ABSTRACT
The aim was to determine whether treatment with BAY 60-2770, a selective activator of oxidized soluble guanylate cyclase (sGC), near the end of an ischemic event would prevent postischemic inflammation and mitochondrial dysfunction in wild-type (WT) and heme oxygenase-1 KO (HO-1(-/-)) mice. This protocol prevented increases in leukocyte rolling (LR) and adhesion (LA) to intestinal venules along with elevated TNFα and circulating neutrophil levels that accompany ischemia-reperfusion (I/R) in both animal models. We further hypothesized that a component of BAY 60-2770 treatment involves maintenance of mitochondrial membrane integrity during I/R. Measurements on isolated enterocytes of calcein fluorescence (mitochondrial permeability) and JC-1 fluorescence ratio (mitochondrial membrane potential) were reduced by I/R, indicating formation of mitochondrial permeability transition pores (mPTP). These effects were abrogated by BAY 60-2770 as well as cyclosporin A and SB-216763, which prevented mPTP opening and inhibited glycogen synthase kinase-3ß (GSK-3ß), respectively. Western blots of WT and HO-1(-/-) enterocytes indicated that GSK-3ß phosphorylation on Ser(9) (inhibitory site) was reduced by half following I/R alone (increased GSK-3ß activity) and increased by one-third (reduced GSK-3ß activity) following BAY 60-2770. Other investigators have associated phosphorylation of the GSK-3ß substrate cyclophilin D (pCyPD) with mPTP formation. We observed a 60% increase in pCyPD after I/R, whereas BAY 60-2770 treatment of sham and I/R groups reduced pCyPD by about 20%. In conclusion, selective activation of oxidized sGC of WT and HO-1(-/-) during ischemia protects against I/R-induced inflammation and preserves mucosal integrity in part by reducing pCyPD production and mPTP formation.
Subject(s)
Enterocytes/metabolism , Ischemia/metabolism , Mitochondria/metabolism , Soluble Guanylyl Cyclase/metabolism , Animals , Benzoates/pharmacology , Biphenyl Compounds/pharmacology , Cells, Cultured , Peptidyl-Prolyl Isomerase F , Cyclophilins/metabolism , Enterocytes/drug effects , Female , Glycogen Synthase Kinase 3 beta/metabolism , Heme Oxygenase-1/genetics , Heme Oxygenase-1/metabolism , Hydrocarbons, Fluorinated/pharmacology , Intestines/blood supply , Intestines/cytology , Membrane Potential, Mitochondrial , Mice , Mitochondrial Membrane Transport Proteins/metabolism , Mitochondrial Permeability Transition PoreABSTRACT
OBJECTIVE: Integrins contribute to vascular morphogenesis through regulation of adhesion and assembly of the extracellular matrix. However, the role of ß1-integrin in the mature vascular wall is less clear. APPROACH AND RESULTS: We sought to determine the function of ß1-integrin in mature smooth muscle cells in vivo using a loss of function approach by crossing a tamoxifen-inducible sm22αCre line to a floxed ß1-integrin transgenic line. Adult mice lacking smooth muscle ß1-integrin survived only 10 weeks post induction. The deletion of ß1-integrin resulted in profound loss of vasomotor control. Histological analysis revealed progressive fibrosis in arteries with associated apoptosis of smooth muscle cells, which was not rescued by adventitial stem cells. Smooth muscle cell apoptosis was detected in arteries with dead cells replaced primarily by collagen. Despite the catastrophic effects on vascular smooth muscle, the deleted visceral smooth muscle remained viable with the exception of a short portion of the colon, indicating that vascular but not visceral smooth muscle is particularly sensitive to changes in ß1-integrin. CONCLUSIONS: This study reveals an essential function of ß1-integrin in the maintenance of vasomotor control and highlights a critical role for ß1-integrin in vascular, but not visceral, smooth muscle survival.
Subject(s)
Integrin beta1/metabolism , Muscle, Smooth, Vascular/metabolism , Myocytes, Smooth Muscle/metabolism , Vasoconstriction , Vasodilation , Adaptation, Physiological , Animals , Apoptosis , Cell Survival , Collagen/metabolism , Dose-Response Relationship, Drug , Fibrosis , Integrin beta1/genetics , Mice , Mice, Knockout , Muscle, Smooth, Vascular/drug effects , Muscle, Smooth, Vascular/pathology , Muscle, Smooth, Vascular/physiopathology , Myocytes, Smooth Muscle/drug effects , Myocytes, Smooth Muscle/pathology , Time Factors , Vasoconstriction/drug effects , Vasoconstrictor Agents/pharmacology , Vasodilation/drug effects , Vasodilator Agents/pharmacologyABSTRACT
Previously we have shown that, unlike wild-type mice (WT), heme oxygenase-1 knockout (HO-1-/-) mice developed nitrate tolerance and were not protected from inflammation caused by ischemia-reperfusion (I/R) when preconditioned with a H2S donor. We hypothesized that stimulation (with BAY 41-2272) or activation (with BAY 60-2770) of soluble guanylate cyclase (sGC) would precondition HO-1-/- mice against an inflammatory effect of I/R and increase arterial nitrate responses. Intravital fluorescence microscopy was used to visualize leukocyte rolling and adhesion to postcapillary venules of the small intestine in anesthetized mice. Relaxation to ACh and BAY compounds was measured on superior mesenteric arteries isolated after I/R protocols. Preconditioning with either BAY compound 10 min (early phase) or 24 h (late phase) before I/R reduced postischemic leukocyte rolling and adhesion to sham control levels and increased superior mesenteric artery responses to ACh, sodium nitroprusside, and BAY 41-2272 in WT and HO-1-/- mice. Late-phase preconditioning with BAY 60-2770 was maintained in HO-1-/- and endothelial nitric oxide synthase knockout mice pretreated with an inhibitor (dl-propargylglycine) of enzymatically produced H2S. Pretreatment with BAY compounds also prevented the I/R increase in small intestinal TNF-α. We speculate that increasing sGC activity and related PKG acts downstream to H2S and disrupts signaling processes triggered by I/R in part by maintaining low cellular Ca²âº. In addition, BAY preconditioning did not increase sGC levels, yet increased the response to agents that act on reduced heme-containing sGC. Collectively these actions would contribute to increased nitrate sensitivity and vascular function.
Subject(s)
Benzoates/pharmacology , Biphenyl Compounds/pharmacology , Enzyme Activators/pharmacology , Heme Oxygenase-1/deficiency , Hydrocarbons, Fluorinated/pharmacology , Inflammation/prevention & control , Intestine, Small/blood supply , Ischemia/drug therapy , Membrane Proteins/deficiency , Mesenteric Vascular Occlusion/drug therapy , Pyrazoles/pharmacology , Pyridines/pharmacology , Receptors, Cytoplasmic and Nuclear/agonists , Reperfusion Injury/prevention & control , Vascular Diseases/drug therapy , Animals , Cyclic GMP-Dependent Protein Kinases/metabolism , Disease Models, Animal , Dose-Response Relationship, Drug , Enzyme Activation , Guanylate Cyclase/metabolism , Heme Oxygenase-1/genetics , Hydrogen Sulfide/metabolism , Inflammation/enzymology , Inflammation/genetics , Inflammation/physiopathology , Inflammation Mediators/metabolism , Ischemia/enzymology , Ischemia/genetics , Ischemia/physiopathology , Leukocyte Rolling/drug effects , Membrane Proteins/genetics , Mesenteric Artery, Superior/drug effects , Mesenteric Artery, Superior/enzymology , Mesenteric Artery, Superior/surgery , Mesenteric Ischemia , Mesenteric Vascular Occlusion/enzymology , Mesenteric Vascular Occlusion/genetics , Mesenteric Vascular Occlusion/physiopathology , Mice , Mice, Inbred C57BL , Mice, Knockout , Nitric Oxide/metabolism , Nitric Oxide Synthase Type III/genetics , Nitric Oxide Synthase Type III/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Reperfusion Injury/enzymology , Reperfusion Injury/genetics , Reperfusion Injury/physiopathology , Signal Transduction/drug effects , Soluble Guanylyl Cyclase , Time Factors , Tumor Necrosis Factor-alpha/metabolism , Vascular Diseases/enzymology , Vascular Diseases/genetics , Vascular Diseases/physiopathology , Vasodilation/drug effects , Venules/drug effects , Venules/enzymologyABSTRACT
We previously demonstrated that preconditioning induced by ethanol consumption at low levels [ethanol preconditioning (EPC)] or with 5-aminoimidazole-4-carboxamide 1-ß-d-ribofuranoside (AICAR-PC) 24 h before ischemia-reperfusion prevents postischemic leukocyte-endothelial cell adhesive interactions (LEI) by a mechanism that is initiated by nitric oxide formed by endothelial nitric oxide synthase. Recent work indicates that 1) ethanol increases the activity of AMP-activated protein kinase (AMPK) and 2) AMPK phosphorylates endothelial nitric oxide synthase at the same activation site seen following EPC (Ser1177). In light of these observations, we postulated that the heterotrimeric serine/threonine kinase, AMPK, may play a role in triggering the development of the anti-inflammatory phenotype induced by EPC. Ethanol was administered to C57BL/6J mice by gavage in the presence or absence of AMPK inhibition. Twenty-four hours later, the numbers of rolling and adherent leukocytes in postcapillary venules of the small intestine were recorded using an intravital microscopic approach. Following 45 min of ischemia, LEI were recorded after 30 and 60 min of reperfusion or at equivalent time points in control animals. Ischemia-reperfusion induced a marked increase in LEI relative to sham-operated control mice. The increase in LEI was prevented by EPC, an effect that was lost with AMPK inhibition during the period of ethanol exposure. Studies conducted in AMPK α(1)- and α(2)-knockout mice suggest that the anti-inflammatory effects of AICAR are not dependent on which isoform of the catalytic α-subunit is present because a deficiency of either isoform results in a loss of protection. In sharp contrast, EPC appears to be triggered by an AMPK α(2)-isoform-dependent mechanism.
Subject(s)
Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Endothelium, Vascular/drug effects , Ischemic Preconditioning/methods , Leukocytes/drug effects , Reperfusion Injury/prevention & control , Aminoimidazole Carboxamide/analogs & derivatives , Aminoimidazole Carboxamide/pharmacology , Animals , Cell Adhesion/drug effects , Ethanol/pharmacology , Intestine, Small/blood supply , Intestine, Small/drug effects , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Reperfusion Injury/drug therapy , Ribonucleotides/pharmacology , Venules/drug effectsABSTRACT
EtOH-PC reduces postischemic neuronal injury in response to cerebral (I/R). We examined the mechanism underlying this protective effect by determining (i) whether it was associated with a decrease in I/R-induced leukocyte-endothelial adhesive interactions in postcapillary venules, and (ii) whether the protective effects were mediated by activation of large conductance, calcium-activated potassium (BK(Ca)) channels. Mice were administered ethanol by gavage or treated with the BK(Ca) channel opener, NS1619, 24 hours prior to I/R with or without prior treatment with the BK(Ca) channel blocker, PX. Both CCA were occluded for 20 minutes followed by two and three hours of reperfusion, and rolling (LR) and adherent (LA) leukocytes were quantified in pial venules using intravital microscopy. The extent of DND, apoptosis and glial activation in hippocampus were assessed four days after I/R. Compared with sham, I/R elicited increases in LR and LA in pial venules and DND and apoptosis as well as glial activation in the hippocampus. These effects were attenuated by EtOH-PC or antecedent NS1619 administration, and this protection was reversed by prior treatment with PX. Our results support a role for BK(Ca) channel activation in the neuroprotective effects of EtOH-PC in cerebral I/R.
Subject(s)
Brain Ischemia/drug therapy , Ethanol/administration & dosage , Large-Conductance Calcium-Activated Potassium Channel alpha Subunits/metabolism , Leukocytes/drug effects , Neurons/drug effects , Neuroprotective Agents/administration & dosage , Reperfusion Injury/drug therapy , Animals , Benzimidazoles/pharmacology , Blood-Brain Barrier/drug effects , Brain Ischemia/blood , Brain Ischemia/pathology , Cell Adhesion/drug effects , Cell Death/drug effects , Cerebrovascular Circulation/drug effects , Endothelial Cells/drug effects , Indoles/pharmacology , Ischemic Preconditioning/methods , Large-Conductance Calcium-Activated Potassium Channel alpha Subunits/antagonists & inhibitors , Male , Mice , Mice, Inbred C57BL , Neurons/pathology , Prosencephalon/blood supply , Prosencephalon/drug effects , Prosencephalon/injuries , Reperfusion Injury/blood , Reperfusion Injury/pathologyABSTRACT
Heme oxygenase-1 knockout, H(mox)1(-/-), mice exhibit exacerbated vascular lesions after ischemia-reperfusion and mechanical injury. Surprisingly, we found no studies that reported contractile responses and sensitivity to vasorelaxants in H(mox)1(-/-) mice. The contractile responses [superior mesenteric arteries (SMA), from female H(mox)1(-/-) mice] exhibited increased sensitivity to phenylephrine (p < 0.001). Cumulative addition of acetylcholine relaxed SMA, with the residual contraction remaining 2 times higher in H(mox)1(-/-) mice (p < 0.001). Sodium nitroprusside (SNP, an NO donor) and 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole [YC-1; acts directly on soluble guanylate cyclase (sGC)] led to further relaxation, yet the residual contraction remained 2 to 3 times higher in H(mox)1(-/-) than H(mox)1(+/+) mice (p < 0.001). Branches from H(mox)1(-/-) mesenteric and renal arteries also showed reduced relaxation (p < 0.025). Relaxation of SMA was measured to 4-({(4-carboxybutyl) [2-(5-fluoro-2-{[4'-(trifluoromethyl) biphenyl-4-yl] methoxy}phenyl)ethyl]amino}benzoic acid (BAY 60-2770), which is a more effective activator of oxidized/heme-free sGC; and to 5-cyclopropyl-2-{1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl}-pyrimidin-4-ylamine (BAY 41-2272), a more effective stimulator of reduced sGC. H(mox)1(-/-) arteries were 15 times more sensitive to BAY 60-2770 (p < 0.025) than were H(mox)1(+/+) arteries. Pretreatment with 1H-[1,2,4]oxadiazolo[3,4-a]quinoxalin-1-one (ODQ), an oxidizer of sGC, predictably shifted the BAY 60-2770 response of H(mox)1(+/+) to the left (p < 0.01) and BAY 41-2272 response to the right (p < 0.01). ODQ had little effect on the responses of H(mox)1(-/-) arteries, indicating that much of sGC was oxidized/heme-free. Western analyses of sGC in SMA indicated that both α1 and ß1 subunit levels were reduced to <50% of H(mox)1(+/+) level (p < 0.025). These findings support the hypothesis that the antioxidant function of H(mox)1 plays a significant role in the maintenance of sGC in a reduced state, which is resistant to degradation and is sensitive to NO. This function may be especially important in reducing vascular damage during ischemia-reperfusion injury.
Subject(s)
Guanylate Cyclase/metabolism , Heme Oxygenase-1/deficiency , Receptors, Cytoplasmic and Nuclear/metabolism , Acetylcholine/metabolism , Acetylcholine/pharmacology , Animals , Arteries/drug effects , Arteries/metabolism , Blotting, Western , Enzyme Activation/drug effects , Enzyme Inhibitors/pharmacology , Gene Expression Regulation, Enzymologic/physiology , Guanylate Cyclase/biosynthesis , Heme/metabolism , Heme Oxygenase-1/antagonists & inhibitors , Heme Oxygenase-1/genetics , Mice , Mice, Knockout , Muscle Relaxation/drug effects , Muscle, Smooth, Vascular/drug effects , Muscle, Smooth, Vascular/metabolism , Nitrates/metabolism , Nitric Oxide Donors/pharmacology , Nitroprusside/pharmacology , Oxidation-Reduction , Phenylephrine/pharmacology , Receptors, Cytoplasmic and Nuclear/biosynthesis , Soluble Guanylyl Cyclase , Vasoconstrictor Agents/pharmacology , Vasodilator Agents/pharmacologyABSTRACT
Potassium channels in vascular smooth muscle (VSM) control vasodilation and are potential regulatory targets. This study evaluated effects of sex differences, exercise training (EX), and high-fat diet (HF) on K(+) currents (I(K)) of coronary VSM cells. Yucatan male and female swine were assigned to either sedentary confinement (SED), 16 wk of EX, 20 wk of HF, or 20 wk of HF with 16 wk of EX (HF-EX). VSM cells of normal-diet SED animals exhibited three components of I(K): 4-aminopyridine-sensitive I(K(KV)), TEA-sensitive I(K(BK)), and 4-aminopyridine + TEA-insensitive I(K). Females exhibited significantly higher basal I(K) than males in the same group. EX increased basal I(K) in males and females. HF reduced I(K) in males and females and nullified effects of EX. Endothelin-1 increased I(K) significantly in males but not in females. In the presence of endothelin-1, 1) I(K(KV)) was similar in SED males and females and EX increased I(K(KV)) to a greater extent in males than in females and 2) I(K(BK)) was greater in SED females than in males and EX increased I(K(BK)) to a greater extent in males, resulting in I(K(BK)) similar to EX females. Importantly, HF nullified effects of EX on I(K(KV)) and I(K(BK)). These data indicate that basal I(K) of SED female swine is inherently greater than that shown in SED males and that males require EX to achieve comparable levels of I(K). Importantly, HF reduced I(K) in males and females and nullified effects of EX, suggesting HF abrogates beneficial effects of EX on coronary smooth muscle.