Mechanisms underlying the attenuation of endothelium-dependent vasodilatation in the mesenteric arterial bed of the streptozotocin-induced diabetic rat

Experiments were designed to investigate the mechanisms underlying the diabetes-related impairment of the vasodilatations of the perfused mesenteric arterial bed induced by acetylcholine (ACh) and K(+). In streptozotocin (STZ)-diabetic rats, the ACh-induced endothelium-dependent vasodilatation was attenuated. The dose-response curves for ACh in control and diabetic rats were each shifted to the right by N(G)-nitro-L-arginine (L-NOARG) and by isotonic high K(+) (60 mM). The ACh dose-response curves under isotonic high K(+) were not different between control and diabetic rats. We also examined the vasodilatation induced by K(+), which is a putative endothelium-derived hyperpolarizing factor (EDHF). The mesenteric vasodilatation induced by a single administration of K(+) was greatly impaired in STZ-induced diabetic rats. Treatment with charybdotoxin plus apamin abolished the ACh-induced vasodilatation but enhanced the K(+)-induced response in controls and diabetic rats. After pretreatment with ouabain plus BaCl(2), the ACh-induced vasodilatation was significantly impaired and the K(+)-induced relaxation was abolished in both control and diabetic rats. The impairment of the endothelium-dependent vasodilatation of the mesenteric arterial bed seen in STZ-induced diabetic rats may be largely due to a defective vascular response to EDHF. It is further suggested that K(+) is one of the endothelium-derived hyperpolarizing factors and that the vasodilatation response to K(+) is impaired in the mesenteric arterial bed from diabetic rats.     

Histamine-induced vasodilatation in the perfused mesenteric arterial bed of diabetic rats

In this study, we have examined the contribution of endothelium-derived nitric oxide (EDNO) and endothelium-derived hyperpolarizing factor (EDHF) to histamine-induced endothelium-dependent relaxation in the perfused mesenteric arterial bed of rats treated with streptozotocin (STZ) to induce diabetes. Histamine (10(-10) to 5 x 10(-6) mol) produced dose-dependent vasodilator response in the perfused mesenteric arterial bed of both control and diabetic animals. In order to isolate the EDHF component of histamine-induced vasodilator response, NG-nitro-L-arginine-methyl ester hydrochloride (L-NAME) (10(-4) M) and indomethacin (10(-6) M) were added to the Krebs solution throughout the experiment. Histamine induced vasodilatation in the perfused mesenteric bed in preparations from both control and diabetic rats. The vasodilator response to histamine was slightly potentiated in the diabetic rat preparations. Sodium nitroprusside (SNP)-induced relaxation was similar in diabetic and control rats. The role of EDNO in histamine-induced vasodilatation was also examined. Vascular preparations were perfused with 20 mM K(+)-Krebs solution to inhibit the EDHF contribution to histamine-induced vasodilatation. Under this condition, histamine induced a vasodilator response in preparations from both control and diabetic rats. However, relative to nondiabetic control animals, histamine-induced maximal response was significantly reduced in preparations from diabetic animals. Pretreatment with L-NAME (10(-4) M) attenuated histamine-induced vasodilatation in both preparations, indicating an NO-mediated vasodilator response. There was a significant attenuation in histamine-induced vasodilatation in the vascular preparations from diabetic rats. The vasodilator effect of calcium ionophore A23187 was investigated in preparations from control and diabetic rats to investigate receptor dysfunction associated with diabetes. A23187 (10(-11) to 10(-7) mol)-induced vasodilator response was not significantly different in the preparations from control and diabetic animals. In conclusion, our results indicated that histamine-induced vasodilation in the perfused mesenteric arterial bed of the STZ-induced diabetic rats is mediated by two vasodilator components, namely EDHF and EDNO. Under diabetic conditions, the EDHF component was potentiated, while histamine-induced vasodilation mediated by the EDNO component was attenuated.     

Dominant role of an endothelium-derived hyperpolarizing factor (EDHF)-like vasodilator in the ciliary vascular bed of the bovine isolated perfused eye

1. The roles of the endothelium-derived nitric oxide, prostacyclin and endothelium-derived hyperpolarizing factor (EDHF) in mediating vasodilator responses to acetylcholine and bradykinin were assessed in the ciliary vascular bed of the bovine isolated perfused eye preparation. 2. Vasodilatation to acetylcholine or bradykinin was unaffected by the nitric oxide synthase inhibitor, L-NAME (100 microM), or the cyclo-oxygenase inhibitor, flurbiprofen (30 microM), but was virtually abolished following treatment with a high concentration of KCl (30 mM), or by damaging the endothelium with the detergent, CHAPS (0.3%, 2 min). 3. Acetylcholine-induced vasodilatation was unaffected by glibenclamide (10 microM), an inhibitor of ATP-sensitive K(+) channels (K(+)(ATP)), but was significantly attenuated by TEA (10 mM), a non-selective inhibitor of K(+) channels. 4. The small conductance calcium-sensitive K(+) channel (SK(+)(Ca)) inhibitor, apamin (100 nM), and the large conductance calcium-sensitive K(+) channel (BK(+)(Ca)) inhibitor, iberiotoxin (50 nM), had no significant effect on acetylcholine-induced vasodilatation. In contrast, the intermediate (IK(+)(Ca))/large conductance calcium-sensitive K(+) channel inhibitor, charybdotoxin (50 nM), powerfully blocked these vasodilator responses, and uncovered a vasoconstrictor response. 5. The combination of apamin (100 nM) with a sub-threshold concentration of charybdotoxin (10 nM) significantly attenuated acetylcholine-induced vasodilatation, but the combination of apamin (100 nM) with iberiotoxin (50 nM) had no effect. 6. In conclusion, blockade by a high concentration of KCl, by charybdotoxin, or by the combination of apamin with a sub-threshold concentration of charybdotoxin, strongly suggests that vasodilatation in the bovine isolated perfused eye is mediated by an EDHF.     

Ascorbate blocks endothelium-derived hyperpolarizing factor (EDHF)-mediated vasodilatation in the bovine ciliary vascular bed and rat mesentery

1. The effects of ascorbate were assessed on vasodilatation mediated by endothelium-derived hyperpolarizing factor (EDHF) in the ciliary vascular bed of the bovine isolated perfused eye and in the rat isolated perfused mesenteric arterial bed. 2. In the bovine eye, EDHF-mediated vasodilator responses induced by acetylcholine or bradykinin were powerfully blocked when ascorbate (50 microM) was included in the perfusion medium for at least 120 min; with acetylcholine a normally-masked muscarinic vasoconstrictor response was also uncovered. 3. The blockade of EDHF-mediated vasodilatation by ascorbate was time-dependent (maximum blockade at 120 min) and concentration-dependent (10 - 150 microM). 4. Ascorbate (50 microM) also blocked acetylcholine-induced, EDHF-mediated vasodilator responses in the rat mesenteric arterial bed in a time-dependent manner (maximum blockade at 180 min). 5. The ability of ascorbate to block EDHF-mediated vasodilatation is likely to result from its reducing properties, since this action was mimicked in the bovine eye by two other reducing agents, namely, N-acetyl-L-cysteine (1 mM) and dithiothreitol (100 microM), but not by the redox-inactive analogue, dehydroascorbate (50 microM). 6. In conclusion, concentrations of ascorbate present in normal plasma block EDHF-mediated vasodilator responses in the bovine eye and rat mesentery. The mechanism and physiological consequences of this blockade remain to be determined.     

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).     

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(+).     

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.     

Functional evidence that K+ is the non-nitric oxide,non-prostanoid endothelium-derived relaxing factor in rat femoral arteries

The mechanisms of K(+)-induced relaxation and of acetylcholine (ACh)-stimulated, endothelium-dependent relaxation were assessed in rat femoral arteries mounted in a myograph. ACh-stimulated (1 nM-1 microM) relaxation of arteries precontracted with 1 microM noradrenaline was mostly resistant to the combination of indomethacin (INDO; 10 microM) and N(omega)-nitro-L-arginine (L-NNA, 100 microM). The remaining relaxation was abolished by 30 mM K(+) or ouabain (1 mM) and significantly reduced by 30 microM Ba(2+) or charybdotoxin (ChTx; 100 nM) plus apamin (100 nM). K(+)-induced relaxation effected by raising [K(+)](o) by 0.5-4 mM was endothelium-independent and inhibited by ouabain and Ba(2+). These results indicate that ACh-stimulated relaxations are effected mainly by a non-prostanoid, non-nitric oxide mechanism, presumably an endothelium-derived hyperpolarising factor (EDHF). Relaxations stimulated by EDHF and K(+) are both mediated by Na(+)-K(+) ATPase and inward rectifier potassium channels (K(IR)). This study provides further functional evidence that EDHF is K(+) derived from endothelial cells that relaxes arterial smooth muscle subsequent to activation of Na(+)-K(+) ATPase and K(IR).     

Role of gap junctions in the responses to EDHF in rat and guinea-pig small arteries

1. In guinea-pig internal carotid arteries with an intact endothelium, acetylcholine (10 microM) and levcromakalim (10 microM) each hyperpolarized the smooth muscle whereas a 5 mM elevation of extracellular K(+) was without effect. 2. Incubation of the carotid artery with the gap junction inhibitors carbenoxolone (100 microM) or gap 27 (500 microM) essentially abolished the hyperpolarization to acetylcholine but it was without effect on that to levcromakalim. Carbenoxolone had no effect on the acetylcholine-induced endothelial cell hyperpolarization but inhibited the smooth muscle hyperpolarization induced by the endothelial cell K(+) channel opener, 1-ethyl-2-benzimidazolinone (600 microM). 3. In rat hepatic and mesenteric arteries with endothelium, carbenoxolone (100 or 500 microM) depolarized the smooth muscle but did not modify hyperpolarizations induced by KCl or levcromakalim. In the mesenteric (but not the hepatic) artery, the acetylcholine-induced hyperpolarization was inhibited by carbenoxolone. 4. Phenylephrine (1 microM) depolarized the smooth muscle cells of intact hepatic and mesenteric arteries, an effect enhanced by carbenoxolone. Gap 27 did not have a depolarizing action. In the presence of phenylephrine, acetylcholine-induced hyperpolarization of both hepatic and mesenteric artery myocytes was partially inhibited by each of the gap junction inhibitors. 5. Collectively, the data suggest that gap junctions play some role in the EDHF (endothelium-derived hyperpolarizing factor) response in rat hepatic and mesenteric arteries. However, in the guinea-pig internal carotid artery, electrotonic propagation of endothelial cell hyperpolarizations via gap junctions may be the sole mechanism underlying the response previously attributed to EDHF.     

Effects of chronic in vivo administration of nitroglycerine on ACh-induced endothelium-dependent relaxation in rabbit cerebral arteries

BACKGROUND AND PURPOSE: In the setting of nitrate tolerance, endothelium-dependent relaxation is reduced in several types of peripheral vessels. However, it is unknown whether chronic in vivo administration of nitroglycerine modulates such relaxation in cerebral arteries. EXPERIMENTAL APPROACH: Isometric force and smooth muscle cell membrane potential were measured in endothelium-intact strips from rabbit middle cerebral artery (MCA) and posterior cerebral artery (PCA). KEY RESULTS: ACh (0.1-10 microM) concentration-dependently induced endothelium-dependent relaxation during the contraction induced by histamine in both MCA and PCA. Chronic (10 days) in vivo administration of nitroglycerine reduced the ACh-induced relaxation in PCA but not in MCA, in the presence of the cyclooxygenase inhibitor diclofenac (3 microM). In the presence of the NO-synthase inhibitor N (omega)-nitro-L-arginine (L-NNA, 0.1 mM) plus diclofenac, in MCA from both nitroglycerine-untreated control and -treated rabbits, ACh (0.1-10 microM) induced a smooth muscle cell hyperpolarization and relaxation, and these were blocked by the small-conductance Ca(2+)-activated K(+)-channel inhibitor apamin (0.1 microM), but not by the large- and intermediate-conductance Ca(2+)-activated K(+)-channel inhibitor charybdotoxin (0.1 microM). In contrast, in PCA, ACh (<3 microM) induced neither hyperpolarization nor relaxation under these conditions, suggesting that the endothelium-derived relaxing factor is NO in PCA, whereas endothelium-derived hyperpolarizing factor (EDHF) plays a significant role in MCA. CONCLUSIONS AND IMPLICATIONS: It is suggested that in rabbit cerebral arteries, the function of the endothelium-derived relaxing factor NO and that of EDHF may be modulated differently by chronic in vivo administration of nitroglycerine.     

Evaluation of potassium ion as the endothelium-derived hyperpolarizing factor (EDHF) in the bovine coronary artery

1. This study explored the role of the potassium ion in endothelium-derived hyperpolarizing factor (EDHF)-mediated vasodilatation in the bovine coronary artery. 2. Bradykinin-induced, EDHF-mediated vasodilatation was blocked by the Na(+)-K(+) ATPase inhibitor, ouabain (1 micro M), in a time-dependent manner, with maximal blockade seen after 90 min. In contrast, the K(IR) channel inhibitor, Ba(2+) (30 micro M), had no effect. 3. When the potassium content of the bathing solution was increased in a single step from 5.9 to 7-19 mM, powerful vasodilatation (max. 75.9+/-3.6%) was observed. Vasodilatation was transient and, consequently, cumulative addition of potassium produced little vasodilatation, with vasoconstriction predominating at the higher concentrations. 4. The magnitude of potassium-induced vasodilatation was similar in endothelium-containing and endothelium-denuded rings, and was unaffected by Ba(2+) (30 micro M), but abolished by ouabain (1 micro M). 5. Ouabain (1 micro M, 90 min) powerfully blocked bradykinin-induced, nitric oxide-mediated vasodilatation as well as that induced by the nitrovasodilator, glyceryl trinitrate, but that induced by the K(ATP) channel opener, levcromakalim, was hardly affected. 6. Thus, activation of Na(+)-K(+) ATPase is likely to be involved in the vasodilator responses of the bovine coronary artery to both nitric oxide and EDHF. These findings, together with the ability of potassium to induce powerful, ouabain- but not Ba(2+)-sensitive, endothelium-independent vasodilatation, are consistent with this ion contributing to the EDHF response in this tissue.     

Depletion of intracellular Ca2+ stores enhances flow-induced vascular dilatation in rat small mesenteric artery

The effect of depleting intracellular Ca2+ stores on flow-induced vascular dilatation and the mechanism responsible for the vasodilatation were examined in rat isolated small mesenteric arteries. The arteries were pressurized to 50 mmHg and preconstricted with phenylephrine. Intraluminal flow reversed the effect of phenylephrine, resulting in vasodilatation. Flow dilatation consisted of an initial transient peak followed by a sustained plateau phase. The magnitude of dilatation was markedly reduced by removing Ca2+ from the intraluminal flow medium.Depletion of intracellular Ca2+ stores with either cyclopiazonic acid (CPA, 2 microM) or 1,4-dihydroxy-2,5-di-tert-butylbenzene (BHQ, 10 microM) significantly augmented the magnitude of flow dilatation. Flow-induced endothelial cell Ca2+ influx was also markedly enhanced in arteries pretreated with CPA or BHQ.Flow-induced dilatation was insensitive to Nw-nitro-L-arginine methyl ester (100 microM) plus indomethacin (3 microM) or to oxyhemoglobin (3 microM), but was markedly reduced by 30 mM extracellular K+ or 2 mM tetrabutylammonium (TBA), suggesting an involvement of EDHF. Catalase at 1200 U ml-1 abolished the flow-induced dilatation, while the application of exogenous H2O2 (90-220 microM) induced relaxation in phenylephrine-preconstricted arteries. Relaxation to exogenous H2O2 was blocked in the presence of 30 mM extracellular K+, and H2O2 (90 microM) hyperpolarized the smooth muscle cells, indicating that H2O2 can act as an EDHF. In conclusion, flow-induced dilatation in rat mesenteric arteries can be markedly enhanced by prior depletion of intracellular Ca2+ stores. Furthermore, these data are consistent with a role for H2O2 as the vasodilator involved.     

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.     

Mechanisms of relaxing response induced by rat/mouse hemokinin-1 in porcine coronary arteries: roles of potassium ion and nitric oxide

Rat and mouse hemokinin-1(r/m hemokinin-1) is a recently described member of the tachykinin family whose cardiovascular functions are not fully understood. In this study, we investigated the mechanisms of the relaxing response induced by r/m hemokinin-1 in isolated porcine coronary arteries by using a specific antagonist of tachykinin NK(1) receptor (SR140333), a nitric oxide synthase inhibitor N(omega)-nitro-L-arginine (L-NNA), and 1H-[1,2,4] Oxadiazolo [4,3-a] quinoxalin-1-one (ODQ), a blocker of cGMP production. r/m Hemokinin-1 (10(-12)-10(-6 )M) evoked a marked endothelium-dependent vasodilatation (E(max)=121.12+/-10.6% and 91.79+/-2.39% in 10(-6) M PGF(2)alpha and 30 mM KCl precontracted arterial rings, respectively) of coronary arteries mediated by activation of endothelial tachykinin NK(1) receptors. Two components contributed to this r/m hemokinin-1-elicited vasodilatation, the first of which was endothelium-derived hyperpolarizing factor (EDHF), which played a major role. This EDHF was identified as a potassium current through certain kinds of potassium channels on the endothelial cell membrane of porcine coronary arteries. Specific antagonists of Ca(2+)-activated K(+) channels (dequalinium and clotrimazole) did not have an inhibitory effect on the r/m hemokinin-1-induced vasodilatation, whereas they did on the substance P-induced vasodilatation. When potassium ion efflux was impaired by a high K(+) concentration (30 mM) or removal of K(+) from the surroundings, NO synthesis was triggered by r/m hemokinin-1 to produce an equivalent EDHF (K(+))-independent vasorelaxation as a compensatory mechanism.     

Histamine-induced vasodilation in the perfused kidney of STZ-diabetic rats: role of EDNO and EDHF

In this study, we have examined the contribution of endothelium-derived nitric oxide (EDNO) and endothelium-derived hyperpolarizing factor (EDHF) to histamine-induced endothelium-dependent relaxation in the perfused kidney of rats treated with streptozotocin (STZ) to induce diabetes. Histamine-induced vasodilatation in the perfused kidney preparations of both control and diabetic animals, which was not significantly different. Sodium nitroprusside (SNP)-induced relaxation was also not affected in diabetic and control rats. In order to isolate the EDHF component of histamine-induced vasodilator response, L-NAME (10(-4)M) and indomethacin (10(-6)M) were added to the Krebs' solution throughout the experiment. TBA (0.5 mM) produced a significant reduction in histamine-induced maximal vasodilator response in both preparations from control and diabetic animals, indicating the involvement of K+ channels in mediating this response. Charybdotoxin (0.05 microM) but not glibenclamide (0.1 microM) produced significant reduction in histamine-induced vasodilator responses. To test the contribution of EDNO in mediating histamine-induced vasodilatation, the vascular preparations were perfused with 20 mM K+ -Krebs' solution to inhibit the EDHF component of the response. Under this condition, histamine-induced vasodilator response was not significantly different in both preparations from control and diabetic rats. Pre-treatment with L-NAME (10(-4)M) attenuated histamine-induced vasodilatation. There was a more significant attenuation in histamine-induced vasodilatation in the vascular preparations from diabetic rats. The vasodilator effect of calcium ionophore A23187 was investigated in preparations from control and diabetic rats to examine receptor dysfunction associated with diabetes. A23187 produced dose-dependent vasodilator response in the preparations from both control and diabetic rats. In conclusion, our results indicate that histamine-induced vasodilatation in the perfused kidney of the STZ-induced diabetic rats is mediated by the two vasodilator components, namely EDHF and EDNO. The EDHF component was not significantly affected by diabetes. However, histamine-induced vasodilatation mediated by the EDNO component was more significantly reduced in diabetic rats. Results have also indicated that the EDHF component of histamine-induced vasodilatation was mediated through Ca2+ -activated K+ channels in perfused kidney preparations from both control and diabetic rats.     

Role of gap junctions in endothelium-derived hyperpolarizing factor responses and mechanisms of K(+)-relaxation

We have examined the effects of ouabain (1 mM), the gap junction inhibitors, 18 alpha-glycyrrhetinic acid (100 microM), N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2, 4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride (SR141716A; 10 microM) and palmitoleic acid (50 microM), and clotrimazole (10 microM) against endothelium-derived hyperpolarizing factor (EDHF)-mediated and K(+)-induced vasorelaxations in the rat mesentery. In the presence of indomethacin (10 microM) and 300-microM N(G)nitro-L-arginine methyl ester (L-NAME), carbachol caused EDHF-mediated relaxations (R(max)=85.3+/-4.0%). In the presence of ouabain, these responses were substantially reduced (R(max)=11.0+/-2.3%). 18 alpha-glycyrrhetinic acid, SR141716A, palmitoleic acid and clotrimazole also significantly inhibited these EDHF-mediated responses. K(+) caused vasorelaxation of preparations perfused with K(+)-free buffer (R(max)=73.7+/-2.4%), which were reduced by 10-microM indomethacin (R(max)=56.4+/-6.2%). K(+) vasorelaxation was essentially abolished by endothelial denudation. Both ouabain and 18 alpha-glycyrrhetinic acid opposed K(+) relaxations, however, neither SR141716A, clotrimazole nor palmitoleic acid had any effect. Direct cell-cell coupling via gap junctions was attenuated by ouabain, clotrimazole and palmitoleic acid. We conclude that: (i) that gap junctional communication plays a major role in EDHF-mediated relaxations, (ii) that K(+)-vasorelaxation is endothelium-dependent (thus, K(+) is unlikely to represent an EDHF), and (iii) that the inhibitory actions of ouabain and clotrimazole on gap junctions might contribute towards their effects against EDHF.     

Suppression of K(+)-induced hyperpolarization by phenylephrine in rat mesenteric artery: relevance to studies of endothelium-derived hyperpolarizing factor

In intact mesenteric arteries, increasing [K(+)]o by 5 mM hyperpolarized both endothelial and smooth muscle cells. Subsequent exposure to 10 microM phenylephrine depolarized both cell types which were then repolarized by a 5 mM increase in [K(+)]o. In endothelium-denuded vessels, increasing [K(+)]o by 5 mM hyperpolarized the smooth muscle but K(+) had no effect after depolarization by 10 microM phenylephrine. On subsequent exposure to iberiotoxin plus 4-aminopyridine, the repolarizing action of 5 mM K(+) was restored. In endothelium-intact vessels exposed to phenylephrine, pretreatment with a gap junction inhibitor (gap 27) reduced K(+)-mediated smooth muscle repolarization without affecting the endothelial cell response. It is concluded that phenylephrine-induced efflux of K(+) via smooth muscle K(+) channels produces a local increase in [K(+)]o which impairs repolarization to added K(+). Thus, studies involving vessels precontracted with agonists which increase [K(+)]o maximize the role of gap junctions and minimize any contribution to the EDHF pathway from endothelium-derived K(+).     

New molecular mechanisms for cardiovascular disease: contribution of endothelium-derived hyperpolarizing factor in the regulation of vasoconstriction in peripheral resistance arteries

Endothelium regulates vascular tone via release of endothelium-derived relaxing factors (EDRF) including nitric oxide (NO), prostaglandin I? (PGI?), and endothelium-derived hyperpolarizing factor (EDHF). The mesenteric vascular bed produces vascular resistance to develop blood pressure and regulate tissue blood flow that plays an important role in maintenance of systemic blood pressure. There is now strong evidence that in these small resistance arteries, EDHF plays a major role in the response to vasoactive substances and regulation of vascular tone. Pharmacological analysis to investigate the role of the vascular endothelium in the regulation of α?-adrenoceptor agonist (methoxamine)-induced vasoconstriction in rat mesenteric vascular beds showed that vasoconstriction induced by continuous perfusion of methoxamine (7 μM), but not high KCl (60 mM), time-dependently decreased to 20% of the initial constriction. The time-dependent reduction of methoxamine-induced vasoconstriction was inhibited by endothelium removal, inhibitor of EDHF (30 mM KCl, K+-channel blockers), and gap-junction inhibitor, but not NO synthase inhibitor and cyclooxygenase inhibitor and ageing. These results suggest that vascular endothelium counteracts to normalize excess vasoconstriction of the mesenteric resistance arteries by releasing EDHF, which is associated with activation of multiple K+-channels and gap junction involvement and markedly decreases with ageing.     

Inhibitory effects of brefeldin A, a membrane transport blocker, on the bradykinin-induced hyperpolarization-mediated relaxation in the porcine coronary artery

1. To elucidate the mechanism of the relaxation mediated by endothelium-derived hyperpolarizing factors (EDHFs), the effect of brefeldin A, a membrane transport blocker, on cytosolic Ca(2+) concentration ([Ca(2+)]i) and tension was determined in the porcine coronary arterial strips. We also examined the effect of brefeldin A on [Ca(2+)]i in the endothelial cells of the porcine aortic valve. 2. In the presence of 10 microM indomethacin and 30 microM N(G)-nitro-L-arginine (L-NOARG), both bradykinin and substance P induced a transient decrease in [Ca(2+)]i and tension in arterial strips contracted with 100 nM U46619 (thromboxane A2 analogue). A 6 h pre-treatment with 20 microg ml(-1) brefeldin A abolished the bradykinin-induced relaxation, while it had no effect on the substance P-induced relaxation. 3. In the absence of indomethacin and L-NOARG, brefeldin A had no effect on the bradykinin-induced relaxation during the contraction induced by U46619 or 118 mM K(+). 4. The indomethacin/L-NOARG-resistant relaxation induced by bradykinin was completely inhibited by 3 mM tetrabutylammonium (non-specific Ca(2+)-activated K(+) channel blocker), while that induced by substance P was not inhibited by 3 mM tetrabutylammonium or 1 mM 4-aminopyridine (voltage-dependent K(+) channels blocker) alone, but completely inhibited by their combination. 5. Brefeldin A had no effect on the [Ca(2+)]i elevation in endothelial cells induced by bradykinin or substance P. 6. In conclusion, bradykinin produce EDHF in a brefeldin A-sensitive mechanism in the porcine coronary artery. However, this mechanism is not active in a substance P-induced production of EDHF, which thus suggests EDHF to be more than a single entity.     

Potassium does not mimic EDHF in rat mesenteric arteries

1. K(+) has been proposed to be EDHF in small arteries. We compared ACh-stimulated, EDHF-mediated dilatation/relaxation with raised [K(+)](o) in rat mesenteric arteries. 2. In pressurized arteries, ACh (10 microM) dilated all arteries. Raising [K(+)](o) from 5.88 to 10. 58 mM only dilated 30% of arteries. Ba(2+) (30 microM) did not affect dilatation to ACh, but abolished 40% of dilatations to raised [K(+)](o). 3. If [K(+)](o) was lowered to 1.18 mM, restoring [K(+)](o) to 5.88 mM produced dilatation which was depressed by Ba(2+) or ouabain (1 mM). Combined application of Ba(2+) and ouabain abolished dilatation. In 1.18 mM K(+), dilatation to ACh was depressed by ouabain, but not by Ba(2+). Combined application of Ba(2+) and ouabain depressed dilatation further. Gap junction inhibitors (Gap-27; 300 microM and 18-alpha-glycyrrhetinic acid; 100 microM) also depressed dilatation to ACh. 4. In arteries mounted isometrically, ACh (1 microM) relaxed endothelium intact (+E), but not endothelium denuded (-E) arteries. Raising [K(+)](o) from 5.9 - 10.9 mM failed to relax all arteries. When [K(+)](o) was lowered to 1 mM, raising [K(+)](o) to 6 mM produced relaxation. In -E arteries, relaxation was unaffected by Ba(2+) but abolished by ouabain. In +E arteries, Ba(2+) depressed and ouabain abolished relaxation. In +E arteries, with 1 mM K(+), ACh relaxation was depressed by ouabain but not Ba(2+). The combined application of Ba(2+) and ouabain further depressed relaxation. 5. In summary, both EDHF and raised [K(+)](o) dilate/relax rat mesenteric arteries, though sensitivities to barium and ouabain differ. K(+) may be a relaxing factor in this tissue, but its characteristics differ from EDHF. Gap junction inhibitors depress EDHF, implying an important role for myo-endothelial gap junctions.