Inhibitory purinergic P2 receptor characterisation in rat distal colon

The aim of this study was to characterise the P2 receptors involved in purinergic relaxant responses in rat distal colon circular muscle. Concentration-response curves for purinergic agonists were constructed on methacholine-precontracted circular muscle strips of rat distal colon in the absence and presence of the nerve blocker TTX and the ecto-nucleotidase inhibitor ARL67156. The effects of the P2 receptor antagonists RB2, PPADS, suramin, MRS2179 and NF279, the NO-synthase inhibitor L-NAME and the small conductance K(+) channel blocker apamin were investigated. The localisation of the different P2 receptors was examined immunocytochemically. Immunocytochemistry demonstrated the expression of P2Y(1), P2Y(6) and P2X(1) receptors on smooth muscle cells and P2Y(2), P2Y(12), P2X(2) and P2X(3) receptors in the myenteric plexus; almost a quarter of the P2Y(2)-immunopositive neurons co-expressed nNOS. The P2X-selective agonist alphabetameATP and the P2Y-selective agonist ADPbetaS were the most potent relaxants; their effects were abolished by apamin. The effect of ADPbetaS was antagonised by the P2Y(1)-selective antagonist MRS2179 pointing to interaction with the muscular P2Y(1)-receptors. The relaxant effect of alphabetameATP was partially reduced by TTX and concentration-dependently antagonised by PPADS, suramin, RB2 and the P2X(1)-selective antagonist NF279; this correlates with an interaction with neuronal P2X(3) and muscular P2X(1) receptors. UTP was the least potent agonist; its effect was markedly increased by ARL67156, nearly abolished by TTX and reduced by L-NAME. This points to interaction with the neuronal P2Y(2)-receptors inducing relaxation, at least partially, by NO release.     

Vasodilator actions of abnormal-cannabidiol in rat isolated small mesenteric artery

1. The nonpsychoactive cannabinoid abnormal-cannabidiol (trans-4-[3-methyl-6-(1-methylethenyl)-2-cyclohexen-1-yl]-5-pentyl-1,3-benzenediol) (abn-cbd) produced concentration-dependent relaxation of methoxamine-precontracted rat small mesenteric artery. Endothelial removal reduced abn-cbd potency six-fold without affecting the maximum relaxation. 2. In endothelium-intact vessels, abn-cbd was less potent under 60 mM KCl-induced tone and inhibited by combination of L-N(G)-nitroarginine methyl ester (L-NAME) (nitric oxide synthase inhibitor; 300 micro M), apamin (small conductance Ca(2+)-activated K(+) channels inhibitor; 50 nM) and charybdotoxin (inhibitor of intermediate conductance Ca(2+)-activated K(+) channels and large conductance Ca(2+)-activated K(+) channels BK(Ca); 50 nM). L-NAME alone or in combination with either toxin alone had little effect. 3. In intact vessels, relaxations to abn-cbd were inhibited by SR 141716A (cannabinoid receptor antagonist; 1 or 3 micro M). Concomitant addition of L-NAME, apamin and charybdotoxin had no further effect. Other cannabinoid receptor antagonists either had little (SR 144528; 1 micro M and AM 251; 1 micro M) or no effect (AM 630; 10 micro M and AM 281; 1 micro M). Inhibition of gap junctions, G(i/o) protein coupling and protein kinase A also had no effect. 4. Endothelium-independent relaxation to abn-cbd was unaffected by L-NAME, apamin plus charybdotoxin or capsaicin (10 micro M). Abn-cbd inhibited CaCl(2)-induced contractions in vessels with depleted intracellular Ca(2+) stores and stimulated with methoxamine or KCl. This was insensitive to SR 141716A (3 micro M) but greatly reduced in vessels stimulated with ionomycin (Ca(2+) ionophore; 1 micro M). 5. We conclude that abn-cbd relaxes the rat small mesenteric artery by endothelium-dependent activation of K(+) channels via SR 141716A-sensitive pathways, which do not involve CB(1) and CB(2) receptors. It also causes endothelium-independent, SR 141716A-insensitive, relaxation by inhibiting Ca(2+) entry through voltage-gated Ca(2+) channels.     

An endothelium-derived factor modulates purinergic neurotransmission to mesenteric arterial smooth muscle of hamster

The interaction between the endothelium and purinergic perivascular nerves was investigated by measuring the changes in amplitude of excitatory junction potential (EJP) of smooth muscle cells in hamster mesenteric arteries (100-350 microm). Uridin-5'-triphosphate (UTP) (100 microM) applied to endothelium-intact preparations evoked a hyperpolarization of 17.0 +/- 0.7 mV (n=46). During this hyperpolarization, the amplitude of electrically evoked EJPs was inhibited to about 50% of that of the control. In endothelium-denuded preparations, UTP (100 microM) neither hyperpolarized the smooth muscle nor inhibited the amplitude of the EJP. Neither a nitric oxide (NO) synthase inhibitor, Nomega-nitro-L-arginine methyl ester (L-NAME) (100 microM), nor a cyclooxygenase inhibitor, indomethacin (1 microM), had an effect on the UTP-evoked hyperpolarization and inhibition of the electrically evoked EJP. The UTP-evoked membrane hyperpolarization and inhibition of the EJP amplitude was antagonized by the P2Y receptor antagonist, cibacron blue (100 microM). Endothelium-derived hyperpolarizing factor (EDHF)-mediated hyperpolarization was inhibited by either adventitial or intimal application of apamin (0.1 micro and charybdotoxin (0.1 microM). However, the EJP inhibition was still present. In apamin- and charybdotoxin-treated preparations, focal application of adenosine 5'-triphosphate (ATP) (10 mM) evoked a depolarization of 15.5 +/- 1.3 mV (n=15). This postjunctional response was not modified by UTP (15.3 +/- 1.7 mV, n=4, P>0.05). These results suggest that exogenously applied UTP activates P2Y receptors of endothelium to release endothelium-derived factors, which in turn inhibit ATP release from purinergic nerves.     

Neurotransmitters involved in the fast inhibitory junction potentials in mouse distal colon

We investigated, in murine colon circular muscle, the role of adenosine 5'-triphosphate (ATP) and pituitary adenylate cyclase activating peptide (PACAP) as inhibitory neurotransmitters of the fast component of nerve-evoked inhibitory junction potential (fast IJP). Fast IJP was antagonised by apamin or suramin, abolished by desensitisation with the P2Y receptor agonist, adenosine 5'-O-2-thiodiphosphate (ADPbetaS), unaffected by desensitisation with P2X receptor agonist, alpha,beta-methylene ATP (alpha,beta-meATP), and reduced by PACAP-(6-38), a PACAP receptor antagonist. ATP induced membrane hyperpolarization resistant to tetrodotoxin, N(omega)-nitro-L-arginine methyl ester (L-NAME) or PACAP-(6-38), but antagonised by apamin, suramin, P2X and P2Y receptor desensitisation. PACAP-(1-27) caused membrane hyperpolarization antagonised by PACAP-(6-38), apamin and P2Y receptor desensitisation, reduced by tetrodotoxin, but not affected by L-NAME and by P2X receptor desensitisation. Therefore, in murine colon circular muscle, an ATP-like endogenous P2Y purinoceptor ligand is the final nonadrenergic, noncholinergic (NANC) inhibitory mediator involved in the generation of fast IJP. A neuromodulator role of PACAP in the inhibitory pathway is supposed.     

Endothelium-dependent relaxation evoked by ATP and UTP in the aorta of P2Y2-deficient mice

Based on pharmacological criteria, we previously suggested that in the mouse aorta, endothelium-dependent relaxation by nucleotides is mediated by P2Y1 (adenosine diphosphate (ADP)), P2Y2 (adenosine triphosphate (ATP)) and P2Y6 (uridine diphosphate (UDP)) receptors. For UTP, it was unclear whether P2Y2, P2Y6 or yet another subtype was involved. Therefore, in view of the lack of selective purinergic agonists and antagonists, we used P2Y2-deficient mice to clarify the action of UTP. Thoracic aorta segments (width 2 mm) of P2Y2-deficient and wild-type (WT) mice were mounted in organ baths to measure isometric force development and intracellular calcium signalling.Relaxations evoked by ADP, UDP and acetylcholine were identical in knockout and WT mice, indicating that the receptors for these agonists function normally. P2Y2-deficient mice showed impaired ATP- and adenosine 5'[gamma-thio] triphosphate (ATPgammaS)-evoked relaxation, suggesting that in WT mice, ATP and ATPgammaS activate predominantly the P2Y2 subtype. The ATP/ATPgammaS-evoked relaxation and calcium signals in the knockout mice were partially rescued by P2Y1, as they were sensitive to 2'-deoxy-N6-methyladenosine 3',5'-bisphosphate (MRS2179), a P2Y1-selective antagonist.In contrast to ATP, the UTP-evoked relaxation was not different between knockout and WT mice. Moreover, the action of UTP was not sensitive to MRS2179. Therefore, the action of UTP is probably mediated mainly by a P2Y6(like) receptor subtype.In conclusion, we demonstrated that ATP-evoked relaxation of the murine aorta is mainly mediated by P2Y2. But this P2Y2 receptor has apparently no major role in UTP-evoked relaxation. The vasodilator effect of UTP is probably mediated mainly by a P2Y6(like) receptor.     

Insulin-induced relaxation of rat mesenteric artery is mediated by Ca(2+)-activated K(+) channels

We tested the hypothesis that relaxation of the rat mesenteric artery in response to insulin is mediated by K(+) channels. Two concentrations of insulin (10 and 100 mU/ml) induced relaxation of the artery by 6+/-1%, 24+/-3% (mean+/-S.E.M.). Denudation of the endothelium or precontraction by KCl (30 mM), clotrimazole (10 microM), a cytochrome P450 inhibitor, charybdotoxin (30 nM) an inhibitor of large-conductance Ca(2+)-activated K(+) channels, abolished the relaxation of the artery in response to insulin. However, N(omega)-nitro-L-arginine methyl ester (L-NAME; 100 microM), an inhibitor of nitric oxide synthase, apamin (1 microM), an inhibitor of small-conductance Ca(2+)-activated K(+) channels, or glibenclamide (10 microM), an ATP-sensitive K(+) channels blocker, did not attenuate the relaxation of the artery caused by insulin. These results suggest that the relaxation of rat mesenteric artery in response to insulin is mediated mostly by large-conductance Ca(2+)-activated K(+) channels, perhaps an endothelium-derived hyperpolarizing factor (EDHF).     

Neurally released ATP mediates endothelium-dependent hyperpolarization in the circular smooth muscle cells of chicken anterior mesenteric artery

The object of the present study was to clarify the neurotransmitter(s) controlling membrane responses to electrical field stimulation (EFS) in the circular smooth muscle cells of first-order branches of chicken anterior mesenteric artery.EFS (five pulses at 20 Hz, 1 ms) evoked a hyperpolarization of amplitude--21.6+/-1.2 mV, total duration 21.8+/-1.2 s and latency 641.7+/-81.9 ms. The response was tetrodotoxin-sensitive and nonadrenergic noncholinergic (NANC) in nature.The NANC response was blocked by the nonspecific purinergic antagonist, suramin, indicating that the response is mediated by the neurotransmitter adenosine 5'-triphosphate (ATP).Either desensitization or blockade of P2Y receptor with its putative agonist 2-methylthioATP (1 microM for 30 min) or with its antagonist cibacron blue F3GA (10 microM), respectively, abolished the purinergic hyperpolarization. PPADS at concentrations up to 100 microM had no effect on the EFS-induced response, indicating that this response is mediated through P2Y, but not P2X, receptor. In addition, the response was completely abolished by two specific P2Y1 receptor antagonists, namely, MRS 2179 (300 nM) and A3P5PS (10 microM). Removal of the endothelium abolished the purinergic hyperpolarization, which was converted, in some preparations, to a small depolarization, indicating that the hyperpolarizing response is endothelium-dependent.The present study suggests that in first-order branches of chicken anterior mesenteric artery, ATP released from perivascular nerves may diffuse to the endothelium-activating P2Y1 receptor to induce release of an inhibitory substance that mediates hyperpolarization in the circular smooth muscle.     

Functional evidence that ATP or a related purine is an inhibitory NANC neurotransmitter in the mouse jejunum: study on the identity of P2X and P2Y purinoceptors involved

1. Conflicting views exist on whether ATP is a neurotransmitter in the enteric nervous system. We investigated the role of ATP in enteric transmission in circular muscle strips of the mouse jejunum. 2. On PGF2alpha-precontracted muscle strips and in the presence of atropine and guanethidine, electrical field stimulation (EFS, 1-8 Hz) of nonadrenergic noncholinergic (NANC) nerves induced transient relaxations that were abolished by the nerve-conductance blocker tetrodotoxin. The NO synthase blocker l-nitroarginine (l-NOARG) partially inhibited the NANC relaxations to EFS, but fast-twitch relaxations to EFS were still observed in the presence of l-NOARG. 3. In the presence of l-NOARG, ATP, the P2X receptor agonist alphabetaMeATP and the P2Y receptor agonist ADPbetaS relaxed jejunal muscle strips. Tetrodotoxin did not affect the relaxation to ATP and ADPbetaS, but inhibited that to alphabetaMeATP. 4. The l-NOARG-resistant NANC relaxations to EFS were almost abolished by apamin, a blocker of small-conductance Ca2+ activated K+ channels, and by suramin and PPADS, blockers of P2 purinoceptors. Relaxations to ATP were almost abolished by apamin and suramin but not affected by PPADS. 5. Desensitisation of alphabetaMeATP-sensitive P2X receptors, the P2X receptor blocker Evans blue and the P2X1,2,3 receptor blocker NF 279 inhibited the l-NOARG-resistant NANC relaxations to EFS and that to alphabetaMeATP without affecting the relaxation to ADPbetaS. Brilliant blue G, a P2X2,5,7 receptor blocker, did not affect the relaxations to EFS. 6. Desensitisation of P2Y receptors and MRS 2179, a P2Y1 receptor blocker, virtually abolished the l-NOARG-resistant NANC relaxations to EFS and the relaxation to ADPbetaS without affecting the relaxation to alphabetaMeATP. 7. Dipyridamole, an adenosine uptake inhibitor, or theophylline and 8-phenyltheophylline, blockers of P1 and A1 purinoceptors, respectively, did not affect the purinergic NANC relaxations to EFS. 8. Our results suggest that ATP or a related purine acts as an inhibitory NANC neurotransmitter in the mouse jejunum, activating P2 but not P1 purinoceptors. Relaxations to the purinergic NANC neurotransmitter mainly involve P2Y receptors of the P2Y1 subtype that are located postjunctionally. Purinergic NANC neurotransmission also involves P2X receptors, most likely of the P2X1 and P2X3 subtype, located pre- and/or postjunctionally.     

P2X receptors counteract the vasodilatory effects of endothelium derived hyperpolarising factor

Dilatory responses of extracellular nucleotides were examined in the precontracted isolated rat mesenteric artery. Dilatation mediated by endothelium-derived hyperpolarising factor (EDHF) was studied in the presence of Nomega-nitro-L-arginine (L-NOARG) and indomethacin, and was most potently induced by the selective P2Y(1) receptor agonist adenosine 5'-O-thiodiphosphate (ADPbetaS), while 2-methylthioadenosine triphosphate (2-MeSATP) and adenosine triphosphate (ATP) were almost inactive. However, after P2X receptor desensitisation (with alphabeta-methylene-adenosine triphosphate, alphabeta-MeATP), 2-MeSATP and ATP potently stimulated EDHF-mediated dilatation. This can be explained by simultaneous activation of endothelial P2Y receptors that release EDHF, and depolarising P2X receptors on smooth muscle cells. Uridine triphosphate (UTP) also induced potent dilatation, suggesting EDHF release via P2Y(2)/P2Y(4) receptors. Uridine diphosphate (UDP) had only minor dilatory effects, and when pretreated with hexokinase it was almost inactive, suggesting a minor role for P2Y(6) receptors. The nitric oxide (NO) mediated dilatation was studied in the presence of charybdotoxin, apamin and indomethacin. ADPbetaS, 2-MeSATP, ATP and UTP were all potent relaxant agonists suggesting NO release via P2Y(1) and P2Y(2)/P2Y(4) receptors, while UDP was much less potent and efficacious. P2X receptor desensitisation had only minor effect on the NO-mediated dilatations. In conclusion, both EDHF and NO-mediated dilatation can be induced by activation of P2Y(1) and P2Y(2)/P2Y(4) receptors. P2X receptor stimulation of smooth muscle cells selectively counteracts the dilatory effect of EDHF.     

Stimulation of P2Y1 receptors causes anxiolytic-like effects in the rat elevated plus-maze: implications for the involvement of P2Y1 receptor-mediated nitric oxide production.

The widespread and abundant distribution of P2Y receptors in the mammalian brain suggests important functions for these receptors in the CNS. To study a possible involvement of the P2Y receptors in the regulation of fear and anxiety, the influences of the P2Y(1,11,12) receptor-specific agonist adenosine 5'-O-(2-thiodiphosphate) (ADPbetaS), the P2X(1,3) receptor agonist alpha,beta-methylene ATP (alpha,betameATP), the unspecific P2 receptor antagonist pyridoxalphosphate-6-azopheny l-2',4'-disulfonic acid (PPADS), and the specific P2Y(1) receptor antagonist N(6)-methyl-2'-deoxyadenosine-3',5'-bisphosphate (MRS 2179) on the elevated plus-maze behavior of the rat were investigated. All tested compounds were given intracerebroventricularly (0.5 microl). ADPbetaS (50 and 500 fmol) produced an anxiolytic-like behavioral profile reflected by an increase of the open arm exploration. The anxiolytic-like effects were antagonized by pretreatment with PPADS (5 pmol) or MRS 2179 (5 pmol). Both compounds caused anxiogenic-like effects when given alone. Furthermore, the anxiolytic-like effects of ADPbetaS could be antagonized by pretreatment with the nitric oxide synthase (NOS) inhibitor N(w)-nitro-L-arginine methyl ester (L-NAME). In addition, the anxiogenic-like effects of PPADS were reversed by the pretreatment with L-arginine (500 pmol), which is the natural substrate for NOS, but not by D-arginine (500 pmol), which is not. Immunofluorescence staining revealed the presence of P2Y(1) receptors on neurons in different brain regions such as hypothalamus, amygdala, hippocampus and the periaqueductal gray. Furthermore, the colocalization of P2Y(1) receptors and neuronal NOS (nNOS) on some neurons in these regions could be demonstrated. The highest density of P2Y(1)- and nNOS-immunoreactivity was detected in the dorsomedial hypothalamic nucleus. Taken together, the present results suggest that P2Y(1) receptors are involved in the modulation of anxiety in the rat. The anxiolytic-like effects after stimulation of P2Y(1) receptors seem to be in close connection with the related nitric oxide production.     

Combination of Ca2+ -activated K+ channel blockers inhibits acetylcholine-evoked nitric oxide release in rat superior mesenteric artery

BACKGROUND AND PURPOSE: The present study investigated whether calcium-activated K+ channels are involved in acetylcholine-evoked nitric oxide (NO) release and relaxation. EXPERIMENTAL APPROACH: Simultaneous measurements of NO concentration and relaxation were performed in rat superior mesenteric artery and endothelial cell membrane potential and intracellular calcium ([Ca2+]i) were measured. KEY RESULTS: A combination of apamin plus charybotoxin, which are, respectively, blockers of small-conductance and of intermediate- and large-conductance Ca2+ -activated K channels abolished acetylcholine (10 microM)-evoked hyperpolarization of endothelial cell membrane potential. Acetylcholine-evoked NO release was reduced by 68% in high K+ (80 mM) and by 85% in the presence of apamin plus charybdotoxin. In noradrenaline-contracted arteries, asymmetric dimethylarginine (ADMA), an inhibitor of NO synthase inhibited acetylcholine-evoked NO release and relaxation. However, only further addition of oxyhaemoglobin or apamin plus charybdotoxin eliminated the residual acetylcholine-evoked NO release and relaxation. Removal of extracellular calcium or an inhibitor of calcium influx channels, SKF96365, abolished acetylcholine-evoked increase in NO concentration and [Ca2+]i. Cyclopiazonic acid (CPA, 30 microM), an inhibitor of sarcoplasmic Ca2+ -ATPase, caused a sustained NO release in the presence, but only a transient increase in the absence, of extracellular calcium. Incubation with apamin and charybdotoxin did not change acetylcholine or CPA-induced increases in [Ca2+]i, but inhibited the sustained NO release induced by CPA. CONCLUSIONS AND IMPLICATIONS: Acetylcholine increases endothelial cell [Ca2+]i by release of stored calcium and calcium influx resulting in activation of apamin and charybdotoxin-sensitive K channels, hyperpolarization and release of NO in the rat superior mesenteric artery.     

The stable pyrimidines UDPbetaS and UTPgammaS discriminate between the P2 receptors that mediate vascular contraction and relaxation of the rat mesenteric artery

The contractile and relaxant effects of the different P2 receptors were characterized in the rat isolated mesenteric artery by use of extracellular nucleotides, including the stable pyrimidines uridine 5'-O-thiodiphosphate (UDPbetaS) and uridine 5'-O-3-thiotriphosphate (UTPgammaS). The selective P2X receptor agonist, alphabeta-methylene-adenosine triphosphate (alphabeta-MeATP) stimulated a potent (pEC(50)=6.0) but relatively weak contraction (E:(max)=57% of 60 mM K(+)). The contractile concentration-response curve of adenosine triphosphate (ATP) was biphasic when added in single concentrations. The first part of the response could be desensitized by alphabeta-MeATP, indicating involvement of P2X receptors, while the second part might be mediated by P2Y receptors. The contractile P2Y receptors were further characterized after P2X receptor desensitization with 10 microM alphabeta-MeATP. Uridine diphosphate (UDP), uridine triphosphate (UTP) and ATP stimulated contraction only in high concentrations (1 - 10 mM). The selective P2Y(6) agonist, UDPbetaS, and the P2Y(2)/P2Y(4)-receptor agonists UTPgammaS and adenosine 5'-O-3-thiotriphosphate (ATPgammaS) were considerably more potent and efficacious (E:(max) approximately 250% of 60 mM K(+)). Adenosine 5'-O-thiodiphosphate (ADPbetaS) was inactive, excluding contractile P2Y(1) receptors. After precontraction with 1 microM noradrenaline, UTP, ADP and ATP induced relaxations with similar potencies (pEC(50) approximately 5.0). UTPgammaS, ADPbetaS and ATPgammaS were approximately one log unit more potent indicating the presence of endothelial P2Y(1) and P2Y(2)/P2Y(4) receptors. The P2Y(6) receptor agonist, UDPbetaS, had no effect. UDPbetaS and UTPgammaS are useful tools when studying P2 receptors in tissue preparations with ectonucleotidase activity. Contractile responses can be elicited by stimulation of P2Y(6) and, slightly less potently, P2Y(2)/P2Y(4) receptors. The P2X response was relatively weak, and there was no P2Y(1) response. Stimulation of P2Y(1) and P2Y(2)/P2Y(4) receptors elicited relaxation, while P2Y(6) did not contribute.     

Relaxation to bradykinin in bovine pulmonary supernumerary arteries can be mediated by both a nitric oxide-dependent and -independent mechanism

1. The aim of the present study was to determine the relative contribution of prostanoids, nitric oxide and K(+) channels in the bradykinin-induced relaxation of bovine pulmonary supernumerary arteries. 2. In endothelium-intact, but not denuded rings, bradykinin produced a concentration-dependent relaxation (pEC(50), 9.6+/-0.1), which was unaffected by the cyclo-oxygenase inhibitor indomethacin. The nitric oxide scavenger hydroxocobalamin (200 micro M, pEC(50), 8.5+/-0.2) and the nitric oxide synthase inhibitor L-NAME (100 micro M, pEC(50), 8.9+/-0.1) and the combination of L-NAME and hydroxocobalamin (pEC(50), 8.1+/-0.2) produced rightward shifts in the bradykinin concentration response curve. 3. The guanylyl cyclase inhibitor ODQ (10 micro M, pEC(50), 9.6+/-0.4) did not affect the response to bradykinin. 4. Elevating the extracellular [K(+)] to 30 mM did not affect the response to bradykinin but abolished the response when ODQ or L-NAME was present. 5. The K(+) channel blocker apamin (100 nM), combined with charybdotoxin (100 nM), produced a small reduction in the maximum response to bradykinin but they abolished the response to bradykinin when ODQ, L-NAME or hydroxocobalamin were present. Apamin (100 nM) combined with iberiotoxin (100 nM) also reduced the response to bradykinin in the presence of hydroxocobalamin or L-NAME. 6. The concentration response curve for sodium nitroprusside-induced relaxation was abolished by ODQ (10 micro M) and shifted to the right by apamin and charybdotoxin. 7. These studies suggest that in bovine pulmonary supernumerary arteries bradykinin can stimulate the formation of nitric oxide and activate an EDHF-like mechanism and that either of these pathways alone can mediate the bradykinin-induced relaxation. In addition nitric oxide, acting through guanylyl cyclase, can activate an apamin/charbydotoxin-sensitive K(+) channel in this tissue.     

Endothelium-dependent vasorelaxation independent of nitric oxide and K(+) release in isolated renal arteries of rats

1. We investigated whether K(+) can act as an endothelium-derived hyperpolarizing factor (EDHF) in isolated small renal arteries of Wistar-Kyoto rats. 2. Acetylcholine (0.001 - 3 microM) caused relaxations that were abolished by removal of the endothelium. However, acetylcholine-induced relaxations were not affected by the nitric oxide (NO) synthase inhibitor N:(omega)-nitro-L-arginine methyl ester (L-NAME, 100 microM), by L-NAME plus the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ, 1 microM) or by L-NAME plus the cyclo-oxygenase inhibitor indomethacin (10 microM). In rings precontracted with high-K(+)(60 mM) physiological salt solution in the presence of L-NAME, acetylcholine-induced relaxations were abolished. 3. L-NAME-resistant relaxations were abolished by the large-conductance Ca(2+)-activated K(+) channel inhibitor charybdotoxin plus the small-conductance Ca(2+)-activated K(+) channel inhibitor apamin, while the inward rectifier K(+) channel inhibitor Ba(2+) or the gap junction inhibitor 18alpha-glycyrrhetinic acid had no effect. Acetylcholine-induced relaxation was unchanged by ouabain (10 microM) but was partially inhibited by a higher concentration (100 microM). 4. In half of the tissues tested, K(+)(10 mM) itself produced L-NAME-resistant relaxations that were blocked by ouabain (10 microM) and partially reduced by charybdotoxin plus apamin, but not affected by 18alpha-glycyrrhetinic acid or Ba(2+). However, K(+) did not induce relaxations in endothelium-denuded tissues. 5. In conclusion, acetylcholine-induced relaxations in this tissue are largely dependent upon hyperpolarization mechanisms that are initiated in the endothelium but do not depend upon NO release. K(+) release cannot account for endothelium-dependent relaxation and cannot be an EDHF in this artery. However, K(+) itself can initiate endothelium-dependent relaxations via a different pathway from acetylcholine, but the mechanisms of K(+)-induced relaxations remain to be clarified.     

Endothelial mediators of the acetylcholine-induced relaxation of the rat femoral artery

This study examined endothelium-derived mediators of acetylcholine-induced relaxation in male rat femoral arteries. Arterial rings were suspended in a myograph for the measurement of isometric force. The generation of hydrogen peroxide (H2O2) in endothelial cells was detected using the fluorescent probe, 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate acetyl ester. N(G)-nitro-L-arginine methyl ester (L-NAME, NOS inhibitor) and 1H-[1,2,4]oxadiazolo[4,2-alpha]quinoxalin-1-one (ODQ, guanylate cyclase inhibitor) alone or in combination with indomethacin (cycloxygenase inhibitor) diminished acetylcholine-induced endothelium-dependent relaxation to a similar extent. A small relaxation to acetylcholine in 60 mM KCl-constricted rings was abolished by L-NAME. Acetylcholine-induced relaxation was reduced by charybdotoxin plus apamin (intermediate- and small-conductance Ca2+-activated K+ channel blockers, respectively) or by 30 mM KCl. Both ouabain (Na+/K+ ATPase inhibitor) and BaCl2 (K(IR) channel blocker) also inhibited the relaxation albeit to a lesser degree. In the presence of L-NAME, ODQ plus indomethacin, charybdotoxin plus apamin or ouabain plus BaCl2 produced further inhibition. Catalase attenuated acetylcholine-induced relaxations and this attenuation was prevented by 3-amino-1,2,4-triazole (catalase inhibitor). Catalase did not affect acetylcholine-induced relaxations in rings treated with L-NAME or ODQ. Acetylcholine increased the dichlorofluorescein fluorescence intensity in native endothelial cells and this effect was abolished by catalase and by L-NAME. Exogenous H2O2 caused endothelium-independent relaxation that was slightly inhibited by iberiotoxin, ODQ or significantly reduced by elevated KCl, and abolished by catalase. The present results indicate that in addition to nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF, sensitive to charybdotoxin plus apamin, ouabain, and BaCl2), the endothelium of rat femoral artery can release H2O2 in response to acetylcholine, which was sensitive to L-NAME. Thus, the eNOS-dependent H2O2 is likely to be the third mediator of acetylcholine-mediated relaxations in rat femoral arteries.     

Smooth muscle mediates circumferential conduction of hyperpolarization and relaxation to focal endothelial cell activation in large coronary arteries

Longitudinal conduction of endothelium-dependent vasodilatation is mediated by intercellular spread of hyperpolarization via gap junctions along the endothelium. If similar electrical signals from the endothelium conduct around the circumference of arteries via smooth muscle cells, then, both longitudinal and circumferential spread of such signals would make it possible for a wide annulus of a large blood vessel like an epicardial coronary artery to dilate to local stimuli. To examine this in vitro, we developed a dual-chambered organ bath in which both membrane potential and force are independently determined in endothelium-intact and -denuded regions of a single annulus of artery. Hyperpolarizations and relaxations to endothelium-dependent vasodilators like bradykinin (BK) and substance P in smooth muscle cells immediately beneath the local endothelium-intact region (local responses) are conducted via smooth muscle cells around the circumference of the artery. The local relaxation was partially inhibited by the nitric oxide synthase inhibitor, N(G)-nitro-L-arginine (L-NOARG), and subsequently abolished by further treatment with a combination of two characteristic inhibitors of endothelium-dependent hyperpolarization-the Ca2+ -activated potassium channel (KCa) channel inhibitors, apamin and charybdotoxin. The conducted hyperpolarizations and relaxations to BK were unaffected by L-NOARG, but were abolished by apamin and charybdotoxin. In conclusion, these studies demonstrate for the first time that NO acts only as a local vasodilator, whereas endothelium-dependent hyperpolarization (EDH) causes local and remote vasodilatation in large coronary arteries. We propose that such a remote EDH-dependent signalling mechanism compensates for the loss of the local NO-dependent vasodilatation in diseased arteries.     

Extracellular nucleotides induce vasodilatation in human arteries via prostaglandins,nitric oxide and endothelium-derived hyperpolarising factor

1. The present study was aimed at examining P2 receptor-mediated vasodilatation in human vessels. The isometric tension was recorded in isolated segments of the human left internal mammary artery branches precontracted with 1 microM noradrenaline. 2. Endothelial denudation abolished the dilator responses. 3. The selective P2Y(1) agonist, 2-MeSADP, induced a potent vasodilatation (pEC(50)=6.9+/-0.1). The P2Y(1) antagonist of 10 microM, MRS 2216, shifted the 2-MeSADP concentration-response curve 1.1 log units to the right. The combined P2Y(1) and P2X agonist, 2-MeSATP, stimulated a dilatation with a potency similar to that of 2-MeSADP. Furthermore, MRS 2216 had a similar antagonistic effect on both 2-MeSATP and 2-MeSADP indicating that P2X receptors do not mediate vasodilatation. 4. Both the P2Y(2/4) agonist, UTPgammaS and the P2Y(6) agonist, UDPbetaS, stimulated potent dilatations (pEC(50)=7.8+/-0.4 for UTPgammaS and 8.4+/-0.2 for UDPbetaS). 5. The 2-MeSADP-induced nitric oxide (NO)-mediated dilatation was studied in the presence of 10 micro M indomethacin, 50 nM charybdotoxin and 1 microM apamin. The involvement of the endothelium-derived hyperpolarising factor (EDHF) was investigated in the presence of 0.1 mM L-NOARG and indomethacin. The involvement of prostaglandins was investigated in the presence of L-NOARG, charybdotoxin and apamin. Both NO, EDHF and prostaglandins mediated 2-MeSADP dilatation with similar efficacy (E(max)=25+/-5% for NO, 25+/-6% for EDHF and 27+/-5% for prostaglandins). 6. In conclusion, extracellular nucleotides induce endothelium-derived vasodilatation in human vessels by stimulating P2Y(1), P2Y(2/4) and P2Y(6) receptors, while P2X receptors are not involved. Endothelial P2Y receptors mediate dilatation by release of EDHF, NO and prostaglandins.     

K(+) channel blockers and cytochrome P450 inhibitors on acetylcholine-induced, endothelium-dependent relaxation in rabbit mesenteric artery

Acetylcholine caused an endothelium-dependent relaxation in isolated rabbit mesenteric small artery in the presence of nitro L-arginine and indomethacin. The acetylcholine-induced relaxation was attenuated by high K(+) solution, suggesting that the response is mediated by a membrane potential-sensitive mechanism, presumably an endothelium-derived hyperpolarizing factor. The acetylcholine-induced relaxation was also inhibited with tetraethylammonium, 4-aminopyridine and charybdotoxin, but not with Ba(2+), apamin, iberiotoxin nor glibenclamide. The relaxation was abolished by a combination of apamin and charybdotoxin, but iberiotoxin could not replace charybdotoxin in this combination. The responses to charybdotoxin and 4-aminopyridine were synergistic but neither apamin nor iberiotoxin increased the effect of 4-aminopyridine. Clotrimazole and proadifen inhibited the acetylcholine-induced relaxation, but these drugs also inhibited the cromakalim-induced relaxation, while protoporphyrin IX inhibited the acetylcholine- but not cromakalim-induced relaxation. 17-Octadecynoic acid and 1-aminobenzotriazole did not affect the response to acetylcholine. Four regioisomers of epoxyeicosatrienoic acids did not relax endothelium-denuded artery. A gap junction inhibitor 18alpha-glycyrrhetinic acid attenuated the relaxation to acetylcholine. It is suggested that in rabbit mesenteric artery, the acetylcholine-induced, nitric oxide- and prostacyclin-independent relaxation is mainly mediated by 4-aminopyridine- and charybdotoxin-sensitive K(+) channels and that the relaxation is not mediated through cytochrome P450 enzyme metabolites. The contribution of heterocellular gap junctional communication to the relaxation is discussed.     

An indirect influence of phenylephrine on the release of endothelium-derived vasodilators in rat small mesenteric artery

1. The possibility that stimulation of smooth muscle alpha(1)-adrenoceptors modulates contraction via the endothelium was examined in rat small mesenteric arteries. 2. N(omega)-nitro-L-arginine methyl ester, (L-NAME, 100 microM to inhibit NO synthase) increased contraction to single concentrations of phenylephrine (1 - 3 microM) by approximately 2 fold (from a control level of 14.2+/-3.0 to 34. 1+/-4.2% of the maximum contraction of the artery, n=20). The action of L-NAME was abolished by disrupting the endothelium. 3. The subsequent addition of apamin (to inhibit small conductance Ca(2+)-activated K(+) channels, 50 nM) further augmented phenylephrine contractions, in an endothelium-dependent manner, to more than 3 fold above control (50.4+/-5.3% of the maximum contraction, n=11). 4.Charybdotoxin (non-selective inhibitor of large conductance Ca(2+)-activated K(+) channels, BK(Ca), 50 nM) plus L-NAME augmented the level of phenylephrine contraction to 4 - 5-fold above control (64.1+/-3.1%, n=5), but this effect was independent of the endothelium. The potentiation of contraction by charybdotoxin could be mimicked with the selective BK(Ca) inhibitor, iberiotoxin,. 5. Apamin together with L-NAME and charybdotoxin further significantly increased the phenylephrine contraction by 5 - 6-fold, to 79.9+/-3.5% of the maximum contraction of the artery (n=13). 6. Phenylephrine failed directly to increase the intracellular Ca(2+) concentration in endothelial cells freshly isolated from the small mesenteric artery. 7. Stimulation of smooth muscle alpha(1)-adrenoceptors in the mesenteric artery induces contraction that is markedly suppressed by the endothelium. The attenuation of contraction appears to reflect both the release of NO from the endothelium and the efflux of K(+) from both endothelial and smooth muscle cells. This suggests that the release of NO and endothelium-derived hyperpolarizing factor can be evoked indirectly by agents which act only on the smooth muscle cells.