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.     

Testosterone suppresses endothelium-dependent dilation of rat middle cerebral arteries

Little is known about vascular effects of testosterone. We previously reported chronic testosterone treatment increases vascular tone in middle cerebral arteries (MCA; 300 microm diameter) of male rats. In the present study, we investigated the hypothesis that physiological levels of circulating testosterone affect endothelial factors that modulate cerebrovascular reactivity. Small branches of MCA (150 microm diameter) were isolated from orchiectomized (ORX) and testosterone-treated (ORX+T) rats. Intraluminal diameters were recorded after step changes in intraluminal pressure (20-100 Torr) in the absence or presence of N(G)-nitro-L-arginine-methyl ester (L-NAME), a nitric oxide synthase (NOS) inhibitor; indomethacin, a cyclooxygenase (COX) inhibitor; and/or apamin and charybdotoxin (CTX); and K(Ca) channel blockers used to inhibit endothelium-derived hyperpolarizing factors (EDHF). At intraluminal pressures >or=60 Torr, arteries from ORX+T developed greater tone compared with ORX arteries. This difference was abolished by removal of the endothelium but remained after treatment of intact arteries with indomethacin or L-NAME. In addition, testosterone treatment had no effect on cerebrovascular production of endothelin-1 or prostacyclin nor did it alter protein levels of endothelial NOS or COX-1. Endothelium removal after L-NAME/indomethacin exposure caused an additional increase in tone. Interestingly, the latter effect was smaller in arteries from ORX+T, suggesting testosterone affects endothelial vasodilators that are independent of NOS and COX. Apamin/CTX, in the presence of L-NAME/indomethacin, abolished the difference in tone between ORX and ORX+T and resulted in vessel diameters similar to those of endothelium-denuded preparations. In conclusion, testosterone may modulate vascular tone in cerebral arteries by suppressing EDHF.     

The permissive role of endothelial NO in CO-induced cerebrovascular dilation

Carbon monoxide (CO) and nitric oxide (NO) are important paracrine messengers in the newborn cerebrovasculature that may act as comessengers. Here, we investigated the role of NO in CO-mediated dilations in the newborn cerebrovasculature. Arteriolar branches of the middle cerebral artery (100-200 microm) were isolated from 3- to 7-day-old piglets and cannulated at each end in a superfusion chamber, and intravascular pressure was elevated to 30 mmHg, which resulted in the development of myogenic tone. Endothelium removal abolished dilations of pressurized pial arterioles to bradykinin and to the CO-releasing molecule Mn(2)(CO)(10) [dimanganese decacarbonyl (DMDC)] but not dilations to isoproterenol. With endothelium intact, N(omega)-nitro-l-arginine (l-NNA), 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ), or tetraethylammonium chloride (TEA(+)), inhibitors of NO synthase (NOS), guanylyl cyclase, and large-conductance Ca(2+)-activated K(+) (K(Ca)) channels, respectively, also blocked dilation induced by DMDC. After inhibition of NOS, a constant concentration of sodium nitroprusside (SNP), a NO donor that only dilated the vessel 6%, returned dilation to DMDC. The stable cGMP analog 8-bromo-cGMP also restored dilation to DMDC in endothelium-intact, l-NNA-treated, or endothelium-denuded arterioles, and this effect was blocked by TEA(+). Similarly, in the continued presence of ODQ, 8-bromo-cGMP restored DMDC-induced dilations. These findings suggest that endothelium-derived NO stimulates guanylyl cyclase in vascular smooth muscle cells and, thereby, permits CO to cause dilation by activating K(Ca) channels. Such a requirement for NO could explain the endothelium dependency of CO-induced dilation in piglet pial arterioles.     

Ca2+-activated K+ channels of small and intermediate conductance control eNOS activation through NAD(P)H oxidase

Ca(2+)-activated K(+) channels (K(Ca)) and NO play a central role in the endothelium-dependent control of vasomotor tone. We evaluated the interaction of K(Ca) with NO production in isolated arterial mesenteric beds of the rat. In phenylephrine-contracted mesenteries, acetylcholine (ACh)-induced vasodilation was reduced by NO synthase (NOS) inhibition with N(ω)-nitro-L-arginine (L-NA), but in the presence of tetraethylammonium, L-NA did not further affect the response. In KCl-contracted mesenteries, the relaxation elicited by 100 nM ACh or 1 μM ionomycin was abolished by L-NA, tetraethylammonium, or simultaneous blockade of small-conductance K(Ca) (SK(Ca)) channels with apamin and intermediate-conductance K(Ca) (IK(Ca)) channels with triarylmethane-34 (TRAM-34). Apamin-TRAM-34 treatment also abolished 100 nM ACh-activated NO production, which was associated with an increase in superoxide formation. Endothelial cell Ca(2+) buffering with BAPTA elicited a similar increment in superoxide. Apamin-TRAM-34 treatment increased endothelial NOS phosphorylation at threonine 495 (P-eNOS(Thr495)). Blockade of NAD(P)H oxidase with apocynin or superoxide dismutation with PEG-SOD prevented the increment in superoxide and changes in P-eNOS(Thr495) observed during apamin and TRAM-34 application. Our results indicate that blockade of SK(Ca) and IK(Ca) activates NAD(P)H oxidase-dependent superoxide formation, which leads to inhibition of NO release through P-eNOS(Thr495). These findings disclose a novel mechanism involved in the control of NO production.     

20-hydroxyeicosatetraenoic acid is a vasoconstrictor in the newborn piglet pulmonary microcirculation

20-Hydroxyeicosatetraenoic acid (20-HETE), a cytochrome p-450 metabolite of arachidonic acid, is a vasoconstrictor in the systemic circulation and a vasodilator in the adult pulmonary circulation. Little is known about the vasoactive properties of 20-HETE in the newborn pulmonary circulation. The objectives of this study were to determine the vascular effects of 20-HETE and to explore the signaling mechanism(s) that mediate these effects in newborn pulmonary resistance-level arteries (PRA). Our findings demonstrate that, in contrast to the adult pulmonary circulation where 20-HETE mediates vasodilation, it causes constriction in newborn PRA at resting tone. Furthermore, inhibition of cyclooxygenase (COX) with indomethacin augments 20-HETE-induced constriction. The enhanced constrictor response to 20-HETE under conditions of COX inhibition is abolished in endothelium-disrupted PRA, suggesting that 20-HETE either stimulates endothelium-derived COX to release a counteracting vasodilator or is rapidly metabolized by COX to a less potent vasoconstrictor. 20-HETE-induced constriction is significantly inhibited by blocking calcium-dependent K(+) (K(Ca)) channels and the thromboxane-PGH(2) receptor. Altogether, our data indicate that the vascular actions of 20-HETE are partially mediated via the activation of K(Ca) channels and are significantly modulated by interactions with the COX-prostaglandin pathway.     

Multiple Ca(2+)-dependent modulators mediate alkalosis-induced vasodilation in newborn piglet lungs

We previously found that alkalosis-induced vasodilation was mediated by endothelium-derived nitric oxide (EDNO) in newborn piglet pulmonary artery and vein rings precontracted with the thromboxane mimetic U-46619. In contrast, prostacyclin or K(+) channel activation contributed to the response in other preparations. This study was undertaken to determine whether EDNO alone also mediates alkalosis-induced pulmonary vasodilation in piglet lungs vasoconstricted with hypoxia and, if not, to identify the mediator(s) involved. Responses to alkalosis were measured during hypoxia under control conditions after blocking nitric oxide synthase (N(omega)-nitro-L-arginine), cyclooxygenase (meclofenamate), or both endothelium-derived modulators (Dual); after blocking voltage-dependent (4-aminopyridine), ATP- dependent (glibenclamide), or Ca(2+)-dependent K(+) (K(Ca); tetraethylammonium) K(+) channels; and after blocking both endothelium-derived modulators and K(Ca) channels (Triple). Vasodilator responses measured after 20 min of alkalosis were blunted in Dual and tetraethylammonium lungs and abolished in Triple lungs. Thus alkalosis-induced vasodilation in hypoxic lungs appeared to be mediated by three Ca(2+)-dependent modulators: EDNO, prostacyclin, and K(Ca) channels. In addition, a transient, unidentified modulator contributed to the nadir of the vasodilator response measured at 10 min of alkalosis. Future studies are needed to identify factors that contribute to the discordance between isolated vessels and whole lungs.     

Rat mesenteric arterial dilator response to 11,12-epoxyeicosatrienoic acid is mediated by activating heme oxygenase

11,12-Epoxyeicosatrienoic acid (11,12-EET), a potent vasodilator produced by the endothelium, acts on calcium-activated potassium channels and shares biological activities with the heme oxygenase/carbon monoxide (HO/CO) system. We examined whether activation of HO mediates the dilator action of 11,12-EET, and that of the other EETs, on rat mesenteric arteries. Dose-response curves (10(-9) to 10(-6) M) to 5,6-EET, 8,9-EET, 11,12-EET, 14,15-EET, and ACh (10(-9) to 10(-4) M) were evaluated in preconstricted (10(-6) mol/l phenylephrine) mesenteric arteries (<350 microm diameter) in the presence or absence of 1) the cyclooxygenase inhibitor indomethacin (2.8 microM), 2) the HO inhibitor chromium mesoporphyrin (CrMP) (15 microM), 3) the soluble guanylyl cyclase (GC) inhibitor ODQ (10 microM), and 4) the calcium-activated potassium channel inhibitor iberiotoxin (25 nM). The vasodilator response to 11,12-EET was abolished by CrMP and iberiotoxin, whereas indomethacin and ODQ had no effect. In contrast, the effect of ACh was attenuated by ODQ but not by CrMP. The vasodilator effect of 8,9-EET, like that of 11,12-EET, was greatly attenuated by HO inhibition. In contrast, the mesenteric vasodilator response to 5,6-EET was independent of both HO and GC, whereas that to 14,15-EET demonstrated two components, an HO and a GC, of equal magnitude. Incubation of mesenteric microvessels with 11,12-EET caused a 30% increase in CO release, an effect abolished by inhibition of HO. We conclude that the rat mesenteric vasodilator action of 11,12-EET is mediated via an increase in HO activity and an activation of calcium-activated potassium channels.     

Role of endothelial intermediate conductance KCa channels in cerebral EDHF-mediated dilations

The present study evaluated the role of endothelial intermediate conductance calcium-sensitive potassium channels (IKCa) in the mechanism of endothelium-derived hyperpolarizing factor (EDHF)-mediated dilations in pressurized cerebral arteries. Male rat middle cerebral arteries (MCA) were mounted in an isolated vessel chamber, pressurized (85 mmHg), and luminally perfused (100 microl/min). Artery diameter was measured simultaneously with either endothelial intracellular Ca2+ concentration ([Ca2+]i; fura-2) or changes in endothelial membrane potential [4-[2-[6-(dioctylamino)-2-naphthalenyl]ethenyl]1-(3-sulfopropyl)-pyridinium (di-8-ANEPPS)]. Nitric oxide synthase and cyclooxygenase inhibitors were present throughout. Luminal application of UTP produced EDHF-mediated dilations that correlated with significant endothelial hyperpolarization. The dilation and endothelial hyperpolarization were virtually abolished by inhibitors of IKCa channels but not by selective inhibitors of small or large conductance KCa channels (apamin and iberiotoxin, respectively). Additionally, direct stimulation of endothelial IKCa channels with 1-ethyl-2-benzimidazolinone (1-EBIO) produced endothelial hyperpolarization and vasodilatation that were blocked by inhibitors of IKCa channels. 1-EBIO hyperpolarized the endothelium but did not affect endothelial [Ca2+]i. We conclude that the mechanism of EDHF-mediated dilations in cerebral arteries requires stimulation of endothelial IKCa channels to promote endothelial hyperpolarization and subsequent vasodilatation.     

Epoxyeicosatrienoic and dihydroxyeicosatrienoic acids dilate human coronary arterioles via BK(Ca) channels: implications for soluble epoxide hydrolase inhibition

Epoxyeicosatrienoic acids (EETs) are metabolized by soluble epoxide hydrolase (sEH) to form dihydroxyeicosatrienoic acids (DHETs) and are putative endothelium-derived hyperpolarizing factors (EDHFs). EDHFs modulate microvascular tone; however, the chemical identity of EDHF in the human coronary microcirculation is not known. We examined the capacity of EETs, DHETs, and sEH inhibition to affect vasomotor tone in isolated human coronary arterioles (HCAs). HCAs from right atrial appendages were prepared for videomicroscopy and immunohistochemistry. In vessels preconstricted with endothelin-1, three EET regioisomers (8,9-, 11,12-, and 14,15-EET) each induced a concentration-dependent dilation that was sensitive to blockade of large-conductance Ca2+-activated K+ (BK(Ca)) channels by iberiotoxin. EET-induced dilation was not altered by endothelial denudation. 8,9-, 11,12-, and 14,15-DHET also dilated HCA via activation of BK(Ca) channels. Dilation was less with 8,9- and 14,15-DHET but was similar with 11,12-DHET, compared with the corresponding EETs. Immunohistochemistry revealed prominent expression of cytochrome P-450 (CYP450) 2C8, 2C9, and 2J2, enzymes that may produce EETs, as well as sEH, in HCA. Inhibition of sEH by 1-cyclohexyl-3-dodecylurea (CDU) enhanced dilation caused by 14,15-EET but reduced dilation observed with 11,12-EET. DHET production from exogenous EETs was reduced in vessels pretreated with CDU compared with control, as measured by liquid chromatography electrospray-ionization mass spectrometry. In conclusion, EETs and DHETs dilate HCA by activating BK(Ca) channels, supporting a role for EETs/DHETs as EDHFs in the human heart. CYP450s and sEH may be endogenous sources of these compounds, and sEH inhibition has the potential to alter myocardial perfusion, depending on which EETs are produced endogenously.     

Epoxyeicosatrienoic acid-induced relaxation is impaired in insulin resistance

We assessed the effect of epoxyeicosatrienoic acids (EETs) in intact mesenteric arteries and Ca(2+)-activated K(+) (BK(Ca)) channels of isolated vascular smooth muscle cells from control and insulin-resistant (IR) rats. The response to 11,12-EET and 14,15-EET was assessed in small mesenteric arteries from control and IR rats in vitro. Mechanistic studies were performed in endothelium intact or denuded arteries and in the presence of pharmacological inhibitors. Moreover, EET-induced activation of the BK(Ca) channel was assessed in myocytes in both the cell-attached and the inside-out (I/O) patch-clamp configurations. In control arteries, both EET isomers induced relaxation. Relaxation was impaired by endothelium denudation, N(omega)-nitro-L-arginine, or iberiotoxin (IBTX), whereas it was abolished by IBTX + apamin or charybdotoxin + apamin. In contrast, the EETs did not relax IR arteries. In control myocytes, the EETs increased BK(Ca) activity in both configurations. Conversely, in the cell-attached mode, EETs had no effect on BK(Ca) channel activity in IR myocytes, whereas in the I/O configuration, BK(Ca) channel activity was enhanced. EETs induce relaxation in small mesenteric arteries from control rats through K(Ca) channels. In contrast, arteries from IR rats do not relax to the EETs. Patch-clamp studies suggest impaired relaxation is due to altered regulatory mechanisms of the BK(Ca) channel.     

Essential role of EDHF in the initiation and maintenance of adrenergic vasomotion in rat mesenteric arteries

The possible roles of endothelial intracellular Ca(2+) concentration ([Ca(2+)](i)), nitric oxide (NO), arachidonic acid (AA) metabolites, and Ca(2+)-activated K(+) (K(Ca)) channels in adrenergically induced vasomotion were examined in pressurized rat mesenteric arteries. Removal of the endothelium or buffering [Ca(2+)](i) selectively in endothelial cells with BAPTA eliminated vasomotion in response to phenylephrine (PE; 10.0 microM). In arteries with intact endothelium, inhibition of NO synthase with N(omega)-nitro-l-arginine methyl ester (l-NAME; 300.0 microM) or N(omega)-nitro-l-arginine (l-NNA; 300.0 microM) did not eliminate vasomotion. Neither inhibition of cGMP formation with 10.0 microM 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) nor inhibition of prostanoid formation (10.0 microM indomethacin) eliminated vasomotion. Similarly, inhibition of AA cytochrome P-450 metabolism with an intraluminal application of 17-octadecynoic acid (17-ODYA) or 6-(2-propargyloxyphenyl)hexanoic acid (PPOH) failed to eliminate vasomotion. In contrast, intraluminal application of the K(Ca) channel blockers apamin (250.0 nM) and charybdotoxin (100.0 nM), together, abolished vasomotion and changed synchronous Ca(2+) oscillations in smooth muscle cells to asynchronous propagating Ca(2+) waves. Apamin, charybdotoxin, or iberiotoxin (100.0 nM) alone did not eliminate vasomotion, nor did the combination of apamin and iberiotoxin. The results show that adrenergic vasomotion in rat mesenteric arteries is critically dependent on Ca(2+)-activated K(+) channels in endothelial cells. Because these channels (small- and intermediate-conductance K(Ca) channels) are a recognized component of EDHF, we conclude therefore that EDHF is essential for the development of adrenergically induced vasomotion.     

Paraoxonase 1 in endothelial cells impairs vasodilation induced by arachidonic acid lactone metabolite

IntroductionParaoxonase 1 (PON1) is a high density lipoprotein (HDL)-associated lactonase, which is known for its antiatherogenic properties. Previous studies in PON1 knockout (PON1KO) mice revealed that PON1KO mice have low blood pressure, which is inversely correlated with the renal levels of the cytochrome P450 -derived arachidonic acid metabolite 5,6-epoxyeicosatrienoic acid (5,6-EET). Our previous studies revealed that 5,6-EET is unstable, transforming to the δ-lactone isomer 5,6-δ-DHTL, an endothelium-derived hyperpolarizing factor (EDHF) that mediates vasodilation, and it is a potential substrate for PON1.AimTo elucidate the role of PON1 in the modulation of vascular resistance via the regulation of the lactone-containing metabolite 5,6-δ-DHTL.ResultsIn mouse resistance arteries, PON1 was found to be present and active in the endothelial layer. Vascular reactivity experiments revealed that 5,6-δ-DHTL dose-dependently dilates PON1KO mouse mesenteric arteries significantly more than wild type (w.t.) resistance arteries. Pre-incubation with HDL or rePON1 reduced 5,6-δ-DHTL-dependent vasodilation. FACS analyses and confocal microscopy experiments revealed that fluorescence-tagged rePON1 penetrates into human endothelial cells' (ECs') in both dose- and time- dependent manner, accumulate in the perinuclear compartment, and retains its lactonase activity in the cells. The presence of rePON1, but not the presence of PON1 loss-of-lactonase-activity mutant, reduced the Ca²⁺ influx in the ECs mediated by 5,6-δ-DHTL.ConclusionPON1 lactonase activity in the endothelium affects vascular dilation by regulating Ca²⁺ influx mediated by the lactone-containing EDHF 5,6-δ-DHTL.     

Endothelium-derived hyperpolarizing factor in coronary microcirculation: responses to arachidonic acid

In coronary resistance vessels, endothelium-derived hyperpolarizing factor (EDHF) plays an important role in endothelium-dependent vasodilation. EDHF has been proposed to be formed through cytochrome P-450 monooxygenase metabolism of arachidonic acid (AA). Our hypothesis was that AA-induced coronary microvascular dilation is mediated in part through a cytochrome P-450 pathway. The canine coronary microcirculation was studied in vivo (beating heart preparation) and in vitro (isolated microvessels). Nitric oxide synthase (NOS) (N(omega)-nitro-L-arginine, 100 microM) and cyclooxygenase (indomethacin, 10 microM) or cytochrome P-450 (clotrimazole, 2 microM) inhibition did not alter AA-induced dilation. However, when a Ca(2+)-activated K(+) channel channel or cytochrome P-450 antagonist was used in combination with NOS and cyclooxygenase inhibitors, AA-induced dilation was attenuated. We also show a negative feedback by NO on NOS-cyclooxygenase-resistant AA-induced dilation. We conclude that AA-induced dilation is attenuated by cytochrome P-450 inhibitors, but only when combined with inhibitors of cyclooxygenase and NOS. Therefore, redundant pathways appear to mediate the AA response in the canine coronary microcirculation.     

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.     

Dietary obesity increases NO and inhibits BKCa-mediated, endothelium-dependent dilation in rat cremaster muscle artery: association with caveolins and caveolae

Obesity is a risk factor for hypertension and other vascular disease. The aim of this study was to examine the effect of diet-induced obesity on endothelium-dependent dilation of rat cremaster muscle arterioles. Male Sprague-Dawley rats (213 ± 1 g) were fed a cafeteria-style high-fat or control diet for 16-20 wk. Control rats weighed 558 ± 7 g compared with obese rats 762 ± 12 g (n = 52-56; P < 0.05). Diet-induced obesity had no effect on acetylcholine (ACh)-induced dilation of isolated, pressurized (70 mmHg) arterioles, but sodium nitroprusside (SNP)-induced vasodilation was enhanced. ACh-induced dilation of arterioles from control rats was abolished by a combination of the K(Ca) blockers apamin, 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34), and iberiotoxin (IBTX; all 0.1 μmol/l), with no apparent role for nitric oxide (NO). In arterioles from obese rats, however, IBTX had no effect on responses to ACh while the NO synthase (NOS)/guanylate cyclase inhibitors N(ω)-nitro-L-arginine methyl ester (L-NAME; 100 μmol/l)/1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 μmol/l) partially inhibited ACh-induced dilation. Furthermore, NOS activity (but not endothelial NOS expression) was increased in arteries from obese rats. L-NAME/ODQ alone or removal of the endothelium constricted arterioles from obese but not control rats. Expression of caveolin-1 and -2 oligomers (but not monomers or caveolin-3) was increased in arterioles from obese rats. The number of caveolae was reduced in the endothelium of arteries, and caveolae density was increased at the ends of smooth muscle cells from obese rats. Diet-induced obesity abolished the contribution of large-conductance Ca(2+)-activated K(+) channel to ACh-mediated endothelium-dependent dilation of rat cremaster muscle arterioles, while increasing NOS activity and inducing an NO-dependent component.     

Increases in endothelial Ca(2+) activate K(Ca) channels and elicit EDHF-type arteriolar dilation via gap junctions

In skeletal muscle arterioles, the pathway leading to non-nitric oxide (NO), non-prostaglandin-mediated endothelium-derived hyperpolarizing factor (EDHF)-type dilations is not well characterized. To elucidate some of the steps in this process, simultaneous changes in endothelial intracellular Ca(2+) concentration ([Ca(2+)](i)) and the diameter of rat gracilis muscle arterioles (approximately 60 microm) to acetylcholine (ACh) were measured by fura 2 microfluorimetry (in the absence of NO and prostaglandins). ACh elicited rapid increases in endothelial [Ca(2+)](i) (101 +/- 7%), followed by substantial dilations (73 +/- 2%, coupling time: 1.3 +/- 0.2 s) that were prevented by endothelial loading of an intracellular Ca(2+) chelator [1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid]. Arteriolar dilations to ACh were also inhibited by intraluminal administration of the Ca(2+)-activated K(+) (K(Ca)) channel blockers charybdotoxin plus apamin or by palmitoleic acid, an uncoupler of myoendothelial gap junctions without affecting changes in endothelial [Ca(2+)](i). The presence of large conductance K(Ca) channels on arteriolar endothelial cells was demonstrated with immunohistochemisty. We propose that in skeletal muscle arterioles, EDHF-type mediation is evoked by an increase in endothelial [Ca(2+)](i), which by activating endothelial K(Ca) channels elicits hyperpolarization that is conducted via myoendothelial gap junctions to the smooth muscle resulting in decreases in [Ca(2+)](i) and consequently dilation.     

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.     

In vivo regulation of endothelium-dependent vasodilation in the rat renal circulation and the effect of streptozotocin-induced diabetes

We assessed the relative contributions of endothelium-derived relaxing factors to renal vasodilation in vivo and determined whether these are altered in established streptozotocin-induced diabetes. In nondiabetic rats, stimulation of the endothelium by locally administered ACh or bradykinin-induced transient renal hyperemia. Neither basal renal blood flow (RBF) nor renal hyperemic responses to ACh or bradykinin were altered by blockade of prostanoid production (indomethacin) or by administration of charybdotoxin (ChTx) plus apamin to block endothelium-derived hyperpolarizing factor (EDHF). In contrast, combined blockade of nitric oxide (NO) synthase, N(omega)-nitro-l-arginine methyl ester (l-NAME), and prostanoid production reduced basal RBF and the duration of the hyperemic responses to ACh and bradykinin and revealed a delayed ischemic response to ACh. Accordingly, l-NAME and indomethacin markedly reduced integrated (area under the curve) hyperemic responses to ACh and bradykinin. Peak increases in RBF in response to ACh and bradykinin were not reduced by l-NAME and indomethacin but were reduced by subsequent blockade of EDHF. l-NAME plus indomethacin and ChTx plus apamin altered RBF responses to endothelium stimulation in a qualitatively similar fashion in diabetic and nondiabetic rats. The integrated renal hyperemic responses to ACh and bradykinin were blunted in diabetes, due to a diminished contribution of the component abolished by l-NAME plus indomethacin. We conclude that NO dominates integrated hyperemic responses to ACh and bradykinin in the rat kidney in vivo. After prior inhibition of NO synthase, EDHF mediates transient renal vasodilation in vivo. Renal endothelium-dependent vasodilation is diminished in diabetes due to impaired NO function.     

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.     

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.