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Angiotensin II (ANG II) plays a key role in regulating blood pressure and inflammation. Prostaglandin E2 (PGE2) signals through four different G protein-coupled receptors, eliciting a variety of effects. We reported that activation of the EP3 receptor reduces cardiac contractility. More recently, we have shown that overexpression of the EP4 receptor is protective in a mouse myocardial infarction model. We hypothesize in this study that the relative abundance of EP3 and EP4 receptors is a major determinant of end-organ damage in the diseased heart. Thus EP3 is detrimental to cardiac function and promotes inflammation, whereas antagonism of the EP3 receptor is protective in an ANG II hypertension (HTN) model. To test our hypothesis, male 10- to 12-wk-old C57BL/6 mice were anesthetized with isoflurane and osmotic minipumps containing ANG II were implanted subcutaneously for 2 wk. We found that antagonism of the EP3 receptor using L798,106 significantly attenuated the increase in blood pressure with ANG II infusion. Moreover, antagonism of the EP3 receptor prevented a decline in cardiac function after ANG II treatment. We also found that 10- to 12-wk-old EP3-transgenic mice, which overexpress EP3 in the cardiomyocytes, have worsened cardiac function. In conclusion, activation or overexpression of EP3 exacerbates end-organ damage in ANG II HTN. In contrast, antagonism of the EP3 receptor is beneficial and reduces cardiac dysfunction, inflammation, and HTN. [ABSTRACT FROM AUTHOR]

The natural peptide N-Acetyl-Seryl-Aspartyl-Lysyl-Proline (Ac-SDKP) decreases inflammation in chronic diseases such as hypertension and heart failure. However, Ac-SDKP effects on acute inflammatory responses during myocardial infarction (MI) are unknown. During the first 72 hours post-MI, neutrophils, M1 macrophages (pro-inflammatory), and M2 macrophages (pro-resolution) and release of myeloperoxidase (MPO) and matrix metalloproteinases (MMP) are involved in cardiac rupture. We hypothesized that in the acute stage of MI, Ac-SDKP decreases the incidence of cardiac rupture and mortality by preventing immune cell infiltration as well as by decreasing MPO and MMP expression. MI was induced by ligating the left descending coronary artery in C57BL/6 mice. Vehicle or Ac-SDKP (1.6 mg/kg/d) was infused via osmotic minipump. Cardiac immune cell infiltration was assessed by flow cytometry, cardiac MPO and MMP levels were measured at 24–48 hrs post-MI. Cardiac rupture and mortality incidence were determined at 7 days post-MI. In infarcted mice, Ac-SDKP significantly decreased cardiac rupture incidence from 51.0% (26 of 51 animals) to 27.3% (12 of 44) and mortality from 56.9% (29 of 51) to 31.8% (14 of 44). Ac-SDKP reduced M1 macrophages in cardiac tissue after MI, without affecting M2 macrophages and neutrophils. Ac-SDKP decreased MMP-9 activation in infarcted hearts with no changes on MPO expression. Ac-SDKP prevents cardiac rupture and decreases mortality post-acute MI. These protective effects of Ac-SDKP are associated with decreased pro-inflammatory M1 macrophage infiltration and MMP-9 activation. [ABSTRACT FROM AUTHOR]

N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is a naturally occurring tetrapeptide that prevents inflammation and fibrosis in hypertension and other cardiovascular diseases. We previously showed that, in angiotensin II-induced hypertension, Ac-SDKP decreased the activation of nuclear transcription factor NF-κB, whereas, in experimental autoimmune myocarditis and hypertension animal models, it also reduced the expression of endothelial leukocyte adhesion molecule ICAM-1. However, the mechanisms by which Ac-SDKP downregulated ICAM-1 expression are still unclear. TNF-α; is a proinflammatory cytokine that induces ICAM-1 expression in various cell types via TNF receptor 1 and activation of the classical NF-κB pathway. We hypothesized that in endothelial cells Ac-SDKP suppresses TNF-α;-induced ICAM-1 expression by decreasing IKK phosphorylation that as a consequence leads to a decrease of IκB phosphorylation and NF-κB activation. To test this hypothesis, human coronary artery endothelial cells were treated with Ac-SDKP and then stimulated with TNF-α;. We found that TNF-α;- induced ICAM-1 expression was significantly decreased by Ac-SDKP in a dose-dependent manner. Ac-SDKP also decreased TNF-α;-induced NF-κB translocation from cytosol to nucleus, as assessed by electrophoretic mobility shift assay, which correlated with a decrease in IκB phosphorylation. In addition, we found that Ac-SDKP decreased TNF-α;-induced IKK phosphorylation and IKK- expression. However, Ac-SDKP had no effect on TNF-α;-induced phosphorylation of p38 MAP kinase or ERK. Thus we conclude that Ac-SDKP inhibition of TNF-α; activation of canonical, i.e., IKK-β-dependent, NF-κB pathway and subsequent decrease in ICAM-1 expression is achieved via inhibition of IKK-β. [ABSTRACT FROM AUTHOR]

ANG II type 2 receptor (AT2) and ANG I-converting enzyme 2 (ACE2) are important components of the renin-ANG system. Activation of AT2 and ACE2 reportedly counteracts proinflammatory effects of ANG II. However, the possible interaction between AT2 and ACE2 has never been established. We hypothesized that activation of AT2 increases ACE2 activity, thereby preventing TNF-α-stimulated ICAM-1 expression via inhibition of NF-κB signaling. Human coronary artery endothelial cells were pretreated with AT2 antagonist PD123319 (PD) or ACE2 inhibitor DX600 and then stimulated with TNF-α in the presence or absence of AT2 agonist CGP42112 (CGP). We found that AT2 agonist CGP increased both ACE2 protein expression and activity. This effect was blunted by AT2 antagonist PD. ICAM-1 expression was very low in untreated cells but greatly increased by TNF-α. Activation of AT2 with agonist CGP or with ANG II under concomitant AT1 antagonist reduced TNF-α-induced ICAM-1 expression, which was reversed by AT2 antagonist PD or ACE2 inhibitor DX600 or knockdown of ACE2 with small interfering RNA. AT2 activation also suppressed TNF-α-stimulated phosphorylation of inhibitory κB (p-IκB) and NF-κB activity. Inhibition of ACE2 reversed the inhibitory effect of AT2 on TNF-α-stimulated p-IκB and NF-κB activity. Our findings suggest that stimulation of AT2 reduces TNF-α-stimulated ICAM-1 expression, which is partly through ACE2-mediated inhibition of NF-κB signaling. [ABSTRACT FROM AUTHOR]

New Findings What is the central question of this study? What is the cardioprotective role of fractalkine neutralization in heart failure and what are the mechanisms responsible?, What is the main finding and its importance? The concentration of fractalkine is increased in the left ventricle of mice with myocardial infarction, similar to the increases in plasma from heart failure patients. The present study shows a clear beneficial effect of neutralizing fractalkine in a model of myocardial infarction, which results in increased survival. Such an approach may be worthwhile in human patients., Concentrations of the chemokine fractalkine (FKN) are increased in patients with chronic heart failure, and our previous studies show that aged mice lacking the prostaglandin E2 EP4 receptor subtype (EP4-KO) have increased cardiac FKN, with a phenotype of dilated cardiomyopathy. However, how FKN participates in the pathogenesis of heart failure has rarely been studied. We hypothesized that FKN contributes to the pathogenesis of heart failure and that anti-FKN treatment prevents heart failure induced by myocardial infarction (MI) more effectively in EP4-KO mice. Male EP4-KO mice and wild-type littermates underwent sham or MI surgery and were treated with an anti-FKN antibody or control IgG. At 2 weeks post-MI, echocardiography was performed and hearts were excised for determination of infarct size, immunohistochemistry and Western blot of signalling molecules. Given that FKN protein levels in the left ventricle were increased to a similar extent in both strains after MI and that anti-FKN treatment improved survival and cardiac function in both strains, we subsequently used only wild-type mice to examine the mechanisms whereby anti-FKN is cardioprotective. Myocyte cross-sectional area and interstitial collagen fraction were reduced after anti-FKN treatment, as were macrophage migration and gelatinase activity. Activation of ERK1/2 and p38 MAPK were reduced after neutralization of FKN. In vitro, FKN increased fibroblast proliferation. In conclusion, increased FKN contributes to heart failure after MI. This effect is not exacerbated in EP4-KO mice, suggesting that there is no link between FKN and lack of EP4. Overall, inhibition of FKN may be important to preserve cardiac function post-MI. [ABSTRACT FROM AUTHOR]

Sperm-associated antigen 6 (SPAG6) is the mammalian orthologue of Chlamydomonas PF16, an axonemal central pair protein involved in flagellar motility. In mice, two Spag6 genes have been identified. The ancestral gene, on mouse chromosome 2, is named Spag6. A related gene originally called Spag6, localized on mouse chromosome 16, evolved from the ancient Spag6 gene. It has been renamed Spag6-like (Spag6l). Spag6 encodes a 1.6 kb transcript consisting of 11 exons, while Spag6l encodes a 2.4 kb transcript which contains an additional non-coding exon in the 3′-end as well as the 11 exons found in Spag6. The two Spag6 genes share high similarities in their nucleotide and amino acid sequences. Unlike Spag6l mRNA, which is widely expressed, Spag6 mRNA expression is limited to a smaller number of tissues, including the testis and brain. In transfected mammalian cells, SPAG6/GFP is localized on microtubules, a similar localization as SPAG6L. A global Spag6l knockout mouse model was generated previously. In addition to a role in modulating the ciliary beat, SPAG6L has many unexpected functions, including roles in the regulation of ciliogenesis/spermatogenesis, hearing, and the immunological synapse, among others. To investigate the role of the ancient Spag6 gene, we phenotyped global Spag6 knockout mice. All homozygous mutant mice were grossly normal, and fertility was not affected in both males and females. The homozygous males had normal sperm parameters, including sperm number, motility, and morphology. Examination of testis histology revealed normal spermatogenesis. Testicular protein expression levels of selected SPAG6L binding partners, including SPAG16L, were not changed in the Spag6 knockout mice, even though the SPAG16L level was significantly reduced in the Spag6l knockout mice. Structural analysis of the two SPAG6 proteins shows that both adopt very similar folds, with differences in a few amino acids, many of which are solvent-exposed. These differences endow the two proteins with different functional characteristics, even though both have eight armadillo repeats that mediate protein–protein interaction. Our studies suggest that SPAG6 and SPAG6L have different functions in vivo, with the evolved SPAG6L protein being more important. Since the two proteins have some overlapping binding partners, SPAG6 could have functions that are yet to be identified. [ABSTRACT FROM AUTHOR]

Ortiz-Capisano MC, Atchison DK, Harding P, Lasley RD, Beierwaltes WH. Adenosine inhibits renin release from juxtaglomerular cells via an A1 receptor-TRPC-mediated pathway. Am J Physiol Renal Physiol 305: F1209-F1219, 2013. First published July 24, 2013; doi:10.1152/ajprenal.00710.2012.--Renin is synthesized and released from juxtaglomerular (JG) cells. Adenosine inhibits renin release via an adenosine A1 receptor (A1R) calcium-mediated pathway. How this occurs is unknown. In cardiomyocytes, adenosine increases intracellular calcium via transient receptor potential canonical (TRPC) channels. We hypothesized that adenosine inhibits renin release via A1R activation, opening TRPC channels. However, higher concentrations of adenosine may stimulate renin release through A2R activation. Using primary cultures of isolated mouse JG cells, immunolabeling demonstrated renin and A1R in JG cells, but not A2R subtypes, although RT-PCR indicated the presence of mRNA of both A2AR and A2BR. Incubating JG cells with increasing concentrations of adenosine decreased renin release. Different concentrations of the adenosine receptor agonist N-ethylcarboxamide adenosine (NECA) did not change renin. Activating A1R with 0.5 µM N6-cyclohexyladenosine (CHA) decreased basal renin release from 0.22 ± 0.05 to 0.14 ± 0.03 µg of angiotensin I generated per milliliter of sample per hour of incubation (AngI/ml/mg prot) (P < 0.03), and higher concentrations also inhibited renin. Reducing extracellular calcium with EGTA increased renin release (0.35 ± 0.08 µg AngI/ml/mg prot; P 0.01), and blocked renin inhibition by CHA (0.28 ± 0.06 µg AngI/ml/mg prot; P < 0. 005 vs. CHA alone). The intracellular calcium chelator BAPTA-AM increased renin release by 55%, and blocked the inhibitory effect of CHA. Repeating these experiments in JG cells from A1R knockout mice using CHA or NECA demonstrated no effect on renin release. However, RT-PCR showed mRNA from TRPC isoforms 3 and 6 in isolated JG cells. Adding the TRPC blocker SKF-96365 reversed CHA-mediated inhibition of renin release. Thus A1R activation results in a calcium-dependent inhibition of renin release via TRPC-mediated calcium entry, but A2 receptors do not regulate renin release. [ABSTRACT FROM AUTHOR]

Atchison DK, Harding P, Beierwaltes WH. Vitamin D increases plasma renin activity independently of plasma Ca2+ via hypovolemia and adrenergic activity. Am J Physiol Renal Physiol 305: F1109 -F1117, 2013. First published August 7, 2013; doi:10.1152/ajprenal.00010.2013.--1, 25-Dihydroxycholechalciferol (calcitriol) and 19-nor-1, 25-dihydroxyvitamin D2 (paricalcitol) are vitamin D receptor (VDR) agonists. Previous data suggest VDR agonists may actually increase renin-angiotensin activity, and this has always been assumed to be mediated by hypercalcemia. We hypothesized that calcitriol and paricalcitol would increase plasma renin activity (PRA) independently of plasma Ca2+ via hypercalciuria-mediated polyuria, hypovolemia, and subsequent increased adrenergic sympathetic activity. We found that both calcitriol and paricalcitol increased PRA threefold (P < 0.01). Calcitriol caused hypercalcemia, but paricalcitol did not. Both calcitriol and paricalcitol caused hypercalciuria (9- and 7-fold vs. control, P < 0.01) and polyuria (increasing 2.6- and 2.2-fold vs. control, P < 0.01). Paricalcitol increased renal calcium-sensing receptor (CaSR) expression, suggesting a potential cause of paricalcitol- mediated hypercalciuria and polyuria. Volume replacement completely normalized calcitriol-stimulated PRA and lowered plasma epinephrine by 43% (P < 0.05). β-Adrenergic blockade also normalized calcitriol-stimulated PRA. Cyclooxygenase-2 inhibition had no effect on calcitriol-stimulated PRA. Our data demonstrate that vitamin D increases PRA independently of plasma Ca2+ via hypercalciuria, polyuria, hypovolemia, and increased β-adrenergic activity. [ABSTRACT FROM AUTHOR]

Myocardial matrix turnover involves a dynamic balance between collagen synthesis and degradation, which is regulated by matrix metalloproteinases (MMPs). N-acetyl-Ser-Asp-Lys-Pro (Ac-SDKP) is a small peptide that inhibits cardiac inflammation and fibrosis. However, its role in MMP regulation is not known. Thus, we hypothesized that Ac-SDKP promotes MMP activation in cardiac fibroblasts and decreases collagen deposition via this mechanism. To that end, we tested the effects of Ac-SDKP on interleukin-1β (IL-1β; 5 ng/ml)-stimulated adult rat cardiac fibroblasts. We measured total collagenase activity, MMP-2, MMP-9, and MMP-13 expressions, and activity along with their inhibitors, tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2. In order to examine the effects of Ac-SDKP on the signaling pathway that controls MMP transcription, we also measured nuclear factor-κB (NFκB) and p42/44 mitogen-activated protein kinase (MAPK) activation. Ac-SDKP did not alter collagenase or gelatinase activity in cardiac fibroblasts under basal conditions, but blunted the IL-1β-induced increase in total collagenase activity. Similarly, Ac-SDKP normalized the IL-1β-mediated increase in MMP-2 and MMP-9 activities and MMP-13 expression. Inhibition of MMPs by Ac-SDKP was associated with increased TIMP-1 and TIMP-2 expressions. Collagen production was not affected by Ac-SDKP, IL-1β, or a combination of both agents. Ac-SDKP blocked IL-1β-induced p42/44 phosphorylation and NFκB activation in cardiac fibroblasts. We concluded that the Ac-SDKP-inhibited collagenase expression and activation was associated with increased expression of TIMP-1 and TIMP-2. These pharmacological effects of Ac-SDKP may be linked to the inhibition of MAPK and NFκB pathway. [ABSTRACT FROM AUTHOR]

Background: Our laboratory reported that male mice with cardiomyocyte-selective knockout of the prostaglandin E2 EP4 receptor sub-type (EP4 KO) exhibit reduced cardiac function. Gene array on left ventricles (LV) showed increased fractalkine, a chemokine implicated in heart failure. We therefore hypothesized that fractalkine is regulated by PGE2 and contributes to depressed contractility via alterations in intracellular calcium. Methods: Fractalkine was measured in LV of 28–32 week old male EP4 KO and wild type controls (WT) by ELISA and the effect of PGE2 on fractalkine secretion was measured in cultured neonatal cardiomyocytes and fibroblasts. The effect of fractalkine on contractility and intracellular calcium was determined in Fura-2 AM-loaded, electrical field-paced cardiomyocytes. Cardiomyocytes (AVM) from male C57Bl/6 mice were treated with fractalkine and responses measured under basal conditions and after isoproterenol (Iso) stimulation. Results: LV fractalkine was increased in EP4 KO mice but surprisingly, PGE2 regulated fractalkine secretion only in fibroblasts. Fractalkine treatment of AVM decreased both the speed of contraction and relaxation under basal conditions and after Iso stimulation. Despite reducing contractility after Iso stimulation, fractalkine increased the Ca2+ transient amplitude but decreased phosphorylation of cardiac troponin I, suggesting direct effects on the contractile machinery. Conclusions: Fractalkine depresses myocyte contractility by mechanisms downstream of intracellular calcium. [ABSTRACT FROM AUTHOR]

Parathyroid hormone (PTH) is positively coupled to the generation of cAMP via its actions on the PTH1R and PTH2R receptors. Renin secretion from juxtaglomerular (JG) cells is stimulated by elevated intracellular cAMP, and every stimulus that increases renin secretion is thought to do so via increasing cAMP. Thus we hypothesized that PTH increases renin release from primary cultures of mouse JG cells by elevating intracellular cAMP via the PTH1R receptor. We found PTH1R, but not PTH2R, mRNA expressed in JG cells. While PTH increased JG cell cAMP content from (log10 means ± SE) 3.27 ± 0.06 to 3.92 ± 0.12 fmol/mg protein (P < 0.001), it did not affect renin release. The PTH1R-specific agonist, parathyroid hormone-related protein (PTHrP), also increased JG cell cAMP from 3.13 ± 0.09 to 3.93 ± 0.09 fmol/mg protein (P < 0.001), again without effect on renin release. PTH2R receptor agonists had no effect on cAMP or renin release. PTHrP increased cAMP in the presence of both low and high extracellular calcium from 3.31 ± 0.17 to 3.83 ± 0.20 fmol/mg protein (P < 0.01) and from 3.29 ± 0.18 to 3.63 ± 0.22 fmol/mg protein (P < 0.05), respectively, with no effect on renin release. PTHrP increased JG cell cAMP in the presence of adenylyl cyclase-V inhibition from 2.85 ± 0.17 to 3.44 ± 0.14 fmol/mg protein (P < 0.001) without affecting renin release. As a positive control, forskolin increased JG cell cAMP from 3.39 ± 0.13 to 4.48 ± 0.07 fmol/mg protein (P < 0.01) and renin release from 2.96 ± 0.10 to 3.29 ± 0.08 ng ANG I.mg prot-1·h-1 (P < 0.01). Thus PTH increases JG cell cAMP via non-calcium-sensitive adenylate cyclases without affecting renin release. These data suggest compartmentalization of cAMP signaling in JG cells. [ABSTRACT FROM AUTHOR]

ANG II type 2 receptors (AT2R) elicit cardioprotective effects in part by stimulating the release of kinins; however, the mechanism(s) responsible have not been fully explored. We demonstrated previously that overexpression of AT2R increased expression of prolylcarboxypeptidase (PRCP; a plasma prekallikrein activator) and release of bradykinin by mouse coronary artery endothelial cells (ECs). In the present study we hypothesized that the AT2R-stimulated increase in PRCP is mediated by the tyrosine phosphatase SHP-1, which in turn activates the PRCP-dependent prekallikrein-kallikrein pathway and releases bradykinin. We found that activation of AT2R using the specific agonist CGP42112A increased SHP-1 activity in ECs, which was blocked by the AT2R antagonist PD123319. Activation of AT2R also enhanced conversion of plasma prekallikrein to kallikrein, and this effect was blunted by a small interfering RNA (siRNA) to SHP-1 and abolished by the tyrosine phosphatase inhibitor sodium orthovanadate. Treating cells with a siRNA to PRCP also blunted AT2R-stimulated prekallikrein activation and bradykinin release. Furthermore, blocking plasma kallikrein with soybean trypsin inhibitor (SBTI) abolished AT2Rstimulated bradykinin release. These findings support our hypothesis that stimulation of AT2R activates a PRCP-dependent plasma prekallikrein pathway, releasing bradykinin. Activation of SHP-1 may also play an important role in AT2R-induced PRCP activation. [ABSTRACT FROM AUTHOR]

Balb/c mice, which are T-helper lymphocyte 2 (Th2) responders, are highly susceptible to infectious and non-infectious heart diseases, whereas C57BL/6 mice (Th1 responders) are not. Angiotensin II (Ang II) is not only a vasopressor but also a pro-inflammatory factor that leads to cardiac hypertrophy, fibrosis and dysfunction. We hypothesized that Ang II exacerbates cardiac damage in Balb/c but not in C57BL/6 mice even though both strains have a similar level of hypertension. Twelve-week-old male C57BL/6J and Balb/c mice received either vehicle or Ang II (1.4 mg kg−1 day−1, s.c. via osmotic minipump) for 8 weeks. At baseline, Balb/c mice exhibited the following: (1) a lower heart rate; (2) an enlarged left ventricular chamber; (3) a lower ejection fraction and shortening fraction; and (4) twice the left ventricular collagen deposition of age-matched C57BL/6J mice. Angiotensin II raised systolic blood pressure (to ∼150 mmHg) and induced cardiomyocyte hypertrophy in a similar manner in both strains. While C57BL/6J mice developed compensatory concentric hypertrophy and fibrosis in response to Ang II, Balb/c mice demonstrated severe left ventricular chamber dilatation, wall thinning and fibrosis, leading to congestive heart failure as evidenced by dramatically decreased ejection fraction and lung congestion (significant increase in lung weight), which are both characteristic of dilated cardiomyopathy. Our study suggests that the Th phenotype plays an active role in cardiac remodelling and function both in basal conditions and in hypertension. Angiotensin II-induced dilated cardiomyopathy in Balb/c mice is an ideal animal model for studying the impact of the adaptive immune system on cardiac remodelling and function and for testing strategies to prevent or treat hypertension-associated heart failure. [ABSTRACT FROM AUTHOR]

Using a line of mice with cardiac-specific knockout (KO) of the EP4 receptor gene, experiments were designed to determine whether a cardiac phenotype developed with age. Cardiac function was assessed by echocardiography in 23to 33-wk-old male and female KO and littermate controls (WT) mice. After echocardiography, hearts were removed to assess weight, and then some were further processed for histology [myocyte cross-sectional area (MCSA), interstitial collagen fraction (ICF), and macrophage infiltrationi and some for extraction of total RNA and protein. Older male KO mice had reduced ejection fraction (EF) coupled with left ventricular dilatation. MCSA and infiltrating macrophages were not different between groups, but ICF increased by 39% in KO mice. In contrast to male KO mice, 30- to 32-wk-old female KO mice had only a slight reduction in EF. To understand gene expression differences between male WT and KO mice, we performed whole genome gene expression profiling (Illumina BeadChips) on hearts of 30-to 32-wk-old mice. Data indicated that 156 genes were overexpressed in the KO hearts more than twofold, including genes involved in remodeling, inflammation, and oxidative stress. Overexpressed chemokines/cytokines were further examined in hearts of 10- to 1 2-wk-old male KO mice, and we found that growth differentiation factor-15 (GDF-15) expression was higher in KO than in WT hearts. In conclusion, EP4 knockdown in cardiac myocytes in aged male KO mice is in part associated with increased fibrosis, reduced EF, and dilated cardiomyopathy. Early overexpression of GDF-15 in hearts of male KO mice may contribute to or be a marker of the disease phenotype. The absence of serious cardiac dysfunction in aged female mice suggests a sexual dimorphism in the phenotype. [ABSTRACT FROM AUTHOR]

We have previously reported that 1) inhibition of cyclooxygenase-2 and PGE2 production reduces hypertrophy after myocardial infarction in mice and 2) PGE2 acting through its EP4 receptor causes hypertrophy of neonatal ventricular myocytes (NVM5) via ERK1/2. It is known that EP4 couples to adenylate cyclase, cAMP, and PKA. The present study was designed to determine interactions between the cAMP-PKA pathway and ERKI/2 and to further characterize events downstream of ERK1/2. We hypothesized that PKA and the small GTPase Rap are upstream of ERKI/2 and that 90-kDa ribosomal S6 kinase (p90RSK) is activated downstream. Treatment of NVMs with PGE2 activated Rap, and this activation was inhibited in part by an EP4 antagonist and PKA inhibition. Transfection of a dominant negative mutant of Rap reduced PGE2 activation of ERK1/2. PGE2 activation of p90RSK was also dependent on EP4, PKA, and Rap. We also tested the involvement of Rap, ERK1/2, and p90RSK in PGE2 regulation of gene expression. PGE2 stimulation of brain natriuretic peptide promoter activity was blocked by either ERK1/2 inhibition or a dominant negative mutation of p90RSK. PGE2 stimulation of c-Fos was dependent on EP4, PKA, ERK1/2, and p90RSK, whereas only the latter two kinases were involved in PGE2 regulation of early growth response-1. Finally, we tested the involvement of EP4-dependent signaling in the NVM growth response and found that the overexpression of EP4 increased NVM cell size. We conclude that EP4-dependent signaling in NVMs in part involves PKA, Rap, ERK1/2, and p90RSK and results in the increased expression of brain natriuretic peptide and c-Fos. [ABSTRACT FROM AUTHOR]

Background/Aims: Glomerular hypertrophy is a feature of many glomerular diseases and is associated with the development of renal failure. We previously demonstrated that the cyclooxygenase 2 inhibitor, NS398, reduced glomerular size after uninephrectomy. Thus, we hypothesized that prostaglandin (PG) E2 would cause mesangial cell hypertrophy in vitro. Methods: We used a mesangial cell line and primary culture of rat mesangial cells. The effects of PGE2 on mesangial cell hypertrophy were determined using immunohistochemistry and image analysis to assess cell size, 3H-leucine incorporation as a measure of protein synthesis and flow cytometry to assess cell cycle status. Western blot was used to examine the effect of PGE2 on expression of cyclin D3, p15, p27 and cyclin-dependent kinase 4 – known regulators of the cell cycle. Results: PGE2 increased cell size by 13% and protein synthesis (3H-leucine incorporation) by 35% over 24 h. By flow cytometry, PGE2 increased the percentage of cells in G0/G1 phase of the cell cycle from 70.13 ± 1.01 to 74.06 ± 1.18% and conversely, decreased the number of cells in S phase from 24.07 ± 1.06 to 22.03 ± 0.78%. The number of cells in G2/M was also reduced. Expression of cyclin D3 was decreased by 60% after treatment with PGE2, while expression of p27 was increased. The effects of PGE2 on cell size and flow cytometry were reproduced by the prostaglandin E2 receptors EP1/EP3 agonist sulprostone. Conclusion: PGE2 induces mesangial cell hypertrophy and cell cycle arrest via its EP1 receptor. Copyright © 2009 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Wind turbines are known to produce shadow flicker by interruption of sunlight by the turbine blades. Known parameters of the seizure provoking effect of flicker, i.e., contrast, frequency, mark-space ratio, retinal area stimulated and percentage of visual cortex involved were applied to wind turbine features. The proportion of patients affected by viewing wind turbines expressed as distance in multiples of the hub height of the turbine showed that seizure risk does not decrease significantly until the distance exceeds 100 times the hub height. Since risk does not diminish with viewing distance, flash frequency is therefore the critical factor and should be kept to a maximum of three per second, i.e., sixty revolutions per minute for a three-bladed turbine. On wind farms the shadows cast by one turbine on another should not be viewable by the public if the cumulative flash rate exceeds three per second. Turbine blades should not be reflective. [ABSTRACT FROM AUTHOR]

High blood pressure (HBP) is an important risk factor for cardiac, renal, and vascular dysfunction. Excess inflammation is the major pathogenic mechanism for HBP-induced target organ damage (TOD). N-acetyl-Ser-Asp-Lys-Pro (Ac-SDKP), a tetrapeptide specifically degraded by angiotensin converting enzyme (ACE), reduces inflammation, fibrosis, and TOD induced by HBP. Our hypothesis is that Ac-SDKP exerts its anti-inflammatory effects by inhibiting: 1) differentiation of bone marrow stem cells (BMSC) to macrophages, 2) activation and migration of macrophages, and 3) release of the proinflammatory cytokine TNF-α by activated macrophages. BMSC were freshly isolated and cultured in macrophage growth medium. Differentiation of murine BMSC to macro- phages was analyzed by flow cytometry using F4180 as a marker of macrophage maturation. Macrophage migration was measured in a modified Boyden chamber. TNF-α release by activated macrophages in culture was measured by ELISA. Myocardial macrophage activation in mice with ANG II-induced hypertension was studied by Western blotting of Mac-2 (galectin-3) protein. Interstitial collagen deposition was measured by picrosirius red staining. We found that Ac-SDKP (10 nM) reduced differentiation of cultured BMSC to mature macrophages by 24.5% [F4180 positivity: 14.09 ± 1.06 mean fluorescent intensity for vehicle and 10.63 ± 0.35 for Ac-SDKP; P < 0.051. Ac-SDKP also decreased galectin-3 and macrophage colony- stimulating factor-dependent macrophage migration. In addition, Ac-SDKP decreased secretion of TNF-α by macrophages stimulated with bacterial LPS. In mice with ANG II-induced hypertension, Ac-SDKP reduced expression of galectin-3, a protein produced by infiltrating macrophages in the myocardium, and interstitial collagen deposition. In conclusion, this study demonstrates that part of the anti-inflammatory effect of Ac-SDKP is due to its direct effect on BMSC and macrophage, inhibiting their differentiation, activation, and cytokine release. These effects explain some of the anti-inflammatory and antifibrotic properties of Ac-SDKP in hypertension. [ABSTRACT FROM AUTHOR]

Several essential components of NADPH oxidase, including p22phox, gp91phox (nox2) and its homologs noxl and nox4, p47phox, p67phox, and rac1, are present in the vasculature. We previously reported that p67phox is essential for adventitial fibroblast NADPH oxidase O(Multiple line equation(s) cannot be represented in ASCII text) production. Thus we postulated that inhibition of adventitial p67phox activity would attenuate angioplasty-induced hyperplasia. To test this hypothesis, we treated the adventitia of carotid arteries with a control adenovirus (Ad-control), a virus expressing dominant-negative p67phox (Ad-p67dn), or a virus expressing a competitive peptide (gp91ds) targeting the p47phox-gp91phox interaction (Ad-gp91ds). Common carotid arteries (CCAs) from male Sprague-Dawley rats were transfected with Ad-control, Ad-p67dn, or Ad-gp91ds in pluronic gel. After 2 days, a 2-F (Fogarty) catheter was used to injure CCAs in vivo. After 14 days, CCAs were perfusion-fixed and analyzed. In 13 experiments, digital morphometry suggested a reduction of neointimal hyperplasia with Ad-p67dn compared with Ad-control; however, the reduction did not reach statistical significance (P = 0.058). In contrast, a significant reduction was achieved with Ad-gp91ds (P = 0.006). No changes in medial area or remodeling were observed with either treatment. Moreover, adventitial fibroblast proliferation in vitro was inhibited by Ad-gp91ds but not by Ad-p67dn, despite confirmation that Ad-p67dn inhibits NADPH oxidase in fibroblasts. These data appear to suggest that a multicomponent vascular NADPH oxidase plays a role in neointimal hyperplasia. However, inhibition of p47phox may be more effective than inhibition of p67phox at attenuating neointimal growth. [ABSTRACT FROM AUTHOR]

Brain natriuretic peptide (BNP) produced by cardiac myocytes has antifibrotic and antigrowth properties and is a marker of cardiac hypertrophy. We previously showed that prostaglandin E2 (PGE2) is the main prostaglandin produced in myocytes treated with proinflammatory stimuli and stimulates protein synthesis by binding to its EP4 receptor. We hypothesized that PGE2, acting through EP4, also regulates BNP gene expression. We transfected neonatal ventricular myocytes with a plasmid encoding the human BNP (hBNP) promoter driving expression of a luciferase reporter gene. PGE2 increased hBNP promoter activity 3.5-fold. An EP4 antagonist reduced the stimulatory effect of PGE2 but not an EP1 antagonist. Because EP4 signaling can involve adenylate cyclase, cAMP, and protein kinase A (PKA), we tested the effect of H-89, a PKA inhibitor, on PGE2 stimulation of the hBNP promoter. H-89 at 5 μM decreased PGE2 stimulation of BNP promoter activity by 100%. Because p42⁄44 MAPK mediates the effect of PGE2 on protein synthesis, we also examined the role of MAPKs in the regulation of BNP promoter activity. PGE2 stimulation of the hBNP promoter was inhibited by a MEK1⁄2 inhibitor and a dominant-negative mutant of Raf, indicating that p42/44 MAPK was involved. In contrast, neither a p38 MAPK inhibitor nor a JNK inhibitor reduced the stimulatory effect of PGE2. Involvement of small GTPases was also studied. Dominant-negative Rap inhibited PGE2 stimulation of the hBNP promoter, but dominant-negative Ras did not. We concluded that PGE2 stimulates the BNP promoter mainly via EP4, PKA, Rap, and p42/44 MAPK. [ABSTRACT FROM AUTHOR]

An anonymous questionnaire on eating behavior and attitudes was administered to the student body of a coeducational private preparatory school. There were 424 males and 337 females, ranging in age from 13 to 19 years and representing 86.5% of the available student body, who completed the questionnaire. Results indicated a high frequency of concern with weight, body image, and dieting, particularly among women. In particular the female subjects reported high frequencies of binge eating, fasting, crash dieting, and feeling obese. A sizable number of women reported self-induced vomiting and amenorrhea secondary to weight loss. These concerns and behaviors were more highly correlated with subjects' opinions about the desirability of their weight rather than with their reported body weight (prorated for height and age). There were 4.0% of women and 0. 8% of men who answered questions that would meet strict DSM III criteria for bulimia. These results provide additional evidence that troubled eating behavior and attitudes towards weight and body image are widespread among adolescent women. [ABSTRACT FROM AUTHOR]

A novel inverse relationship exists between intracellular calcium and cAMP formation in parathyroid glands, cAMP is the second messenger regulating PTH secretion. It is not known how increased intracellular calcium inhibits PTH secretion. However, there are two isoforms adenylyl cyclase (AC) which are inhibited by calcium; types-5 and 6. We hypothesized that in the rat, the parathyroid gland expresses one (or both) of the calcium-inhibitable isoforms of AC, either type-5 or 6, such that increased intracellular calcium would inhibit AC activity. Parathyroid glands were dissected from male Sprague-Dawley rats. Western Blot analysis was performed on rat parathyroid homogenates using monoclonal antibodies against either AC-5, AC-6 or a polyclonal antibody against both AC-5 and 6. Positive controls were run using heart and brain. We did not detect either AC-5 or AC-6 in rat parathyroid by Western blot analysis. Additionally, reverse transcription polymerase chain reaction (RT-PCR) performed with AC-5 specific primers on total RNA extracted from rat parathyroid did not indicate that AC-5 was expressed in rat parathyroid gland. Thus, contrary to our hypothesis, we do not find evidence that either calcium-inhibitable isoform of AC expressed in rat parathyroid. [ABSTRACT FROM AUTHOR]

Calcium-sensing receptors (CaSR) are present in many cells, and sense and translate micro molar changes in extracellular calcium into intracellular signals, including parallel changes in intracellular calcium. In the juxtaglomerular (JG) cell, increased intracellular calcium suppresses the calcium-inhibitable adenylyl cyclase type-V (AC-V), reducing cAMP and inhibiting renin release. We hypothesized that 1) JG cells express CaSR, and 2) changes in AC-V activity and renin release might be mediated by activation of CaSR. We used primary cultures of isolated mouse JG cells. RT-PCR on total RNA extracted from JG cells was run 40 cycles with CaSR-specific primers, giving a positive band at 151 bp, consistent with CaSR. Next, in JG cells, immunofluorescent labeling by an antibody specific for CaSR showed significant fluorescence compared to negative controls (0 vs. 167± 8.5 AU, p<0.001) Finally, treating primary cultures of isolated JG cells with the calcium sensing receptor agonist Cinacalcet (Amgen) at 50 and 1000 nM to stimulate the CaSR in the presence of IBMX decreased cAMP to 85.8±27.2% and 79.1±12.7% of basal, respectively, and decreased renin release from 450.1±81.7 to 350.2±83.8 and 222.6±56.2 ng ANGI/ml/hr/mg protein (p<0.01), respectively. We conclude that 1) the CaSR is expressed in JG cells and 2) these CaSR mediate changes in cAMP formation and renin release from JG cells. [ABSTRACT FROM AUTHOR]