum contraction. Prior calcium feeding along with simultaneous high salt (sodium chloride) intake appears to interfere with the enhancement of vascular contractility associated with high salt intake. Ca2+ would therefore be relevant in the prevention and treatment of salt-induced hypertension in pregnancy.

Moving pieces in a cryptomic puzzle: Cryptide from Tityus serrulatus Ts3 Nav toxin as potential agonist of muscarinic receptors

Cryptome is as a subset of a given proteome containing bioactive cryptides embedded in larger peptides or proteins. We pinpointed a striking sequence similarity between two peptides from the Tityus serrulatus venom: Ts10 (KKDGYPVEYDRAY) and the N-terminal of Ts3 (KKDGYPVEYDNCAY). Ts3 (former Tityustoxin or TsIV) is an α-neurotoxin acting on voltage-gated sodium channels while Ts10 (former Peptide T) is a bradykinin-potentiating peptide and was originally reported as inhibitor of the angiotensin-converting enzyme (ACEi). Thus, the goal of this study was to evaluate whether such peptide hidden in the N-terminal of Ts3 (Ts31-14[C12S]) was able to mimic known effects of Ts10 as well as to expand the current knowledge of the vascular effects and molecular targets of these peptides. Similar to Ts10, Ts31-14[C12S] was able to potentiate the hypotensive effect of bradykinin (BK). However, none of these peptides was able to induce a long-lasting BK-potentiating effect, suggesting that this effect may not be their main biological outcome. On the other hand, we report that Ts10 and mainly Ts31-14[C12S] induced a strong vasodilation effect depending on the presence of functional endothelium and nitric oxide (NO) production. Unlike previously reported, Ts10 was not able to inhibit ACE activity (similar result was observed for Ts31-14[C12S]). On the other hand, we report that Ts31-14[C12S] induces vasodilation via the activation of muscarinic acetylcholine receptors (mAChRs) M2 and M3 while only the activation of mAChR M2 seems to be required for Ts10-induced vasodilation.

GW24-e1139 Vasorelaxant and antihypertensive effects of 7, 8-dihydroxyflavone in rats

Objectives7,8-Dihydroxyflavone (7,8-DHF) is a new potent agonist for TrkB receptors. 7, 8-DHF exerts protecting effects on neurons in Parkinson’s and Alzheimer’s diseases and stroke, improves memory and antagonises depression. Its...

Dexmedetomidine Induces Both Relaxations and Contractions, via Different α2-Adrenoceptor Subtypes, in the Isolated Mesenteric Artery and Aorta of the Rat

Dexmedetomidine is an α(2)-adrenoceptor agonist and anesthetic. The present study was designed to characterize the receptor subtypes and the downstream mechanisms of the vascular effects of dexmedetomidine in small (mesenteric artery) and large (aorta) arteries ex vivo. Isometric tension was measured in Sprague-Dawley rat mesenteric and aortic rings (with or without endothelium). To study relaxations, cumulative concentrations of dexmedetomidine, 5-bromo-N-(2-imidazolin-2-yl)-6-quinoxalinamine, (UK14304), or clonidine were added to rings contracted with 9,11-dideoxy-9α,11α-methanoepoxy prostaglandin F(2α) (U46619) in the presence or absence of indomethacin; N(ω)-nitro-l-arginine methyl ester hydrochloride (l-NAME); 2-[2H-(1-methyl-1,3-dihydroisoindole)methyl]-4,5-dihydroimidazole maleate (BRL44408); 2-[2-(4-(2-methoxyphenyl)piperazin-1-yl)ethyl]-4,4-dimethyl-1,3-(2H,4H)-isoquinolindione dihydrochloride (ARC239); l-657,743, (2S-trans)-1,3,4,5',6,6',7,12b-octahydro-1',3'-dimethyl-spiro[2H-benzofuro[2,3-a]quinolizine-2,4'(1'H)-pyrimidin]-2'(3'H)-one hydrochloride hydrate (MK912); rauwolscine; prazosin; or pertussis toxin. To study contractions, dexmedetomidine was added to quiescent rings without endothelium or after incubation with l-NAME, rauwolscine, prazosin, indomethacin, or 3-[(6-amino-(4-chlorobenzensulfonyl)-2-methyl-5,6,7,8-tetrahydronaphth)-1-yl]propionic acid (S18886). Dexmedetomidine evoked relaxation at low concentrations (10 pM-30 nM) followed by contraction at higher concentrations (>30 nM) in the mesenteric artery. In the aorta, the relaxation was significantly smaller. The relaxation to dexmedetomidine depended on nitric oxide, endothelium, and G(i) protein, and it was mediated by α(2A/D)-adrenoceptors and possibly α(2B)-adrenoceptors. The contraction was mediated mainly by α(2B)- and α(1)-adrenoceptors and involved the action of prostanoids. UK14304 and clonidine induced greater and smaller relaxations, respectively, than dexmedetomidine. In conclusion, depending on the concentration used and the presence of functional endothelium, dexmedetomidine may evoke both relaxation and contraction in isolated arteries. The vascular effects also vary depending on the blood vessel studied. Its vascular effect is different from that of clonidine and UK14304.

The flavonoid dioclein is a selective inhibitor of cyclic nucleotide phosphodiesterase type 1 (PDE1) and a cGMP-dependent protein kinase (PKG) vasorelaxant in human vascular tissue

The inhibitory effect of the flavonoid dioclein was assessed on purified vascular cyclic nucleotide phosphodiesterase isoforms (EC 3.1.4.17, PDE1-5) in comparison with 8-methoxymethyl-isobutylmethylxanthine (8-MM-IBMX) and vinpocetine which are currently used as PDE1 inhibitors. The mechanism underlying the vasorelaxant effect of dioclein was investigated in human saphenous vein. Dioclein inhibited PDE1 more selectively than vinpocetine and 8-MM-IBMX, with IC 50 values of 2.47 ± 0.26 and 1.44 ± 0.35 µM, respectively in basal- and calmodulin-activated states. Dioclein behaved as a competitive inhibitor for cGMP hydrolysis by PDE1 in basal- and calmodulin-activated states ( K i = 0.62 ± 0.14 and 0.55 ± 0.07 µM, respectively), indicating this inhibitory effect to be independent of calmodulin interactions. In addition, dioclein induced a concentration-dependent relaxation of human saphenous vein which was independent on the presence of functional endothelium (EC 50 values of 7.3 ± 3.1 and 11 ± 2.7 µM, respectively with and without endothelium). 8-MM-IBMX relaxed human saphenous vein with an EC 50 = 31 ± 16 µM, whereas vinpocetine did not cause any vasorelaxation at concentrations up to 100 µM. Rp-8-pCPT-cGMPS, which inhibits cGMP-dependent protein kinase (PKG), blocked the vasodilator effect of dioclein, whereas H-89, which is a cAMP-dependent protein kinase (PKA) inhibitor, had a minor inhibitory effect. Our data show that dioclein is a potent calmodulin-independent selective inhibitor of PDE1 and that inhibition of PDE1 is involved in the PKG-mediated vasorelaxant effect of dioclein in human saphenous vein. Furthermore, dioclein may represent a new archetype to develop more specific PDE1 inhibitors.

Endothelial COX-1 and -2 differentially affect reactivity of MVB in portal hypertensive rats.

Expression of constitutive and inducible cyclooxygenase (COX-1 and COX-2, respectively) and the role of prostanoids were investigated in the aorta and mesenteric vascular bed (MVB) from the portal vein-ligated rat (PVL) as a model of portal hypertension. Functional experiments were carried out in MVB from PVL and sham-operated rats in the absence or presence of the nonselective COX inhibitor indomethacin or the selective inhibitors of COX-1 (SC-560) or COX-2 (NS-398). Western blots of COX-1 and COX-2 proteins were evaluated in aorta and MVB, and PGI(2) production by enzyme immunoassay of 6-keto-PGF(1alpha) was evaluated in the aorta. In the presence of functional endothelium, decreased contraction to norepinephrine (NE) and increased vasodilatation to ACh were observed in MVB from PVL. Exposure of MVB to indomethacin, SC-560, or NS-398 reversed the hyporeactivity to NE and the increased endothelial vasodilatation to ACh in PVL, with NS-398 being more potent than the other two inhibitors. Upregulation of COX-1 and COX-2 expressions was detected in aorta and MVB from PVL portal hypertensive rats, and increased production of 6-keto-PGF(1alpha) was observed in aorta from portal hypertensive rats. These results suggest that generation of endothelial vasodilator prostanoids, from COX-1 and COX-2 isoforms, accounts for the increased mesenteric blood flow in portal hypertension.

Low 4-aminopyridine concentration-induced contraction is mediated by neuronal noradrenaline in canine saphenous vein

4-Aminopyridine (4-AP), a known inhibitor of the voltage-dependent potassium channels, is able to increase the basal tone of different types of blood vessel preparations. In order to determine the efficiency of 4-AP in veins and to clarify its possible mechanism of action, the aim of the present study was to determine the basal tone and release of radiolabelled tissue noradrenaline (NA) after administration of low 4-AP concentrations. Experiments were performed in canine saphenous vein in the absence and presence of functional endothelium. 4-AP (0.012–5 μM) enhanced the basal tone of venous rings without and with endothelium (maximum tone at 5 μM 4-AP: 2.20±1.29 and 1.3±0.57 mN, respectively). NA stores of the venous tissue were loaded by adding 1 mM NA to the tissue for 10 min and then washed out. After loading the NA-stores of venous tissue, 4-AP-induced contractions were significantly increased both in the absence and presence of endothelium (maximum tone at 5 μM 4-AP after loading with NA: 10.51±3.64 and 10.52±4.69 mN, respectively). Following NA loading, chemical denervation of the endothelium denuded venous preparations by 0.5 mM 6-hydroxydopamine (6-OHDA) completely abolished the contractions evoked by 4-AP. After incubation of the saphenous preparations with 3H-NA, 5 μM 4-AP significantly increased tritium-efflux from the tissue. These results provide evidence for the efficiency of 4-AP on the basal tone of isolated canine saphenous vein when applied in low concentrations. Furthermore, it is suggested that this action of 4-AP may considerably depend on the release of NA from the perivascular nerve endings.

Discussion

Discussion

Different types of antagonism by losartan and irbesartan on the effects of angiotensin II and its degradation products in rabbit arteries.

A previous study by our group has demonstrated that the selective AT1-receptor antagonist losartan behaves as a noncompetitive antagonist in rabbit isolated renal artery (RA). In the present investigation, the influence of losartan and irbesartan on the contractile effects of angiotensin II (AII) and its degradation products angiotensin III (AIII) and angiotensin IV (AIV) was determined in the rabbit isolated RA and femoral artery (FA). The arteries were set up in organ chambers and changes in isometric force were recorded. In both rabbit isolated RA and FA preparations, AII, AIII and AIV elicited significant contractile responses with a similar efficacy. These effects were impaired by the presence of functional endothelium in RA preparations but not in FA preparations. In both preparations studied, the effects of AII, AIII and AIV were influenced neither by the aminopeptidase-A and -M inhibitor amastatin (10 microM), nor by the aminopeptidase-B and -M inhibitor bestatin (10 microM). In endothelium-denuded FA preparations, preincubation with losartan (3-300 nM) antagonized AII-, AIII- and AIV-induced contractions in a competitive manner. However, in endothelium-denuded RA preparations, losartan depressed the maximal contractile responses induced by AII but not those induced by AIII and AIV. In the same preparations, preincubation of another selective AT1-receptor antagonist irbesartan (3-30 nM) concentration-dependently shifted AII and AIII curves to the right in an insurmountable manner. The reduction of the maximal response of AII is more potent when compared to that of AIII (47.7 +/- 1.51% vs. 66.7 +/- 1.88%, percentage of the initial maximal response; P < 0.05; n=5). The selective AT2-receptor antagonist PD123177 (1 microM) did not influence the responses to all three peptides in both RA and FA preparations. These heterogeneous antagonistic effects of the two AT1-receptor antagonists studied with respect to the contractile actions of AII, AIII and AIV suggest the possible existence of multiple, functionally relevant AT1-receptor subtypes in rabbit RA preparations.

Mechanisms of the contractile effect of the hydroalcoholic extract of Cissampelos sympodialis Eichl. in the rat aorta.

In the present work the effect of the aqueous fraction of the ethanolic extract of the leaves (AFL) of Cissampelos sympodialis Eichl. was investigated in the rat aorta. In the presence of functional endothelium, AFL produced concentration-dependent contractions (EC50 value of 76.6 +/- 17.8 micrograms/ml). In the absence of functional endothelium, the concentration-response curves to AFL were significantly shifted to the left (EC50 values of 1.3 +/- 0.9 micrograms/ml) without modification of its maximal contractile effect. In the presence of L-NAME (300 microM) and of indomethacin (10 mM), the concentration-response curves produced by AFL were also shifted to the left (EC50 values of 21.8 +/- 6.2 and 24.3 +/- 13.2 micrograms/ml, respectively). The treatment of the aortas with L-NAME (300 microM) plus indomethacin (10 microM) produced a significant shift to the left of the concentration-dependent curves of AFL (EC50 value of 4.9 +/- 2.2 micrograms/ml), similar to that observed in the absence of the vascular endothelium. In addition, AFL-induced contraction was abolished in the presence of prazosin (1 microM), and significantly shifted to the right in the presence of yohimbine (EC50 value of 723.6 +/- 76.4 micrograms/ml). Thus, based on these results, it can be concluded that contractions induced by AFL in the rat aorta were due to activation of alpha-adrenoceptors. Furthermore, these results also showed that the AFL-induced contractions were modulated by the endothelium, via the release of NO and of a cyclooxygenase-derived relaxant product. Finally, it can be concluded that the contractile effects of AFL on vascular smooth muscle may play an important role in the hypertensive effects of this plant in vivo.

Endothelin-1 is not involved in hemoglobin associated vasoactivities.

This study tested the hypothesis: is hemoglobin (Hb) associated vasoconstriction mediated by endothelins (ET)? Two millimeter segments of the thoracic aorta with intact endothelium were obtained from Sprague-Dawley rats (200-350g) and suspended in oxygenated Krebs buffer (37C, pH 7.4). The vessels were equilibrated at 2g of imposed tension for 1 hour. After submaximal tone was induced with norepinephrine (50nM), the presence of functional endothelium was verified by an acetylcholine (33uM) dilation test. The vessel rings were then treated with either ET-1 (3nM) or human stroma-free Hb (SFH; 2.2uM) and vascular response characteristics observed. Subsequently, the vessel rings were treated with BQ-123 (15uM), an ET-1 receptor antagonist, to test whether the ET-1 pathway is involved in the Hb associated vasoconstriction. Both ET-1 and SFH elicited contractory responses in aortic rings; the maximal tension increases at the doses tested were 54.8 +/- 8.5% and 39.2 +/- 22.5%, respectively, over the pretreatment values. The contractory response characteristics were, however, distinct; ET-1 caused a slow (time to maximal response; TMR = 28.8 +/- 6.4 min, N = 6) but prolonged contraction (> 30 min) while SFH caused a faster contraction (TMR = 7.2 +/- 1.7 min, N = 6) with a shorter duration (< 30 min). The TMR of ET-1 treated rings were significantly longer than that of SFH (P < 0.0005, t-test). Treatment with BQ-123 caused an immediate reversal of the SFH induced contraction but had no significant effect on the SFH induced contraction. If the Hb associated vasoconstriction were mediated by ET-1, BQ-123 should have also reversed the contraction. These results suggest that ET-1 pathway is not involved in the Hb mediated vasoconstriction.

Alterations of cyclo-oxygenase products and NO in responses to angiotensin II of resistance arteries from the spontaneously hypertensive rat.

1. The involvement of cyclo-oxygenase (COX) products and nitric oxide (NO) in contractile responses of resistance arteries to angiotensin II (AII) were investigated in small mesenteric arteries from spontaneously hypertensive rats (SHR) and Wistar Kyoto (WKY) rats. 2. In endothelium intact vessels, AII induced concentration-dependent responses without any significant difference between the two strains. However, removal of functional endothelium resulted in enhanced sensitivity to AII, the pD2 value increasing from 8.4 +/- 0.2 to 8.9 +/- 0.2 (P < 0.05) in WKY and from 8.2 +/- 0.1 to 8.6 +/- 0.1 (P < 0.05) in SHR (not significantly different between strains, n = 9 - 12). In addition, endothelium removal enhanced maximal contractions elicited by AII in SHR (1.4 +/- 0.1 to 2.1 +/- 0.2 mN mm-1, n = 5; P < 0.05) but not in WKY (1.0 +/- 0.1 to 1.2 +/- 0.1 mN mm-1, n = 5) vessels. 3. In the absence of functional endothelium, the COX inhibitor indomethacin (10(-5) M) reduced contractile responses elicited by AII in SHR arteries, resulting in 33 +/- 5% (n = 5) decrease in maximal contraction. However, it produced minimal if any, effect on responses of WKY vessels. In both strains, the TP receptor antagonist GR32191 B (3 x 10(-6) M) did not modify contractions elicited by AII in these conditions. 4. In the presence of functional endothelium, indomethacin (10(-5) M) almost abolished the responses to AII in both strains. GR32191 B (3 x 10(-6) M) reduced the sensitivity of WKY arteries to AII (pD2 = 8.1 +/- 0.1, P < 0.01) without any effect on maximal contraction. In SHR arteries, it markedly reduced maximal contraction (47 +/- 3.5%). 5. In both strains, the NO synthase inhibitor NG-nitro-L-arginine methy lester (L-NAME; 10(-4) M) had no effect in the absence of functional endothelium but it markedly reduced the inhibitory influence of endothelium on contractile responses to AII. Furthermore, in arteries with endothelium, it reduced the effect of both indomethacin and GR32191 B to the same level as observed in vessels without functional endothelium. 6. The results suggest that enhanced contraction caused by COX products was counteracted by enhanced relaxation caused by endothelium-derived NO in resistance mesenteric arteries of the SHR exposed to AII, compared to WKY arteries. The COX products involved in alterations of SHR responses comprised an endothelium-derived prostaglandin activating TP receptors and another nonendothelial unidentified vasoconstrictor compound which did not activate these receptors.

Reciprocal inhibition of nitric oxide and prostacyclin synthesis in human saphenous vein.

1. Angiotensin II (AII) causes contraction of isolated rings of human saphenous vein, responses that are attenuated by the presence of functional endothelium. In this study, we have investigated the mechanisms controlling the release by AII of two endothelial-derived vasorelaxants, prostacyclin (PGI2) and nitric oxide (NO). 2. Myotropic and biochemical changes were measured in response to AII. The biochemical responses measured were the output of PGI2 (as 6-oxo-PGF1 alpha) and of NO (as cyclic GMP). Inhibitors of cyclo-oxygenase (COX; piroxicam) or NO synthase (NOS; L-NAME), were added to the system to determine the influence of endogenous prostaglandins and NO on both myotropic and biochemical responses. Furthermore, to mimic the effects of endogenous, PGI2 or NO, exogenous forms of these relaxants were added, during inhibition of their endogenous release. 3. Contractions of the rings of saphenous vein in response to AII (1-100 nM) were unaffected by treatment with either piroxicam (5 microM) or L-NAME (200 microM) individually. However, when these two inhibitors were used together, there was an increase in the contractions in response to AII. 4. Biochemical analyses revealed that during stimulation by AII, levels of PGI2 and NO were enhanced when synthesis of the other vasodilator was inhibited, suggesting that endogenous NO inhibits PGI2 synthesis and endogenous, PGI2 or another vasorelaxant PG can inhibit NO synthesis. 5. Exogenous PGI2 (as iloprost) or NO (from glyceryl trinitrate) inhibited the increased output of endogenous NO or PGI2 respectively. 6. These results demonstrate the presence, in human saphenous vein, of a mechanism which ensures that levels of vasodilatation are maintained through a compensatory increase in one relaxant agonist when output of the other is decreased. If present in vivo such a mechanism would be important in maintaining saphenous vein graft patency as both PGI2 and NO are not only vasodilators, but inhibit platelet aggregation and myoinitimal hyperplasia, processes implicated in degeneration of graft function.

Vasodilatation produced by adenosine in isolated rat perfused mesenteric artery: a role for endothelium

Adenosine and adenosine triphosphate (ATP) induced vasodilatation was studied in isolated rat perfused mesenteric artery at constant flow. Decrease in perfusion pressure was measured after induction of tone by continuous infusion with phenylephrine (5-7 microM). Adenosine and ATP caused dose-dependent vasodilation. Following infusion with selective A2 adenosine receptor antagonist, 3,7-dimethyl-1-propargylxanthine (DMPX) (10 microM), or non-selective adenosine receptor antagonist, theophylline (30 microM), vasodilation produced by adenosine were significantly reduced at lower doses. Responses to adenosine were not affected by pretreatment of tissues with either the P2-purinoceptor desensitizing agent, alpha, beta methylene ATP (30 microM), or the P2-purinoceptor antagonist, suramin (10 microM). In contrast, both alpha, beta methylene ATP and suramin significantly attenuate relaxation produced by ATP. Further, it was found that relaxation elicited by either adenosine or ATP was not significantly affected by the presence of glibenclamide (30 microM). Vasodilatation induced by adenosine and ATP was greatly reduced in denuded arteries but more so for ATP than adenosine. It is concluded that adenosine-mediated vasodilatation may hardly be due to the stimulation of A2 adenosine receptors and is strongly dependent on the presence of functional endothelium whereas ATP-mediated vasodilator responses were mediated via the activation of P2-purinoceptors and appeared to be entirely dependent upon the presence of functional endothelium. Further, vasodilator responses to neither adenosine nor ATP were sensitive to inhibition by the potassium channel blocker glibenclamide, in isolated mesenteric perfused bed. This would imply that ATP-sensitive potassium channels were not involved in adenosine and ATP mediated vasodilatation.

Rhythmic contractions in isolated small arteries of rat: role of K+ channels and the Na+,K(+)-pump.

Small mesenteric arteries from Wistar rats display rhythmic tension oscillations, associated with oscillations in membrane potential, when stimulated with noradrenaline. The purpose of this study was to investigate the role of potassium conductance and Na+, K(+)-pump activity in the generation of these oscillations. The effect on the rhythmic contractions of several agents, interacting with K+ channels, was studied. Application of apamin, pinacidil or glibenclamide did not affect the rhythmic activity. Tetraethylammonium (TEA) increased the frequency of the rhythmic contractions, while application of 4-aminopyridine (4-AP) increased the amplitude by approximately 50%, with no changes in frequency. Ba2+, on the other hand, impaired the rhythmic contractions or converted them to irregular oscillations in the presence of functional endothelium, but did not affect oscillations in endothelium-denuded vessels. Ouabain or exposure to K(+)-free solution, procedures known to inhibit the Na+,K(+)-pump, abolished the rhythmic contractions. This effect was immediate, suggesting that it was due to elimination of the electrogenic action of the Na+,K(+)-ATPase, rather than to a change in intracellular ion concentrations. Exposure to an extracellular potassium concentration of more than 20 mM also inhibited the oscillation activity. The results suggest that the oscillations are not caused by, but may be modulated by, variations in potassium conductance. The Na+,K(+)-pump seems to play an important role in the generation of rhythmic contractions in these vessels.

Endothelium-dependent and independent relaxation of the rat aorta by cyclic nucleotide phosphodiesterase inhibitors.

1. The effects of selective inhibitors of adenosine 3':5'-cyclic monophosphate (cyclic AMP) and guanosine 3':5'-cyclic monophosphate (cyclic GMP) phosphodiesterases (PDEs) were investigated on PDEs isolated from the rat aorta and on relaxation of noradrenaline (1 microM) precontracted rat aortic rings, with and without functional endothelium. 2. Four PDE forms were isolated by DEAE-sephacel chromatography from endothelium-denuded rat aorta: a calmodulin-activated PDE (PDE I) which hydrolyzed preferentially cyclic GMP, two cyclic AMP PDEs (PDE III and PDE IV) and one cyclic GMP-specific PDE (PDE V). The latter was selectively and potently inhibited by zaprinast. The two cyclic AMP PDEs were discriminated by specific inhibitors: one was inhibited by cyclic GMP (PDE III) and by new cardiotonic agents (milrinone, CI 930, LY 195115 and SK&F 94120); the other was inhibited by denbufylline and rolipram (PDE IV). None of these drugs significantly inhibited PDE I. 3. The PDE III inhibitors caused endothelium-independent relaxations of rat aortic rings with the following EC50 values (microM concentration producing 50% relaxation): LY 195115: 3.4, milrinone: 5.7, CI 930; 7.8, SK&F 94120: 14.7. Neither NG-monomethyl-L-arginine (L-NMMA, 300 microM), an inhibitor of the L-arginine-NO pathway, nor L-arginine (1 mM) modified the effect of PDE III inhibitors. However, methylene blue (10 microM) an inhibitor of soluble guanylate cyclase abolished relaxation induced by PDE III inhibitors except in the case of compound CI 930. 4. The specific PDE IV and PDE V inhibitors both produced endothelium-dependent relaxations which were inhibited by L-NMMA and by methylene blue (10 microM). In the presence of L-NMMA, relaxation was restored by subsequent addition of L-arginine. 5. The relaxant effects of denbufylline and rolipram were studied in the presence of drugs stimulating either adenylate cyclase (forskolin and isoprenaline) or soluble guanylate cyclase (sodium nitroprusside, SNP), or inhibiting PDE III (milrinone). In endothelium-denuded rings, a relaxing effect of both denbufylline and rolipram was found in the presence of milrinone (EC5o values 1.7 and 12 microM, respectively) or SNP (EC50 values 12.3 and 124 microM, respectively), but not in the presence of forskolin or isoprenaline. However in the presence of functional endothelium, relaxations produced by PDE IV inhibitors were significantly potentiated by forskolin, isoprenaline, milrinone and SNP (respective EC50 values for denbufylline: 2, 2, 0.4 and 0.7 microM and for rolipram: 7, 13, 7 and 1.2 microM). 6. These results indicate that the relaxant effects of inhibitors of the cyclic AMP-specific PDE IV are markedly enhanced by cyclic GMP elevating agents and by the PDE III inhibitor milrinone. They support the hypothesis that cyclic GMP enhances cyclic AMP-mediated relaxation, possibly through the inhibition of the cyclic GMP-inhibited PDE III.

Endothelium‐dependent modulation of the pressor activity of arginine vasopressin in the isolated superior mesenteric arterial bed of the rat

1. Pressor responses to arginine vasopressin (AVP) were determined in the rat isolated superior mesenteric arterial bed perfused with Krebs-Henseleit solution and compared with those to noradrenaline. 2. In control preparations the maximum pressor response to the peptide was 34 +/- 3 mmHg and the ED50 was 21 +/- 4 mu (n = 11). The maximal pressor response to noradrenaline (30 micrograms) was 100 +/- 6 mmHg (n = 8). After removal of the functional endothelium with the detergent CHAPS, the maximum pressor response to AVP increased to 64 +/- 4 mmHg and the ED50 decreased to 7.7 +/- 2.0 mu (n = 11) but the response to 10 micrograms noradrenaline was unaffected. 3. Nordihydroguaiaretic acid (2.5 microM) significantly increased the maximum pressor response to AVP from 41 +/- 2 mmHg to 86 +/- 8 mmHg (n = 9); the ED50 was unchanged. Methylene blue (50 microM) also increased the maximum response from 41 +/- 3 mmHg to 87 +/- 13 mmHg (n = 8) without affecting the ED50. Neither treatment significantly affected the response to 10 micrograms noradrenaline. 4. Neither indomethacin (10 microM) nor BW755C (10 microM) had significant effects upon either the maximal response or ED50 for AVP nor did they affect the response to 10 micrograms noradrenaline. 5. In 6 preparations SKF-525A significantly increased both the ED50, from 9.8 +/- 2.1 to 22 +/- 2 mu, and the maximum response, from 36 +/- 2 to 70 +/- 3 mmHg. 6. It is concluded that the pressor response to AVP in this vascular bed is modulated, in the presence of functional endothelium, by the simultaneous release of endothelium-derived relaxing factor.