ß Adrenergic Receptor Kinase C-Terminal Peptide Gene-Therapy Improves ß2-Adrenergic Receptor-Dependent Neoangiogenesis after Hindlimb Ischemia.

After hindlimb ischemia (HI), increased catecholamine levels within the ischemic muscle can cause dysregulation of ß2-adrenergic receptor (ß2AR) signaling, leading to reduced revascularization. Indeed, in vivo ß2AR overexpression via gene therapy enhances angiogenesis in a rat model of HI. G protein-coupled receptor kinase 2 (GRK2) is a key regulator of ßAR signaling, and ß adrenergic receptor kinase C-terminal peptide (ßARKct), a peptide inhibitor of GRK2, has been shown to prevent ßAR down-regulation and to protect cardiac myocytes and stem cells from ischemic injury through restoration of ß2AR protective signaling (i.e., protein kinase B/endothelial nitric oxide synthase). Herein, we tested the potential therapeutic effects of adenoviral-mediated ßARKct gene transfer in an experimental model of HI and its effects on ßAR signaling and on endothelial cell (EC) function in vitro. Accordingly, in this study, we surgically induced HI in rats by femoral artery resection (FAR). Fifteen days of ischemia resulted in significant ßAR down-regulation that was paralleled by an approximately 2-fold increase in GRK2 levels in the ischemic muscle. Importantly, in vivo gene transfer of the ßARKct in the hindlimb of rats at the time of FAR resulted in a marked improvement of hindlimb perfusion, with increased capillary and ßAR density in the ischemic muscle, compared with control groups. The effect of ßARKct expression was also assessed in vitro in cultured ECs. Interestingly, ECs expressing the ßARKct fenoterol, a ß2AR-agonist, induced enhanced ß2AR proangiogenic signaling and increased EC function. Our results suggest that ßARKct gene therapy and subsequent GRK2 inhibition promotes angiogenesis in a model of HI by preventing ischemia-induced ß2AR down-regulation.

AAV6-ßARKct gene delivery mediated by molecular cardiac surgery with recirculating delivery (MCARD) in sheep results in robust gene expression and increased adrenergic reserve.

OBJECTIVE: Genetic modulation of heart function is a novel therapeutic strategy. We investigated the effect of molecular cardiac surgery with recirculating delivery (MCARD)-mediated carboxyl-terminus of the ß-adrenergic receptor kinase (ßARKct) gene transfer on cardiac mechanoenergetics and ß-adrenoreceptor (ßAR) signaling. METHODS: After baseline measurements, sheep underwent MCARD-mediated delivery of 10(14) genome copies of self-complimentary adeno-associated virus (scAAV6)-ßARKct. Four and 8 weeks after MCARD, mechanoenergetic studies using magnetic resonance imaging were performed. Tissues were analyzed with real-time quantitative polymerase chain reaction (RT-qPCR) and Western blotting. ßAR density, cyclic adenosine monophosphate levels, and physiologic parameters were evaluated. RESULTS: There was a significant increase in dP/dt(max) at 4 weeks: 1384 ± 76 versus 1772 ± 182 mm Hg/s; and the increase persisted at 8 weeks in response to isoproterenol (P < .05). Similarly, the magnitude of dP/dt(min) increased at both 4 weeks and 8 weeks with isoproterenol stimulation (P < .05). At 8 weeks, potential energy was conserved, whereas in controls there was a decrease in potential energy (P < .05) in response to isoproterenol. RT-qPCR confirmed robustness of ßARKct expression throughout the left ventricle and undetectable expression in extracardiac tissues. Quantitative Western blot data confirmed higher expression of ßARKct in the left ventricle: 0.46 ± 0.05 versus 0.00 in lung and liver (P < .05). Survival was 100% and laboratory parameters of major organ function were within normal limits. CONCLUSIONS: MCARD-mediated ßARKct delivery is safe, results in robust cardiac-specific gene expression, enhances cardiac contractility and lusitropy, increases adrenergic reserve, and improves energy utilization efficiency in a preclinical large animal model.

Pasireotide and octreotide stimulate distinct patterns of sst2A somatostatin receptor phosphorylation.

Pasireotide (SOM230) is currently under clinical evaluation as a successor compound to octreotide for the treatment of acromegaly, Cushing's disease, and carcinoid tumors. Whereas octreotide acts primarily via the sst(2A) somatostatin receptor, pasireotide was designed to exhibit octreotide-like sst(2A) activity combined with enhanced binding to other somatostatin receptor subtypes. In the present study, we used phophosite-specific antibodies to examine agonist-induced phosphorylation of the rat sst(2A) receptor. We show that somatostatin and octreotide stimulate the complete phosphorylation of a cluster of four threonine residues within the cytoplasmic (353)TTETQRT(359) motif in a variety of cultured cell lines in vitro as well as in intact animals in vivo. This phosphorylation was mediated by G protein-coupled receptor kinases (GRK) 2 and 3 and followed by rapid cointernalization of the receptor and ss-arrestin into the same endocytic vesicles. In contrast, pasireotide failed to promote substantial phosphorylation and internalization of the rat sst(2A) receptor. In the presence of octreotide or SS-14, SOM230 showed partial agonist behavior, inhibiting phosphorylation, and internalization of sst(2A). Upon overexpression of GRK2 or GRK3, pasireotide stimulated selective phosphorylation of Thr356 and Thr359 but not of Thr353 or Thr354 within the (353)TTETQRT(359) motif. Pasireotide-mediated phosphorylation led to the formation of relatively unstable beta-arrestin-sst(2A) complexes that dissociated at or near the plasma membrane. Thus, octreotide and pasireotide are equally active in inducing classical G protein-dependent signaling via the sst(2A) somatostatin receptor. Yet, we find that they promote strikingly different patterns of sst(2A) receptor phosphorylation and, hence, stimulate functionally distinct pools of beta-arrestin.

Negative regulation of Ca(2+) influx during P2Y(2) purinergic receptor activation is mediated by Gbetagamma-subunits.

We have previously reported that P2Y(2) purinoceptors and muscarinic M(3) receptors trigger Ca(2+) responses in HT-29 cells that differ in their timecourse, the Ca(2+) response to P2Y(2) receptor activation being marked by a more rapid decline of intracellular Ca(2+) concentration ([Ca(2+)](i)) after the peak response and that this rapid decline of [Ca(2+)](i) was slowed in cells expressing heterologous beta-adrenergic receptor kinase (betaARK). In the present study, we demonstrate that, during P2Y(2) receptor activation, betaARK expression increases the rate of Gd(3+)-sensitive Mn(2+) influx, a measure of the rate of store-operated Ca(2+) entry from the extracellular space, during P2Y(2) activation and that this effect of betaARK is mimicked by exogenous alpha-subunits of G(q), G(11) and G(i2). The effect of betaARK on the rate of Mn(2+) influx is thus attributable to its ability to scavenge G protein betagamma-subunits released during activation of P2Y(2) receptor. We further find that the effect of betaARK on the rate of Mn(2+) influx during P2Y(2) receptor activation can be overcome by arachidonic acid. In addition, the UTP-induced Mn(2+) influx rate was significantly increased by inhibitors of phospholipase A(2) (PLA(2)) and an siRNA directed against PLA(2)beta, but not by an siRNA directed against PLA(2)alpha or by inhibitors of arachidonic acid metabolism. These findings provide evidence for the existence of a P2Y(2) receptor-activated signalling system that acts in parallel with depletion of intracellular Ca(2+) stores to inhibit Ca(2+) influx across the cell membrane. This signalling process is mediated via Gbetagamma and involves PLA(2)beta and arachidonic acid.

Identification of Nogo as a novel indicator of heart failure.

Numerous genetically engineered animal models of heart failure (HF) exhibit multiple characteristics of human HF, including aberrant beta-adrenergic signaling. Several of these HF models can be rescued by cardiac-targeted expression of the Gbetagamma inhibitory carboxy-terminus of the beta-adrenergic receptor kinase (betaARKct). We recently reported microarray analysis of gene expression in multiple animal models of HF and their betaARKct rescue, where we identified gene expression patterns distinct and predictive of HF and rescue. We have further investigated the muscle LIM protein knockout model of HF (MLP-/-), which closely parallels human dilated cardiomyopathy disease progression and aberrant beta-adrenergic signaling, and their betaARKct rescue. A group of known and novel genes was identified and validated by quantitative real-time PCR whose expression levels predicted phenotype in both the larger HF group and in the MLP-/- subset. One of these novel genes is herein identified as Nogo, a protein widely studied in the nervous system, where it plays a role in regeneration. Nogo expression is altered in HF and normalized with rescue, in an isoform-specific manner, using left ventricular tissue harvested from both animal and human subjects. To investigate cell type-specific expression of Nogo in the heart, immunofluorescence and confocal microscopy were utilized. Nogo expression appears to be most clearly associated with cardiac fibroblasts. To our knowledge, this is the first report to demonstrate the relationship between Nogo expression and HF, including cell-type specificity, in both mouse and human HF and phenotypic rescue.

Confirmation of an association between the TNF(-308) promoter polymorphism and stroke risk in children with sickle cell anemia.

BACKGROUND AND PURPOSE: The etiology of stroke in children with sickle cell anemia (SCA) is complex and poorly understood. Growing evidence suggests that genetic factors beyond the sickle cell mutation influence stroke risk in SCA. We previously reported risk associations with polymorphisms in several proinflammatory genes in SCA children with ischemic stroke. The aim of this replication study was to confirm our previous findings of associations between the TNF(-308) G/A, IL4R 503 S/P, and ADRB2 27 Q/E polymorphisms and large vessel stroke risk. METHODS: Using previously collected MRA data, we assessed an independent population of SCA children from the multicenter Stroke Prevention Trial in Sickle Cell Anemia (STOP) for the presence or absence of large vessel stenosis. Samples were genotyped for 104 polymorphisms among 65 candidate vascular disease genes. Genotypic associations with risk of large vessel stroke were screened using univariable analysis and compared with results from our original study. Joint analysis of the 2 study populations combined was performed using multivariable logistic regression. RESULTS: A total of 96 children (49 MRA-positive, 47 MRA-negative) were included in this study. Of the SNP associations previously identified in the original study, the TNF(-308) G/A association with large vessel stroke remained significant and the IL4R 503 S/P variant approached significance in the joint analysis of the combined study populations. Consistent with our original findings, the TNF(-308) GG genotype was associated with a >3-fold increased risk of large vessel disease (OR=3.27; 95% CI=1.6, 6.9; P=0.006). Unadjusted analyses also revealed a previously unidentified association between the LTC4S(-444) A/C variant and large vessel stroke risk. CONCLUSIONS: Similar findings in 2 independent study populations strongly suggest that the TNF(-308) G/A promoter polymorphism is a clinically important risk factor for large vessel stroke in children with SCA. The previously observed association with the IL4R 503 S/P variant and the novel association with the LTC4S(-444) A/C variant suggest that these loci may also contribute to large vessel stroke risk in children with SCA.

Substrate specificities of g protein-coupled receptor kinase-2 and -3 at cardiac myocyte receptors provide basis for distinct roles in regulation of myocardial function.

The closely related G protein-coupled receptor kinases GRK2 and GRK3 are both expressed in cardiac myocytes. Although GRK2 has been extensively investigated in terms of regulation of cardiac beta-adrenergic receptors, the substrate specificities of the two GRK isoforms at G protein-coupled receptors (GPCR) are poorly understood. In this study, the substrate specificities of GRK2 and GRK3 at GPCRs that control cardiac myocyte function were determined in fully differentiated adult cardiac myocytes. Concentration-effect relationships of GRK2, GRK3, and their respective competitive inhibitors, GRK2ct and GRK3ct, at endogenous endothelin, alpha(1)-adrenergic, and beta(1)-adrenergic receptor-generated responses in cardiac myocytes were achieved by adenovirus gene transduction. GRK3 and GRK3ct were highly potent and efficient at the endothelin receptors (IC(50) for GRK3, 5 +/- 0.7 pmol/mg of protein; EC(50) for GRK3ct, 2 +/- 0.2 pmol/mg of protein). The alpha(1)-adrenergic receptor was also a preferred substrate of GRK3 (IC(50),7 +/- 0.4 pmol/mg of protein). GRK2 lacked efficacy at both endothelin and alpha(1)-adrenergic receptors despite massive overexpression. On the contrary, both GRK2ct and GRK3ct enhanced beta(1)-adrenergic receptor-induced cAMP production with comparable potencies. However, the potency of GRK3ct at beta(1)-adrenergic receptors was at least 20-fold lower than that at endothelin receptors. In conclusion, this study demonstrates distinct substrate specificities of GRK2 and GRK3 at different GPCRs in fully differentiated adult cardiac myocytes. As inferred from the above findings, GRK2 may play its primary role in regulation of cardiac contractility and chronotropy by controlling beta(1)-adrenergic receptors, whereas GRK3 may play important roles in regulation of cardiac growth and hypertrophy by selectively controlling endothelin and alpha(1)-adrenergic receptors.

The pharmacogenetics of lithium response depends upon clinical co-morbidity.

BACKGROUND: Based on results from randomized, controlled clinical trials, lithium monotherapy or lithium with the addition of an antipsychotic remains a first-line treatment option for both acute and long-term mood stabilization in bipolar mania. However, response to lithium is poor in bipolar patients who exhibit clinical characteristics such as rapid cycling and mixed manic states, suggesting that they may have a biologically and genetically distinct form of bipolar disorder. A test that could predict response to lithium based upon genetic factors would have significant clinical value. METHODS: Eight clinical characteristics were assessed in 92 lithium responders and 92 nonresponders; all probands were from families recruited for linkage studies. Lithium response was rated retrospectively from a standardized interviews and medical records. Eight candidate genes were selected from those reported to be associated with susceptibility to illness, lithium response, or lithium mechanism of action. Sixty-seven single nucleotide polymorphisms (SNPs) were genotyped in these subjects and analyzed for association with the defined clinical characteristics. RESULTS: Using q-value analysis for multiplicity correction, we found significant interactions between lithium response and SNPs (rs1387923 and rs1565445) in the gene encoding neurotrophic tyrosine kinase receptor type 2 (NTRK2) and suicidal ideation, and between SNP rs2064721 in the gene encoding inositol polyphosphate-1-phosphatase (INPP1) and post-traumatic stress disorder. CONCLUSION: These data support the idea that response to lithium has a multi-genetic etiology dependent upon manifestations of other clinical co-diagnoses.

The G protein-coupled receptor regulatory kinase GPRK2 participates in Hedgehog signaling in Drosophila.

Signaling by Smoothened (Smo) plays fundamental roles during animal development and is deregulated in a variety of human cancers. Smo is a transmembrane protein with a heptahelical topology characteristic of G protein-coupled receptors. Despite such similarity, the mechanisms regulating Smo signaling are not fully understood. We show that Gprk2, a Drosophila member of the G protein-coupled receptor kinases, plays a key role in the Smo signal transduction pathway. Lowering Gprk2 levels in the wing disc reduces the expression of Smo targets and causes a phenotype reminiscent of loss of Smo function. We found that Gprk2 function is required for transducing the Smo signal and that when Gprk2 levels are lowered, Smo still accumulates at the cell membrane, but its activation is reduced. Interestingly, the expression of Gprk2 in the wing disc is regulated in part by Smo, generating a positive feedback loop that maintains high Smo activity close to the anterior-posterior compartment boundary.

The membrane-proximal portion of CD3 epsilon associates with the serine/threonine kinase GRK2.

The activation of protein kinases is one of the primary mechanisms whereby T cell receptors (TCR) propagate intracellular signals. To date, the majority of kinases known to be involved in the early stages of TCR signaling are protein-tyrosine kinases such as Lck, Fyn, and ZAP-70. Here we report a constitutive association between the TCR and a serine/threonine kinase, which was mediated through the membrane-proximal portion of CD3 epsilon. Mass spectrometry analysis of CD3 epsilon-associated proteins identified G protein-coupled receptor kinase 2 (GRK2) as a candidate Ser/Thr kinase. Transient transfection assays and Western blot analysis verified the ability of GRK2 to interact with the cytoplasmic domain of CD3 epsilon within a cell. These findings are consistent with recent reports demonstrating the ability of certain G protein-coupled receptors (GPCR) and G proteins to physically associate with the alpha/beta TCR. Because GRK2 is primarily involved in arresting GPCR signals, its interaction with CD3 epsilon may provide a novel means whereby the TCR can negatively regulate signals generated through GPCRs.

Taxol normalizes the impaired agonist-induced beta2-adrenoceptor internalization in splenocytes from GRK2+/- mice.

G protein-coupled receptor kinase 2 (GRK2) is involved in the agonist-induced desensitization of beta2-adrenoceptors. In addition, GRK2 is capable of binding and phosphorylating tubulin. Interestingly, microtubule dynamics profoundly affect agonist-induced internalization of beta2-adrenoceptors. Here, we analyzed agonist-induced beta2-adrenoceptor internalization and signaling in splenocytes from GRK2+/- mice that have a approximately 50% lower level of GRK2 protein compared to wild type (WT) mice. In addition, we investigated the role of microtubule stability in these processes. Splenocytes from GRK2+/- mice express approximately 50% less beta2-adrenoceptors on the cell surface and show impaired agonist-induced beta2-adrenoceptor internalization. Disruption of microtubules using colchicine reduces agonist-induced beta2-adrenoceptor internalization in cells from WT, but not in cells from GRK2+/- mice. Importantly, increasing tubulin stability by taxol almost completely restores the defective agonist-induced beta2-adrenoceptor internalization in cells from GRK2+/- animals, without affecting WT cells. Despite lower surface receptor numbers, cells of GRK2+/- mice show normal beta2-adrenoceptor agonist-induced cAMP responses. Although interfering with microtubule stability has major effects on agonist-induced receptor internalization in GRK2+/- cells, microtubule dynamics do not influence cAMP responses. Our data suggest that cells with low GRK2 adapt to the lower GRK2 level by decreasing the number of beta2-adrenoceptors on the cell surface. In addition, the cellular GRK2 level determines the extent of agonist-induced beta2-adrenoceptor internalization via a mechanism involving microtubule stability. Importantly, however, normalization of agonist-induced receptor internalization by taxol is not sufficient to alter receptor signaling.

Extracellular signal-regulated kinase 1/2-mediated transcriptional regulation of G-protein-coupled receptor kinase 3 expression in neuronal cells.

Relatively small changes in G-protein-coupled receptor kinase (GRK) 3 expression (approximately 2-fold) profoundly affect alpha2-adrenergic receptor (AR) function and preferentially regulate neuronal alpha2A- and alpha2B-AR signaling. In the present study, we provide evidence that epinephrine (EPI)-induced up-regulation of GRK3 protein expression in two neuronal cell lines, BE(2)-C cells (endogenously express alpha2A- and beta2AR) and BN17 cells [endogenously express alpha2B (NG108) and transfected to express beta2-AR] is due in part to increased GRK3 gene expression. In both cell lines, the increase in GRK3 transcription occurred via an extracellular signal-regulated kinase (ERK) 1/2-dependent mechanism because the increase in GRK3 mRNA is eliminated in the presence of the mitogen-activated protein kinase/ERK kinase 1/2 inhibitor, U0126 [1,4-diamino-2,3-dicyano-1,4-bis (2-amino phenylthiobutadiene)]. EPI-induced GRK3 mRNA up-regulation also is prevented in the presence of propranolol or phentolamine. Moreover, GRK3 mRNA did not increase in response to EPI treatment in NG108 cells (endogenously express alpha2B-AR with no beta2-AR). Both these results suggest that simultaneous activation of alpha2- and beta2-AR by EPI is required for the ERK1/2-dependent increase in GRK3 mRNA. The EPI-induced increase in GRK3 mRNA was unaffected in the presence of the protein kinase C inhibitor, chelerythrine chloride. Finally, EPI treatment resulted in increased nuclear translocation and accumulation of the transcription factors, Sp-1 and Ap-2, in BE(2)-C cells. Taken together, our results demonstrate the involvement of the ERK1/2 pathway in selective up-regulation of GRK3 mRNA expression, possibly via activation of Sp-1 and Ap-2 transcription factors in neuronal cells.

Expression analysis of imbalanced genes in prostate carcinoma using tissue microarrays.

To identify candidate genes relevant for prostate tumour prognosis and progression, we performed an exhaustive gene search in seven previously described genomic-profiling studies of 161 prostate tumours, and four expression profiling studies of 61 tumours. From the resulting list of candidate genes, six were selected for protein-expression analysis based on the availability of antibodies applicable to paraffinised tissue: fatty acid synthase (FASN), MYC, beta-adrenergic receptor kinase 1 (BARK1, GRK2) the catalytic subunits of protein phosphatases PP1alpha (PPP1CA) and PP2A (PPP2CB) and metastasis suppressor NM23-H1. These candidates were analysed by immunohistochemistry (IHC) on a tissue microarray containing 651 cores of primary prostate cancer samples and benign prostatic hyperplasias (BPH) from 175 patients. In univariate analysis, expression of PP1alpha (P=0.001) was found to strongly correlate with Gleason score. MYC immunostaining negatively correlated with both pT-stage and Gleason score (P<0.001 each) in univariate as well as in multivariate analysis. Furthermore, a subgroup of patients with high Gleason scores was characterised by a complete loss of BARK1 protein (P=0.023). In conclusion, our study revealed novel molecular markers of potential diagnostic and therapeutic relevance for prostate carcinoma.

Protein kinase A-mediated phosphorylation contributes to enhanced contraction observed in mice that overexpress beta-adrenergic receptor kinase-1.

Transgenic mice with cardiac specific overexpression of beta-adrenergic receptor kinase-1 (betaARK-1) exhibit reduced contractility in the presence of adrenergic stimulation. However, whether contractility is altered in the absence of exogenous agonist is not clear. Effects of betaARK-1 overexpression on contraction were examined in mouse ventricular myocytes, studied at 37 degrees C, in the absence of adrenergic stimulation. In myocytes voltage-clamped with microelectrodes (18-26 MOmega; 2.7 M KCl) to minimize intracellular dialysis, contractions were significantly larger in betaARK-1 cells than in wild-type myocytes. In contrast, when cells were dialyzed with patch pipette solution (1-3 MOmega; 0 mM NaCl, 70 mM KCl, 70 mM potassium aspartate, 4 mM MgATP, 1 mM MgCl(2), 2.5 mM KH(2)PO(4), 0.12 mM CaCl(2), 0.5 mM EGTA, and 10 mM HEPES), the extent of cell shortening was similar in wild-type and betaARK-1 myocytes. Furthermore, when cells were dialyzed with solutions that contained phosphodiesterase-sensitive sodium-cAMP (50 microM), the extent of cell shortening was similar in wild-type and betaARK-1 myocytes. However, when patch solutions were supplemented with phosphodiesterase-resistant 8-bromo-cAMP (50 muM), contractions were larger in betaARK-1 than wild-type cells. This difference was eliminated by the protein kinase A inhibitor N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline (H89). Interestingly, Ca(2+) current amplitudes and inactivation rates were similar in betaARK-1 and wild-type cells in all experiments. These results suggest components of the adenylyl cyclase-protein kinase A pathway are sensitized by chronically increased betaARK-1 activity, which may augment contractile function in the absence of exogenous agonist. Thus, changes in contractile function in myocytes from failing hearts may reflect, in part, effects of chronic up-regulation of betaARK-1 on the cAMP-protein kinase A pathway.

Knock-out mice reveal the contributions of P2Y and P2X receptors to nucleotide-induced Ca2+ signaling in macrophages.

Immune cell function is modulated by changes in extracellular nucleotide levels. Here we used reverse transcription-PCR analyses, single cell Ca2+ imaging, and knock-out mice to define the receptors mediating nucleotide-induced Ca2+ signaling in resident peritoneal macrophages. In Ca2+-free buffer, the potent (K0.5<1 microm) stimulatory effect of UTP (or ATP) on endoplasmic reticulum (ER) Ca2+ release was abolished in cells isolated from P2Y2/P2Y4 double knock-out mice. Moreover, P2Y4(0/-), but not P2Y2-/-, macrophages responded to UTP. In P2Y2-/- macrophages, we could elicit Ca2+ responses to "pure" P2X receptor activation by applying ATP in buffer containing Ca2+. Purified UDP and ADP were ineffective agonists, although modest UDP-induced Ca2+ responses could be elicited in macrophages after "activation" with lipopolysaccharide and interferon-gamma. Notably, in Ca2+-free buffer, UTP-induced Ca2+ transients decayed within 1 min, and there was no response to repeated agonist challenge. Measurements of ER [Ca2+] with mag-fluo-4 showed that ER Ca2+ stores were depleted under these conditions. When extracellular Ca2+ was available, ER Ca2+ stores refilled, but Ca2+ increased to only approximately 40% of the initial value upon repeated UTP challenge. This apparent receptor desensitization persisted in GRK2+/- and GRK6-/- macrophages and after inhibition of candidate kinases protein kinase C and calmodulin-dependent kinase II. Initial challenge with UTP also reduced Ca2+ mobilization by complement component C5a (and vice versa). In conclusion, homologous receptor desensitization is not the major mechanism that rapidly dampens Ca2+ signaling mediated by P2Y2, the sole Gq-coupled receptor for UTP or ATP in macrophages. UDP responsiveness (P2Y6 receptor expression) increases following macrophage activation.

Decreased expression of G protein-coupled receptor kinases in the detrusor smooth muscle of human urinary bladder with outlet obstruction.

AIM: We examine the expression of mRNA of G protein-coupled receptor kinase (GRK) subtypes and muscarinic acetylcholine receptor (M) subtypes in the detrusor smooth muscle of the human urinary bladder. Furthermore, we confirm the presence and the localization of GRK proteins in the detrusor smooth muscle of the obstructed bladder in comparison with the control bladder. METHODS: Detrusor smooth muscle tissues of the human urinary bladder were obtained from 12 male patients; 6 patients did not have bladder outlet obstruction, and the other 6 patients had bladder outlet obstruction. Portions of the dome or anterior wall of the urinary bladder were used for the present study. Reverse transcription/polymerase chain reaction for GRK2, M2 and M3 was performed using total RNA extracted from human urinary bladder detrusor. Antibodies to GRK2, GRK3 and GRK4 were used to confirm the presence of the protein product in the human urinary bladder using immunohistochemical staining and the western blotting technique. RESULTS: All complementary DNA (cDNA) transcribed from three different mRNA (M2, M3 and GRK2) were successfully amplified and size-fractionated. The expression of GRK2 protein was strong in the human bladder detrusor, but was significantly weakened by western blotting in obstructed bladder in comparison with control bladder. CONCLUSIONS: Failure in desensitization mechanisms of muscarinic acetylcholine receptors might be related to storage symptom elicited by overactivity in obstructed bladder with benign prostatic hyperplasia.

Smoothened signal transduction is promoted by G protein-coupled receptor kinase 2.

Deregulation of the Sonic hedgehog pathway has been implicated in an increasing number of human cancers. In this pathway, the seven-transmembrane (7TM) signaling protein Smoothened regulates cellular proliferation and differentiation through activation of the transcription factor Gli. The activity of mammalian Smoothened is controlled by three different hedgehog proteins, Indian, Desert, and Sonic hedgehog, through their interaction with the Smoothened inhibitor Patched. However, the mechanisms of signal transduction from Smoothened are poorly understood. We show that a kinase which regulates signaling by many "conventional" 7TM G-protein-coupled receptors, G protein-coupled receptor kinase 2 (GRK2), participates in Smoothened signaling. Expression of GRK2, but not catalytically inactive GRK2, synergizes with active Smoothened to mediate Gli-dependent transcription. Moreover, knockdown of endogenous GRK2 by short hairpin RNA (shRNA) significantly reduces signaling in response to the Smoothened agonist SAG and also inhibits signaling induced by an oncogenic Smoothened mutant, Smo M2. We find that GRK2 promotes the association between active Smoothened and beta-arrestin 2. Indeed, Gli-dependent signaling, mediated by coexpression of Smoothened and GRK2, is diminished by beta-arrestin 2 knockdown with shRNA. Together, these data suggest that GRK2 plays a positive role in Smoothened signaling, at least in part, through the promotion of an association between beta-arrestin 2 and Smoothened.

Impaired neutrophil chemotaxis in sepsis associates with GRK expression and inhibition of actin assembly and tyrosine phosphorylation.

The deregulation of inflammatory response during sepsis seems to reflect the overproduction of mediators, which suppress leukocyte functions. We investigated the intracellular mechanisms underlying the inability of neutrophils from severe septic patients to migrate toward chemoattractants. Patients with sepsis (52) and 15 volunteers were prospectively enrolled. Patients presented increased circulating levels of tumor necrosis factor-alpha, interferon-gamma, interleukin (IL)-8, and IL-10. Patients showed reduced neutrophil chemotaxis to formyl-methionyl-leucyl-phenylalanine (FMLP), leukotriene B4 (LTB4) or IL-8. No difference in the transcription or expression of the IL-8 receptor, CXCR1, was detected in neutrophils from controls and patients. However, septic neutrophils failed to increase tyrosine phosphorylation and actin polymerization in response to IL-8 or LTB4. In contrast, septic neutrophils, similar to controls, showed phagocytic activity that induced actin polymerization and augmented phosphotyrosine content. Treatment of control neutrophils with cytokines and lipopolysaccharide (LPS) to mimic endogenous septic environment inhibited actin polymerization and tyrosine phosphorylation in response to IL-8 or LTB4. High expression of G protein-coupled receptor kinase 2 (GRK2) and GRK5 was detected in septic neutrophils and control cells treated with cytokines plus LPS. Data suggest that endogenous mediators produced during sepsis might continually activate circulating neutrophils, leading to GRK activation, which may induce neutrophil desensitization to chemoattractants.

Competitive displacement of phosphoinositide 3-kinase from beta-adrenergic receptor kinase-1 improves postinfarction adverse myocardial remodeling.

Adverse remodeling after myocardial infarction (MI) determines the progression of heart failure. Failing hearts are characterized by downregulation of beta-adrenergic receptor (beta-AR) signaling in part because of increased beta-AR kinase 1 activity. Our previous studies have shown that overexpression of the phosphoinositide kinase (PIK) domain of phosphoinositide 3-kinase (PI3K), prevents beta-AR downregulation and enhances adrenergic agonist responsiveness by inhibiting the targeting of PI3K to the beta-AR complex. To investigate whether preventing beta-AR downregulation in the heart ameliorates cardiac function post-MI, transgenic mice with cardiac-specific overexpression of the PIK domain peptide (TgPIK) underwent left coronary artery ligation and were subsequently followed by serial echocardiography at 4, 8, 12, 16, and 20 wk. Despite having similar infarction sizes, TgPIK mice showed better systolic function, less cardiac dilatation, and improved hemodynamic response to dobutamine compared with littermate controls after MI. To test that displacement of PI3K from the beta-AR complex, but not the total loss of PI3K-gamma, is critical for amelioration of cardiac function, mice lacking the PI3K-gamma (PI3K-gamma-KO) underwent MI, and their cardiac function was assessed 20 wk post-MI. Serial echocardiographic measurements showed severe reduction in contractile performance in PI3K-gamma-KO compared with TgPIK mice. Furthermore, significant beta-AR downregulation and desensitization were only seen in infarcted wild-type and PI3K-gamma-KO mice and not in TgPIK mice. Together, these results demonstrate that adverse remodeling of the ventricle after MI can be attenuated by a strategy that prevents recruitment of PI3K to the plasma membrane and restores normal beta-AR function.

Beta-adrenergic receptor trafficking by exercise in rat adipocytes: roles of G-protein-coupled receptor kinase-2, beta-arrestin-2, and the ubiquitin-proteasome pathway.

The effect of exercise on beta-adrenergic receptor (beta-AR) trafficking was investigated in rat adipocytes. The binding sites of a hydrophilic ligand, [(3)H]CGP12177, increased immediately (0 h) and at 3 h after exercise (3 h) but decreased at 24 h after exercise (24 h). The data of immunoblotting revealed that the alterations in the binding sites mainly paralleled the alterations in the beta2-AR proteins in membrane fractions. The protein expressions of both G-protein-coupled receptor kinase (GRK)-2 and beta-arrestin-2 were reduced, with a decline in beta2-AR ubiquitination at 0 h and 3 h. The protein expressions of beta2-AR, GRK-2, beta-arrestin-2, the beta2-AR/beta-arrestin-2 complex, and beta2-AR ubiquitination returned to their respective control levels at 24 h, whereas the beta2-AR mRNA level was reduced. Administration of either lactacystin or propranolol did not alter GRK-2 and beta2-AR protein expressions after exercise. Thus, the mechanism underlying the increased density of beta2-AR up to at least 3 h may involve alterations in a multistep event involving the coordinate interaction among proteins mediating beta2-AR trafficking, in which both the receptor-agonist interactions and ubiquitin-proteasome pathway have a key role. However, the decreased protein expression of beta2-AR at 24 h might be due to some change occurring at the translational levels.