Rap1b in smooth muscle and endothelium is required for maintenance of vascular tone and normal blood pressure.

OBJECTIVE: Small GTPase Ras-related protein 1 (Rap1b) controls several basic cellular phenomena, and its deletion in mice leads to several cardiovascular defects, including impaired adhesion of blood cells and defective angiogenesis. We found that Rap1b(-/-) mice develop cardiac hypertrophy and hypertension. Therefore, we examined the function of Rap1b in regulation of blood pressure. APPROACH AND RESULTS: Rap1b(-/-) mice developed cardiac hypertrophy and elevated blood pressure, but maintained a normal heart rate. Correcting elevated blood pressure with losartan, an angiotensin II type 1 receptor antagonist, alleviated cardiac hypertrophy in Rap1b(-/-) mice, suggesting a possibility that cardiac hypertrophy develops secondary to hypertension. The indices of renal function and plasma renin activity were normal in Rap1b(-/-) mice. Ex vivo, we examined whether the effect of Rap1b deletion on smooth muscle-mediated vessel contraction and endothelium-dependent vessel dilation, 2 major mechanisms controlling basal vascular tone, was the basis for the hypertension. We found increased contractility on stimulation with a thromboxane analog or angiotensin II or phenylephrine along with increased inhibitory phosphorylation of myosin phosphatase under basal conditions consistent with elevated basal tone and the observed hypertension. Cyclic adenosine monophosphate-dependent relaxation in response to Rap1 activator, Epac, was decreased in vessels from Rap1b(-/-) mice. Defective endothelial release of dilatory nitric oxide in response to elevated blood flow leads to hypertension. We found that nitric oxide-dependent vasodilation was significantly inhibited in Rap1b-deficient vessels. CONCLUSIONS: This is the first report to indicate that Rap1b in both smooth muscle and endothelium plays a key role in maintaining blood pressure by controlling normal vascular tone.

A role for the low-affinity A2B adenosine receptor in regulating superoxide generation by murine neutrophils.

The formation of adenosine dampens inflammation by inhibiting most cells of the immune system. Among its actions on neutrophils, adenosine suppresses superoxide generation and regulates chemotactic activity. To date, most evidence implicates the G(s) protein-coupled A(2A) adenosine receptor (AR) as the primary AR subtype responsible for mediating the actions of adenosine on neutrophils by stimulating cAMP production. Given that the A(2B)AR is now known to be expressed in neutrophils and that it is a G(s) protein-coupled receptor, we examined in this study whether it signals to suppress neutrophil activities by using 2-[6-amino-3,5-dicyano-4-[4-(cyclopropylmethoxy)phenyl]pyridin-2-ylsulfanyl]acetamide (BAY 60-6583), a new agonist for the human A(2B)AR that was confirmed in preliminary studies to be a potent and highly selective agonist for the murine A(2B)AR. We found that treating mouse neutrophils with low concentrations (10(-9) and 10(-8) M) of BAY 60-6583 inhibited formylated-methionine-leucine-phenylalanine (fMLP)-stimulated superoxide production by either naive neutrophils, tumor necrosis factor-α-primed neutrophils, or neutrophils isolated from mice treated systemically with lipopolysaccharide. This inhibitory action of BAY 60-6583 was confirmed to involve the A(2B)AR in experiments using neutrophils obtained from A(2B)AR gene knockout mice. It is noteworthy that BAY 60-6583 increased fMLP-stimulated superoxide production at higher concentrations (>1 µM), which was attributed to an AR-independent effect. In a standard Boyden chamber migration assay, BAY 60-6583 alone did not stimulate neutrophil chemotaxis or influence chemotaxis in response to fMLP. These results indicate that the A(2B)AR signals to suppress oxidase activity by murine neutrophils, supporting the idea that this low-affinity receptor for adenosine participates along with the A(2A)AR in regulating the proinflammatory actions of neutrophils.

Evidence that the acute phase of ischemic preconditioning does not require signaling by the A 2B adenosine receptor.

Ischemic preconditioning (IPC) is a protective phenomenon in which brief ischemia renders the myocardium resistant to subsequent ischemic insults. Here, we used A(2B)AR gene knock-out (A(2B)KO)/ß-galactosidase reporter gene knock-in mice and the A(2B)AR antagonist ATL-801 to investigate the potential involvement of the A(2B)AR in IPC, focusing on the acute phase of protection. Cardioprotection provided by acute IPC elicited by two 3-min occlusion/3-min reperfusion cycles was readily apparent in an isolated, Langendorff-perfused mouse heart model in studies using hearts from A(2B)KO mice. IPC equivalently improved the recovery of contractile function following 20 min of global ischemia and 45 min of reperfusion in both WT and A(2B)KO hearts by ~30-40%, and equivalently decreased the release of cardiac troponin I during the reperfusion period (from 5969 ± 925 to 1595 ± 674 ng/g and 4376 ± 739 to 2278 ± 462 ng/g using WT and A(2B)KO hearts, respectively). Similarly, the infarct size-reducing capacity of acute IPC in an in vivo model of infarction was fully manifested in experiments using A(2B)KO mice, as well as in experiments using rats pretreated with ATL-801. We did observe, however, a marked reduction in infarct size in rats following administration of the selective A(2B)AR agonist BAY 60-6583 (~25% reduction at a dose of 1.0mg/kg). While supportive of its concept as a cardioprotective receptor, these experiments indicate that the mechanism of the early phase of IPC is not dependent on signaling by the A(2B)AR. We present the idea that the A(2B)AR may contribute to the later stages of IPC dependent on the induction of stress-responsive genes.

A(3) adenosine receptor activation during reperfusion reduces infarct size through actions on bone marrow-derived cells.

The goal of this study was to examine whether the A(3) adenosine receptor (A(3)AR) agonist Cl-IB-MECA protects against myocardial ischemia/reperfusion injury when administered at the time of reperfusion in an in vivo mouse model of infarction induced by 30min of coronary occlusion and 24h of reperfusion. Treating B6 wild-type with Cl-IB-MECA during the reperfusion phase (100microg/kg i.v. bolus+0.3microg/kg/min subcutaneously via implantation of Alzet mini-osmotic pumps) reduced myocardial infarct size approximately 37% from 50.1+/-2.5% in vehicle-treated mice to 31.6+/-2.8% in Cl-IB-MECA-treated mice, and significantly reduced the number of leukocytes that infiltrated into the ischemic-reperfused myocardium. Cl-IB-MECA did not reduce infarct size or limit leukocyte accumulation in studies using B6 congenic A(3)AR gene "knock-out" mice or in chimeric mice lacking the expression of A(3)ARs in bone marrow (BM)-derived cells. Subsequent mechanistic studies demonstrated that Cl-IB-MECA inhibited migration of mouse neutrophils isolated from BM towards the chemotactic substance c5a in trans-well migration assays, and inhibited leukocyte migration into the peritoneal cavity in a mouse model of thioglycollate-induced peritonitis. We conclude that treating with the A(3)AR agonist Cl-IB-MECA at the time of reperfusion provides effective protection from ischemia/reperfusion injury in the heart through activation of the A(3)AR expressed in BM-derived cells, potentially by suppressing the robust inflammatory reaction that occurs during reperfusion and neutrophil-mediated tissue injury.

Characterization of the A2B adenosine receptor from mouse, rabbit, and dog.

We have cloned and pharmacologically characterized the A(2B) adenosine receptor (AR) from the dog, rabbit, and mouse. The full coding regions of the dog and mouse A(2B)AR were obtained by reverse transcriptase-polymerase chain reaction, and the rabbit A(2B)AR cDNA was obtained by screening a rabbit brain cDNA library. It is noteworthy that an additional clone was isolated by library screening that was identical in sequence to the full-length rabbit A(2B)AR, with the exception of a 27-base pair deletion in the region encoding amino acids 103 to 111 (A(2B)AR(103-111)). This 9 amino acid deletion is located in the second intracellular loop at the only known splice junction of the A(2B)AR and seems to result from the use of an additional 5' donor site found in the rabbit and dog but not in the human, rat, or mouse sequences. [(3)H]3-Isobutyl-8-pyrrolidinoxanthine and 8-[4-[((4-cyano-[2,6-(3)H]-phenyl)carbamoylmethyl)oxy]phenyl]-1,3-di(n-propyl)xanthine ([(3)H]MRS 1754) bound with high affinity to membranes prepared from human embryonic kidney (HEK) 293 cells expressing mouse, rabbit, and dog A(2B)ARs. Competition binding studies performed with a panel of agonist (adenosine and 2-amino-3,5-dicyano-4-phenylpyridine analogs) and antagonist ligands identified similar potency orders for the A(2B)AR orthologs, although most xanthine antagonists displayed lower binding affinity for the dog A(2B)AR compared with A(2B)ARs from rabbit and mouse. No specific binding could be detected with membranes prepared from HEK 293 cells expressing the rabbit A(2B)AR(103-111) variant. Furthermore, the variant failed to stimulate adenylyl cyclase or calcium mobilization. We conclude that significant differences in antagonist pharmacology of the A(2B)AR exist between species and that some species express nonfunctional variants of the A(2B)AR due to "leaky" splicing.

The DNA sequence of human chromosome 7.

Human chromosome 7 has historically received prominent attention in the human genetics community, primarily related to the search for the cystic fibrosis gene and the frequent cytogenetic changes associated with various forms of cancer. Here we present more than 153 million base pairs representing 99.4% of the euchromatic sequence of chromosome 7, the first metacentric chromosome completed so far. The sequence has excellent concordance with previously established physical and genetic maps, and it exhibits an unusual amount of segmentally duplicated sequence (8.2%), with marked differences between the two arms. Our initial analyses have identified 1,150 protein-coding genes, 605 of which have been confirmed by complementary DNA sequences, and an additional 941 pseudogenes. Of genes confirmed by transcript sequences, some are polymorphic for mutations that disrupt the reading frame.

A physical map of the mouse genome.

A physical map of a genome is an essential guide for navigation, allowing the location of any gene or other landmark in the chromosomal DNA. We have constructed a physical map of the mouse genome that contains 296 contigs of overlapping bacterial clones and 16,992 unique markers. The mouse contigs were aligned to the human genome sequence on the basis of 51,486 homology matches, thus enabling use of the conserved synteny (correspondence between chromosome blocks) of the two genomes to accelerate construction of the mouse map. The map provides a framework for assembly of whole-genome shotgun sequence data, and a tile path of clones for generation of the reference sequence. Definition of the human-mouse alignment at this level of resolution enables identification of a mouse clone that corresponds to almost any position in the human genome. The human sequence may be used to facilitate construction of other mammalian genome maps using the same strategy.