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Activation of the ?-common receptor by erythropoietin impairs acetylcholine-mediated vasodilation in mouse mesenteric arterioles.

ABSTRACT: Clinically, erythropoietin (EPO) is known to increase systemic vascular resistance and arterial blood pressure. However, EPO stimulates the production of the potent vasodilator, nitric oxide (NO), in culture endothelial cells. The mechanism by which EPO causes vasoconstriction despite stimulating NO production may be dependent on its ability to activate two receptor complexes, the homodimeric EPO (EPOR2 ) and the heterodimeric EPOR/?-common receptor (?CR). The purpose of this study was to investigate the contribution of each receptor to the vasoactive properties of EPO. First-order, mesenteric arteries were isolated from 16-week-old male C57BL/6 mice, and arterial function was studied in pressure arteriographs. To determine the contribution of each receptor complex, EPO-stimulating peptide (ESP), which binds and activates the heterodimeric EPOR/?CR complex, and EPO, which activates both receptors, were added to the arteriograph chamber 20 min prior to evaluation of endothelium-dependent (acetylcholine, bradykinin, A23187) and endothelium-independent (sodium nitroprusside) vasodilator responses. Only ACh-induced vasodilation was impaired in arteries pretreated with EPO or ESP. EPO and ESP pretreatment abolished ACh-induced vasodilation by 100% and 60%, respectively. EPO and ESP did not affect endothelium-independent vasodilation by SNP. Additionally, a novel ?CR inhibitory peptide (?IP), which was computationally developed, prevented the impairment of acetylcholine-induced vasodilation by EPO and ESP, further implicating the EPOR/?CR complex. Last, pretreatment with either EPO or ESP did not affect vasoconstriction by phenylephrine and KCl. Taken together, these findings suggest that acute activation of the heterodimeric EPOR/?CR in endothelial cells leads to a selective impairment of ACh-mediated vasodilator response in mouse mesenteric resistance arteries.

SUBMITTER: Kilar CR

PROVIDER: S-EPMC6016622 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

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Activation of the β-common receptor by erythropoietin impairs acetylcholine-mediated vasodilation in mouse mesenteric arterioles.

Kilar Cody R CR Diao YanPeng Y Sautina Larysa L Sekharan Sivakumar S Keinan Shahar S Carpino Bianca B Conrad Kirk P KP Mohandas Rajesh R Segal Mark S MS

Physiological reports 20180601 12

Clinically, erythropoietin (EPO) is known to increase systemic vascular resistance and arterial blood pressure. However, EPO stimulates the production of the potent vasodilator, nitric oxide (NO), in culture endothelial cells. The mechanism by which EPO causes vasoconstriction despite stimulating NO production may be dependent on its ability to activate two receptor complexes, the homodimeric EPO (EPOR<sub>2</sub> ) and the heterodimeric EPOR/β-common receptor (βCR). The purpose of this study wa ...[more]

PMID: 29939494

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Project description:Active neurons communicate to intracerebral arterioles in part through an elevation of cytosolic Ca(2+) concentration ([Ca(2+)](i)) in astrocytes, leading to the generation of vasoactive signals involved in neurovascular coupling. In particular, [Ca(2+)](i) increases in astrocytic processes ("endfeet"), which encase cerebral arterioles, have been shown to result in vasodilation of arterioles in vivo. However, the spatial and temporal properties of endfoot [Ca(2+)](i) signals have not been characterized, and information regarding the mechanism by which these signals arise is lacking. [Ca(2+)](i) signaling in astrocytic endfeet was measured with high spatiotemporal resolution in cortical brain slices, using a fluorescent Ca(2+) indicator and confocal microscopy. Increases in endfoot [Ca(2+)](i) preceded vasodilation of arterioles within cortical slices, as detected by simultaneous measurement of endfoot [Ca(2+)](i) and vascular diameter. Neuronal activity-evoked elevation of endfoot [Ca(2+)](i) was reduced by inhibition of inositol 1,4,5-trisphosphate (InsP(3)) receptor Ca(2+) release channels and almost completely abolished by inhibition of endoplasmic reticulum Ca(2+) uptake. To probe the Ca(2+) release mechanisms present within endfeet, spatially restricted flash photolysis of caged InsP(3) was utilized to liberate InsP(3) directly within endfeet. This maneuver generated large amplitude [Ca(2+)](i) increases within endfeet that were spatially restricted to this region of the astrocyte. These InsP(3)-induced [Ca(2+)](i) increases were sensitive to depletion of the intracellular Ca(2+) store, but not to ryanodine, suggesting that Ca(2+)-induced Ca(2+) release from ryanodine receptors does not contribute to the generation of endfoot [Ca(2+)](i) signals. Neuronally evoked increases in astrocytic [Ca(2+)](i) propagated through perivascular astrocytic processes and endfeet as multiple, distinct [Ca(2+)](i) waves and exhibited a high degree of spatial heterogeneity. Regenerative Ca(2+) release processes within the endfeet were evident, as were localized regions of Ca(2+) release, and treatment of slices with the vasoactive neuropeptides somatostatin and vasoactive intestinal peptide was capable of inducing endfoot [Ca(2+)](i) increases, suggesting the potential for signaling between local interneurons and astrocytic endfeet in the cortex. Furthermore, photorelease of InsP(3) within individual endfeet resulted in a local vasodilation of adjacent arterioles, supporting the concept that astrocytic endfeet function as local "vasoregulatory units" by translating information from active neurons into complex InsP(3)-mediated Ca(2+) release signals that modulate arteriolar diameter.

Project description:1. Activation of PAR2 in second-order mesenteric arteriole (MA) rings from C57BL/6J, NOS3 (-/-) and PAR2 (-/-) mice was assessed for the contributions of NO, cyclo-oxygenases, guanylyl cyclase, adenylyl cyclase, and of K(+) channel activation to vascular smooth muscle relaxation. 2. PAR2 agonist, SLIGRL-NH(2) (0.1 to 30 microM), induced relaxation of cirazoline-precontracted MA from C57BL/6J and NOS3 (-/-), but not PAR2 (-/-) mice. Maximal relaxation (E(max)) was partially reduced by a combination of L-(G)N-nitroarginine methyl ester (L-NAME), 1H-[1,2,4]-oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and indomethacin. An ODQ/L-NAME/indomethacin resistant relaxation was also caused by trypsin (30 nM) in PAR2 (+/+), but not in PAR2 (-/-) mice. Relaxation was endothelium-dependent and inhibited by either 30 mM KCl-precontraction, or pretreatment with apamin, charybdotoxin, and their combination; iberiotoxin did not substitute for charybdotoxin nor did scyllatoxin substitute fully for apamin. 3. Tetraethylammonium (TEA), glibenclamide, tetrodotoxin, 17-octadecynoic acid, carboxy-2-phenyl-4,4,5,5,-tetramethyl-imidazoline-1-oxyl-3-oxide, SQ22536, carbenoxolone, arachidonyl trifluoromethyl ketone, 7-nitroindazole, N-(3-(aminomethyl)benzyl)acetamidine (1400W), N-(2-cyclohexyloxy-4-nitrophenyl)-methanesulfonamide (NS-398) and propanolol did not inhibit relaxation. 4-aminopyridine significantly increased the potency of SLIGRL-NH(2). A combination of 30 microM BaCl(2) and 10 microM ouabain significantly reduced the potency for relaxation, and in the presence of L-NAME, ODQ and indomethacin, E(max) was reduced. 4. We conclude PAR2-mediated relaxation of mouse MA utilizes multiple mechanisms that are both NO-cGMP-dependent, and -independent. The data are also consistent with a role for endothelium-dependent hyperpolarization of vascular smooth muscle that involves the activation of an apamin/charybdotoxin-sensitive K(+) channel(s) and, in part, may be mediated by K(+).

Project description:Several pathological conditions predict the use of glucocorticoids for the management of the inflammatory response; however, chronic or high dose glucocorticoid treatment is associated with hyperglycemia, hyperlipidemia, and insulin resistance and can be considered a risk factor for cardiovascular disease. Therefore, we investigated the mechanisms involved in the vascular responsiveness and inflammatory profile of mesenteric arteries of rats treated with high doses of glucocorticoids. Wistar rats were divided into a control (CO) group and a dexamethasone (DEX) group, that received dexamethasone for 7 days (2mg/kg/day, i.p.). Blood samples were used to assess the lipid profile and insulin tolerance. Vascular reactivity to Phenylephrine (Phe) and insulin, and O2•-production were evaluated. The intracellular insulin signaling pathway PI3K/AKT/eNOS and MAPK/ET-1 were investigated. Regarding the vascular inflammatory profile, TNF-?, IL-6, IL-1? and IL-18 were assessed. Dexamethasone-treated rats had decreased insulin tolerance test and endothelium-dependent vasodilation induced by insulin. eNOS inhibition caused vasoconstriction in the DEX group, which was abolished by the ET-A antagonist. Insulin-mediated relaxation in the DEX group was restored in the presence of the O2.- scavenger TIRON. Nevertheless, in the DEX group there was an increase in Phe-induced vasoconstriction. In addition, the intracellular insulin signaling pathway PI3K/AKT/eNOS was impaired, decreasing NO bioavailability. Regarding superoxide anion generation, there was an increase in the DEX group, and all measured proinflammatory cytokines were also augmented in the DEX group. In addition, the DEX-group presented an increase in low-density lipoprotein cholesterol (LDL-c) and total cholesterol (TC) and reduced high-density lipoprotein cholesterol (HDL-c) levels. In summary, treatment with high doses of dexamethasone promoted changes in insulin-induced vasodilation, through the reduction of NO bioavailability and an increase in vasoconstriction via ET-1 associated with generation of O2•- and proinflammatory cytokines.

Project description:1. In this study, intracellular Ca(2+) was measured as the Fura-2 ratio (R) of fluorescence excited at 340 and 380 nm (F(340)/F(380)) in nonpressurized rat mesenteric small arterioles ( (lumen diameter) 10-25 microm). 2. The response to depolarization using 75 mm KCl was an increase in R from a baseline of 0.96+/-0.01 ([Ca(2+)](i) approximately 74 nm) to 1.04+/-0.01 ( approximately 128 nm) (n=80). The response to 75 mm K(+) was reversibly abolished in Ca(2+)-free physiological saline solution, whereas phentolamine (10 microm) or tetrodotoxin (1 microm) had no effects. LaCl(3) (200 microm) inhibited 61+/-9% of the response. 3. A [K(+)]-response curve indicated that the Ca(2+) response was activated between 15 and 25 mm K(+). The data suggest that the Ca(2+) response was caused by the activation of voltage-dependent Ca(2+) channels. 4. Mibefradil use dependently inhibited the Ca(2+) response to 75 mm K(+) by 29+/-2% (100 nm), 73+/-7% (1 microm) or 89+/-7% (10 microm). Pimozide (500 nm) use dependently inhibited the Ca(2+) response by 85+/-1%. 5. Nifedipine (1 microm) inhibited the Ca(2+) response to 75 mm K(+) by 41+/-12%. The response was not inhibited by calciseptine (500 nm), omega-agatoxin IVA (100 nm), omega-conotoxin MVIIA (500 nm), or SNX-482 (100 nm). 6. Using reverse transcriptase-polymerase chain reaction, it was shown that neither Ca(V)2.1a (P-type) nor Ca(V)2.1b (Q-type) voltage-dependent Ca(2+) channels were expressed in mesenteric arterioles, whereas the Ca(V)3.1 (T-type) channel was expressed. Furthermore, no amplification products were detected when using specific primers for the beta(1b), beta(2), or beta(3) auxiliary subunits of high-voltage-activated Ca(2+) channels. 7. The results suggest that the voltage-dependent Ca(2+) channel activated by sustained depolarization in mesenteric arterioles does not classify as any of the high-voltage-activated channels (L-, P/Q-, N-, or R-type), but is likely to be a T-type channel. The possibility that the sustained Ca(2+) influx observed was the result of a T-type window current is discussed.

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Dataset Information

Activation of the ?-common receptor by erythropoietin impairs acetylcholine-mediated vasodilation in mouse mesenteric arterioles.

Publications

Activation of the β-common receptor by erythropoietin impairs acetylcholine-mediated vasodilation in mouse mesenteric arterioles.

Similar Datasets

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