Cultured vascular smooth muscle cells from porcine coronary artery possess A1 and A2 adenosine receptor activity

This study tested the hypothesis that an A1 adenosine receptor capable of inhibiting adenylate cyclase activity is present in porcine coronary vascular smooth muscle cells. In the absence of blockade of the A2 adenosine receptor, the A1 adenosine receptor agonists phenylisopropyladenosine (PIA) and cyclopentyladenosine (CPA) (10(-9) M) failed to inhibit Gpp(NH)p stimulated adenylate cyclase activity. However, after blockade of the A2 adenosine receptor with 30 nM CGS 15943A, cyclopentyladenosine (10(-9) M) inhibited Gpp(NH)p stimulated adenylate cyclase activity by 27 +/- 3% (4.3 +/- 0.7, Mean +/- SEM; pmoles/min/mg vs 5.9 +/- 0.8, P less than .05). The data demonstrate that both A1 and A2 adenosine receptors are present in coronary vascular smooth muscle. The results indicate that adenosine may mediate both vasodilation and vasoconstriction in the coronary circulation via A2 and A1 adenosine receptors, respectively.     

Trypanosoma cruzi: aberrant expression of class II major histocompatibility complex molecules in skeletal and heart muscle cells of chronically infected mice

In isolated skeletal, heart, and smooth muscle cells from BALB/c and C3H/HeJ mice infected with different strains of Trypanosoma cruzi the presence of class II MHC molecules was investigated by immunocytochemical techniques. We employed single muscle fibers instead of conventional cryostat sections to obtain a more accurate antigen localization. Approximately half of the skeletal muscle cells isolated from the rectus femoris expressed Ia antigens on their surface, irrespective of the mouse or parasite strain combination. Ia expression was apparent only at 30 days postinfection and thereafter. The heart muscle cells expressed class II molecules only at 1 and 3 months postinfection. In no case did the smooth muscle cells from infected mice express Ia antigens. Studies of the same molecules in the noninfected animals gave constantly negative results. We conclude that in the course of the chronic infection of mice with T. cruzi, ectopic expression of class II MHC molecules occurs at the surface of skeletal and heart muscle cells, providing a possible mechanism for explaining the anti-striped muscle autoreactivity present in Chagas' disease.     

Expression of adenosine A2b receptor in rat type II and III taste cells

We previously demonstrated that equilibrative nucleoside transporter 1 was expressed in taste cells, suggesting the existence of an adenosine signaling system, but whether or not the expression of an adenosine receptor occurs in rat taste buds remains unknown. Therefore, we examined the expression profiles of adenosine receptors and evaluated their functionality in rat circumvallate papillae. Among adenosine receptors, the mRNA for an adenosine A2b receptor (A2bR) was expressed by the rat circumvallate papillae, and its expression level was significantly greater in the circumvallate papillae than in the non-taste lingual epithelium. A2bR-immunoreactivity was detected primarily in type II taste cells, and partial, but significant expression was also observed in type III ones, but there was no immunoreactivity in type I ones. The cAMP generation in isolated epithelium containing taste buds treated with 500 μM adenosine or 10 μM BAY60-6583 was significantly increased compared to in the controls. These findings suggest that adenosine plays a role in signaling transmission via A2bR between taste cells in rats.     

Isolation and characterization of coronary endothelial and smooth muscle cells from A1 adenosine receptor-knockout mice

Mice have been used widely in in vivo and in vitro cardiovascular research. The availability of knockout mice provides further clues to the physiological significance of specific receptor subtypes. Adenosine A(1) receptor (A(1)AR)-knockout (A(1)KO) mice and their wild-type (A(1)WT) controls were employed in this investigation. The heart and aortic arch were carefully removed and retroinfused with enzyme solution (1 mg/ml collagenase type I, 0.5 mg/ml soybean trypsin inhibitor, 3% BSA, and 2% antibiotics) through the aortic arch. The efflux was collected at 30-, 60-, and 90-min intervals. The cells were centrifuged, and the pellets were mixed with medium [medium 199-F-12 medium with 10% FBS and 2% antibiotics (for endothelial cells) and advanced DMEM with 10% FBS, 10% mouse serum, 2% GlutaMax, and 2% antibiotics (for smooth muscle cells)] and plated. Endothelial cells were characterized by a cobblestone appearance and positive staining with acetylated LDL labeled with 1,1'-dioctadecyl-3,3,3',3-tetramethylindocarbocyanine perchlorate. Smooth muscle cells were characterized by positive staining of smooth muscle alpha-actin and smooth muscle myosin heavy chain. Homogeneity of the smooth muscle cells was approximately 91%. Western blot analysis showed expression of smoothelin in the cells from passages 3, 7, and 11 in A(1)WT and A(1)KO mice. Furthermore, the A(1)AR was characterized by Western blot analysis using an A(1)AR-specific antibody. To our knowledge, this is the first isolation and successful characterization of smooth muscle cells from the mouse coronary system.     

Aberrant expression of Fas ligand in mice deficient for the MHC class II transactivator

The MHC class II transactivator (CIITA) is a critical regulator of MHC class II genes and other genes involved in the Ag presentation pathway. CIITA-deficient mice lack MHC class II expression on almost all APCs. In this study, we show that these mice also have aberrant Fas ligand expression on both CD4 T cells and B cells. We found that Fas ligand expression was greatly increased on CIITA-deficient CD4 T cells during the Th1 differentiation process. However, both CIITA-deficient and control Th1 effector cells up-regulated Fas ligand to similar levels if cells were reactivated. The introduction of CIITA into primary CD4 T cells via retroviral infection resulted in a reduction in the level of Fas ligand and delay in apoptosis after activation. Interestingly, activated B cells from the CIITA-deficient mice also showed increased levels of Fas ligand that could be to some degree inhibited by the introduction of IL-4.     

Signal transduction mechanism via adenosine A1 receptor in the cat esophageal smooth muscle cells

We investigated what adenosine receptor type exists and the signaling pathways on the contraction of circular muscle cells isolated by enzymatic digestion from the cat esophagus. Adenosine or the selective A1 receptor agonist R-PIA causes a concentration-dependent contraction. After pretreatment with A1 receptor antagonist, DPCPX, adenosine-mediated contraction was abolished. Adenosine-induced contraction was significantly increased when A1 receptors were preserved by pretreatment with DPCPX followed by inactivation of all unprotected receptors with N-ethylmaleimide. Adenosine- or R-PIA-induced contraction was significantly augmented in the preserved cells and the increase was abolished in the presence of the A1 receptor antagonist DPCPX. PTX abolished contraction induced by adenosine or R-PIA, implying that contraction activated by A1 receptor was coupled to a pertussis toxin (PTX)-sensitive G(i) protein. After permeabilization, contraction was inhibited by G(i2), but not by G(i1) and G(i3), antibodies. These data suggest that adenosine-induced contraction of esophagus depends on PTX-sensitive G(i2.) Adenosine- or R-PIA-induced contraction of esophageal smooth muscle cells was not affected by the phospholipase D (PLD) inhibitor rho-chloromercuribenzoic acid (rhoCMB), phospholipase A(2) (PLA(2)) inhibitor DEDA or PKC antagonist chelerythrine, but was significantly abolished by phospholipase C (PLC) inhibitor, neomycin. PLC-beta3 antibody inhibited R-PIA-induced contraction. R-PIA-induced contraction of esophageal muscle cells was inhibited by IP(3) receptor antagonist heparin, which suggests that the contraction of esophageal smooth muscle cells is dependent on phosphatidylinositol-specific phospholipase (PI-PLC) and IP(3). In conclusion, adenosine- and R-PIA-induced contraction in cat esophageal smooth muscle cell was mediated by A1 receptor. A1 receptor is coupled to PTX-sensitive G protein G(i2), which results in the activation of PI-PLC-beta3. PI hydrolysis by PI-PLC forms IP(3), which binds to IP(3) receptor on endoplasmic reticulum, resulting in the release of intracellular Ca(2+).     

Major histocompatibility complex class II A gene polymorphism in the striped bass

Adaptions of the polymerase chain reaction were used to isolate cDNA sequences encoding the Major histocompatibility complex(Mhc) class II A gene(s) of the striped bass (Morone saxatilis). Four complete Mhc class II A genes were cloned and sequenced from a specimen originating in the Roanoke River, North Carolina, and another three A genes from a specimen originating from the Santee-Cooper Reservoir, South Carolina, identifying a total of seven unique sequences. The sequence suggests the presence of at least two Mhc class II A loci. The extensive sequence variability observed between the seven different Mhc class II clones was concentrated in the 1 encoding domain. The encoded 2, transmembrane, and cytoplasmic regions of all seven striped genes correlated well with those of known vertebrate Mhc class II proteins. Overall, the striped bass sequences showed greatest similarity to the Mhc class II A genes of the zebrafish. Southern blot analysis demonstrated extensive polymorphism in the Mhc class II A genes in members of a Roanoke river-caught population of striped bass versus a lesser degree of polymorphism in an aquacultured Santee-Cooper population of striped bass.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers (Mosa-A-S5) L35062, (Mosa-A-S8) L35066, (Mosa-A-R7) L35067, and (Mosa-A-S7) L35072 L35066, (Mossa-A-R7) L35067, and (Mosa-A-S7) L35072     

Selective down-regulation of angiotensin II receptor type 1A signaling by protein tyrosine phosphatase SHP-2 in vascular smooth muscle cells

The heptahelical AT(1) G-protein-coupled receptor lacks inherent tyrosine kinase activity. Angiotensin II binding to AT(1) nevertheless activates several tyrosine kinases and stimulates both tyrosine phosphorylation and phosphatase activity of the SHP-2 tyrosine phosphatase in vascular smooth muscle cells. Since a balance between tyrosine kinase and tyrosine phosphatase activities is essential in angiotensin II signaling, we investigated the role of SHP-2 in modulating tyrosine kinase signaling pathways by stably transfecting vascular smooth muscle cells with expression vectors encoding wild-type SHP-2 protein or a catalytically inactive SHP-2 mutant. Our data indicate that SHP-2 is an efficient negative regulator of angiotensin II signaling. SHP-2 inhibited c-Src catalytic activity by dephosphorylating a positive regulatory tyrosine 418 within the Src kinase domain. Importantly, SHP-2 expression also abrogated angiotensin II-induced activation of ERK, whereas expression of catalytically inactive SHP-2 caused sustained ERK activation. Thus, SHP-2 likely regulates angiotensin II-induced MAP kinase signaling by inactivating c-Src. These SHP-2 effects were specific for a subset of angiotensin II signaling pathways, since SHP-2 overexpression failed to influence Jak2 tyrosine phosphorylation or Fyn catalytic activity. These data show SHP-2 represents a critical negative regulator of angiotensin II signaling, and further demonstrate a new function for this phosphatase in vascular smooth muscle cells.     

Major histocompatibility complex class II expression and hemagglutinin subtype influence the infectivity of type A influenza virus for respiratory dendritic cells

Dendritic cells (DC) play a key role in antiviral immunity, functioning both as innate effector cells in early phases of the immune response and subsequently as antigen-presenting cells that activate the adaptive immune response. In the murine respiratory tract, there are several respiratory dendritic cell (RDC) subsets, including CD103(+) DC, CD11b(hi) DC, monocyte/macrophage DC, and plasmacytoid DC. However, little is known about the interaction between these tissue-resident RDC and viruses that are encountered during natural infection in the respiratory tract. Here, we show both in vitro and in vivo that the susceptibility of murine RDC to infection with type A influenza virus varies with the level of MHC class II expression by RDC and with the virus strain. Both CD103(+) and CD11b(hi) RDC, which express the highest basal level of major histocompatibility complex (MHC) class II, are highly susceptible to infection by type A influenza virus. However, efficient infection is restricted to type A influenza virus strains of the H2N2 subtype. Furthermore, enhanced infectivity by viruses of the H2N2 subtype is linked to expression of the I-E MHC class II locus product. These results suggest a potential novel role for MHC class II molecules in influenza virus infection and pathogenesis in the respiratory tract.     

Ca2+-sensing transgenic mice: postsynaptic signaling in smooth muscle

Genetically encoded signaling proteins provide remarkable opportunities to design and target the expression of molecules that can be used to report critical cellular events in vivo, thereby markedly extending the scope and physiological relevance of studies of cell function. Here we report the development of a transgenic mouse expressing such a reporter and its use to examine postsynaptic signaling in smooth muscle. The circularly permutated, Ca2+-sensing molecule G-CaMP (Nakai, J., Ohkura, M., and Imoto, K. (2001) Nat. Biotechnol. 19, 137-141) was expressed in vascular and non-vascular smooth muscle and functioned as a lineage-specific intracellular Ca2+ reporter. Detrusor tissue from these mice was used to identify two separate types of postsynaptic Ca2+ signals, mediated by distinct neurotransmitters. Intrinsic nerve stimulation evoked rapid, whole-cell Ca2+ transients, or "Ca2+ flashes," and slowly propagating Ca2+ waves. We show that Ca2+ flashes occur through P2X receptor stimulation and ryanodine receptor-mediated Ca2+ release, whereas Ca2+ waves arise from muscarinic receptor stimulation and inositol trisphosphate-mediated Ca2+ release. The distinct ionotropic and metabotropic postsynaptic Ca2+ signals are related at the level of Ca2+ release. Importantly, individual myocytes are capable of both postsynaptic responses, and a transition between Ca2+ -induced Ca2+ release and inositol trisphosphate waves occurs at higher synaptic inputs. Ca2+ signaling mice should provide significant advantages in the study of processive biological signaling.     

Molecular cloning and expression of an adenosine A2b receptor from human brain.

A novel receptor cDNA was isolated from a human hippocampal cDNA library. The encoded polypeptide contains structural features consistent with its classification as a G protein-coupled receptor and shares 45% homology with the human A1 and A2a adenosine receptors. Chinese hamster ovary K1 cells expressing this receptor showed marked stimulation of adenylate cyclase when treated with 1mM adenosine. There was no response to ligands selective for A1 and A2a receptors but the general adenosine agonist N-ethylcarboxyamidoadenosine (NECA) caused a 10 fold increase in cyclic AMP accumulation with an EC50 of approximately 0.9 microM. This effect was inhibited by the adenosine receptor antagonist theophylline. Specific binding of A1 and A2a selective agonists and NECA was not detected. It is proposed that the novel receptor is a human brain adenosine A2b receptor subtype.     

Serum cholesterol and expression of ApoAI, LXRβ and SREBP2 in vitamin D receptor knock-out mice

Vitamin D insufficiency has been reported to be associated with increased blood cholesterol concentrations. Here we used two strains of VDR knock-out (VDR-KO) mice to study whether a lack of vitamin D action has any effect on cholesterol metabolism. In 129S1 mice, both in male and female VDR-KO mice serum total cholesterol levels were significantly higher than those in wild type (WT) mice (20.7% (P = 0.05) and 22.2% (P = 0.03), respectively). In addition, the serum high-density lipoprotein-bound cholesterol (HDL-C) level was 22% (P = 0.03), respectively higher in male VDR-KO mice than in WT mice. The mRNA expression levels of five cholesterol metabolism related genes in livers of 129S1 mice were studied using quantitative real-time PCR (QRT-PCR): ATP-binding cassette transporter A1 (ABCA1), regulatory element binding protein (SREBP2), apolipoprotein A-I (ApoAI), low-density lipoprotein receptor (LDLR) and liver X receptor beta (LXRβ). In the mutant male mice, the mRNA level of ApoAI and LXRβ were 49.2% (P = 0.005) and 38.8% (P = 0.034) higher than in the WT mice. These changes were not observed in mutant female mice, but the female mutant mice showed 52.5% (P = 0.006) decrease of SREBP2 mRNA expression compared to WT mice. Because the mutant mice were fed with a special rescue diet, we wanted to test whether the increased cholesterol levels in mutant mice were due to the diet. Both the WT and mutant NMRI mice were given the same diet for 3 weeks before the blood sampling. No difference in cholesterol or in HDL-C between WT and mutant mice was found. The results suggest that the food, gender and genetic background have an effect on the cholesterol metabolism. Although VDR seems to regulate some of the genes involved in cholesterol metabolism, its role in the regulation of serum cholesterol seems to be minimal.