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Endothelium-derived hyperpolarizing factor: a cousin to nitric oxide and prostacyclin.

There is now strong evidence that an endothelial mechanism, other than nitric oxide or prostacyclin, exists for dilating arteries and arterioles. This third pathway has been named endothelium-derived hyperpolarizing factor (EDHF) and should not be confused with endothelium-derived relaxing factor, which is nitric oxide. Currently, there are several ideas for the mechanism of EDHF, which may vary among vessels of different organs and species. During some pathologic states, EDHF can be up-regulated. This up-regulation often occurs as the dilator effects of endothelium-derived nitric oxide are suppressed. The up-regulated EDHF may serve in a protective capacity to help maintain blood flow to organs and tissues during these stressful states. Many anesthetics attenuate the dilator actions of EDHF; however, the full clinical implications of this anesthetic-related attenuation are not known. Like its cousins, nitric oxide and prostacyclin, EDHF is an important regulator of blood flow and should prove to be an important clinical consideration as we gain more knowledge of its mechanisms of action.

P2u receptor-mediated release of endothelium-derived relaxing factor/nitric oxide and endothelium-derived hyperpolarizing factor from cerebrovascular endothelium in rats.

BACKGROUND AND PURPOSE Stimulation of P2u purinoceptors by UTP on endothelium dilates the rat middle cerebral artery (MCA) through the release of endothelium-derived relaxing factor/nitric oxide (EDRF/NO) and an unknown relaxing factor. The purpose of this study was to determine whether this unknown relaxing factor is endothelium-derived hyperpolarizing factor (EDHF). METHODS Rat MCAs were isolated, cannulated, pressurized, and luminally perfused. UTP was added to the luminal perfusate to elicit dilations. RESULTS Resting outside diameter of the MCAs in one study was 209+/-7 micrometer (n=10). The MCAs showed concentration-dependent dilations with UTP administration. Inhibition of NO synthase with NG-nitro-L-arginine methyl ester (L-NAME) (1 micromol/L to 1 mmol/L) did not diminish the maximum response to UTP but did shift the concentration-response curve to the right. Scavenging NO with hemoglobin (1 or 10 micromol/L) or inhibition of guanylate cyclase with ODQ (1 or 10 micromol/L) had effects on the UTP-mediated dilations similar to those of L-NAME. In the presence of L-NAME, dilations induced by 10 micromol/L UTP were accompanied by 13+/-2 mV (P<0.009) hyperpolarization of the vascular smooth muscle membrane potential (-28+/-2 to -41+/-1 mV). Iberiotoxin (100 nmol/L), blocker of the large-conductance calcium-activated K channels, sometimes blocked the dilation, but its effects were variable. Charybdotoxin (100 nmol/L), also a blocker of the large-conductance calcium-activated K channels, abolished the L-NAME-insensitive component of the dilation to UTP. CONCLUSIONS Stimulation of P2u purinoceptors on the endothelium of the rat MCA released EDHF, in addition to EDRF/NO, and dilated the rat MCA by opening an atypical calcium-activated K channel.

Characterisation of an ATP receptor mediating mitogenesis in vascular smooth muscle cells

Adenosine triphosphate (ATP), a co-transmitter in sympathetic nerves and released from platelets, has recently been shown to stimulate growth of vascular smooth muscle cells. It might therefore contribute to the development of vascular hypertrophy seen in hypertension and atherosclerosis. We aimed at characterising the receptor mediating this mitogenic effect in rat aorta smooth muscle cells. The potency of agonists indicates a P2 purinoceptor since ATP > or = ADP >> AMP, adenosine. The P2x-receptor subtype, which is responsible for ATP induced vasoconstriction in rat aorta, does not mediate the mitogenic effect since alpha, beta-methyleneATP had no effect and beta, gamma-methyleneATP had lower potency than ATP. The P2Y-receptor subtype was excluded since the selective agonist 2-methylthioATP had weak effect with lower potency than ATP. When we studied the involvement of other nucleotides similar effects were seen of the purines ATP, GTP and ITP; also the pyrimidine UTP had powerful mitogenic effects (Emax = 52% of ATP) with similar potency. Nucleotides with fewer phosphate groups showed a stepwise fall in mitogenic effect. This indicates involvement of a nucleotide-receptor (P2U). Ap4A were of equal potency and effect as ATP. There was strong correlation between the mitogenic effects of the nucleotides and analogues with both 45Ca(2+)-influx and inositol phosphate (IP) production, indicating that they may participate in mediating the mitogenic response. This is the first study describing the potencies for the mitogenic effects of the selective ATP-analogues and other nucleotides in vascular smooth muscle cells. The receptor characterisation indicates a nucleotide-receptor similar to the receptor which stimulates 45Ca(2+)-influx and inositol phosphate-formation in rat aorta smooth muscle cells. Substances related to ATP such as GTP, ITP, UTP and Ap4A which also can be released extracellularly in vivo stimulate mitogenesis of rat aorta smooth muscle cells through the same receptor.

Neuropeptide Y-mediated constriction and dilation in rat middle cerebral arteries

Neuropeptide Y (NPY) is an important vasoconstrictor in the cerebral circulation. Its constrictor response is because of activation of NPY receptors on the vascular smooth muscle (VSM). Little is known regarding the effects of NPY on the endothelium. In the current study, the authors tested the hypothesis that NPY can either constrict or dilate rat middle cerebral arteries (MCAs). Constriction is elicited by stimulating receptors on the VSM; dilation is elicited by stimulating receptors on the endothelium. Middle cerebral arteries were isolated, cannulated with micropipettes, pressurized to 85 mm Hg, and luminally perfused. The extraluminal application of NPY (mixed agonist), [Leu31, Pro34]-NPY (Y1 agonist), or NPY-[13–36] (Y2 agonist) produced concentration-dependent constrictions. BIBP 3226 (Y1 selective antagonist) significantly attenuated the NPY-and [Leu31, Pro34]-NPY–induced constrictions. The luminal application of NPY, [Leu31, Pro34]-NPY, and NPY-[13–36] produced concentration-dependent dilations of MCAs. The maximum dilation produced by the NPY receptor agonists was approximately 40% of the dilation elicited by the luminal administration of 10−5 mol/L ATP. Dilations elicited by luminal NPY, [Leu31, Pro34]-NPY, or NPY-[13–36] were abolished by inhibition of nitric oxide synthase with 10−5 mol/L Nω-nitro-L-arginine methyl ester (L-NAME) or removal of the endothelium. Dilations produced by luminal NPY or luminal [Leu31, Pro34]-NPY were not affected by BIBP 3226. Stimulation of NPY receptors on vascular smooth muscle constricted MCAs. Stimulation of an NPY receptor other than the Y1 subtype on endothelium dilated the MCAs by releasing nitric oxide.

Characterization of endothelin-A receptors in the cerebral circulation

ENDOTHELIN-1 (ET-1), a 21-amino acid peptide produced by the vascular endothelium, mediates contraction. In the present study we demonstrate that both ET-1 (Emax: 238 × 29% of potassium contraction) and ET-2 (Emax: 231 × 36%) produce strong concentration-dependent contractions of circular segments of guinea-pig middle cerebral artery, whereas ET-3 has only weak effects (Emax: 32 × 13%). FR 139317 (10–6 M), a selective endothelinA (ETA) receptor antagonist, shifted the ET-1 response curve to the right (pD2:7.86 × 0.09 with and 8.76 × 0.09 without the antagonist) in a competitive manner (pA2 = 6.83). These findings are the first to show the presence of ETA receptors in cerebral vessels.

Potentiated Endothelium-Derived Hyperpolarizing Factor–Mediated Dilations in Cerebral Arteries Following Mild Head Injury

Evidence in the literature suggests that endothelium-derived hyperpolarizing factor (EDHF) may act in a compensatory manner such that during conditions of compromised nitric oxide (NO), EDHF serves as a back-up mechanism. Given that constitutive NO synthase is chronically downregulated after head trauma, we tested the hypothesis that EDHF is potentiated following injury. Male adult rats were subjected to either sham injury (n = 27) or mild controlled cortical impact (CCI) injury (n = 26). Branches of the middle cerebral artery (MCA) directly within the contusion site were harvested either 1 or 24 h later, pressurized to 60 mm Hg in a vessel chamber and allowed to develop spontaneous tone. Relaxation to luminal application of adenosine triphosphate (ATP) was similar in all groups. Relaxation to ATP in the presence of L-NAME (N(G)-nitro-L-arginine methyl ester) and indomethacin was similar in all groups except for vessels isolated at 24 h following mild CCI injury. In this case, L-NAME and indomethacin had no effect on the ATP-mediated dilation. The ATP-mediated dilation in L-NAME and indomethacin-treated MCA branches was inhibited by charybdotoxin, an inhibitor of large conductance Ca2+-sensitive K+ channels. These findings suggest that there is a significant potentiation of the EDHF-mediated dilation to ATP in cerebral arteries isolated at 24 h following mild CCI injury.

Role of cytoplasmic phospholipase A2 in endothelium-derived hyperpolarizing factor dilations of rat middle cerebral arteries

Very little is known regarding the mechanism of action for the endothelium-derived hyperpolarizing factor (EDHF) response in cerebral vessels. The authors tested two hypotheses: (1) activation of the cytoplasmic form of phospholipase A2 (cPLA2) is involved with EDHF-mediated dilations in rat middle cerebral arteries; and (2) activation of the cPLA2 involves an increase in endothelial Ca2+ through activation of phospholipase C. Middle cerebral arteries were isolated from the rat, pressurized to 85 mm Hg, and luminally perfused. The EDHF response was elicited by luminal application of uridine triphosphate (UTP) after NO synthase and cyclooxygenase inhibition (10−5 mol/L N-nitro-l-arginine methyl ester and 10−5 mol/L indomethacin, respectively). AACOCF3 and PACOCF3, inhibitors of cPLA2 (Ca2+-sensitive) and Ca2+-insensitive PLA2 (iPLA2), dose dependently attenuated the EDHF response. A selective inhibitor for iPLA2, haloenol lactone suicide substrate, had no effect on the EDHF response. The EDHF response elicited by UTP was accompanied by an increase in endothelial Ca2+ (144 to 468 nmol/L), and the EDHF dilation was attenuated with U73122, a phospholipase C inhibitor. The authors conclude that the EDHF response elicited by luminal UTP in rat middle cerebral arteries involved activation of phospholipase C, an increase in endothelial Ca2+, and activation of cPLA2.

Characterization of Neuropeptide Y Receptors Mediating Contraction, Potentiation and Inhibition of Relaxation

In addition to its direct vasoconstrictive effect, neuropeptide Y (NPY) potentiates noradrenaline-(NA) induced contraction and inhibits acetylcholine-(ACh) induced relaxation: The aim of the present study was to elucidate the NPY receptor subtypes responsible for mediating these three responses. NPY, peptide YY (PYY) and pro34NPY (a NPY Y1 receptor agonist) induced equipotent and equally strong concentration-dependent contractions of guinea pig basilar arteries. NPY13-36 (a NPY Y2 receptor agonist), however, caused only weak contraction with significantly lower potency. The NPY-induced contraction was significantly inhibited by the selective NPY Y1 receptor antagonist BIBP3226 (1 microM). NPY, PYY and pro34NPY but not NPY13-36 significantly potentiated the NA-induced contraction in guinea pig mesenteric arteries. The potentiation was significantly inhibited by BIBP3226 (1 microM). In precontracted guinea pig basilar arteries, ACh induced a concentration-dependent relaxation which was significantly inhibited by NPY, PYY and NPY13-36 but not by pro34NPY. BIBP3226 had no significant effect on the NPY-induced inhibition of the relaxation. These results suggests that the NPY Y1 receptors mediate the direct contraction and the potentiation of the NA-induced contraction but not the inhibition of the ACh-induced relaxation. This effect seems to be mediated by another NPY receptor subtype, presumably by the Y2 receptor, as judged from the agonist potency order.

Peptidergic innervation of guinea-pig brain vessels : comparison with immunohistochemistry and in vitro pharmacology in rostrally and caudally located arteries

The peptidergic innervation of the guinea-pig basilar artery and the posterior, middle and anterior cerebral arteries were studied by means of immunohistochemical and image analysis techniques using whole mount preparations. An in vitro pharmacological study was performed to correlate the distribution of peptide-containing nerves and the action of neuropeptides on vessel segments from the same vascular regions. The overall distribution of perivascular nerve fibres was demonstrated using an antiserum to the general neuronal marker protein gene product 9.5 (PGP 9.5) and the percentage immunostained area of total vessel wall area occupied by PGP-containing nerves, in each of the basilar, posterior and middle cerebral arteries, was set at 100% and used to determine the relative density of specific populations of autonomic and sensory nerve fibres. In all four cerebral arteries, the majority of nerve fibres possessed neuropeptide Y (NPY) and tyrosine hydroxylase (TH) immunoreactivity, occupying 6.2-13.3% and 5.8-7.5% of the total vessel wall area, respectively. Vasoactive intestinal peptide (VIP), substance P (SP) and calcitonin-gene-related peptide (CGRP) were detected at lower densities. The pharmacological study performed on small circular segments with an intact endothelium revealed that, in all four cerebral arteries, NPY was a more potent constrictor than noradrenaline (NA). The rank order of potency for relaxant agents was CGRP = SP > VIP > ACh in the PCA and MCA, and SP = CGRP > VIP > ACh in the BA and ACA. The correlation between immunostained nerve area and the agonist potency suggested that the denser the peptidergic nerve-supply, the lower the sensitivity to the agonist.

alpha-Trinositol: a functional (non-receptor) neuropeptide Y antagonist in vasculature

Neuropeptide Y is a sympathetic co‐neurotransmitter released with noradrenaline upon sympathetic nerve stimulation. This study describes the ability of a synthetic inositol phosphate, α‐trinositol (d‐myo‐inositol 1,2,6−triphosphate; PP 56) to antagonize vasoconstrictor responses to neuropeptide Y in‐vitro as well as in‐vivo.