Abstract-Agonist-induced Ca 2ϩ entry is important for the synthesis and release of vasoactive factors in endothelial cells. The transient receptor potential vanilloid type 4 (TRPV4) channel, a Ca 2ϩ -permeant cation channel, is expressed in endothelial cells and involved in the regulation of vascular tone. Here we investigated the role of TRPV4 channels in acetylcholine-induced vasodilation in vitro and in vivo using the TRPV4 knockout mouse model. The expression of TRPV4 mRNA and protein was detected in both conduit and resistance arteries from wild-type mice. In small mesenteric arteries from wild-type mice, the TRPV4 activator 4␣-phorbol-12,13-didecanoate increased endothelial [Ca 2ϩ ] i in situ, which was reversed by the TRPV4 blocker ruthenium red. In wild-type animals, acetylcholine dilated small mesenteric arteries that involved both NO and endothelium-derived hyperpolarizing factors. In TRPV4-deficient mice, the NO component of the relaxation was attenuated and the endothelium-derived hyperpolarizing factor component was largely eliminated. Compared with their wild-type littermates, TRPV4-deficient mice demonstrated a blunted endothelial Ca 2ϩ response to acetylcholine in mesenteric arteries and reduced NO release in carotid arteries. Acetylcholine (5 mg/kg, IV) decreased blood pressure by 37.0Ϯ6.2 mm Hg in wild-type animals but only 16.6Ϯ2.7 mm Hg in knockout mice. We conclude that acetylcholine-induced endothelium-dependent vasodilation is reduced both in vitro and in vivo in TRPV4 knockout mice. These findings may provide novel insight into mechanisms of variety of agonists such as acetylcholine, bradykinin, and even mechanical stimuli induce a rapid increase in endothelial Ca 2ϩ , leading to the synthesis and release of relaxing factors, including NO, prostacyclin, and endothelium-derived hyperpolarizing factors (EDHFs). 1 In endothelial and other mammalian cells, the Ca 2ϩ increase is usually a consequence of Ca 2ϩ release from intracellular stores of the endoplasmic reticulum and Ca 2ϩ influx through Ca 2ϩ -permeable cation channels in the plasma membrane via store-operated or receptor-operated mechanisms. 2 The influx of Ca 2ϩ from the extracellular space contributes to the sustained increase of the cytosolic Ca 2ϩ concentration. Despite the importance of calcium entry in the synthesis of endothelial relaxing factors, the proximate cause of this critical signaling event remains elusive.The discovery of transient receptor potential (TRP) channels provides new insights into potential mechanisms of Ca 2ϩ entry in endothelial cells. TRP channel-mediated Ca 2ϩ entry has been implicated in diverse responses, including changes in vascular permeability, angiogenesis, vascular remodeling, and vasorelaxation. 3,4 Of many subtypes of TRP channels expressed in endothelial cells, TRP vanilloid type 4 (TRPV4) channels have received increasing attention. These channels are widely expressed in vascular endothelial cells of several species and activated by both chemical and physical stimuli, including hypotonic...

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We used pharmacological agents and genetic methods to determine whether the potent A 3 adenosine receptor (AR) agonist 2-chloro-N 6 -(3-iodobenzyl)adenosine-5Ј-N-methylcarboxamide (Cl-IB-MECA) protects against myocardial ischemia/ reperfusion injury in mice via the A 3 AR or via interactions with other AR subtypes. Pretreating wild-type (WT) mice with Cl-IB-MECA reduced myocardial infarct size induced by 30 min of coronary occlusion and 24 h of reperfusion at doses (30 and 100 g/kg) that concomitantly reduced blood pressure and stimulated systemic histamine release. The A 3 AR-selective antagonist MRS 1523 [3-propyl-6-ethyl-5[(ethylthio) To further examine the selectivity of Cl-IB-MECA, we assessed its cardioprotective effectiveness in A 3 AR gene "knock-out" (A 3 KO) mice. Cl-IB-MECA did not reduce myocardial infarct size in A 3 KO mice in vivo and did not protect isolated perfused hearts obtained from A 3 KO mice from injury induced by global ischemia and reperfusion. Additional studies using WT mice treated with compound 48/80 [condensation product of p-methoxyphenethyl methylamine with formaldehyde] to deplete mast cell contents excluded the possibility that Cl-IB-MECA was cardioprotective by releasing mediators from mast cells. These data demonstrate that Cl-IB-MECA protects against myocardial ischemia/reperfusion injury in mice principally by activating the A 3 AR.Several different A 3 adenosine receptor (AR) agonists, including the N 6 -benzyl adenosine-5Ј-N-methylcarboxamide derivatives IB-MECA, Cl-IB-MECA, and CB-MECA, have been shown to be effective at protecting against myocardial ischemia/reperfusion injury in animal models of infarction and myocardial stunning (Auchampach et al., 1997b(Auchampach et al., , 2003Tracey et al., 1997Tracey et al., , 1998Tracey et al., , 2003Jordan et al., 1999; Thourani et al., 1999a,b;Kodani et al., 2001;Takano et al., 2001). However, it remains uncertain whether these agents are effective by activating the A 3 AR or by nonspecific interactions with other AR subtypes. This issue has been difficult to address, because useful A 3 AR antagonists have only recently been developed.The goal of this investigation was to test the cardioprotective effectiveness of Cl-IB-MECA in an in vivo mouse model of infarction and in an isolated mouse heart model of global ischemia and reperfusion. A second goal of this investigation was to establish definitively whether Cl-IB-MECA exerts cardioprotection by activating A 3 ARs. Our experimental approach involved the use of the rodent A 3 AR antagonist MRS 1523 (Li et al., 1998), the potent A 2A AR antagonist ZM This work was supported by National Institutes of Health Grants R01 HL60051, R01 HL07707, and T32 HL73643 and by American Heart Association Research Fellowships 0320019Z and 0315274Z.Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

We examined the cardioprotective profile of the new A 3 adenosine receptor (AR) agonist 903 [N 6 -(2,5-dichlorobenzyl)-3Ј-aminoadenosine-5Ј-N-methylcarboxamide] in an in vivo mouse model of infarction and an isolated heart model of global ischemia/reperfusion injury. In radioligand binding and cAMP accumulation assays using human embryonic kidney 293 cells expressing recombinant mouse ARs, CP-532,903 was found to bind with high affinity to mouse A 3 ARs (K i ϭ 9.0 Ϯ 2.5 nM) and with high selectivity versus mouse A 1 AR (100-fold) and A 2A ARs (1000-fold). In in vivo ischemia/reperfusion experiments, pretreating mice with 30 or 100 g/kg CP-532,903 reduced infarct size from 59.2 Ϯ 2.1% of the risk region in vehicle-treated mice to 42.5 Ϯ 2.3 and 39.0 Ϯ 2.9%, respectively. Likewise, treating isolated mouse hearts with CP-532,903 (10, 30, or 100 nM) concentration dependently improved recovery of contractile function after 20 min of global ischemia and 45 min of reperfusion, including developed pressure and maximal rate of contraction/relaxation. In both models of ischemia/reperfusion injury, CP-532,903 provided no benefit in studies using mice with genetic disruption of the A 3 AR gene, A 3 knockout (KO) mice. In isolated heart studies, protection provided by CP-532,903 and ischemic preconditioning induced by three brief ischemia/ reperfusion cycles were lost in Kir6.2 KO mice lacking expression of the pore-forming subunit of the sarcolemmal ATPsensitive potassium (K ATP ) channel. Whole-cell patch-clamp recordings provided evidence that the A 3 AR is functionally coupled to the sarcolemmal K ATP channel in murine cardiomyocytes. We conclude that CP-532,903 is a highly selective agonist of the mouse A 3 AR that protects against ischemia/reperfusion injury by activating sarcolemmal K ATP channels.A 3 adenosine receptor (AR) agonists have been shown to effectively limit infarct size and reduce contractile dysfunction in several different animal models of ischemia/reperfusion injury (Auchampach et al., 1997b(Auchampach et al., , 2003Tracey et al., 1997Tracey et al., , 1998Tracey et al., , 2003Jordan et al., 1999;Thourani et al., 1999;Ge et al., 2004Ge et al., , 2006. A 3 AR agonists are attractive as cardioprotective agents because they do not alter systemic hemodynamic parameters in nonrodent species and are effective if administered before the ischemic event or only during reperfusion (Auchampach et al

Abstract-We used a genetic approach to determine whether increasing the level of A 3 adenosine receptors (A 3 ARs) expressed in the heart confers protection against ischemia without causing cardiac pathology. We generated mice carrying one (A 3 tg.1) or six (A 3 tg.6) copies of a transgene consisting of the cardiomyocyte-specific ␣-myosin heavy chain gene promoter and the A 3 AR cDNA. A 3 tg.1 and A 3 tg.6 mice expressed 12.7Ϯ3.15 and 66.3Ϯ9.4 fmol/mg of the high-affinity G protein-coupled form of the A 3 AR in the myocardium, respectively. Extensive morphological, histological, and functional analyses demonstrated that there were no apparent abnormalities in A 3 tg.1 transgenic mice compared with nontransgenic mice. In contrast, A 3 tg.6 mice exhibited dilated hearts, expression of markers of hypertrophy, bradycardia, hypotension, and systolic dysfunction. When A 3 tg mice were subjected to 30 minutes of coronary occlusion and 24 hours of reperfusion, infarct size was reduced Ϸ30% in A 3 tg.1 mice and Ϸ40% in A 3 tg.6 mice compared with nontransgenic littermates. The reduction in infarct size in the transgenic mice was not related to differences in risk region size, systemic hemodynamics, or body temperature, indicating that the cardioprotection was a result of increased A 3 AR signaling in the ischemic myocardium. The results demonstrate that low-level expression of A 3 ARs in the heart provides effective protection against ischemic injury without detectable adverse effects, whereas higher levels of A 3 AR expression lead to the development of a dilated cardiomyopathy.

Objective: It is controversial whether endothelium-dependent relaxation resistance to inhibitors of nitric oxide (NO) and prostacyclin 1 synthases is completely attributed to endothelium-derived hyperpolarizing factor (EDHF). This study examined NO release and K G channels involved in endothelium-dependent relaxation and hyperpolarization resistance to N -nitro-L-arginine (L-NNA) and indomethacin in coronary arteries with emphasis on the microarteries. Methods: NO release, isometric force, and membrane potential of porcine coronary arteries were measured using a NO-specific electrode, wire myograph, and microelectrode, respectively. The endothelium relaxes vascular smooth muscle cells produced by the action of prostacyclin synthase on endoby at least three factors: nitric oxide (NO) [1], prostacyclin peroxides, the production of which are induced by cyclo-(PGI ) [2], and endothelium-derived hyperpolarizing facoxygenase (COX). This factor evokes vasodilation via the 2 activation of adenylate cyclase. In contrast, the enzyme responsible for EDHF production is unknown. However, it

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Background The role of endothelial nitric oxide synthase (eNOS) in isoflurane postconditioning (IsoPC)-elicited cardioprotection is poorly understood. We addressed this issue using eNOS-/- mice. Methods In vivo or Langendorff-perfused mouse hearts underwent 30 min of ischemia followed by 2 h of reperfusion in the presence and absence of postconditioning produced with isoflurane 5 min before ischemia and 3 min after reperfusion. Ca2+-induced mitochondrial permeability transition pore opening was assessed in isolated mitochondria. Echocardiography was used to evaluate ventricular function. Results Postconditioning with 0.5, 1.0, and 1.5 minimum alveolar concentrations of isoflurane decreased infarct size from 56 ± 10% (n = 10) in control to 48 ± 10%, 41 ± 8% (n = 8, P < 0.05), and 38 ± 10% (n = 8, P < 0.05), respectively and improved cardiac function in wild-type mice. Improvement in cardiac function by IsoPC was blocked by NG-nitro-L-arginine methyl ester (a nonselective NOS inhibitor) administered either prior to ischemia or at the onset of reperfusion. Mitochondria isolated from postconditioned hearts required significantly higher in vitro Ca2+ loading than control (78 ± 29 vs. 40 ± 25 μM CaCl2 mg protein-1, n = 10, P < 0.05) to open the mitochondrial permeability transition pore. Hearts from eNOS-/- mice displayed no marked differences in infarct size, cardiac function, and sensitivity of mitochondrial permeability transition pore to Ca2+, compared to the wild-type hearts. However, IsoPC failed to alter infarct size, cardiac function or the amount of Ca2+ necessary to open the mitochondrial permeability transition pore in mitochondria isolated from the eNOS-/- hearts compared to control hearts. Conclusions IsoPC protects mouse hearts from reperfusion injury by preventing MPT pore opening in an eNOS-dependent manner. Nitric oxide functions as both a trigger and a mediator of cardioprotection produced by IsoPC.