The contribution of d-tubocurarine-sensitive and apamin-sensitive K-channels to EDHF-mediated relaxation of mesenteric arteries from eNOS-/- mice

The nature of the potassium channels involved in determining endothelium-derived hyperpolarizing factor-mediated relaxation was investigated in first-order small mesenteric arteries from male endothelial nitric oxide synthase (eNOS-/-)-knockout and control (+/+) mice. Acetylcholine-induced endothelium-dependent relaxation of small mesenteric arteries of eNOS-/- was resistant to N-nitro-L-arginine and indomethacin and the guanylyl cyclase inhibitor, 1H-(1,2,4) oxadiazolo (4,3-a) quinoxalin-1-one. Apamin and the combination of apamin and iberiotoxin or apamin and charybdotoxin induced a transient endothelium-dependent contraction of small mesenteric arteries from both eNOS-/- and +/+ mice. Acetylcholine-induced relaxation in eNOS-/- mice was unaffected by charybdotoxin or apamin alone but significantly inhibited by the combination of these agents. However, the combination of scyllatoxin and iberiotoxin did not mimic the inhibitory effect of the apamin/charybdotoxin combination. Tubocurarine alone completely blocked acetylcholine-induced relaxation in eNOS-/- mice. Single channel analysis of myocytes from small mesenteric arterioles revealed a large conductance calcium-activated potassium channel that was sensitive to iberiotoxin, charybdotoxin, and tetraethylammonium. Tubocurarine blocked this channel from the cytosolic side but not when applied extracellularly. Solutions of nitric oxide (NO) gas also relaxed small mesenteric arteries that had been contracted with cirazoline in a concentration-dependent manner, and the sensitivity to NO was reduced by iberiotoxin and the combination of apamin, scyllatoxin, or tubocurarine with charybdotoxin but not by apamin, charybdotoxin, scyllatoxin, or tubocurarine alone. These data indicate that acetylcholine-induced endothelium-derived hyperpolarizing factor-mediated relaxation in small mesenteric arteries from eNOS-/- involved the activation of tubocurarine and apamin-/charybdotoxin-sensitive K-channels. In eNOS+/+ mice, the acetylcholine-induced response was primarily mediated by NO and was sensitive to iberiotoxin and the combination of apamin and charybdotoxin.     

Different contribution of endothelial nitric oxide in the relaxation of human coronary arteries of ischemic and dilated cardiomyopathic hearts

Coronary artery disease and congestive heart failure (CHF) have been associated with a reduction in nitric oxide (NO) release or bioavailability from the vascular endothelium. The objectives of this study were to compare the role of NO in human coronary vessels isolated from nonischemic dilated (DCM) (n = 10) and ischemic (ICM) (n = 12) cardiomyopathic hearts. Segments were mounted on a wire myograph to record changes in isometric tension. All experiments were performed in the presence of indomethacin (10 microM). Contractions induced by angiotensin II (0.1 microM) or a depolarizing physiologic solution containing 40 mM KCl, were of similar amplitude in DCM and ICM. In vessels precontracted with angiotensin II, acetylcholine (1 microM) caused an endothelium-dependent relaxation of rings from DCM but a paradoxical contraction of rings from ICM; NO synthase inhibition with Nomega-nitro-L-arginine (L-NNA, 100 microM) did not affect acetylcholine-induced relaxation or contraction of DCM or ICM vessels, respectively. By contrast, substance P (0.1 microM) induced an endothelium-dependent relaxation in both groups of vessels; this relaxation was prevented (p < 0.05) by L-NNA in vessels from ICM hearts but only reduced (p < 0.05) by L-NNA in vessels from DCM hearts. In depolarized conditions, acetylcholine contracted (p < 0.05) whereas substance P induced a complete relaxation (p < 0.05) of vessels from both groups: substance P-induced relaxation was abolished (p < 0.05) by L-NNA. Our data suggest that in the presence of indomethacin, NO does not contribute to acetylcholine-induced relaxation of human epicardial coronary arteries isolated from DCM hearts. Furthermore, whereas NO and a secondary endothelium-derived relaxing factor sensitive to high K+ contribute to substance P-induced relaxation of rings from DCM hearts, only NO is involved in ICM hearts.     

Flow- and acetylcholine-induced dilatation in small arteries from rats with renovascular hypertension--effect of tempol treatment

We investigated whether renovascular hypertension alters vasodilatation mediated by nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) and the influence of the superoxide dismutase mimetic tempol on vasodilatation. One-kidney one-clip hypertensive Sprague-Dawley rats, treated with either vehicle or tempol (from weeks 5 to 10 after placement of the clip), and uninephrectomized control rats were investigated. In renal hypertensive rats systolic blood pressure increased to 171+/-6 mmHg (n=10), while in tempol-treated rats systolic blood pressure remained normal (139+/-7 mmHg, n=5). In isolated pressurized mesenteric small arteries NO-mediated dilatation was obtained by increasing flow rate and EDHF-mediated dilatation by acetylcholine. In arteries from hypertensive rats, flow-induced dilatation was blunted, as compared to normotensive and tempol-treated rats, while acetylcholine-induced dilatation remained normal. Measured by dihydroethidium staining there was an increased amount of superoxide in arteries from vehicle-treated rats, but not from tempol-treated rats. Expression by immunoblotting of endothelial NO synthase and the NAD(P)H oxidase subunit p47phox remained unaffected by high blood pressure and tempol treatment. Simultaneous measurements of NO-concentration and relaxation were performed in isolated coronary arteries from the same animals. As compared to vehicle-treated rats, both acetylcholine-induced relaxation and NO-concentration increased in arteries from tempol-treated animals, while only the relaxation was improved by the NO donor, S-nitroso-N-acetylpenicillamine (SNAP). In conclusion renovascular hypertension selectively inhibits flow-induced NO-mediated vasodilatation, while EDHF-type vasodilatation remains unaffected, suggesting that high blood pressure leads to increased generation of superoxide contributing to decreased NO bioavailability. Furthermore, the abnormal endothelium function can be corrected by tempol treatment, but this seems to involve mechanisms partly independent of NO.     

Alterations in EDHF-mediated hyperpolarization and relaxation in mesenteric arteries of female rats in long-term deficiency of oestrogen and during oestrus cycle

This study was undertaken to determine whether endothelium-dependent relaxations are altered in mesenteric arteries from young female rats during oestrus cycle and after castration. The contractile response to phenylephrine (Phe) was significantly enhanced in arteries from rats subjected to ovariectomy than in those from sham-operated (control) rats. Treatment of ovariectomized rats with 17beta-oestradiol returned the Phe response to the control level. Arteries from rats at the diestrus stage also exhibited greater contraction in response to Phe. In the presence of 100 microM N(G)-nitro-L-arginine (L-NOARG), the enhancement of the Phe contractile response associated with oestrogen deficiency was not observed. Endothelium-dependent relaxations elicited by acetylcholine (ACh) in arteries precontracted with Phe were significantly reduced in ovariectomized and diestrus rats regardless of whether endothelium-derived nitric oxide (NO) was blocked with L-NOARG. Treatment with 17beta-oestradiol prevented the reduced vascular relaxant response to ACh in ovariectomized rats. The reduction in the ACh responses observed in ovariectomized and diestrus rats was eliminated when 500 nM apamin and 100 nM charybdotoxin were present. ACh-induced endothelium-dependent hyperpolarizations were depressed in arteries from ovariectomized and diestrus rats. The hyperpolarizing response to ACh was significantly improved when ovariectomized rats were treated with 17beta-oestradiol. The resting membrane potentials and pinacidil-induced hyperpolarizations were unaffected by ovariectomy or the diestrus stage. These results suggest that oestrogen-deficient states of both short and long duration reduce the basal release of NO from the endothelium and specifically attenuate endothelium-dependent hyperpolarization and relaxation transduced by endothelium-derived hyperpolarizing factor.     

Different role of nitric oxide and endothelium-derived hyperpolarizing factor in endothelium-dependent hyperpolarization and relaxation in porcine coronary arterial and venous system

OBJECTIVE: We compared the basal and bradykinin (BK)-induced NO release and endothelium-derived hyperpolarizing factor-mediated function between coronary arteries and cardiac veins. METHODS AND RESULTS: Isolated coronary vessels (large arteries, cardiac veins as well as microvessels) were studied. An NO-specific electrode and a conventional intracellular glass microelectrode were used to directly measure NO released from endothelial cells and hyperpolarization of smooth muscle cells in conduit coronary vessels. The basal and BK-induced release of NO was 14.2 +/- 2.0 nmol/L and 237.1 +/- 27.2 nmol/L (n = 8) in the artery, significantly greater than in veins (8.0 +/- 1.1 nmol/L and 135.6 +/- 14.5 nmol/L, n = 8, P < 0.01). The BK-induced hyperpolarization was significantly reduced by N-nitro-L-arginine, indomethacin, and hemoglobin in both arteries and veins and was greater in the arteries. The EDHF-mediated relaxation was significantly higher in the arteries than in veins, greater in microveins than in large veins, and almost abolished by charybdotoxin and apamin. CONCLUSIONS: Both NO and EDHF are involved in the regulation of the vascular tone in the coronary arterial and venous systems but the amount of NO release and the EDHF-mediated relaxation and associated hyperpolarization are less significant in the vein than in the artery in the coronary system.     

Comparison of the pharmacological properties of EDHF-mediated vasorelaxation in guinea-pig cerebral and mesenteric resistance vessels

In the presence of L-NNA (100 microM), indomethacin (10 microM) and ODQ (10 microM), acetylcholine induced a concentration-dependent vasorelaxation of guinea-pig mesenteric and middle cerebral arteries precontracted with cirazoline or histamine, but not with high K(+), indicating the contribution of an endothelium-derived hyperpolarizing factor (EDHF). In cerebral arteries, charybdotoxin (ChTX; 0.1 microM) completely inhibited the indomethacin, L-NNA and ODQ-insensitive relaxation; iberiotoxin (IbTX, 0.1 microM), 4-aminopyridine (4-AP, 1 mM), or barium (30 microM) significantly reduced the response; in the mesenteric artery, ChTX and IbTX also reduced this relaxation. Glibenclamide (10 microM) had no affect in either the mesenteric or cerebral artery. Neither clotrimazole (1 microM) nor 7-ethoxyresorufin (3 microM) affected EDHF-mediated relaxation in the mesenteric artery, but abolished or attenuated EDHF-mediated relaxations in the cerebral artery. AM404 (30 microM), a selective anandamide transport inhibitor, did not affect the vasorelaxation response to acetylcholine in the cerebral artery, but in the mesenteric artery potentiated the vasorelaxation response to acetylcholine in an IbTX, and apamin-sensitive, but SR 141816A-insensitive manner. Ouabain (100 microM) almost abolished EDHF-mediated relaxation in the mesenteric artery, but enhanced the relaxation in the cerebral artery whereas the addition of K(+) (5 - 20 mM) to precontracted guinea-pig cerebral or mesenteric artery induced further vasoconstriction. These data suggest that in the guinea-pig mesenteric and cerebral arteries different EDHFs mediate acetylcholine-induced relaxation, however, EDHF is unlikely to be mediated by K(+).     

Potassium- and acetylcholine-induced vasorelaxation in mice lacking endothelial nitric oxide synthase

1. The contribution of an endothelium-derived hyperpolarizing factor (EDHF) was investigated in saphenous and mesenteric arteries from endothelial nitric oxide synthase (eNOS) (-/-) and (+/+) mice. 2. Acetylcholine-induced endothelium-dependent relaxation of saphenous arteries of eNOS(-/-) was resistant to N(omega)-nitro-L-arginine (L-NNA) and indomethacin, as well as the guanylyl cyclase inhibitor, 1H-(1,2,4)oxadiazolo(4,3-a) quinoxalin-1-one(ODQ). 3. Potassium (K(+)) induced a dose-dependent vasorelaxation which was endothelium-independent and unaffected by either L-NNA or indomethacin in both saphenous and mesenteric arteries from eNOS(-/-) or (+/+) mice. 4. Thirty microM barium (Ba(2+)) and 10 microM ouabain partially blocked potassium-induced, but had no effect on acetylcholine-induced vasorelaxation in saphenous arteries. 5. Acetylcholine-induced relaxation was blocked by a combination of charybdotoxin (ChTX) and apamin which had no effect on K(+)-induced relaxation, however, iberiotoxin (IbTX) was ineffective against either acetylcholine- or K(+)-induced relaxation. 6. Thirty microM Ba(2+) partially blocked both K(+)- and acetylcholine-induced relaxation of mesenteric arteries, and K(+), but not acetylcholine-induced relaxation was totally blocked by the combination of Ba(2+) and ouabain. 7. These data indicate that acetylcholine-induced relaxation cannot be mimicked by elevating extracellular K(+) in saphenous arteries from either eNOS(-/-) or (+/+) mice, but K(+) may contribute to EDHF-mediated relaxation of mesenteric arteries.     

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.     

Evidence for selective inhibition by lysophosphatidylcholine of acetylcholine-induced endothelium-dependent hyperpolarization and relaxation in rat mesenteric artery.

The effects of lysophosphatidylcholine (LPC) on acetylcholine-induced hyperpolarization and relaxation were examined in rat mesenteric arteries. LPC (3-10 microM) reversibly inhibited endothelium-dependent hyperpolarization by acetylcholine in a concentration-dependent manner. LPC (10 microM) inhibited only partially endothelium-dependent relaxation by acetylcholine. However, acetylcholine-induced relaxation obtained in the presence of 100 microM NG-nitro-L-arginine was almost completely eliminated by 10 microM LPC. These results indicate that LPC inhibits hyperpolarization and relaxation due to endothelium-derived hyperpolarizing factor more specifically than the relaxation due to endothelium-derived nitric oxide.     

Relaxation induced by acetylcholine involves endothelium-derived hyperpolarizing factor in 2-kidney 1-clip hypertensive rat carotid arteries

Acetylcholine induced relaxation in a concentration-dependent way in isolated phenylephrine-contracted carotid artery rings from normotensive two-kidney (2K) and hypertensive two-kidney one-clip (2K-1C) rats. In the presence of the nitric oxide (NO) synthase inhibitor NG-nitro-L-arginine (L-NOARG, 100 micromol/l), the relaxation stimulated with acetylcholine was blocked in 2K arteries. However, in 2K-1C arteries, the relaxation was only partially inhibited. Indomethacin (3 micromol/l) had no effect in both groups. In 2K arteries, the combination of L-NOARG and indomethacin had similar effects to L-NOARG alone. On the other hand, in 2K-1C arteries, indomethacin further inhibited the maximum effect induced by acetylcholine. Endothelium-dependent relaxation induced by acetylcholine was markedly reduced in 2K arteries contracted with 90 mmol/l KCl, and it was abolished in 2K-1C arteries. The remaining response to acetylcholine in 2K arteries was blocked by L-NOARG. Thus, in addition to NO, a relaxing factor sensitive to extracellular K+ changes in the membrane potential contributes to endothelium-dependent relaxation in 2K-1C rat carotid artery. On the other hand, in arteries from 2K rats, only NO is involved in the relaxation induced by acetylcholine. The combination of 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ, 3 micromol/l), indomethacin (3 micromol/l) and L-NOARG (100 micromol/l) reduced the relaxation induced by acetylcholine in arteries from 2K-1C rats contracted with phenylephrine. On the other hand, in 2K arteries, the relaxation induced by acetylcholine was abolished. The combination of ODQ and K+ channel blockers charybdotoxin (100 nmol/l), apamin (500 nmol/l) and 4-aminopyridine (1 micromol/l) abolished the relaxation induced by acetylcholine in 2K and 2K-1C carotid arteries. These data indicate that the endothelium-derived relaxing factors that contribute to relaxation induced by acetylcholine are different in 2K and 2K-1C arteries. In 2K arteries, the only factor is NO, which involves the activation of K+ channels and the cGMP pathway. However, in 2K-1C arteries, the relaxation induced by acetylcholine is dependent on NO in addition to another factor, which is insensitive to indomethacin, but also activates the K+ channels and the cGMP pathway, presumably by membrane hyperpolarization through endothelium-derived hyperpolarizing factor.     

Stress susceptibility as a determinant of the response to adrenergic stimuli in mesenteric resistance arteries of the rat

Characterized by the behavioral response to apomorphine, two outbred lines of Wistar rats can be recognized with constitutionally determined high (apomorphine susceptible, APO-SUS) or low (apomorphine unsusceptible, APO-UNSUS) adrenal responses to similar environmental stress. Within the accumbens nucleus, the APO-SUS and APO-UNSUS rats differ in alpha -adrenergic receptor responsiveness. This study explored whether these differences in adrenergic receptor sensitivity also exist in mesenteric resistance arteries. A Mulvany myograph was used to study the vasomotor responses of isolated mesenteric resistance arteries to adrenergic receptor stimulation. Phenylephrine (alpha1-agonist)-induced vasoconstriction did not differ between the two lines (pEC : 5.8 +/- 0.05 microM versus 5.8 +/- 0.04 microM and Emax: 36 +/- 2 kPa versus 33 +/- 1 kPa for APO-SUS, n = 9, and APO-UNSUS, n = 11, respectively, p > 0.1). After precontraction with phenylephrine, salbutamol (beta -agonist)-induced relaxation was less in APO-SUS rats (pEC50 4.9 +/- 0.06 versus 5.3 +/- 0.06M for APO-SUS, n = 9, and APO-UNSUS, n = 7, respectively, p < 0.001). Likewise, clonidine (alpha2-agonist)-induced relaxation was reduced in APO-SUS rats (pEC50: 6.7 +/- 0.07 versus 7.0 +/- 0.04, for APO-SUS, n = 9, and APO-UNSUS, n = 8, respectively; p < 0.01). In conclusion, constitutionally determined high susceptibility to stress is accompanied by an impaired vasorelaxation to adrenergic stimuli whereas vasoconstriction is unaffected. An unopposed vasoconstrictor action of norepinephrine may place the APO-SUS rats at increased risk for the development of hypertension, insulin resistance, and atherosclerosis.     

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.     

Acetylcholine-induced relaxation in bovine isolated mesenteric arteries is suppressed by polymorphonuclear leukocytes.

1. The endothelium plays a critical role in maintaining vascular tone via generation of potent vasoconstrictor and dilator substances. We examined the effect of bovine purified polymorphonuclear leukocytes (PMN) on the endothelium-dependent relaxation to acetylcholine in isolated mesenteric arteries. 2. In the presence of PMN (2.5 x 10(6) cells ml-1) the maximal relaxation to acetylcholine was decreased from 76.1 +/- 2.4% to 44.9 +/- 7.4% of the precontraction (P < 0.001). This effect was inhibited by superoxide dismutase and NG-mono-methyl-L-arginine, but not by catalase or indomethacin. 3. PMN were not able to influence significantly the endothelium-independent relaxation to nitroprusside. 4. Removal of PMN after preincubation and prior to precontraction and relaxation did not influence the acetylcholine-induced relaxation, indicating that no irreversible vascular damage had occurred. 5. Superoxide anion production by unstimulated PMN was less than 10% compared to phorbol myristate acetate-activated PMN, measured by chemiluminescence and reduction of ferricytochrome c. 6. We conclude that small amounts of superoxide anions produced by unstimulated PMN contribute to a decrease in relaxation to acetylcholine by interfering with endothelium-derived nitric oxide.     

Endothelium-dependent relaxation in isolated renal arteries of diabetic rabbits

1 In this study, we have investigated the vasodilator response to acetylcholine under diabetes conditions in isolated renal arteries of rabbits. We have also examined the contribution of endothelium-derived nitric oxide (EDNO) and endothelium-derived hyperpolarizing factor (EDHF) to the endothelium-dependent relaxation caused by acetylcholine in the renal arteries of alloxan-induced diabetic rabbits. 2 Acetylcholine (10(-10) - 10(-4) M) produced cumulative concentration-response curve in the renal arteries of both control and diabetic rabbits. The EC50 values and maximal responses to acetylcholine were not significantly different relative to diabetic conditions. In order to isolate the EDHF component of acetylcholine-induced vasodilator response, L-nitro-methyl arginine ester (L-NAME, 10(-4) M) and indomethacin (10(-6) M) were added to the Krebs' solution throughout the experiment. Under these conditions, acetylcholine induced vasodilatation in the isolated renal arteries from both control and diabetic rabbits. The vasodilator response to acetylcholine was not affected under diabetic conditions. 3 Sodium nitroprusside (SNP)-induced relaxation was increased in the diabetic rabbits compared with the control animals. 4 Tetrabutyl ammonium (TBA, 0.5 mM) produced a significant reduction in acetylcholine-induced vasodilatation in both preparations from control and diabetic animals, consistent with involvement of K+ channels in mediating this response. Glibenclamide (1 microM) attenuated acetylcholine-induced vasodilatation in preparations from control animals only, while iberiotoxin (0.05 microM) significantly reduced the vasodilator response to acetylcholine in preparations from both control and diabetic animals. 5 The role of EDNO in mediating acetylcholine-induced vasodilatation was examined. The vascular preparations were incubated with 20 mM K(+)-Krebs' solution to inhibit the EDHF contribution to acetylcholine-induced vasodilatation. Under this condition, acetylcholine induced a vasodilator response in both preparations from control and diabetic rats. Pretreatment with L-NAME (10(-4) M) attenuated acetylcholine-induced vasodilatation in both preparations, indicating an nitric oxide-mediated vasodilator response. 6 Our results indicated that acetylcholine-induced vasodilatation in the isolated renal arteries of alloxan-induced diabetic rabbits was not affected under diabetic conditions. Acetylcholine-induced vasodilatation is mediated by two vasodilator components; namely, EDHF and EDNO. The contribution of EDHF and EDNO to acetylcholine-induced vasodilatation was not affected under diabetic conditions and there was no indication of endothelial dysfunction associated with diabetes. EDHF component was found to act mainly through high conductance Ca(2+)-activated K+ channels under normal and diabetic conditions, while the adenosine triphosphate-dependent K+ channels were involved in mediating acetylcholine vasodilator response in the control preparations only.     

Time-dependent reduction of acetylcholine-induced relaxation in corpus cavernosum of cholestatic rats: role of nitric oxide and cyclooxygenase pathway

The endothelium-dependent relaxation of corpus cavernosum smooth muscle and the roles of nitric oxide (NO) and arachidonic acid products of cyclooxygenase were investigated in non-operated, SHAM-operated, and bile duct-ligated rats. We further investigated the time-dependent alterations of corpus cavernosum relaxation in 2-, 7-, and 14-day bile duct-ligated animals. Acetylcholine produced concentration-dependent relaxation in phenylephrine-precontracted strips of corpus cavernosum. A significant reduction in the acetylcholine-induced relaxation was observed 2 days after bile duct ligation, and a greater reduction was observed on subsequent days. Incubation with 20 microM indomethacin reduced the acetylcholine-induced relaxation of the corpus cavernosum of unoperated rats while it had no effect in the corpus cavernosum of bile duct-ligated rats. Chronic treatment with Nomega-Nitro-L-Arginine Methyl Ester (L-NAME, 3 mg/kg/day, intraperitoneally) reduced the relaxation responses in the unoperated group while it had no effect in the bile duct-ligated group. These results show that acetylcholine-induced corporal relaxation is impaired in cholestatic rats, and this may be related to deficient nitric oxide production by the endothelium. The involvement of prostaglandins in this impairment seems unlikely.     

DIFFERENT RESPONSES TO ACETYLCHOLINE IN THE PRESENCE OF NITRIC OXIDE INHIBITOR IN RAT AORTAE AND MESENTERIC ARTERIES

1. This study compared the relaxation induced by acetylcholine (ACh) in aortic and mesenteric arterial rings from Sprague-Dawley (SD) rats in the presence and absence of inhibitors of the known endothelium-derived relaxing factors. 2. ACh-induced relaxations were completely blocked by methylene blue and N”-nitro-L-arginine (LNNA) in aortae, whereas these were only partially attenuated by methylene blue and LNNA in mesenteric arteries. 3. This methylene blue-resistant relaxation of ACh was partly attenuated by potassium channel blockers (tetraethylammonium and barium) but not affected by LNNA, indomethacin and calcium-free solution. 4. These results suggest that there may be another endothelial relaxing factor which is not nitric oxide (NO), prostanoids or endothelium-derived hyperpolarizing factor (EDHF) in mesenteric arteries but not in aortae. This unknown factor seems to be extracellular calcium ([Ca²⁺]_o)-independent.     

Role of membrane potential in endothelium-dependent relaxation of isolated mouse main pulmonary artery

The physiology of smooth muscle and endothelial cells of a particular vascular bed and from different species differs from each other. Acetylcholine causes an endothelium-dependent relaxation of preconstricted pulmonary arteries from the rat. This relaxation is mediated by nitric oxide (NO) plus a yet-unidentified endothelium-derived hyperpolarizing factor, which relaxes the smooth muscles by hyperpolarizing them. Our aim is to test whether these observations could be generalized to the smooth muscle cells from the mouse pulmonary artery. Smooth muscle or endothelial cell membrane potential of strips of murine pulmonary artery were measured simultaneously with the force developed by the strip. Acetylcholine hyperpolarized the endothelial cells. However, acetylcholine did not induce an endothelium-dependent hyperpolarization of the smooth muscle, while it relaxed the strip in an endothelium-dependent manner. This relaxation was abolished by an inhibitor of NO synthesis, nitro-L-arginine. Moreover, nitroglycerin relaxed the strips without changing the membrane potential of the smooth muscle cells. Injection of Lucifer yellow into the endothelial cells and the smooth muscle cells did not show heterocellular dye coupling. Furthermore, electron microscopy did not show gap junction plate at the myoendothelial junctions. We conclude that in the mouse main pulmonary artery, NO alone is responsible for the acetylcholine-induced endothelium-dependent vasodilatation, whereas the phenomenon called endothelium-derived hyperpolizing factor is not present. Therefore, caution should be taken when comparing different animal models to study pulmonary circulation and its reactivity.     

Regulation of vascular tone by endothelium-derived hyperpolarizing factor

1. Endothelium-derived hyperpolarizing factor (EDHF) mediates the nitric oxide (NO)-independent component of the relaxation in rat mesenteric arteries. The relationship between hyperpolarization and vascular tone was studied by simultaneous recording of membrane potential with intracellular microelectrodes and tension in ring segments of rat mesenteric arteries. 2. By depolarizing arteries with high potassium solutions, it was determined that the threshold for contraction is approximately -46 mV. Maximum contraction was attained when the arteries were depolarized to -20 mV. Thus, 1 mV depolarization resulted in an approximate 4% increase in tone. This relationship was not altered in spontaneously hypertensive rats. 3. Noradrenaline (0.3 mumol/L) caused contraction and depolarized arteries by 13 mV. Acetylcholine caused endothelium-dependent relaxation and hyperpolarization up to 14 mV. In the presence of N omega-nitro-L-arginine, the EDHF-mediated relaxation was correlated to hyperpolarization. A hyperpolarization of 1 mV corresponded to a 4.3% decrease of the induced tone. 4. At concentrations (10 mumol/L) causing total relaxation, the maximum hyperpolarization induced by NO was only 7.6 mV. 5. A maximum relaxation of 88% was observed with pinacidil (3 mumol/L), despite a 25 mV hyperpolarization. Relaxations to NO and pinacidil were not correlated with hyperpolarization. At similar levels of hyperpolarization, NO and pinacidil elicited more relaxation than EDHF. 6. These studies show that vascular tone is very sensitive to membrane potential change in the range between -46 and -20 mV in the rat mesenteric artery. The relaxation response to EDHF, unlike that to NO and pinacidil, can be accounted for solely by its effect on the membrane potential.     

内皮依赖性超极化因子在血管舒张中的作用

目的研究内皮依赖性超极化因子(EDHF)在血管舒张中的作用及机制。方法测定各种内皮依赖性舒张因子抑制剂、钾通道抑制因子、细胞色素P450单氧化酶抑制剂作用下的血管环张力。结果EDHF的血管舒张作用在大鼠肠系膜微动脉明显大于胸主动脉。一氧化氮(NO)合成受到慢性抑制时,胸主动脉的EDHF作用有增加趋势,在肠系膜微动脉投药后3 d和1周的EDHF作用明显增加。ChTx部分抑制、TBA明显抑制EDHF在肠系膜微动脉的舒张作用。结论EDHF在大鼠肠系膜微动脉的内皮依赖性舒张反应中起主要作用;在NO合成受抑制时其作用明显增加;其作用介导于K_Ca通道。     

Involvement of EDHF in the hypotension and increased gastric mucosal blood flow caused by PAR-2 activation in rats

1. Agonists for protease-activated receptor-2 (PAR-2) cause hypotension and an increase in gastric mucosal blood flow (GMBF) in vivo. We thus studied the mechanisms underlying the circulatory modulation by PAR-2 activation in vivo, especially with respect to involvement of endothelium-derived hyperpolarizing factor (EDHF). 2. Arterial blood pressure and GMBF were measured in anesthetized rats in vivo. Vascular relaxation was assessed in the precontracted rat gastric arterial rings in vitro. 3. The PAR-2-activating peptide SLIGRL-NH2 and/or trypsin, administered i.v., produced largely NO-independent hypotension and increase in GMBF accompanied by decreased gastric mucosal vascular resistance (GMVR) in rats. 4. Combined administration of apamin and charybdotoxin, but not each of them, specifically abolished the hypotension, increased GMBF and decreased GMVR caused by the PAR-2 agonists. 5. In the isolated rat gastric artery, SLIGRL-NH2 elicited endothelium-dependent relaxation even in the presence of an NO synthase inhibitor and indomethacin, which was abolished by apamin plus charybdotoxin. 6. Our data suggest involvement of apamin/charybdotoxin-sensitive K+ channels in the PAR-2-triggered hypotension and increased GMBF, predicting a role of EDHF-like factors.