Temporal correlation between wall shear stress and in-stent stenosis after Wingspan stent in swine model.

BACKGROUND AND PURPOSE: A recent randomized clinical trial on intracranial atherosclerosis was discontinued because of the higher frequency of stroke and death in the angioplasty and stent placement group than in the medical treatment group. An in-depth understanding of the relationship between biologic responses and flow dynamics is still required to identify the current limitations of intracranial stent placement. MATERIALS AND METHODS: Five Wingspan stents were deployed in tapered swine ascending pharyngeal arteries. Temporal wall shear stress distributions and in-stent stenosis were evaluated at days 0, 7, 14, and 28 after stent placement. The physiologic role of wall shear stress was analyzed regarding its correlation with in-stent stenosis. RESULTS: In-stent stenosis reached a peak of nearly 40% at day 14 and decreased mainly at the distal stent segment until day 28. The wall shear stress demonstrated a characteristic pattern with time on the basis of the in-stent stenosis change. The wall shear stress gradient increased from the proximal to distal segment until day 14. At day 28, the trend was reversed dramatically, decreasing from the proximal to the distal segment. A significant correlation between the in-stent stenosis growth until day 14 and low wall shear stress values just after stent placement was detected. In-stent stenosis regression between days 14 and 28 was also associated with the high wall shear stress values at day 14. CONCLUSIONS: These data suggest that the physiologic wall shear stress can control the biphasic in-stent stenosis change in tapered arteries.

Real-time G-protein-coupled receptor imaging to understand and quantify receptor dynamics.

Understanding the trafficking of G-protein-coupled receptors (GPCRs) and their regulation by agonists and antagonists is fundamental to develop more effective drugs. Optical methods using fluorescent-tagged receptors and spinning disk confocal microscopy are useful tools to investigate membrane receptor dynamics in living cells. The aim of this study was to develop a method to characterize receptor dynamics using this system which offers the advantage of very fast image acquisition with minimal cell perturbation. However, in short-term assays photobleaching was still a problem. Thus, we developed a procedure to perform a photobleaching-corrected image analysis. A study of short-term dynamics of the long isoform of the dopamine type 2 receptor revealed an agonist-induced increase in the mobile fraction of receptors with a rate of movement of 0.08 µm/s For long-term assays, the ratio between the relative fluorescence intensity at the cell surface versus that in the intracellular compartment indicated that receptor internalization only occurred in cells co-expressing G protein-coupled receptor kinase 2. These results indicate that the lateral movement of receptors and receptor internalization are not directly coupled. Thus, we believe that live imaging of GPCRs using spinning disk confocal image analysis constitutes a powerful tool to study of receptor dynamics.

[Diagnosis and treatment of osteoporosis. Position of the Mexican College of Orthopedics and Traumatology]. / Diagnóstico y tratamiento de la osteoporosis. Posición del Colegio Mexicano de Ortopedia y Traumatología.

Our current in Mexico is that it represents a serious health problem not yet recognized as low-energy fractures in older adults account for approximately 10% of subjects over 65 years (compared with 29% in Japan) about 4.4 million fractures in patients over 70 years, taking into account that we are a nation of 112 million, the problem is minor compared with other diseases in this and other population groups. In the Mexican health system, orthopedic services instead share with other health priorities, so that the authorities do not understand osteoporosis as a health problem, not observe increased morbidity and mortality that implicitly leads, there are few centers to support the diagnosis of osteoporosis (densitometers do not have), and recruitment, diagnosis and management of patients who have suffered a broken ground mechanically compromised. Have increased the frequency of fractures in osteoporotic ground, and institutional level has only treatments based on calcitriol and calcium to maintain bone mineral density. In the Mexican health system, orthopedic services instead share with other health priorities, so that the authorities do not understand osteoporosis as a health problem, not observe increased morbidity and mortality that implicitly leads, there are few centers to support the diagnosis of osteoporosis (we don't count with densitometers), and recruitment, diagnosis and management of patients who have suffered a broken ground mechanically compromised. Have increased the frequency of fractures in osteoporotic ground, and institutional level has only treatments based on calcitriol and calcium to maintain bone mineral density.

GRK2-dependent desensitization downstream of G proteins.

G protein-coupled receptor kinases (GRKs) are serine/threonine kinases first discovered by its role in receptor desensitization. Phosphorylation of the C-terminal tail of GPCRs by GRKs triggers the docking of beta-arrestins and the functional uncoupling of G proteins and receptors. In addition, we and others have uncovered new direct ways by which GRKs could impinge into intracellular signalling pathways independently of receptor phosphorylation. In particular, we have characterized that elevated GRK2 levels can reduce CCR2-mediated activation of the ERK MAPK route in a manner that is independent of kinase activity and also of G proteins. This inhibition of ERK occurred in the absence of any reduction on MEK phosphorylation, what implicates that GRK2 is acting at the level of MEK or at the MEK-ERK interface to achieve a downregulation of ERK phosphorylation. In fact, we describe here that a direct association between GRK2 and MEK proteins can be detected in vitro. p38 MAPK pathway also appears to be regulated directly by GRK2 in a receptor-independent manner. p38 can be phosphorylated by GRK2 in threonine 123, a residue sitting at the entrance of a docking groove by which this MAPK associates to substrates and upstream activators. The T123phospho-mimetic mutant of p38 shows a reduced ability to bind to MKK6, concomitant with an impaired p38 activation, and a decreased phosphorylation of downstream substrates such as MEF2, MK2 and ATF2. Elevated levels of GRK2 downregulate p38-dependent cellular responses, such as differentiation of preadipocytic cells, while LPS-induced cytokine release is enhanced in macrophages from GRK2 (+/-) mice. In sum, we describe in this article different ways by which GRK2 directly regulates MAPK-mediated cellular events. This regulation of the MAPK modules by GRK2 could be relevant in pathological situations where the levels of this kinase are altered, such as during inflammatory diseases or cardiovascular pathologies.

Hydrogen peroxide impairs GRK2 translation via a calpain-dependent and cdk1-mediated pathway.

Oxidative mechanisms of injury are involved in many neurodegenerative diseases such as stroke, ischemia-reperfusion injury and multiple sclerosis. G protein-coupled receptor kinase 2 (GRK2) plays a key role in G protein-coupled receptor (GPCR) signaling modulation, and its expression levels are decreased after brain hypoxia/ischemia and reperfusion as well as in several inflammatory conditions. We report here that hydrogen peroxide downregulates GRK2 expression in C6 rat glioma cells. The hydrogen peroxide-induced decrease in GRK2 is prevented by a calpain protease inhibitor, but does not involve increased GRK2 degradation or changes in GRK2 mRNA level. Instead we show that hydrogen peroxide treatment impairs GRK2 translation in a process that requires Cdk1 activation and involves the mTOR pathway. This novel mechanism for the control of GRK2 expression in glial cells upon oxidative stress challenge may contribute to the modulation of GPCR signaling in different pathological conditions.

Beta-arrestin- and c-Src-dependent degradation of G-protein-coupled receptor kinase 2.

G-protein-coupled receptor kinase 2 (GRK2) plays a key role in the regulation of G-protein-coupled receptors (GPCRs). GRK2 expression is altered in several pathological conditions, but the molecular mechanisms that modulate GRK2 cellular levels are largely unknown. We recently have described that GRK2 is degraded rapidly by the proteasome pathway. This process is enhanced by GPCR stimulation and is severely impaired in a GRK2 mutant that lacks kinase activity (GRK2-K220R). In this report, we find that beta-arrestin function and Src-mediated phosphorylation of GRK2 are critically involved in GRK2 proteolysis. Overexpression of beta-arrestin triggers GRK2-K220R degradation based on its ability to recruit c-Src, since this effect is not observed with beta-arrestin mutants that display an impaired c-Src interaction. The presence of an inactive c-Src mutant or of tyrosine kinase inhibitors strongly inhibits co-transfected or endogenous GRK2 turnover, respectively, and a GRK2 mutant with impaired phosphorylation by c-Src shows a markedly retarded degradation. This pathway for the modulation of GRK2 protein stability puts forward a new feedback mechanism for regulating GRK2 levels and GPCR signaling.

Effect of hypothyroidism on G protein-coupled receptor kinase 2 expression levels in rat liver, lung, and heart.

GRK2 is a member of the G protein-coupled receptor kinase family that phosphorylates the activated form of beta-adrenergic and other G protein-coupled receptors and plays an important role in their desensitization and modulation. Alterations in thyroid hormone levels have been reported to lead to important changes in adrenergic receptor responsiveness and signaling in a variety of tissues. In this context, we have explored the effects of experimental hypothyroidism on GRK2 protein levels in rat heart, lung, and liver using a specific antibody. Hypothyroid animals show significant up-regulation ( approximately 50% increase compared with controls) in GRK2 levels in heart and lung at 60 days after birth, whereas a 50% reduction is detected in the liver at this stage. These alterations are selective, as beta-adrenergic receptors or other G protein-coupled receptor regulatory proteins, such as G protein-coupled receptor kinase 5 or beta-arrestin-1, display a different pattern of expression changes in the hypothyroid animals. The reported changes in GRK2 levels and in the receptor/kinase ratio predict alterations in adrenergic receptor desensitization and signal transduction efficacy consistent with those observed in thyroid disorders, thus suggesting a relevant role for the modulation of GRK2 expression in this physiopathological condition.

Expression of the G protein-coupled receptor kinase 2 during early mouse embryogenesis.

Whilst several G protein-coupled receptors (GPCRs) have been shown to play important roles during development, little study has been carried out on the G protein-coupled receptor kinases (GRKs) that modulate their activity in embryos. Here, we have analyzed the expression of GRK2, the predominant GRK expressed during embryogenesis. We show that at early stages, the expression of GRK2 is restricted to populations of cells that are undifferentiated, multipotent and in many cases, migratory. As such, GRK2 transcripts were found in the early mesoderm and neural crest as they migrate from the primitive streak and the neural tube, respectively. In the limb bud, GRK2 transcripts were observed in cells of the progress zone and in the interdigital areas. At later stages, the expression in the heart is compatible with the phenotype observed in the GRK2 deficient mice.

Expression patterns of the regulatory proteins G protein-coupled receptor kinase 2 and beta-arrestin 1 during rat postnatal brain development: effect of hypothyroidism.

G protein-coupled receptor kinase 2 (GRK2) and beta-arrestin 1 are key regulatory proteins that modulate the desensitization and resensitization of a wide variety of G protein-coupled receptors (GPCRs) involved in brain functions. In this report, we describe the postnatal developmental profile of the mRNA and protein levels of GRK2 and beta-arrestin 1 in rat brain. The expression levels of GRK2 and beta-arrestin 1 display a marked increase at the second and third week after birth, respectively, consistent with an involvement of these proteins in brain maturation processes. However, the expression attained at birth and during the first postnatal week with respect to adult values (45-70% for GRK2, approximately 30% for beta-arrestin 1) is relatively high compared to that reported for several GPCRs, indicating the existence of changes in the ratio of receptors to their regulatory proteins during brain development. On the other hand, we report that experimental hypothyroidism results in changes in the patterns of expression of GRK2 and beta-arrestin 1 in cerebral cortex, leading to a 25-30% reduction in GRK2 levels at several stages of development. Such changes could help to explain the alterations in GPCR signaling that occur during this pathophysiological condition.

Phosphorylation of phosducin and phosducin-like protein by G protein-coupled receptor kinase 2.

G protein-coupled receptor kinase 2 (GRK2) is able to phosphorylate a variety of agonist-occupied G protein-coupled receptors (GPCR) and plays an important role in GPCR modulation. However, recent studies suggest additional cellular functions for GRK2. Phosducin and phosducin-like protein (PhLP) are cytosolic proteins that bind Gbetagamma subunits and act as regulators of G-protein signaling. In this report, we identify phosducin and PhLP as novel GRK2 substrates. The phosphorylation of purified phosducin and PhLP by recombinant GRK2 proceeds rapidly and stoichiometrically (0.82 +/- 0.1 and 0.83 +/- 0.09 mol of P(i)/mol of protein, respectively). The phosphorylation reactions exhibit apparent K(m) values in the range of 40-100 nm, strongly suggesting that both proteins could be endogenous targets for GRK2 activity. Our data show that the site of phosducin phosphorylation by GRK2 is different and independent from that previously reported for the cAMP-dependent protein kinase. Analysis of GRK2 phosphorylation of a variety of deletion mutants of phosducin and PhLP indicates that the critical region for GRK2 phosphorylation is localized in the C-terminal domain of both phosducin and PhLP (between residues 204 and 245 and 195 and 218, respectively). This region is important for the interaction of these proteins with G beta gamma subunits. Phosphorylation of phosducin by GRK2 markedly reduces its G beta gamma binding ability, suggesting that GRK2 may modulate the activity of the phosducin protein family by disrupting this interaction. The identification of phosducin and PhLP as new substrates for GRK2 further expands the cellular roles of this kinase and suggests new mechanisms for modulating GPCR signal transduction.

Analysis of the human G protein-coupled receptor kinase 2 (GRK2) gene promoter: regulation by signal transduction systems in aortic smooth muscle cells.

BACKGROUND: Desensitization of G protein-coupled receptors (GPCR) is emerging as an important feature of several cardiovascular diseases. G protein-coupled receptor kinase 2 (GRK2) plays a key role in the regulation of a variety of these receptors, and its cardiac expression levels are altered in pathological situations such as chronic heart failure. However, very little is known about the signals and mechanisms that modulate GRK2 expression in cardiovascular cells. METHODS AND RESULTS: We have studied the transcriptional activity of the 1.6-kb-long proximal genomic region of the human GRK2 gene. In an aortic smooth muscle cell line, agents that lead to physiological vasoconstriction and hypertrophy, such as phorbol esters, increased GRK2 promoter activity. Activation of signaling pathways by cotransfected G(alphaq) subunits or alpha(1)-adrenergic receptors also markedly enhanced the expression of the GRK2 promoter constructs. Conversely, proinflammatory cytokines, such as interleukin-1beta, tumor necrosis factor-alpha, or interferon-gamma, led to the opposite effect, decreasing the activity of the GRK2 promoter. CONCLUSIONS: Our results suggest that the expression of GRK2 in vascular cells is tightly controlled at the transcriptional level by the interplay between several extracellular messengers, which may trigger alterations of normal GRK2 levels in some physiopathological circumstances, thus promoting changes in the efficacy of the GPCR signal transduction.

Agonist-dependent modulation of G protein-coupled receptor kinase 2 by mitogen-activated protein kinases.

A variety of G protein-coupled receptors (GPCRs) are phosphorylated by G protein-coupled receptor kinase 2 (GRK2). This event promotes the binding of regulatory proteins termed beta-arrestins to GPCRs, leading to uncoupling from G proteins and receptor internalization. Recent data indicate that GRK2 and beta-arrestins also play an important role in the stimulation of the extracellular signal-regulated kinases (ERK)/mitogen-activated protein kinase (MAPK) cascade by GPCRs. In this report, we have investigated the existence of functional interactions between GRK2 and MAPK. We show that activation of beta(2)-adrenergic receptors (beta(2)-AR) promotes the rapid association of GRK2 and MAPK in living cells, as assessed by coimmunoprecipitation experiments in COS-7 cells transfected with beta(2)-AR, GRK2, and an epitope-tagged MAPK. Coimmunoprecipitation of MAPK and GRK2 is blocked by inhibition of the MAPK cascade and is not observed upon activation of MAPK in the absence of beta(2)-AR stimulation, thus indicating that both an active MAPK and agonist occupancy of GPCR are required for the association to occur. Interestingly, we have found that purified ERK1/MAPK can directly phosphorylate the C-terminal domain of GRK2, and that the phosphorylation process is favored by the presence of Gbetagamma-subunits or an activated receptor. Furthermore, GRK2 phosphorylation by MAPK leads to a decreased activity of GRK2 toward GPCR. Taken together, our results suggest that stimulation of GPCRs promotes the rapid association of GRK2 and MAPK leading to modulation of GRK2 functionality, thus putting forward a new feedback mechanism for the regulation of GPCR signaling.

Agonist-dependent phosphorylation of the G protein-coupled receptor kinase 2 (GRK2) by Src tyrosine kinase.

GRK2 is a member of the G protein-coupled receptor kinase (GRK) family, which phosphorylates the activated form of a variety of G protein-coupled receptors (GPCR) and plays an important role in GPCR modulation. It has been recently reported that stimulation of the mitogen-activated protein kinase cascade by GPCRs involves tyrosine phosphorylation of docking proteins mediated by members of the Src tyrosine kinase family. In this report, we have investigated the possible role of c-Src in modulating GRK2 function. We demonstrate that c-Src can directly phosphorylate GRK2 on tyrosine residues, as shown by in vitro experiments with purified proteins. The phosphorylation reaction exhibits an apparent K(m) for GRK2 of 12 nM, thus suggesting a physiological relevance in living cells. Consistently, overexpression of the constitutively active c-Src Y527F mutant in COS-7 cells leads to tyrosine phosphorylation of co-expressed GRK2. In addition, GRK2 can be detected in phosphotyrosine immunoprecipitates from HEK-293 cells transiently transfected with this Src mutant. Interestingly, phosphotyrosine immunoblots reveal a rapid and transient increase in GRK2 phosphorylation upon agonist stimulation of beta(2)-adrenergic receptors co-transfected with GRK2 and wild type c-Src in COS-7 cells. This tyrosine phosphorylation is maximal within 5 min of isoproterenol stimulation and reaches values of approximately 5-fold over basal conditions. Furthermore, GRK2 phosphorylation on tyrosine residues promotes an increased kinase activity toward its substrates. Our results suggest that GRK2 phosphorylation by c-Src is inherent to GPCR activation and put forward a new mechanism for the regulation of GPCR signaling.

Degradation of the G protein-coupled receptor kinase 2 by the proteasome pathway.

GRK2 is a ubiquitous member of the G protein-coupled receptor kinase (GRK) family and has been shown to play a key role in determining the desensitization and resensitization patterns of a variety of G protein-coupled receptors. In this report, we show that GRK2 is actively degraded by the proteasome proteolytic pathway, unveiling a new mechanism for the rapid regulation of its expression levels. Interestingly, activation of beta2-adrenergic receptors (beta2AR) markedly increases GRK2 ubiquitination and degradation through the proteasome pathway. In addition, blocking GRK2 degradation notably alters beta2AR signaling and internalization, consistent with a relevant physiological role for GRK2 proteasomal degradation. Activity-dependent modulation of GRK2 cellular levels emerges as an important mechanism for modulating the cellular response to agonists acting through G protein-coupled receptors.

The subcellular and cellular distribution of G protein-coupled receptor kinase 2 in rat brain.

G protein-coupled receptor kinase 2 has been found to phosphorylate and thus regulate the activity of several G protein-coupled receptors implicated in neuronal signalling pathways. Although this kinase was initially described as a soluble protein, our laboratory has recently found that a significant amount of G protein-coupled receptor kinase 2 is associated with microsomal membranes in liver and different types of cultured cells. In the present report we show that high G protein-coupled receptor kinase 2 specific activity and protein levels are present in microsomal fractions of rat brain homogenates. On the other hand, immunochemical detection using a new antibody raised against the N-terminus of the kinase revealed a specific and widely distributed staining in different areas of the central nervous system, and the association of G protein-coupled receptor kinase 2 with intracellular structures in nervous cells. Our results further suggest that this receptor kinase may be involved in the modulation of G protein-coupled receptor-mediated neurotransmission and that association with microsomal membranes may play a role in G protein-coupled receptor kinase 2 functions in the brain.

G protein-coupled receptor kinase 2 (GRK2): mechanisms of regulation and physiological functions.

G protein-coupled receptor kinase 2 (GRK2) plays a key role in determining the rate and extent of G protein-coupled receptor (GPCR) desensitization and resensitization. Recent data indicate that GRK2 activity, subcellular distribution and expression are tightly regulated. The important physiological function of GRK2 as a modulator of the efficacy of GPCR signal transduction systems is exemplified by its relevance in cardiovascular physiopathology as well as by its emerging role in the regulation of chemokine receptors.

The chemokine monocyte chemotactic protein 1 triggers Janus kinase 2 activation and tyrosine phosphorylation of the CCR2B receptor.

The chemokines are a growing family of low m.w., 70- to 80-residue proinflammatory cytokines that operate by interacting with G protein-coupled receptors. Chemokines are involved in cell migration and in the activation of specific leukocyte subsets. Using the Mono Mac 1 monocytic cell line, we show that monocyte chemotactic protein 1 (MCP-1) triggers activation of the Janus kinase 2 (JAK2)/STAT3 pathway and CCR2 receptor tyrosine phosphorylation. Both Ca2+ mobilization and cell migration are blocked in Mono Mac 1 cells by tyrphostin B42, a specific JAK2 kinase inhibitor. Within seconds of MCP-1 activation, JAK2 phosphorylates CCR2 at the Tyr139 position and promotes JAK2/STAT3 complex association to the receptor. This MCP-1-initiated phosphorylation and association to JAK2 is also observed in CCR2B-transfected HEK293 cells. In contrast, when a CCR2B Tyr139Phe mutant is expressed in HEK293 cells, it is not phosphorylated in tyrosine and triggers neither JAK2/STAT3 activation nor Ca2+ mobilization in response to MCP-1. These results implicate the tyrosine kinase pathway in early chemokine signaling, suggesting a key role for this kinase in later events.

Monocyte chemoattractant protein-1-induced CCR2B receptor desensitization mediated by the G protein-coupled receptor kinase 2.

Monocyte chemoattractant protein 1 (MCP-1) is a member of the chemokine cytokine family, whose physiological function is mediated by binding to the CCR2 and CCR4 receptors, which are members of the G protein-coupled receptor family. MCP-1 plays a critical role in both activation and migration of leukocytes. Rapid chemokine receptor desensitization is very likely essential for accurate chemotaxis. In this report, we show that MCP-1 binding to the CCR2 receptor in Mono Mac 1 cells promotes the rapid desensitization of MCP-1-induced calcium flux responses. This desensitization correlates with the Ser/Thr phosphorylation of the receptor and with the transient translocation of the G protein-coupled receptor kinase 2 (GRK2, also called beta-adrenergic kinase 1 or betaARK1) to the membrane. We also demonstrate that GRK2 and the uncoupling protein beta-arrestin associate with the receptor, forming a macromolecular complex shortly after MCP-1 binding. Calcium flux responses to MCP-1 in HEK293 cells expressing the CCR2B receptor were also markedly reduced upon cotransfection with GRK2 or the homologous kinase GRK3. Nevertheless, expression of the GRK2 dominant-negative mutant betaARK-K220R did not affect the initial calcium response, but favored receptor response to a subsequent challenge by agonists. The modulation of the CCR2B receptor by GRK2 suggests an important role for this kinase in the regulation of monocyte and lymphocyte response to chemokines.

Regulation of G protein-coupled receptor kinase 2 in brains of opiate-treated rats and human opiate addicts.

The effects of opiate drugs (heroin, morphine, and methadone) on the levels of G protein-coupled receptor kinase 2 (GRK2) were studied in rat and human brain frontal cortices. The density of brain GRK2 was measured by immunoblot assays in acute and chronic opiate-treated rats as well as in opiate-dependent rats after spontaneous or naloxone-precipitated withdrawal and in human opiate addicts who had died of an opiate overdose. In postmortem brains from human addicts, total GRK2 immunoreactivity was not changed significantly, but the level of the membrane-associated kinase was modestly but significantly increased (12%) compared with matched controls. In rats treated chronically with morphine or methadone modest increases of the enzyme levels (only significant after methadone) were observed. Acute treatments with morphine and methadone induced dose- and time-dependent increases (8-22%) in total GRK2 concentrations [higher increases were observed for the membrane-associated enzyme (46%)]. Spontaneous and naloxone-precipitated withdrawal after chronic morphine or methadone induced a marked up-regulation in the levels of total GRK2 in the rat frontal cortex (18-25%). These results suggest that GRK2 is involved in the short-term regulation of mu-opioid receptors in vivo and that the activity of this regulatory kinase in brain could have a relevant role in opiate tolerance, dependence, and withdrawal.