NADPH oxidase pathway is involved in aortic contraction induced by A3 adenosine receptor in mice.

The NADPH oxidase (Nox) subunits 1, 2 (gp91 phox), and 4 are the major sources for reactive oxygen species (ROS) in vascular tissues. In conditions such as ischemia-reperfusion and hypoxia, both ROS and adenosine are released, suggesting a possible interaction. Our aim in this study was to examine the A(3) adenosine receptor (A(3)AR)-induced vascular effects and its relation to ROS and Nox1, 2, and 4 using aortic tissues from wild-type (WT) and A(3)AR knockout (A(3)KO) mice. The selective A(3)AR agonist 2-chloro-N(6)-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (Cl-IBMECA) (10(-10)-10(-5) M) induced contraction of the aorta from WT but not from A(3)KO mice, and this contraction was inhibited by the Nox inhibitor apocynin (10(-5) M) and the ROS scavengers superoxide dismutase-polyethylene glycol and catalase-polyethylene glycol (100 U/ml each). Cl-IBMECA-induced contraction was not affected by the mast cell degranulator compound 48/80 (100 µg/ml) or the stabilizer cromolyn sodium (10(-4) M). In addition, Cl-IBMECA (10(-7) M) increased intracellular ROS generation by 35 ± 14% in WT but not in A(3)KO aorta, and this increase was inhibited by apocynin (10(-5) M), diphenyleneiodonium chloride (10(-5) M), and the A(3)AR antagonist 3-propyl-6-ethyl-5-[(ethylthio)carbonyl]-2 phenyl-4-propyl-3-pyridine carboxylate (MRS1523) (10(-5) M). Furthermore, Cl-IBMECA selectively increased the protein expression of the Nox2 subunit by 150 ± 15% in WT but not in A(3)KO mice without affecting either Nox1 or 4, and this increase was inhibited by apocynin. The mRNA of Nox2 was unchanged by Cl-IBMECA in either WT or A(3)KO aortas. In conclusion, A(3)AR enhances ROS generation, possibly through activation of Nox2, with subsequent contraction of the mouse aorta.

A(1) adenosine receptor-mediated PKC and p42/p44 MAPK signaling in mouse coronary artery smooth muscle cells.

The A(1) adenosine receptor (A(1)AR) is coupled to G(i)/G(o) proteins, but the downstream signaling pathways in smooth muscle cells are unclear. This study was performed in coronary artery smooth muscle cells (CASMCs) isolated from the mouse heart [A(1)AR wild type (A(1)WT) and A(1)AR knockout (A(1)KO)] to delineate A(1)AR signaling through the PKC pathway. In A(1)WT cells, treatment with (2S)-N(6)-(2-endo-norbornyl)adenosine (ENBA; 10(-5)M) increased A(1)AR expression by 150%, which was inhibited significantly by the A(1)AR antagonist 1,3-dipropyl-8-cyclopentylxanthine (10(-6)M), but not in A(1)KO CASMCs. PKC isoforms were identified by Western blot analysis in the cytosolic and membrane fractions of cell homogenates of CASMCs. In A(1)WT and A(1)KO cells, significant levels of basal PKC-alpha were detected in the cytosolic fraction. Treatment with the A(1)AR agonist ENBA (10(-5)M) translocated PKC-alpha from the cytosolic to membrane fraction significantly in A(1)WT but not A(1)KO cells. Phospholipase C isoforms (betaI, betaIII, and gamma(1)) were analyzed using specific antibodies where ENBA treatment led to the increased expression of PLC-betaIII in A(1)WT CASMCs while having no effect in A(1)KO CASMCs. In A(1)WT cells, ENBA increased PKC-alpha expression and p42/p44 MAPK (ERK1/2) phospohorylation by 135% and 145%, respectively. These effects of ENBA were blocked by Gö-6976 (PKC-alpha inhibitor) and PD-98059 (p42/p44 MAPK inhibitor). We conclude that A(1)AR stimulation by ENBA activates the PKC-alpha signaling pathway, leading to p42/p44 MAPK phosphorylation in CASMCs.

Involvement of Ca2+ channels in endothelin-1-induced MAP kinase phosphorylation, myosin light chain phosphorylation and contraction in rabbit iris sphincter smooth muscle.

The purpose of the present study was to investigate the role and type of Ca2+ channels involved in the stimulatory effects of endothelin-1 (ET-1) on the Ca2+-dependent functional responses, p42/p44 MAP kinase phosphorylation, 20-kDa myosin light chain (MLC) phosphorylation and contraction, in rabbit iris sphincter, a nonvascular smooth muscle. ET-1 induced inositol phosphates production, MAP kinase phosphorylation, MLC phosphorylation (MLC20-P plus MLC20-2P) and contraction in a concentration-dependent manner with EC50 values of 71, 8, 6 and 25 nM, respectively. ET-1-induced MAP kinase phosphorylation, MLC phosphorylation and contraction were not significantly affected by nifedipine (1-60 microM), an L-type Ca2+ channel blocker, or by LOE 908 (1-100 microM), a blocker of Ca2+-permeable nonselective cation channels. However, SKF96365, a receptor-operated Ca2+ channel (ROCC) blocker, inhibited MAP kinase phosphorylation, MLC phosphorylation and contraction in a concentration-dependent manner with IC50 values of 28, 30 and 42 microM, respectively. 2-APB, a store-operated Ca2+ channel (SOCC) blocker, inhibited ET-1-induced MLC phosphorylation and contraction in a concentration-dependent manner with IC50 values of 12.7 and 19 microM, respectively, but was without effect on MAP kinase phosphorylation. The combined effects of submaximal concentrations of SKF96365 and 2-APB on ET-1-induced MLC phosphorylation and contraction were not additive, implying that their inhibitory actions could be mediated through a common Ca2+ entry channel. PD98059, a MAP kinase inhibitor, had no effect on ET-1-induced MLC phosphorylation and contraction, suggesting that these ET-1 effects in the rabbit iris muscle are MAP kinase-independent. In conclusion, the present study demonstrated for the first time that in rabbit iris sphincter (a) ET-1, through the ETA receptor, stimulates MAP kinase phosphorylation, MLC phosphorylation and contraction in a concentration-dependent manner, (b) that these Ca2+-dependent functional responses are not significantly affected by nifedipine or LOE908, and (c) that ET-1-induced MLC phosphorylation and contraction are inhibited by SKF96365 and 2-APB, suggesting that these effects are mainly due to store- and/or receptor Ca2+ entry.

Effects of prostaglandin F2alpha and latanoprost on phosphoinositide turnover, myosin light chain phosphorylation and contraction in cat iris sphincter.

The effects of the ocular hypotensive agents prostaglandin F(2alpha) (PGF(2alpha)) and its analog latanoprost on intraocular pressure (IOP) in both animals and human have been investigated extensively in the last two decades. While there is general agreement that application of these PGs to the eye alters IOP by altering the aqueous humor outflow of the eye via the uveoscleral and trabecular meshwork pathways, the mechanism of action of these agents on IOP lowering remains unclear. There is evidence which suggests that myosin light kinase (MLC kinase) may be involved in the IOP-lowering effects of these agents. Thus, the purpose of the present work was to investigate in cat iris sphincter the effects of these PGs on the MLC kinase signaling pathway, inositol phosphates production, MLC phosphorylation and contraction, in order to gain more information about the mechanism through which these agents modulate smooth muscle function and lower IOP. [(3)H]myo-inositol phosphates production was measured by ion-exchange chromatography, MLC kinase activity was measured by incorporation of (32)Pi into MLC, and changes in muscle tension were recorded isometrically. PGF(2alpha) and latanoprost induced contraction in a concentration-dependent manner with EC(50) values of 18.6 and 29.9 nM, respectively, and increased inositol phosphates production in a concentration-dependent manner. At 1 microM, PGF(2alpha) and latanoprost increased inositol phosphates formation by 125 and 102% over basal, respectively. PGF(2alpha) and latanoprost increased MLC phosphorylation in a concentration- and time-dependent manner, at 1 microM and 5 min incubation, the PGs increased the MLC response by 181 and 176% over basal, respectively. In general, PGF(2alpha) was slightly more potent in inducing the biochemical and pharmacological responses. Wortmannin, ML-7 and ML-9, selective inhibitors of MLC kinase, inhibited significantly PGF(2alpha)- and latanoprost-induced MLC phosphorylation and contraction. These results demonstrate for the first time involvement of the MLC kinase pathway in the FP receptor function of this ocular tissue. Contraction-relaxation of smooth muscle alters the shape and stiffness of smooth muscle cells and MLC kinase, through myosin phosphorylation and dephosphorylation, has been shown to be involved in cytoskeletal remodeling, cytoskeletal alterations, and IOP lowering. In light of these reports and the findings presented here we suggest that alterations in the MLC kinase signaling pathway and its derived second messengers, which leads to changes in contraction-relaxation of the smooth muscles of the anterior segment, could facilitate aqueous humor outflow and thus contribute to the IOP-lowering effects of the FP-class PGs.

Human ciliary muscle cell responses to FP-class prostaglandin analogs: phosphoinositide hydrolysis, intracellular Ca2+ mobilization and MAP kinase activation.

Phospholipase C induced phosphoinositide (PI) turnover, intracellular Ca(2+) ([Ca(2+)](i)) mobilization and mitogen-activated protein (MAP) kinase activation by FP-class prostaglandin analogs was studied in normal human ciliary muscle (h-CM) cells. Agonist potencies obtained in the PI turnover assays were: travoprost acid ((+)-fluprostenol; EC(50) = 2.6 +/- 0.8 nM) > bimatoprost acid (EC(50) = 3.6 +/- 1.2 nM) > (+/-)-fluprostenol (EC(50) = 4.3 +/- 1.3 nM) >> prostaglandin F(2 alpha) (PGF(2 alpha)) (EC(50) = 134 +/- 17 nM) > latanoprost acid (EC(50) = 198 +/- 83 nM) > S-1033 (EC(50) = 2930 +/- 1420 nM) > unoprostone (EC(50) = 5590 +/- 1490 nM) > bimatoprost (EC(50) = 9600 +/- 1100 nM). Agonist potencies in h-CM cells correlated well with those previously obtained for the cloned human ciliary body-derived FP receptor (r = 0.96, p< 0.001) and that present on h-TM cells (r = 0.94, p< 0.0001). Travoprost acid, PGF(2 alpha) and unoprostone also stimulated [Ca(2+)](i) mobilization in h-CM cells with travoprost acid being the most potent agonist. MAP kinase activity was stimulated in the h-CM cells with the following rank order of activity (at 100 nM): travoprost acid > PGF(2 alpha) > latanoprost acid > PGD(2) > bimatoprost > latanoprost = bimatoprost acid = fluprostenol > PGE(2) = S-1033 > unoprostone > PGI(2). The PI turnover, [Ca(2+)](i) mobilization and MAP kinase activation induced by several of these agonists was blocked by the FP receptor antagonist, AL-8810 (11 beta-fluoro-15-epiindanyl PGF(2 alpha)) (e.g. K(i) = 5.7 microM versus PI turnover). These studies have characterized the biochemical and pharmacological properties of the native FP prostaglandin receptor present on h-CM cells using three signal transduction mechanism assays and a broad panel of FP-class agonist analogs (including free acids of bimatoprost, travoprost and latanoprost) and the FP receptor antagonist, AL-8810.

A(3) adenosine receptor: a plausible therapeutic target for cardio-protection in diabetes.

Diabetes mellitus categorized as type I and II, is a disease of pancreatic insulin, affecting blood glucose level in the body. Recent evidence suggests that cardiac diseases such as hypertension, coronary artery disease, congestive heart failure, and diabetic cardiomyopathy are associated with diabetes and hyperglycemia. The adenosine receptors (AR) have been reported to play an important role in the regulation of these diseases. Four adenosine receptors have been cloned and characterized from several different mammalian species. The receptors are named adenosine A(1), A(2A), A(2B), and A(3). The A(2A) and A(2B) receptors preferably interact with members of the Gs family of G proteins and the A(1) and A(3) receptors with Gi/o proteins. The ubiquitous levels of adenosine are found in each cell in normal conditions but in disease conditions its level has been shown to increase and activate G-protein mediated signaling pathway leading to artery constriction in cardiovascular diseases and diabetes. Various studies have demonstrated that A(3)AR is a potent cardioprotectant during myocardial ischemeia/ischemic reperfusion. Role of A(3)AR receptor as a possible cardioprotectant in diabetes is under investigation and studies have verified the involvement of cyclooxygenases (COXs) and NADPH oxidase pathways. This review summarizes the possible role of A(3)AR in cardiovascular disease and discusses advancement in the development of therapeutic agents targeting cardioprotection with discussion on recent patents on A(3) agonists that are being utilized in the clinical setting. We anticipate that detailed pharmacological studies of adenosine A(3) receptors could help in understanding the link between cardiovascular disease and diabetes and this can be utilized to develop newer therapies that selectively target A(3) receptor to overcome cardiac challenges.

P1 (adenosine) purinoceptor assays.

P1 purinoceptors, or adenosine (ADO) receptors, mediate the biological effects of the endogenous nucleoside, ADO and its analogs. ADO works through four receptor subtypes: A(1), A(2A), A(2B), and A(3). Isolated tissue assays used for the pharmacological characterization of ADO receptors based on functional responses are described in this unit. The guinea pig atrium, pig coronary artery, guinea pig aorta ,and mouse aorta have been used for the characterization of ADO receptor subtypes.

Role of CYP epoxygenases in A2A AR-mediated relaxation using A2A AR-null and wild-type mice.

We hypothesized that A2A adenosine receptor (A2A AR) activation causes vasorelaxation through cytochrome P-450 (CYP) epoxygenases and endothelium-derived hyperpolarizing factors, whereas lack of A2A AR activation promotes vasoconstriction through Cyp4a in the mouse aorta. Adenosine 5'-N-ethylcarboxamide (NECA; 10(-6) M), an adenosine analog, caused relaxation in wild-type A2A AR (A2A AR+/+; +33.99 +/- 4.70%, P < 0.05) versus contraction in A2A AR knockout (A2A AR(-/-); -27.52 +/- 4.11%) mouse aortae. An A2A AR-specific antagonist (SCH-58261; 1 microM) changed the NECA (10(-6) M) relaxation response to contraction (-35.82 +/- 4.69%, P < 0.05) in A2A AR+/+ aortae, whereas no effect was noted in A2A AR(-/-) aortae. Significant contraction was seen in the absence of the endothelium in A2A AR+/+ (-2.58 +/- 2.25%) aortae compared with endothelium-intact aortae. An endothelial nitric oxide synthase inhibitor (N-nitro-L-arginine methyl ester; 100 microM) and a cyclooxygenase inhibitor (indomethacin; 10 microM) failed to block NECA-induced relaxation in A2A AR+/+ aortae. A selective inhibitor of CYP epoxygenases (methylsulfonyl-propargyloxyphenylhexanamide; 10 microM) changed NECA-mediated relaxation (-22.74 +/- 5.11% at 10(-6) M) and CGS-21680-mediated relaxation (-18.54 +/- 6.06% at 10(-6) M) to contraction in A2A AR+/+ aortae, whereas no response was noted in A2A AR(-/-) aortae. Furthermore, an epoxyeicosatrienoic acid (EET) antagonist [14,15-epoxyeicosa-5(Z)-enoic acid; 10 microM] was able to block NECA-induced relaxation in A2A AR+/+ aortae, whereas omega-hydroxylase inhibitors (10 microM dibromo-dodecenyl-methylsulfimide and 10 microM HET-0016) changed contraction into relaxation in A2A AR(-/-) aorta. Cyp2c29 protein was upregulated in A2A AR+/+ aortae, whereas Cyp4a was upregulated in A2A AR(-/-) aortae. Higher levels of dihydroxyeicosatrienoic acids (DHETs; 14,15-DHET, 11,12-DHET, and 8,9-DHET, P < 0.05) were found in A2A AR+/+ versus A2A AR(-/-) aortae. EET levels were not significantly different between A2A AR+/+ and A2A AR(-/-) aortae. It is concluded that CYP epoxygenases play an important role in A2A AR-mediated relaxation, and the deletion of the A2A AR leads to contraction through Cyp4a.

Evidence for the involvement of nitric oxide in A2B receptor-mediated vasorelaxation of mouse aorta.

We have investigated the role of adenosine and its analogs on vasorelaxation of mouse aorta in intact endothelium with rank order of potency as follows: 5'-N-ethylcarboxamidoadenosine (NECA) > 2-chloroadenosine > adenosine >> CGS-21680, which is consistent with the profile of A(2B)-adenosine receptor (A(2B)AR). In endothelium-intact tissues, acetylcholine produced relaxation ranging from 65 to 80% in phenylephrine (PE, 10(-7) M)-precontracted mouse aorta, whereas no relaxation was observed in endothelium-denuded tissues. The A(2B)AR antagonist alloxazine (10(-5) M) shifted concentration-response curve for NECA (EC(50) = 0.005 x 10(-5) M) to the right with an EC(50) of 2.8 x 10(-5) M, demonstrating that this relaxation is partially dependent on functional endothelium mediated predominantly via A(2B)AR in this tissue. This conclusion was further supported by the following findings: 1) in the endothelium-intact mouse aorta, the EC(50) values for NECA and adenosine were found to be 0.05 and 1.99 x 10(-4) M, respectively; however, in denuded endothelium, these values were 0.098 and 3.55 x 10(-4) M, respectively; 2) NECA-induced relaxation was significantly blocked by N(G)-nitro-l-arginine methyl ester (l-NAME; 10(-4) M) in endothelium-intact tissues, which was reversed by pretreatment with l-arginine (10(-4) M), whereas no significant inhibition was found in endothelium-denuded tissues; 3) total nitrites and nitrates (NOx) in intact endothelium with l-NAME (10(-4) M) alone and in combination with l-arginine were 59% (P < 0.05) and 96%, respectively, in comparison with control (PE + NECA); and 4) endothelial nitric oxide synthase gene expression was found to be 67% (P < 0.05) less in endothelium-denuded as opposed to endothelium-intact mouse aorta. Thus these data demonstrate that adenosine-mediated vasorelaxation is partially dependent on A(2B)AR in mouse aorta.

Enhanced airway reactivity and inflammation in A2A adenosine receptor-deficient allergic mice.

A(2A) adenosine receptor (A(2A)AR) has potent anti-inflammatory properties, which may be important in the regulation of airway reactivity and inflammation. Inflammatory cells that possess A(2A)AR also produce nitrosative stress, which is associated with pathophysiology of asthma, so we hypothesized that A(2A)AR deficiency may lead to increased airway reactivity and inflammation through nitrosative stress. Thus the present study was carried out to investigate the role of A(2A)AR on airway reactivity, inflammation, NF-kappaB signaling, and nitrosative stress in A(2A)AR knockout (KO) and wild-type (WT) mice using our murine model of asthma. Animals were sensitized intraperitoneally on days 1 and 6 with 200 microg of ragweed, followed by aerosolized challenges with 0.5% ragweed on days 11, 12, and 13, twice a day. On day 14, airway reactivity to methacholine was assessed as enhanced pause (Penh) using whole body plethysmography followed by bronchoalveolar lavage (BAL) and lung collection for various analyses. Allergen challenge caused a significant decrease in expression of A(2A)AR in A(2A) WT sensitized mice, with A(2A)AR expression being undetected in A(2A) KO sensitized group leading to decreased lung cAMP levels in both groups. A(2A)AR deletion/downregulation led to an increase in Penh to methacholine and influx of total cells, eosinophils, lymphocytes, and neutrophils in BAL with highest values in A(2A) KO sensitized group. A(2A) KO sensitized group further had increased NF-kappaB expression and nitrosative stress compared with WT sensitized group. These data suggest that A(2A)AR deficiency leads to airway inflammation and airway hyperresponsiveness, possibly via involvement of nitrosative stress in this model of asthma.

Involvement of COX-1 in A3 adenosine receptor-mediated contraction through endothelium in mice aorta.

We investigated whether A(3) adenosine receptor (A(3)AR) is involved in endothelium-mediated contraction through cyclooxygenases (COXs) with the use of wild-type (WT) and A(3) knockout (A(3)KO) mice aorta. A(3)AR-selective agonist, Cl-IBMECA, produced a concentration-dependent contraction (EC(50): 2.9 +/- 0.2 x 10(-9) M) in WT mouse aorta with intact endothelium (+E) and negligible effects in A(3)KO +E aorta. At 10(-7) M, contractions produced by Cl-IBMECA were 29% in WT +E, while being insignificant in A(3)KO +E aorta. Cl-IBMECA-induced responses were abolished in endothelium-denuded tissues (-E), in both WT and A(3)KO aorta. A(3)AR gene and protein expression were reduced by 74 and 72% (P < 0.05), respectively, in WT -E compared with WT +E aorta, while being undetected in A(3)KO +E/-E aorta. Indomethacin (nonspecific COXs blocker, 10(-5) M), SC-560 (specific COX-1 blocker, 10(-8) M), SQ 29549 (thromboxane prostanoid receptor antagonist, 10(-6) M), and furegrelate (thromboxane synthase inhibitor, 10(-5) M) inhibited Cl-IBMECA-induced contraction significantly. Cl-IBMECA-induced thromboxane B(2) production was also attenuated significantly by indomethacin, SC-560, and furegrelate in WT +E aorta, while having negligible effects in A(3)KO +E aorta. NS-398 (specific COX-2 blocker) produced negligible inhibition of Cl-IBMECA-induced contraction in both WT +E and A(3)KO +E aorta. Cl-IBMECA-induced increase in COX-1 and thromboxane prostanoid receptor expression were significantly inhibited by MRS1523, a specific A(3)AR antagonist in WT +E aorta. Expression of both A(3)AR and COX-1 was located mostly on endothelium of WT and A(3)KO +E aorta. These results demonstrate for the first time the involvement of COX-1 pathway in A(3)AR-mediated contraction via endothelium.

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.

Involvement of p38-mitogen-activated protein kinase in adenosine receptor-mediated relaxation of coronary artery.

The purpose of this study was to explore the involvement of adenosine receptor(s) in porcine coronary artery (PCA) relaxation and to define the role of MAPK signaling pathways. Isometric tensions were recorded in denuded PCA rings. 5'-(N-ethylcarboxamido)adenosine (NECA), a nonselective adenosine receptor agonist, induced a concentration-dependent relaxation (EC(50) = 16.8 nM) of PGF(2alpha) (10 microM)-preconstricted arterial rings. NECA-induced relaxation was completely blocked by 0.1 microM SCH-58261 (A(2A) antagonist) at lower doses (1-40 nM) but not at higher doses (80-1,000 nM). MRS-1706 (1 microM, A(2B) antagonist) was able to shift the NECA concentration-response curve to the right. CGS-21680 (selective A(2A) agonist) induced responses similarly to NECA, whereas N(6)-cyclopentyladenosine (A(1) agonist) and Cl-IB-MECA (A(3) agonist) did not. Furthermore, the effect of NECA was attenuated by the addition of SB-203580 (10 microM, p38 MAPK inhibitor) but not by PD-98059 (10 microM, MEK inhibitor). Interestingly, SB-203580 had no effect on CGS-21680-induced relaxation. Western blot analysis demonstrated that PGF(2alpha) and adenosine agonists stimulated p38 MAPK at a concentration of 40 nM in PCA smooth muscle cells. MRS-1706 (1 microM) significantly reduced NECA-induced p38 MAPK phosphorylation. Addition of NECA and SB-203580 alone or in combination inhibited PGF(2alpha)-induced p38 MAPK. Western blot data were further confirmed by p38 MAPK activity measurement using activating transcription factor-2 assay. Our results suggest that the adenosine receptor subtype involved in causing relaxation of porcine coronary smooth muscle is mainly A(2A) subtype, although A(2B) also may play a role, possibly through p38 MAPK pathway.

Effects of prostaglandin F2alpha, latanoprost and carbachol on phosphoinositide turnover, MAP kinases, myosin light chain phosphorylation and contraction and functional existence and expression of FP receptors in bovine iris sphincter.

A potential role for myosin light chain kinase (MLCK) in regulating intraocular pressure and outflow function has recently been reported in living monkey eye and rabbit eye. There is little information about the effects of the ocular hypotensive agents, prostaglandin F2alpha (PGF2alpha) and latanoprost on this signaling pathway in ocular tissues. The aim of this study was to determine the agonist activity of PGF2alpha, latanoprost and carbachol (CCh) on the MLCK pathway in isolated bovine iris sphincter and furthermore to investigate the existence of the FP receptor in this tissue. In the present studies on the MLCK pathway four signal transduction mechanism assays were employed, phosphoinositide (PI) turnover, p42/p44 MAP kinase phosphorylation and activation, MLC phosphorylation and contraction. In the studies on the existence of the FP receptor in the bovine iris sphincter, the pharmacology and expression of the FP receptor protein, using a polyclonal anti-FP-receptor antibody and Western blot analysis, were determined. The data obtained on the MLCK pathway showed that the three agonists stimulated the biochemical and pharmacological responses in a concentration and time-dependent manner and that the order of potency and efficacy is PGF2alpha>latanoprost>CCh. The EC50 values in the PI turnover, MAP kinase phosphorylation, MLC phosphorylation and contraction assays were for PGF2alpha: 9, 42, 200 and 140 nM, respectively, for latanoprost: 13, 59, 250 and 828 nM, respectively, and for CCh: 22, 200, 630 and 910 nM, respectively. Wortmannin, a selective inhibitor of MLCK, dose-dependently inhibited MLC phosphorylation and contraction induced by PGF2alpha, demonstrating a close relationship between activation of the MLCK pathway and contraction. The pharmacological studies showed that in the concentration range of 1 nM to 10 microM, the FP-receptor agonists caused concentration-response curves with the following order of potencies: 17-phenyl trinor PGF2alpha (bimatoprost acid)>PGF2alpha>cloprostenol>latanoprost>latanoprost acid>bimatoprost amide>>fluprostenol. Immunoblot analysis of the FP receptor demonstrated expression of the prostaglandin FP receptor protein in this smooth muscle. These results clearly indicate that the MLCK signaling pathway is involved in the FP-receptor function of the bovine iris sphincter and furthermore demonstrate that functional FP receptors exist and are expressed in this tissue.

Retinol enhances differentiation of the gastric parietal cell lineage in developing rabbits.

In the gastric glands, parietal cells are the targets for anti-ulcer drugs because they contain the proton pump or HK-ATPase responsible for acid secretion. Little is known about factors influencing developmental expression and activity of HK-ATPase. In this study, the parietal cell lineage was investigated in rabbits at post-natal days 0 (P0) to P60 by using morphological and biochemical methods. Immunohistochemical and ultrastructural studies show that the HK-ATPase-expressing cells that appear at P0 and P3 are pre-parietal cells. However, terminally differentiated, mature parietal cells make their appearance at P7. These data correlate with the activity of HK-ATPase, measured as K(+)-dependent hydrolysis of p-nitrophenyl phosphate. Three-day-retinol treatment of P3-P30 rabbits induced an increase in the (i) production of parietal cells, (ii) intensity of the HK-ATPase immunostaining per cell, (iii) activity of HK-ATPase and (iv) amount of HK-ATPase protein measured by Western blotting. In conclusion, retinol upregulates the development of HK-ATPase in rabbits, perhaps due to precocious acceleration of the differentiation program of parietal cell lineage.

A transgenic mouse model of metastatic carcinoma involving transdifferentiation of a gastric epithelial lineage progenitor to a neuroendocrine phenotype.

Human neuroendocrine cancers (NECs) arise in various endoderm-derived epithelia, have diverse morphologic features, exhibit a wide range of growth phenotypes, and generally have obscure cellular origins and ill-defined molecular mediators of initiation and progression. We describe a transgenic mouse model of metastatic gastric cancer initiated by expressing simian virus 40 large tumor antigen (SV40 TAg), under control of regulatory elements from the mouse Atp4b gene, in the progenitors of acid-producing parietal cells. Parietal cells normally do not express endocrine or neural features, and Atp4b-Cre bitransgenic mice with a Cre reporter confirmed that the Atp4b regulatory elements are not active in gastric enteroendocrine cells. GeneChip analyses were performed on laser capture microdissected SV40 TAg-expressing cells in preinvasive foci and invasive tumors. Genes that distinguish invasive from preinvasive cells were then hierarchically clustered with DNA microarray datasets obtained from human lung and gastric cancers. The results, combined with immunohistochemical and electron microscopy studies of Apt4b-SV40 TAg stomachs, revealed that progression to invasion was associated with transdifferentiation of parietal cell progenitors to a neuroendocrine phenotype, and that invasive cells shared molecular features with NECs arising in the human pulmonary epithelium, including transcription factors that normally regulate differentiation of various endocrine lineages and maintain neural progenitors in an undifferentiated state. The 399 mouse genes identified as regulated during acquisition of an invasive phenotype and concomitant neuroendocrine transdifferentiation, plus their human orthologs associated with lung NECs, provide a foundation for molecular classification of NECs arising in other tissues and for genetic tests of the molecular mechanisms underlying NEC pathogenesis.