Contributions of endothelium-derived relaxing factors to control of hindlimb blood flow in the mouse in vivo

We determined the contributions of various endothelium-derived relaxing factors to control of basal vascular tone and endothelium-dependent vasodilation in the mouse hindlimb in vivo. Under anesthesia, catheters were placed in a carotid artery, jugular vein, and femoral artery (for local hindlimb circulation injections). Hindlimb blood flow (HBF) was measured by transit-time ultrasound flowmetry. N(omega)-nitro-L-arginine methyl ester (L-NAME, 50 mg/kg plus 10 mg x kg(-1) x h(-1)), to block nitric oxide (NO) production, altered basal hemodynamics, increasing mean arterial pressure (30 +/- 3%) and reducing HBF (-30 +/- 12%). Basal hemodynamics were not significantly altered by indomethacin (10 mg x kg(-1) x h(-1)), charybdotoxin (ChTx, 3 x 10(-8) mol/l), apamin (2.5 x 10(-7) mol/l), or ChTx plus apamin (to block endothelium-derived hyperpolarizing factor; EDHF). Hyperemic responses to local injection of acetylcholine (2.4 microg/kg) were reproducible in vehicle-treated mice and were not significantly attenuated by L-NAME alone, indomethacin alone, L-NAME plus indomethacin with or without co-infusion of diethlyamine NONOate to restore resting NO levels, ChTx alone, or apamin alone. Hyperemic responses evoked by acetylcholine were reduced by 29 +/- 11% after combined treatment with apamin plus charybdotoxin, and the remainder was virtually abolished by additional treatment with L-NAME but not indomethacin. None of the treatments altered the hyperemic response to sodium nitroprusside (5 microg/kg). We conclude that endothelium-dependent vasodilation in the mouse hindlimb in vivo is mediated by both NO and EDHF. EDHF can fully compensate for the loss of NO, but this cannot be explained by tonic inhibition of EDHF by NO. Control of basal vasodilator tone in the mouse hindlimb is dominated by NO.     

Role of endothelium-derived relaxing factors in the renal response to vasoactive agents in hypothyroid rats

This study analyzed the role of nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) in the abnormal renal vascular reactivity of hypothyroid rats. Renal responses to vasoconstrictors [VC: phenylephrine (PHE) and ANG II] and vasodilators [VD: ACh, sodium nitroprusside (SNP), and papaverine (PV)] were studied in kidneys from control and hypothyroid rats under normal conditions and after NO or EDHF blockade. NO was blocked by the administration of Nomega-nitro-l-arginine methyl ester (l-NAME) and EDHF by the administration of tetraethylammonium (TEA) or by an increased extracellular K+. The response to VC was also evaluated after endothelium removal. Hypothyroid kidneys showed reduced responsiveness to PHE and a normal response to ANG II. l-NAME and TEA administration produced an increased sensitivity to PHE and to ANG II in control preparations. l-NAME also increased the response to PHE in hypothyroid kidneys, but the differences between control and hypothyroid kidneys were maintained. TEA administration did not change the response to either VC in hypothyroid preparations. In endothelium-removed preparations, TEA was unable to increase pressor responsiveness to VC. Hypothyroid kidneys showed reduced responsiveness to ACh and SNP and normal response to PV. The differences between hypothyroid and control preparations in the responses to ACh and SNP were maintained after l-NAME or increased K+. In conclusion, this study shows that 1) the attenuated response to PHE in hypothyroidism is not related to an increased production of endothelium-derived relaxing factors NO and EDHF; 2) the response to VC in hypothyroid preparations is insensitive to EDHF blockade; and 3) hypothyroid preparations have a reduced reactivity to the NO donor, and NO-independent vasodilatation remains unaffected.     

Tranilast Increases Vasodilator Response to Acetylcholine in Rat Mesenteric Resistance Arteries through Increased EDHF Participation

Background and Purpose

Tranilast, in addition to its capacity to inhibit mast cell degranulation, has other biological effects, including inhibition of reactive oxygen species, cytokines, leukotrienes and prostaglandin release. In the current study, we analyzed whether tranilast could alter endothelial function in rat mesenteric resistance arteries (MRA).

Experimental Approach

Acetylcholine-induced relaxation was analyzed in MRA (untreated and 1-hour tranilast treatment) from 6 month-old Wistar rats. To assess the possible participation of endothelial nitric oxide or prostanoids, acetylcholine-induced relaxation was analyzed in the presence of L-NAME or indomethacin. The participation of endothelium-derived hyperpolarizing factor (EDHF) in acetylcholine-induced response was analyzed by preincubation with TRAM-34 plus apamin or by precontraction with a high K⁺ solution. Nitric oxide (NO) and superoxide anion levels were measured, as well as vasomotor responses to NO donor DEA-NO and to large conductance calcium-activated potassium channel opener NS1619.

Key Results

Acetylcholine-induced relaxation was greater in tranilast-incubated MRA. Acetylcholine-induced vasodilation was decreased by L-NAME in a similar manner in both experimental groups. Indomethacin did not modify vasodilation. Preincubation with a high K⁺ solution or TRAM-34 plus apamin reduced the vasodilation to ACh more markedly in tranilast-incubated segments. NO and superoxide anion production, and vasodilator responses to DEA-NO or NS1619 remained unmodified in the presence of tranilast.

Conclusions and Implications

Tranilast increased the endothelium-dependent relaxation to acetylcholine in rat MRA. This effect is independent of the nitric oxide and cyclooxygenase pathways but involves EDHF, and is mediated by an increased role of small conductance calcium-activated K+ channels.     

In vivo mechanisms of acetylcholine-induced vasodilation in rat sciatic nerve

We examined the importance of nitric oxide (NO), endothelium-derived hyperpolarizing factor (EDHF), and neurogenic activity in agonist-induced vasodilation and baseline blood flow [i.e., nerve microvascular conductance (NMVC)] in rat sciatic nerve using laser Doppler flowmetry. Agonists were acetylcholine (ACh) and 3-morpholinosydnonimine (SIN-1). Vasodilation occurring despite NO synthase (NOS) and cyclooxygenase inhibition and showing dependence on K(+) channel activity was taken as being mediated by EDHF. NOS and cyclooxygenase inhibition with N(omega)-nitro-L-arginine (L-NNA) + indomethacin (Indo) revealed two phases of ACh-induced vasodilation: an initial, transient L-NNA + Indo-resistant vasodilation, peaking at 23 +/- 6 s and lasting 145 +/- 69 s, followed by sustained L-NNA + Indo-sensitive vasodilation. L-NNA alone did not affect sustained ACh-induced vasodilation but decreased baseline NMVC by 55%. In the presence of L-NNA + Indo, the K(+) channel blocker tetraethylammonium (TEA) inhibited transient ACh-induced vasodilation by 58% and reduced baseline NMVC by 25%. SIN-1-induced vasodilation increased fourfold in the presence of L-NNA, whereas the specific guanylyl cyclase inhibitor 1H-(1, 2, 4)oxadiazolo(4,3-alpha)quinoxalin-1-one abolished it. However, in homogenates of rat sciatic nerve, SIN-1-stimulated soluble guanylyl cyclase (sGC) activity was unaffected by L-NNA. TTX affected neither SIN-1- nor ACh-induced vasodilation. In conclusion, ACh-induced vasodilation consisted of two components, the first partially mediated by EDHF and the second by a vasodilatory prostanoid + NO. Baseline NMVC was dependent on NO and EDHF. Although L-NNA enhanced SIN-1-induced vasodilation, it had no effect on sGC-activity.     

Effects of Trientine,a Metal Chelator,on Defective Endothelium-dependent Relaxation in the Mesenteric Vasculature of Diabetic Rats

Diabetes mellitus compromises endothelium-dependent relaxation of blood vessels. This has been linked to the generation of reactive oxygen species (ROS), which neutralise nitric oxide (NO) and interfere with vasodilator function. Experiments using chelators have emphasised the importance of ROS produced by transition metal catalysed reactions. However, particularly for the small arteries and arterioles that control microcirculatory blood flow, NO is not the only endothelium-derived mediator; endothelium-derived hyperpolarizing factor (EDHF) has a major role. EDHF-mediated vasodilation is severely curtailed by diabetes; however, little information exists on the underlying pathophysiology. Deficits in the EDHF system, alone or in combination with the NO system, are crucial for the development of diabetic microvascular complications. To further elucidate the mechanisms involved, the aim was to examine the effects of diabetes and preventive and intervention chelator therapy with trientine on a preparation that has well-defined NO and EDHF-mediated responses, the rat mesenteric vascular bed. In phenylephrine-preconstricted preparations, maximum vasodilation to acetylcholine was reduced by 35 and 44% after 4 and 8 weeks of streptozotocin-induced diabetes, respectively. Trientine treatment over the first 4 weeks gave 72% protection; intervention therapy over the final 4 weeks prevented deterioration and corrected the initial deficit by 68%. These responses depend on both NO and EDHF. When the latter mechanism was isolated by NO synthase inhibition, diabetic deficits of 53.4 (4 weeks) and 65.4% (8 weeks) were revealed, that were 65% prevented and 50% corrected by trientine treatment. Neither diabetes nor trientine altered vascular smooth muscle responses to the NO donor, sodium nitroprusside (SNP). Thus, the data suggest that metal catalysed ROS production makes a substantial contribution to defects in both the EDHF and NO endothelial mechanisms in diabetes, which has therapeutic implications for microvascular complications.     

Effects of trientine,a metal chelator,on defective endothelium-dependent relaxation in the mesenteric vasculature of diabetic rats

Diabetes mellitus compromises endothelium-dependent relaxation of blood vessels. This has been linked to the generation of reactive oxygen species (ROS), which neutralise nitric oxide (NO) and interfere with vasodilator function. Experiments using chelators have emphasised the importance of ROS produced by transition metal catalysed reactions. However, particularly for the small arteries and arterioles that control microcirculatory blood flow, NO is not the only endothelium-derived mediator; endothelium-derived hyperpolarizing factor (EDHF) has a major role. EDHF-mediated vasodilation is severely curtailed by diabetes; however, little information exists on the underlying pathophysiology. Deficits in the EDHF system, alone or in combination with the NO system, are crucial for the development of diabetic microvascular complications. To further elucidate the mechanisms involved, the aim was to examine the effects of diabetes and preventive and intervention chelator therapy with trientine on a preparation that has well-defined NO and EDHF-mediated responses, the rat mesenteric vascular bed. In phenylephrine-preconstricted preparations, maximum vasodilation to acetylcholine was reduced by 35 and 44% after 4 and 8 weeks of streptozotocin-induced diabetes, respectively. Trientine treatment over the first 4 weeks gave 72% protection; intervention therapy over the final 4 weeks prevented deterioration and corrected the initial deficit by 68%. These responses depend on both NO and EDHF. When the latter mechanism was isolated by NO synthase inhibition, diabetic deficits of 53.4 (4 weeks) and 65.4% (8 weeks) were revealed, that were 65% prevented and 50% corrected by trientine treatment. Neither diabetes nor trientine altered vascular smooth muscle responses to the NO donor, sodium nitroprusside (SNP). Thus, the data suggest that metal catalysed ROS production makes a substantial contribution to defects in both the EDHF and NO endothelial mechanisms in diabetes, which has therapeutic implications for microvascular complications.     

Nitric oxide-dependent and -independent mechanisms in the relaxation elicited by acetylcholine in fetal rat aorta

The aim of the present study was to analyze the mechanisms involved in the relaxation induced by 1 microM acetylcholine (ACh) in aortic segments from fetal rats at term precontracted with 3 microM prostaglandin F2alpha (PGF2alpha) and incubated with 1 microM indomethacin. The endothelium-dependent relaxation caused by ACh was reduced by the nitric oxide (NO) synthase inhibitor NG-monomethyl-L-arginine (L-NMMA, 0.1 mM), such an effect was reversed by 0.1 mM L-arginine (L-Arg). After precontraction of segments with 50 mM KCl the relaxant response to ACh was smaller than that after precontraction with PGF2alpha; this reduction was increased by L-NMMA, whereas L-NMMA plus L-Arg potentiated the relaxation. Thiopentone sodium (0. 1 mM), ouabain (10 microM), tetraethylammonium (TEA, 0.5 mM) and apamin (1 microM), inhibitors of cytochrome P450 monooxygenases, Na+ pump, Ca2+-activated (KCa) and small-conductance (SKCa) K+ channels, respectively, reduced the relaxation to ACh, which was unaffected by charybdotoxin (0.1 microM) and glibenclamide (1 microM), inhibitors of large-conductance BKCa and ATP-sensitive K+ channels. The L-NMMA/indomethacin-resistant relaxation to ACh was markedly reduced by thiopentone sodium, and similarly decreased by either ouabain or TEA. The endothelium-independent relaxation induced by exogenous NO (10 microM) in segments precontracted with PGF2alpha was unaltered by ouabain, glibenclamide, TEA and after precontraction with 50 mM KCl, and potentiated by L-NMMA. The potentiation of NO responses by L-NMMA was also observed in segments precontracted with KCl. These results suggest that ACh relaxes the fetal rat aorta by endothelial release of both NO and endothelium-derived hyperpolarizing factor (EDHF), a metabolite derived from cytochrome P450 monooxygenases, that hyperpolarizes smooth muscle cells by activation of KCa, essentially SKCa channels, and Na+ pump. It seems that when the effect of EDHF is abolished, the formation of NO could be increased.     

EDHF-mediated vasodilation involves different mechanisms in normotensive and hypertensive rat lungs

The role of endothelium-derived hyperpolarizing factor (EDHF) in regulating the pulmonary circulation and the participation of cytochrome P-450 (CYP450) activity and gap junction intercellular communication in EDHF-mediated pulmonary vasodilation are unclear. We tested whether tonic EDHF activity regulated pulmonary vascular tone and examined the mechanism of EDHF-mediated pulmonary vasodilation induced by thapsigargin in salt solution-perfused normotensive and hypoxia-induced hypertensive rat lungs. After blockade of both cyclooxygenase and nitric oxide synthase, inhibition of EDHF with charybdotoxin plus apamin did not affect either normotensive or hypertensive vascular tone or acute hypoxic vasoconstriction but abolished thapsigargin vasodilation in both groups of lungs. The CYP450 inhibitors 7-ethoxyresorufin and sulfaphenazole and the gap junction inhibitor palmitoleic acid, but not 18alpha-glycyrrhetinic acid, inhibited thapsigargin vasodilation in normotensive lungs. None of these agents inhibited the vasodilation in hypertensive lungs. Thus tonic EDHF activity does not regulate either normotensive or hypertensive pulmonary vascular tone or acute hypoxic vasoconstriction. Whereas thapsigargin-induced EDHF-mediated vasodilation in normotensive rat lungs involves CYP450 activity and might act through gap junctions, the mechanism of vasodilation is apparently different in hypertensive lungs.     

Responses of cerebral arterioles to ADP: eNOS-dependent and eNOS-independent mechanisms

ADP mediates platelet-induced relaxation of blood vessels and may function as an important intercellular signaling molecule in the brain. We used pharmacological and genetic approaches to examine mechanisms that mediate responses of cerebral arterioles to ADP, including the role of endothelial nitric oxide synthase (eNOS). We examined responses of cerebral arterioles (control diameter approximately 30 microm) in anesthetized wild-type (WT, eNOS+/+) and eNOS-deficient (eNOS-/-) mice using a cranial window. In WT mice, local application of ADP produced vasodilation that was not altered by indomethacin but was reduced by approximately 50% by NG-nitro-L-arginine (L-NNA) or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (inhibitors of NOS and soluble guanylate cyclase, respectively). In eNOS-/- mice, responses to ADP were largely preserved, and a significant component of the response was resistant to L-NNA (a finding similar to that in WT mice treated with L-NNA). In the absence of L-NNA, responses to ADP were markedly reduced by charybdotoxin plus apamin [inhibitors of Ca2+-dependent K+ channels and responses mediated by endothelium-derived hyperpolarizing factor (EDHF)] in both WT and eNOS-/- mice. Thus pharmacological and genetic evidence suggests that a significant portion of the response to ADP in cerebral microvessels is mediated by a mechanism independent of eNOS. The eNOS-independent mechanism is functional in the absence of inhibited eNOS and most likely is mediated by an EDHF.     

Endothelium-derived relaxing factors in the kidney of spontaneously hypertensive rats

Acetylcholine (ACh)-induced vasodilation is mainly due to endothelium-derived nitric oxide (EDNO) and hyperpolarizing factor (EDHF). To explore the mechanisms underlying attenuated endothelium-dependent vasodilation in hypertensive arteries, we measured the EDNO released from isolated kidneys of spontaneously hypertensive rats (SHR) using a sensitive chemiluminescence assay system of NO. ACh-induced renal vasodilation was significantly smaller in SHR than in the normotensive control, Wistar-Kyoto rats (WKY). However, ACh-induced NO release did not differ between SHR and WKY (10⁻⁷ M: SHR +37 ± 2 [SE] vs. WKY +32 ± 4 fmol/min/g kidney). Perfusion with a 20 mEq/L high-K⁺ buffer, which is reported to inhibit action of EDHF, significantly reduced ACh-induced vasorelaxation in WKY but not in SHR, resulting in identical renal perfusion pressure in SHR and wKY under these conditions. These results indicate that attenuated ACh-induced vasorelaxation in the SHR kidney may be attributed to a decrease in EDHF rather than that in EDNO.     

EDHF contributes to strain-related differences in pulmonary arterial relaxation in rats

Pulmonary arteries from the Madison (M) strain relax more in response to acetylcholine (ACh) than those from the Hilltop (H) strain of Sprague-Dawley rats. We hypothesized that differences in endothelial nitric oxide (NO) synthase (eNOS) expression and function, metabolism of ACh by cholinesterases, release of prostacyclin, or endothelium-derived hyperpolarizing factor(s) (EDHF) from the endothelium would explain the differences in the relaxation response to ACh in isolated pulmonary arteries. eNOS mRNA and protein levels as well as the NO-dependent relaxation responses to thapsigargin in phenylephrine (10(-6) M)-precontracted pulmonary arteries from the M and H strains were identical. The greater relaxation response to ACh in M compared with H rats was also observed with carbachol, a cholinesterase-resistant analog of ACh, a response that was not modified by pretreatment with meclofenamate (10(-5) M). N(omega)-nitro-L-arginine (10(-4) M) completely abolished carbachol-induced relaxation in H rat pulmonary arteries but not in M rat pulmonary arteries. Precontraction with KCl (20 mM) blunted the relaxation response to carbachol in M rat pulmonary arteries and eliminated differences between the M and H rat pulmonary arteries. NO-independent relaxation present in the M rat pulmonary arteries was significantly reduced by 17-octadecynoic acid (2 microM) and was completely abolished by charybdotoxin plus apamin (100 nM each). These findings suggest that EDHF, but not NO, contributes to the strain-related differences in pulmonary artery reactivity. Also, EDHF may be a metabolite of cytochrome P-450 that activates Ca(2+)-dependent K(+) channels.     

Elucidation of the temporal relationship between endothelial-derived NO and EDHF in mesenteric vessels

Although the endothelium co-generates both nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF), the relative contribution from each vasodilator is not clear. In studies where the endothelium is stimulated acutely, EDHF responses predominate in small arteries. However, the temporal relationship between endothelial-derived NO and EDHF over more prolonged periods is unclear but of major physiological importance. Here we have used a classical pharmacological approach to show that EDHF is released transiently compared with NO. Acetylcholine (3 x 10(-6) mol/l) dilated second- and/or third-order mesenteric arteries for prolonged periods of up to 1 h, an effect that was reversed fully and immediately by the subsequent addition of L-NAME (10(-3) mol/l) but not TRAM-34 (10(-6) mol/l) plus apamin (5 x 10(-7) mol/l). When vessels were pretreated with L-NAME, acetylcholine induced relatively transient dilator responses (declining over approximately 5 min), and vessels were sensitive to TRAM-34 plus apamin. When measured in parallel, the dilator effects of acetylcholine outlasted the smooth muscle hyperpolarization. However, in the presence of L-NAME, vasodilatation and hyperpolarization followed an identical time course. In vessels from NOSIII(-/-) mice, acetylcholine induced small but detectable dilator responses that were transient in duration and blocked by TRAM-34 plus apamin. EDHF responses in these mouse arteries were inhibited by an intracellular calcium blocker, TMB-8, and the phospholipase A(2) inhibitor AACOCF(3), suggesting a role for lipid metabolites. These data show for the first time that EDHF is released transiently, whereas endothelial-derived NO is released in a sustained manner.     

Nitric oxide exerts feedback inhibition on EDHF-induced coronary arteriolar dilation in vivo

We tested the hypothesis that nitric oxide (NO) inhibits endothelium-derived hyperpolarizing factor (EDHF)-induced vasodilation via a negative feedback pathway in the coronary microcirculation. Coronary microvascular diameters were measured using stroboscopic fluorescence microangiography. Bradykinin (BK)-induced dilation was mediated by EDHF, when NO and prostaglandin syntheses were inhibited, or by NO when EDHF and prostaglandin syntheses were blocked. Specifically, BK (20, 50, and 100 ng. kg(-1). min(-1) ic) caused dose-dependent vasodilation similarly before and after administration of N(G)-monomethyl-L-arginine (L-NMMA) (3 micromol/min ic for 10 min) and indomethacin (Indo, 10 mg/kg iv). The residual dilation to BK with L-NMMA and Indo was completely abolished by suffusion of miconazole or an isosmotic buffer containing high KCl (60 mM), suggesting that this arteriolar vasodilation is mediated by the cytochrome P-450 derivative EDHF. BK-induced dilation was reduced by 39% after inhibition of EDHF and prostaglandin synthesis, and dilation was further inhibited by combined blockade with L-NMMA to a 74% reduction in the response. This suggests an involvement for NO in the vasodilation. After dilation to BK was assessed with L-NMMA and Indo, sodium nitroprusside (SNP, 1-3 microgram. kg(-1). min(-1) ic), an exogenous NO donor, was administered in a dose to increase the diameter to the original control value. Dilation to BK was virtually abolished when administered concomitantly with SNP during L-NMMA and Indo (P < 0.01 vs. before SNP), suggesting that NO inhibits EDHF-induced dilation. SNP did not affect adenosine- or papaverine-induced arteriolar dilation in the presence of L-NMMA and Indo, demonstrating that the effect of SNP was not nonspecific. In conclusion, our data are the first in vivo evidence to suggest that NO inhibits the production and/or action of EDHF in the coronary microcirculation.     

Role of 5,6-epoxyeicosatrienoic acid in the regulation of newborn piglet pulmonary vascular tone

We examined the responses of newborn piglet pulmonary resistance arteries (PRAs) to 5,6-epoxyeicosatrienoic acid (5,6-EET), a cytochrome P-450 metabolite of arachidonic acid. In PRAs preconstricted with a thromboxane A(2) mimetic, 5,6-EET caused a concentration-dependent dilation. This dilation was partially inhibited by the combination of charybdotoxin (CTX) and apamin, inhibitors of large and small conductance calcium-dependent potassium (K(Ca)) channels, and was abolished by depolarization of vascular smooth muscle with KCl. Disruption of the endothelium significantly attenuated the dilation, suggesting involvement of one or more endothelium-derived vasodilator pathways in this response. The dilation was partially inhibited by nitro-L-arginine (L-NA), an inhibitor of nitric oxide synthase (NOS), but was unaffected by indomethacin, a cyclooxygenase (COX) inhibitor. The combined inhibition of NOS and K(Ca) channels with L-NA, CTX, and apamin abolished 5,6-EET-mediated dilation. Similarly, combined inhibition of NOS and COX abolished the response. We conclude that 5,6-EET is a potent vasodilator in newborn piglet PRAs. This dilation is mediated by redundant pathways that include release of nitric oxide (NO) and COX metabolites and activation of K(Ca) channels. The endothelium dependence of this response suggests that 5,6-EET is not itself an endothelium-derived hyperpolarizing factor (EDHF) but may induce the release of one or more endothelium-derived relaxing factors, such as NO and/or EDHF.     

Connexin 43 mediates endothelium-derived hyperpolarizing factor-induced vasodilatation in subcutaneous resistance arteries from healthy pregnant women

The role of gap junctions in endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxation of human arteries was assessed using connexin mimetic peptides (CMPs) designated (37,43)Gap27, (40)Gap27, and (43)Gap26 according to homology with the major vascular connexins (Cx37, Cx40, and Cx43). Resistance arteries were obtained from subcutaneous fat biopsies of healthy pregnant women undergoing elective cesarean section. Endothelium-dependent vasodilatation to bradykinin (BK) was assessed using wire myography. N(omega)-nitro-l-arginine methyl ester (l-NAME) and indomethacin (nitric oxide synthase and cyclooxygenase inhibitors, respectively) attenuated maximal relaxation to BK (R(max)) by approximately 50%. Coincubation with l-NAME, indomethacin, and the combined CMPs ((37,43)Gap27, (40)Gap27, and (43)Gap26) almost abolished relaxation to BK (R(max) = 12.2 +/- 3.7%). In arteries incubated with l-NAME and indomethacin, the addition of either (37,43)Gap27 or (40)Gap27 had no significant effect on R(max), whereas (43)Gap26 caused marked inhibition (R(max) = 21 +/- 6.4%, P = 0.005 vs. l-NAME plus indomethacin alone) that was similar to that of the triple combination. Endothelium-independent vasorelaxation was unaffected by CMPs, l-NAME, or indomethacin. Immunohistochemistry demonstrated Cx37, Cx40, and Cx43 expression in the endothelium and vascular smooth muscle. In pregnant women, EDHF-mediated vasorelaxation of subcutaneous resistance arteries is dependent on Cx43 and gap junctions.     

Nascent EDHF-mediated cerebral vasodilation in ovariectomized rats is not induced by eNOS dysfunction

In estrogen-depleted [i.e., ovariectomized (Ovx)] animals, an endothelium-derived hyperpolarizing factor (EDHF)-like mechanism may arise to, at least partially, replace endothelial nitric oxide (NO) synthase (eNOS)-derived NO in modulating cerebral arteriolar tone. Additional findings show that eNOS expression and function is restored in estrogen-treated Ovx female rats, while the nascent EDHF-like activity disappears. Because NO has been linked to repression of EDHF activity in the periphery, the current study was undertaken to examine whether the nascent EDHF role in cerebral vessels of Ovx females relates to a chronically repressed eNOS-derived NO-generating function. We compared the effects of chronic NOS inhibition with Nomega-nitro-L-arginine-methyl ester (L-NAME; 100 mg. kg-1. day-1 for 3 wk) on EDHF-mediated pial arteriolar vasodilation in anesthetized intact, Ovx, and 17beta-estradiol-treated (0.1 mg. kg-1. day-1 ip, 1 wk) Ovx (OVE) female rats as well as in male rats that were prepared with closed cranial windows. In the chronic NOS inhibition groups, pial arteriolar responses were monitored in the absence (all groups) and presence (females only) of indomethacin (Indo; 10 mg/kg iv). Finally, the gap junction inhibitory peptide Gap 27 (300 muM) was applied to block EDHF-related vasodilation. NO donor (S-nitroso-N-acetyl-penicillamine) responses were similar in all rats studied. Acetylcholine (ACh) reactivity was virtually absent in control Ovx rats and chronically NOS-inhibited intact female, OVE, and male rats. However, a partial recovery of ACh reactivity was seen in L-NAME-treated Ovx females. In addition, in the presence of L-NAME, a normal CO2 reactivity was observed in all females, whereas a 50% reduction in CO2 reactivity was seen in males. In intact and OVE rats, both chronic and acute (NG-nitro-L-arginine suffusion) NOS inhibition, combined with Indo, depressed ADP-induced dilation by > or =50%, and subsequent application of Gap 27 had no further effect on ADP-induced vasodilation. ADP reactivity was retained in Ovx rats after combined chronic NOS inhibition and acute Indo, but was attenuated significantly by Gap 27. In males, Gap 27 had no effect on arteriolar reactivity. Taken together, our data demonstrate that in the cerebral microcirculation, NO does not have an inhibitory effect on EDHF production or action. The increased EDHF-like function in chronic estrogen-depleted animals is not due to eNOS deficiency, suggesting a more direct effect of estrogen in modulating EDHF-mediated cerebral vasodilation.     

Diabetes impairs endothelium-dependent relaxation of human penile vascular tissues mediated by NO and EDHF

Standard treatments for erectile dysfunction (ED) (i.e., PDE5 inhibitors) are less effective in diabetic patients for unknown reasons. Endothelium-dependent relaxation (EDR) of human corpus cavernosum (HCC) depends on nitric oxide (NO), while in human penile resistance arteries (HPRA) endothelium-derived hyperpolarizing factor (EDHF) and NO participate. Here we show that diabetes significantly reduced EDR induced by acetylcholine (ACh) in HCC and HPRA. Relaxation attributed to EDHF was also impaired in HPRA from diabetic patients. The PDE5 inhibitor, sildenafil (10nM), reversed diabetes-induced endothelial dysfunction in HCC, but not in HPRA. Calcium dobesilate (DOBE; 10 microM) fully reversed diabetes-induced endothelial dysfunction in HPRA by specifically potentiating the EDHF-mediated component of EDR. Impairment by diabetes of NO and EDHF-dependent responses precluded the complete recovery of endothelial function in HPRA by sildenafil. This could explain the poor clinical response to PDE5 inhibitors of diabetic men with ED and suggests that a pharmacological approach that combines enhancement of NO/cGMP and EDHF pathways could be necessary to treat ED in many diabetic men.     

15-Lipoxygenase metabolites contribute to age-related reduction in acetylcholine-induced hypotension in rabbits

Arachidonic acid (AA) metabolites from the 15-lipoxygenase-1 (15-LO-1) pathway, trihydroxyeicosatrienoic acids (THETAs) and hydroxy-epoxyeicosatrienoic acids (HEETAs), are endothelium-derived hyperpolarizing factors (EDHFs) and relax rabbit arteries. Rabbit vascular 15-LO-1 expression, THETA and HEETA synthesis, and nitric oxide and prostaglandin-independent relaxations to acetylcholine (ACh) and AA decreased with age (neonates to 16-wk-old). We characterized age-dependent ACh-hypotensive responses in vivo in 1-, 4-, 8-, and 16-wk-old rabbits and the contribution of THETAs and HEETAs to these responses. In anesthetized rabbits, blood pressure responses to ACh (4-4,000 ng/kg) were determined in the presence of vehicle or various inhibitors. ACh responses decreased with age (P > 0.001). In the absence or presence of N(omega)-nitro-l-arginine methyl ester (l-NAME) and indomethacin (Indo), maximum responses in 1 (-54.7 +/- 7.4 and -37.9 +/- 3.9%)- and 4 (-48.8 +/- 2.4 and -35.5 +/- 7.8%)-wk-old rabbits were higher than 8 (-30.0 +/- 2.8 and -26.6 +/- 4.4%)- and 16 (-36.7 +/- 3.5 and -27.3 +/- 10%)-wk-old rabbits. A lipoxygenase inhibitor, BW755C, reduced THETA and HEETA synthesis in mesenteric arteries. In the presence of Indo and N(omega)-nitro-l-arginine, ACh relaxations were reduced by BW755C to a greater extent in the mesenteric arteries from the younger rabbits. In 4-wk-old rabbits treated with l-NAME and Indo, the maximum ACh hypotension was reduced by the potassium channel inhibitors apamin and charybdotoxin to -6.9 +/- 0.9%, by apamin alone to -19.5 +/- 1.4%, and by BW755C to -18.8 +/- 3.5%. The present study indicates that the age-related decrease in ACh-induced hypotension is mediated by the decreased synthesis of the 15-LO-1 metabolites THETAs and HEETAs.     

Receptor-activating peptides for PAR-1 and PAR-2 relax rat gastric artery via multiple mechanisms

Receptor-activating peptides for protease-activated receptors (PARs) 1 or 2 enhance gastric mucosal blood flow (GMBF) and protect against gastric mucosal injury in rats. We thus examined and characterized the effects of PAR-1 and PAR-2 agonists on the isometric tension in isolated rat gastric artery. The agonists for PAR-2 or PAR-1 produced vasodilation in the endothelium-intact arterial rings, which was abolished by removal of the endothelium. The mechanisms underlying the PAR-2- and PAR-1-mediated relaxation involved NO, endothelium-derived hyperpolarizing factor (EDHF) and prostanoids, to distinct extent, as evaluated by use of inhibitors of NO synthase, cyclo-oxygenase and Ca2+-activated K+ channels. The EDHF-dependent relaxation responses were significantly attenuated by gap junction inhibitors. These findings demonstrate that endothelial PAR-1 and PAR-2, upon activation, dilate the gastric artery via NO and prostanoid formation and also EDHF mechanisms including gap junctions, which would enhance GMBF.     

Mechanisms of bradykinin-mediated dilation in newborn piglet pulmonary conducting and resistance vessels

Bradykinin (BK) is a potent dilator of the perinatal pulmonary circulation. We investigated segmental differences in BK-induced dilation in newborn pig large conducting pulmonary artery and vein rings and in pressurized pulmonary resistance arteries (PRA). In conducting pulmonary arteries and veins, BK-induced relaxation is abolished by endothelial disruption and by inhibition of nitric oxide (NO) synthase with nitro-L-arginine (L-NA). In PRA, two-thirds of the dilation response is L-NA insensitive. Charybdotoxin plus apamin and depolarization with KCl abolish the L-NA-insensitive dilations, findings that implicate the release of endothelium-derived hyperpolarizing factor (EDHF). However, endothelium-disrupted PRA retain the ability to dilate to BK but not to ACh or A-23187. In endothelium-disrupted PRA, dilation was inhibited by charybdotoxin. Thus in PRA, BK elicits dilation by multiple and duplicative signaling pathways. Release of NO and EDHF contributes to the response in endothelium-intact PRA; in endothelium-disrupted PRA, dilation occurs by direct activation of vascular smooth muscle calcium-dependent potassium channels. Redundant signaling pathways mediating pulmonary dilation to BK may be required to assure a smooth transition to extrauterine life.