Retrospective analysis of real-world prescribing data for managing cisplatin-based chemotherapy-induced nausea and vomiting in China.

BACKGROUND: The current utilization of neurokinin-1 receptor antagonists (NK1RAs) and the impact of updated guidelines on prescription patterns of antiemetic drugs among Chinese patients receiving highly emetogenic chemotherapy (HEC) remain undetermined. This study aims to analyze the present situation of Chinese cancer patients using antiemetic drugs and assess the appropriateness of antiemetic regimens. METHODS: Prescription data were collected between January 2015 and December 2020 from cancer patients receiving cisplatin-based chemotherapy at 76 hospitals in six major cities in China. Trends in the use of antiemetic drugs, prescribing patterns and adherence to antiemetic guidelines were assessed. RESULTS: Among the 108,611 patients included in this study, 6 classes and 17 antiemetic drugs were identified as monotherapy or combination therapy in 93,872 patients (86.4%), whereas 14,739 patients (13.6%) were administered no antiemetic treatment. 5-hydroxytryptamine 3 receptor antagonists (5-HT3RAs) and glucocorticoids were the two most frequently used classes of antiemetics, followed by metoclopramide. NK1RAs were underused across the six cities, only 9332 (8.6%) and 1655 (1.5%) cisplatin-based treatments were prescribed aprepitant and fosaprepitant, respectively. Prescriptions of olanzapine and lorazepam were very low throughout the study period. In prescribing patterns of antiemetic drugs, dual combination regimens were the most common (40.0%), followed by triple combination therapy and monotherapy (25.8% and 15.1%, respectively). Overall, the adherence to antiemetic guidelines for patients undergoing cisplatin-based regimens was only 8.1% due to inadequate prescription of antiemetic drugs. Finally, our study also revealed that 5-HT3RAs and glucocorticoids were overprescribed in 8.8% and 1.6% of patients, respectively. CONCLUSIONS: The current study reveals suboptimal utilization of recommended antiemetic drugs for managing cisplatin-based HEC-induced nausea and vomiting in China. Improving the management of CINV is crucial, and these findings provide valuable insights into optimizing antiemetic drug practices.

Comparison of clinical and economic evaluation between selected generic and original febuxostat tablets in Chinese gout patients with hyperuricemia: A real-world multicenter retrospective study.

Generic febuxostat tablets were listed in China's third-round centralized drug procurement program. However, there are no sufficient data available on the use of febuxostat in a real-world setting. This study aimed to compare the efficacy, safety, and cost of selected generic febuxostat with original febuxostat in primary gout and hyperuricemia. Medical records at 3 tertiary hospitals from January 2014 to February 2022 were retrospectively analyzed. Propensity score matching was used to balance the distribution of baseline characteristics. The proportion of patients achieving target serum uric acid (SUA) levels at 12 weeks, the percent changes from baseline in SUA, adverse drug reactions, and the cost of febuxostat therapy were assessed. A total of 221 patients were recruited and 57 pairs of patients were 1:1 matched in the 2 groups. There was no statistically significant difference in the proportion of patients achieving a target SUA levels below 300 µmol/L, the percent changes of SUA decreased from baseline, and the incidence of adverse drug reactions between the 2 groups (all P > .05). The daily febuxostat cost in the generic group were significantly lower than that in original group (P < .05). Based on the results of this study, the clinical efficacy of selected generic febuxostat is comparable to that of original febuxostat for gout with hyperuricemia. No serious adverse reactions were reported in the 2 groups, and generic febuxostat is more economical than the original febuxostat.

Correlation of functional and radioligand binding characteristics of GPER ligands confirming aldosterone as a GPER agonist.

Aldosterone exerts some of its effects not by binding to mineralocorticoid receptors, but rather by acting via G protein-coupled estrogen receptors (GPER). To determine if aldosterone binds directly to GPER, we studied the ability of aldosterone to compete for the binding of [3 H] 2-methoxyestradiol ([3 H] 2-ME), a high potency GPER-selective agonist. We used GPER gene transfer to engineer Sf9-cultured insect cells to express GPER. We chose insect cells to avoid interactions with any intrinsic mammalian receptors for aldosterone. [3 H] 2-ME binding was saturable and reversible to a high-affinity population of receptors with Kd  = 3.7 nM and Bmax  = 2.2 pmol/mg. Consistent with agonist binding to G Protein-coupled receptors, [3 H] 2-ME high-affinity state binding was reduced in the presence of the hydrolysis-resistant GTP analog, GppNHp. [3 H] 2-ME binding was competed for by the GPER agonist G1, the GPER antagonist G15, estradiol (E2), as well as aldosterone (Aldo). The order of potency for competing for [3 H] 2-ME binding, namely 2ME > Aldo > E2 ≥ G1, paralleled the orders of potency for inhibition of cell proliferation and inhibition of ERK phosphorylation by ligands acting at GPER. These data confirm the ability of aldosterone to interact with the GPER, consistent with the interpretation that aldosterone likely mediates its GPER-dependent effects by direct binding to the GPER. SIGNIFICANCE STATEMENT: Despite the growing evidence for aldosterone's actions via G protein-coupled estrogen receptors (GPER), there remains significant skepticism that aldosterone can directly interact with GPER. The current studies are the first to demonstrate directly that aldosterone indeed is capable of binding to the GPER and thus likely mediates its GPER-dependent effects by direct binding to the receptor.

Evaluation of Activated Carbon Treatment on Overcoming the Interference of Rivaroxaban on the Detection of Functional Lupus Anticoagulant.

OBJECTIVE: The aim of this study was to review the role of activated carbon (AC) in eliminating the interference of rivaroxaban in the detection of lupus anticoagulants (LAs). METHODS: Normal pooled plasma was obtained as group N1, group N2 took 1 mL plasma from N1 and added AC, group N3 was prepared by mixing normal plasma with rivaroxaban, and group N3 was treated with AC according to our procedure, as group N4. Plasma from 22 patients was collected before and 6-12 h after rivaroxaban therapy, described as group P1 and group P2, respectively, and 1 mL plasma was taken from group P2 and treated with AC, as group P3. Anti-Xa and diluted Russell's viper venom time (dRVVT)/silica clotting time (SCT) index in each group were measured and compared. RESULTS: Rivaroxaban concentrations and anti-Xa had high intercorrelations in group N3, and the levels of anti-Xa and dRVVT/SCT index had high intercorrelations. After treatment with AC, influence of rivaroxaban was removed, with LA and coagulation factor assays not influenced. Rivaroxaban administration could affect LA assay results in patients, with all LA results increased. After treatment with AC, results of anti-Xa and LA tests recovered to the level before rivaroxaban therapy. CONCLUSIONS: We proposed a reference procedure for the LA detection of patients using rivaroxaban by AC, and activated carbon was proven to be a simple product to eliminate the interference of rivaroxaban.

Extent of Vascular Remodeling Is Dependent on the Balance Between Estrogen Receptor α and G-Protein-Coupled Estrogen Receptor.

Estrogens are important regulators of cardiovascular function. Some of estrogen's cardiovascular effects are mediated by a G-protein-coupled receptor mechanism, namely, G-protein-coupled estrogen receptor (GPER). Estradiol-mediated regulation of vascular cell programmed cell death reflects the balance of the opposing actions of GPER versus estrogen receptor α (ERα). However, the significance of these opposing actions on the regulation of vascular smooth muscle cell proliferation or migration in vitro is unclear, and the significance in vivo is unknown. To determine the effects of GPER activation in vitro, we studied rat aortic vascular smooth muscle cells maintained in primary culture. GPER was reintroduced using adenoviral gene transfer. Both estradiol and G1, a GPER agonist, inhibited both proliferation and cell migration effects that were blocked by the GPER antagonist, G15. To determine the importance of the GPER-ERα balance in regulating vascular remodeling in a rat model of carotid ligation, we studied the effects of upregulation of GPER expression versus downregulation of ERα. Reintroduction of GPER significantly attenuated the extent of medial hypertrophy and attenuated the extent of CD45 labeling. Downregulation of ERα expression comparably attenuated the extent of medial hypertrophy and inflammation after carotid ligation. These studies demonstrate that the balance between GPER and ERα regulates vascular remodeling. Receptor-specific modulation of estrogen's effects may be an important new approach in modifying vascular remodeling in both acute settings like vascular injury and perhaps in longer term regulation like in hypertension.

Nosocomial transmission of Clostridium difficile ribotype 027 in a Chinese hospital, 2012-2014, traced by whole genome sequencing.

BACKGROUND: The rapid spread of Clostridium difficile NAP1/BI/027 (C. difficile 027) has become one of the leading threats of healthcare-associated infections worldwide. However, C. difficile 027 infections have been rarely reported in Asia, particularly in China. RESULTS: In this study, we identified a rare C. difficile bloodstream infection (BSI) from three isolates of a patient during repeated hospital admission. This finding triggered a retrospective epidemiological study to scan all cases and strains emerged from this ward during the past three years. Using medical personnel interviews, medical record reviews and the genomic epidemiology, two outbreaks in 2012 and 2013-2014 were identified. Through using whole genome sequencing, we succeeded to trace the origin of the BSI strain. Surprisingly, we found the genome sequences were similar to C. difficile 027 strain R20291, indicating the occurrence of a rare C. difficile 027 strain in China. Integrated epidemiological investigation and whole genome sequencing of all strains, we constructed a nosocomial transmission map of these two C. difficile 027 outbreaks and traced the origin of the infection. CONCLUSIONS: By genome sequencing, spatio-temporal analysis and field epidemiology investigation, we can estimate their complex transform network and reveal the possible modes of transmission in this ward. Based on their genetic diversity, we can assume that the toilets, bathroom, and janitor's equipment room may be contaminated area, which may be suggested to improve infection control measures in the following health care.

Aldosterone mediates metastatic spread of renal cancer via the G protein-coupled estrogen receptor (GPER).

Although aldosterone is a known regulator of renal and cardiovascular function, its role as a regulator of cancer growth and spread has not been widely considered. This study tested the hypothesis that aldosterone regulates cancer cell growth/spread via G protein-coupled estrogen receptor (GPER) activation. In vitro in murine renal cortical adenocarcinoma (RENCA) cells, a widely used murine in vitro model for the study of renal cell adenocarcinoma, aldosterone increased RENCA cell proliferation to a maximum of 125 ± 3% of control at a concentration of 10 nM, an effect blocked by the GPER antagonist G15 or by GPER knockdown using short interfering (sh) RNA techniques. Further, aldosterone increased RENCA cell migration to a maximum of 170 ± 20% of control at a concentration of 100 nM, an effect also blocked by G15 or by GPER down-regulation. In vivo, after orthotopic RENCA cell renal transplantation, pulmonary tumor spread was inhibited by pharmacologic blockade of aldosterone effects with spironolactone (percentage of lung occupied by metastasis: control = 68 ± 13, spironolactone = 26 ± 8, P < 0.05) or inhibition of aldosterone synthesis with a high dietary salt diet (percentage of lung: control = 44 ± 6, high salt = 12 ± 3, P < 0.05), without reducing primary tumor size. Additionally, adrenalectomy significantly reduced the extent of pulmonary tumor spread, whereas aldosterone infusion recovered pulmonary metastatic spread toward baseline levels. Finally, inhibition of GPER either with the GPER antagonist G15 or by GPER knockdown comparably inhibited RENCA cell pulmonary metastatic cancer spread. Taken together, these findings provide strong evidence for aldosterone serving a causal role in renal cell cancer regulation via its GPER receptor; thus, antagonism of GPER represents a potential new target for treatment to reduce metastatic spread.-Feldman, R. D., Ding, Q., Hussain, Y., Limbird, L. E., Pickering, J. G., Gros, R. Aldosterone mediates metastatic spread of renal cancer via the G protein-coupled estrogen receptor (GPER).

G-protein estrogen receptor as a regulator of low-density lipoprotein cholesterol metabolism: cellular and population genetic studies.

OBJECTIVE: Estrogen deficiency is linked with increased low-density lipoprotein (LDL) cholesterol. The hormone receptor mediating this effect is unknown. G-protein estrogen receptor (GPER) is a recently recognized G-protein-coupled receptor that is activated by estrogens. We recently identified a common hypofunctional missense variant of GPER, namely P16L. However, the role of GPER in LDL metabolism is unknown. Therefore, we examined the association of the P16L genotype with plasma LDL cholesterol level. Furthermore, we studied the role of GPER in regulating expression of the LDL receptor and proprotein convertase subtilisin kexin type 9. APPROACH AND RESULTS: Our discovery cohort was a genetically isolated population of Northern European descent, and our validation cohort consisted of normal, healthy women aged 18 to 56 years from London, Ontario. In addition, we examined the effect of GPER on the regulation of proprotein convertase subtilisin kexin type 9 and LDL receptor expression by the treatment with the GPER agonist, G1. In the discovery cohort, GPER P16L genotype was associated with a significant increase in LDL cholesterol (mean±SEM): 3.18±0.05, 3.25±0.08, and 4.25±0.33 mmol/L, respectively, in subjects with CC (homozygous for P16), CT (heterozygotes), and TT (homozygous for L16) genotypes (P<0.05). In the validation cohort (n=339), the GPER P16L genotype was associated with a similar increase in LDL cholesterol: 2.17±0.05, 2.34±0.06, and 2.42±0.16 mmol/L, respectively, in subjects with CC, CT, and TT genotypes (P<0.05). In the human hepatic carcinoma cell line, the GPER agonist, G1, mediated a concentration-dependent increase in LDL receptor expression, blocked by either pretreatment with the GPER antagonist G15 or by shRNA-mediated GPER downregulation. G1 also mediated a GPER- and concentration-dependent decrease in proprotein convertase subtilisin kexin type 9 expression. CONCLUSIONS: GPER activation upregulates LDL receptor expression, probably at least, in part, via proprotein convertase subtilisin kexin type 9 downregulation. Furthermore, humans carrying the hypofunctional P16L genetic variant of GPER have increased plasma LDL cholesterol. In aggregate, these data suggest an important role of GPER in the regulation of LDL receptor expression and consequently LDL metabolism.

A common hypofunctional genetic variant of GPER is associated with increased blood pressure in women.

AIMS: Activation of vascular GPER has been linked to vasodepressor effects in animals. However, the significance of GPER regulation on chronic blood pressure control in humans is unknown. METHODS: To examine this question we determined the functional significance of expression of a common missense single nucleotide variant of GPER, P16L in vascular smooth muscle cells, and its association with blood pressure in humans. Further, to validate the importance of carrying GPER P16L in the development of hypertension we assessed allele frequency in a cohort of hard-to-treat hypertensive patients referred to a tertiary care clinic. RESULTS: Expression of the GPER P16L variant (V) vs. wild type (WT) in rat aortic vascular smooth muscle cells, was associated with a significant decrease in G1 (1 µm, a GPER agonist)-mediated ERK phosphorylation (slope of the function of G1-stimulated ERK phosphorylation: GPER content WT: 16.2, 95% CI 9.9, 22.6; V: 5.0, 95% CI 1.0, 9.0; P < 0.005) and apoptosis (slope of the function of G1-stimulated apoptosis: GPER content: WT: 4.4, 95% CI: 3.4, 5.4; V: 2.5, 95% CI 1.6, 2.3 P < 0.005). Normotensive female subjects, but not male subjects, carrying this hypofunctional variant (allele frequency 22%) have increased blood pressure [mean arterial pressure: P16/P16: 80 ± 1 mmHg (n = 204) vs. P16L carriers: 82 ± 1 mmHg (n = 127), 95% CI for difference: 0.6, 4.0 mmHg, P < 0.05], [systolic blood pressure: P16/P16: 105 ± 1 mmHg vs. P16L carriers: 108 ± 1 mmHg, 95% CI for difference:1.0, 5.1 mmHg, P < 0.05], [diastolic blood pressure: P16/P16: 66 ± 0.5 mmHg vs. P16L carriers 68 ± 0.7, 95% CI for difference: 0.2, 3.6 mmHg, P < 0.05]. Further, the P16L allele frequency was almost two-fold higher in female vs. male hypertensive patients (31% vs. 16%, allele ratio 0.5, 95% CI 0.32, 0.76, P < 0.05). CONCLUSIONS: The common genetic variant, GPER P16L, is hypofunctional and female carriers of this allele have increased blood pressure. There was an increased prevalence in a population of hard-to-treat hypertensive female patients. Cumulatively, these data suggest that in females, impaired GPER function might be associated with increased blood pressure and risk of hypertension.

Aldosterone mediates its rapid effects in vascular endothelial cells through GPER activation.

The importance of the rapid vascular effects of aldosterone is increasingly appreciated. Through these rapid pathways, aldosterone has been shown to regulate vascular contractility, cell growth, and apoptosis. In our most recent studies, we demonstrated the effects of aldosterone on cell growth and contractility in vascular smooth muscle cells. We showed that these effects could occur via activation of the classic mineralocorticoid receptor, as well the recently characterized G protein-coupled estrogen receptor (GPER), initially characterized as an estrogen-specific receptor. However, the mechanisms underlying aldosterone's endothelium-dependent actions are unknown. Furthermore, the ERK regulatory and proapoptotic effects of aldosterone mediated by GPER activation in cultured vascular smooth muscle cells were only apparent when GPER was reintroduced into these cells by gene transfer. Whether GPER activation via aldosterone might be an important regulator in native vascular cells has been questioned. Therefore, to determine the role of GPER in mediating aldosterone's effects on cell growth and vascular reactivity in native cells, we examined rat aortic vascular endothelial cells, a model characterized by persistent robust expression of GPER, but without detectable mineralocorticoid receptor expression. In these endothelial cells, the GPER agonist G1 mediates a rapid increase in ERK phosphorylation that is wholly GPER-dependent, paralleling the actions of aldosterone. The effects of G1 and aldosterone to stimulate ERK phosphorylation paralleled their proapoptotic and antiproliferative effects. In previous studies, we reported that aldosterone mediates a rapid endothelium-dependent vasodilatory effect, antagonistic to its direct vasoconstrictor effect in endothelium-denuded preparations. Using a rat aortic ring/organ bath preparation to determine the GPER dependence of aldosterone's endothelium-dependent vasodilator effects, we demonstrate that aldosterone inhibits phenylephrine-mediated contraction. This vasodilator effect parallels the actions of the GPER agonist G1. Furthermore, the effects of aldosterone were completely ablated by the GPER antagonist G15. These data support an important role of GPER activation in aldosterone-mediated regulation of endothelial cell growth, as well as in aldosterone's endothelium-mediated regulation of vasoreactivity.

Human papillomavirus 18 E1

The human papilloma virus E4 protein is highly expressed in late times of infection. Evidence to date suggests that E4 is essential for amplification of the viral genome and that it can influence cell cycle. Examination of the sequences encoding the E4 proteins from several genotypes of human papillomavirus revealed the presence of RXL-containing motifs reminiscent of the cyclin-binding motifs that have been identified in several cyclin-binding proteins. When baculovirus-produced human cyclin E and cyclin A with cdk2 were incubated in vitro with a GST-E4 fusion protein, both cyclin E and A stably interacted with the GST-E4 protein containing the full E4 sequence from HPV18. The interaction was not dependent on the presence of the kinase subunit but was dependent on the integrity of the RXL motif in E4. When incubated with cell extracts from the C33A human cervical carcinoma cell line or when expressed in C33A cells, the GST-E4 protein formed interactions with cyclin A and cdk2 and kinase activity could be demonstrated in the GST-E4 complex. In contrast to the baculovirus-produced cyclin E, cellular cyclin E failed to detectably interact with GST-E4 suggesting that the HPV18 E4 sequences are capable of interacting only with cyclin A in mammalian cells. These observations suggest that human papillomavirus E4 proteins can interact with cyclin A/cdk2, which may contribute to viral manipulation of the host cell cycle.

miR-146a-Mediated extracellular matrix protein production in chronic diabetes complications.

OBJECTIVE: MicroRNAs (miRNAs), through transcriptional regulation, modulate several cellular processes. In diabetes, increased extracellular matrix protein fibronectin (FN) production is known to occur through histone acetylator p300. Here, we investigated the role of miR-146a, an FN-targeting miRNA, on FN production in diabetes and its relationship with p300. RESEARCH DESIGN AND METHODS: miR-146a expressions were measured in endothelial cells from large vessels and retinal microvessels in various glucose levels. FN messenger RNA expression and protein levels with or without miR-146a mimic or antagomir transfection were examined. A luciferase assay was performed to detect miR-146a's binding to FN 3'-untranslated region (UTR). Likewise, retinas from type 1 diabetic rats were studied with or without an intravitreal injection of miR-146a mimic. In situ hybridization was used to localize retinal miR-146a. Cardiac and renal tissues were analyzed from type 1 and type 2 diabetic animals. RESULTS: A total of 25 mmol/L glucose decreased miR-146a expression and increased FN expression compared with 5 mmol/L glucose in both cell types. miR-146a mimic transfection prevented such change, whereas miR-146a antagomir transfection in the cells in 5 mmol/L glucose caused FN upregulation. A luciferase assay confirmed miR-146a's binding to FN 3'-UTR. miR-146a was localized in the retinal endothelial cells and was decreased in diabetes. Intravitreal miR-146a mimic injection restored retinal miR-146a and decreased FN in diabetes. Additional experiments showed that p300 regulates miR-146a. Similar changes were seen in the retinas, kidneys, and hearts in type 1 and type 2 diabetic animals. CONCLUSIONS: These studies showed a novel, glucose-induced molecular mechanism in which miR-146a participates in the transcriptional circuitry regulating extracellular matrix protein production in diabetes.

Delineating the receptor mechanisms underlying the rapid vascular contractile effects of aldosterone and estradiol.

It is increasingly appreciated that steroid hormones such as aldosterone and estradiol can mediate important cardiovascular effects. Many of these effects occur over a time course not consistent with the genomic actions of these hormones acting through classical nuclear receptors / transcription factors. Further, multiple receptors have been implicated in mediating these rapid effects of both aldosterone and estradiol, including a newly appreciated G-protein-coupled receptor, GPR30. In previous studies we demonstrated that both aldosterone and estradiol mediate contraction in vascular smooth muscle cells, as assessed in single cell assays. However, the receptor mechanisms underlying these effects remained unclear. Therefore, we studied the actions of estradiol and aldosterone on rat aortic vascular smooth muscle cells. Both aldosterone and estradiol mediated a concentration-dependent increase in contraction, as assessed in substrate deformation assays with EC(50)s in the range of nanomoles per litre. These effects paralleled increased myosin light chain phosphorylation. The effects of aldosterone were inhibited by the mineralocorticoid selective antagonist eplerenone. Further, aldosterone's contractile effects were enhanced by increased expression of the mineralocorticoid receptor. The contractile effects of estradiol were inhibited by estrogen receptor (ER)-selective antagonists, tamoxifen, and ICI 182780, as well as eplerenone. Further, estradiol's effects were enhanced by the increased expression of both ERα and the mineralocorticoid receptor (MR). To assess the potential role of GPR30 in mediating the effects of aldosterone and estradiol, GPR30 was re-introduced, since these cells lose endogenous GPR30 expression in culture. Re-expression of GPR30 enhanced both estradiol- and aldosterone-mediated contraction. These studies demonstrate that in rat aortic vascular smooth muscle cells, both aldosterone and estradiol mediate vascular smooth muscle contraction and that these effects can be mediated by MR, ERα, and by GPR30.

GPR30 expression is required for the mineralocorticoid receptor-independent rapid vascular effects of aldosterone.

It has been increasingly appreciated that steroids elicit acute vascular effects through rapid, so-called nongenomic signaling pathways. Though aldosterone, for example, has been demonstrated to mediate rapid vascular effects via both mineralocorticoid receptor-dependent and -independent pathways, the mechanism(s) of this mineralocorticoid receptor-independent effect of aldosterone is yet to be determined. For estrogen, its rapid effects have been reported to be, at least in part, mediated via the 7-transmembrane-spanning, G protein-coupled receptor GPR30. Previous studies have demonstrated common response outcomes in response to both aldosterone and estrogen on GPR30 expression, ie, activation of phosphatidylinositol 3-kinase-dependent contraction and extracellular signal-regulated kinase activation in vascular smooth muscle cells. The present studies were undertaken to test the hypothesis that the rapid response to aldosterone in smooth muscle is dependent on the availability of a GPR30-dependent signaling pathway. These findings not only reconcile differences in the literature for aldosterone response in freshly isolated versus cultured aortic smooth muscle cells but also suggest alternative therapeutic strategies for modulating aldosterone actions on the vasculature in vivo.

Estradiol-mediated ERK phosphorylation and apoptosis in vascular smooth muscle cells requires GPR 30.

Recent studies suggest that the rapid and nongenomic effects of estradiol may be mediated through the G protein-coupled receptor dubbed GPR30 receptor. The present study examines the role of GPR30 versus a classical estrogen receptor (ERalpha) in mediating the growth regulatory effects of estradiol. GPR30 is readily detectable in freshly isolated vascular tissue but barely detectable in cultured vascular smooth muscle cells (VSMC). In freshly isolated aortic tissue, estradiol stimulated extracellular signal-regulated kinases (ERK) phosphorylation. In contrast, in cultured VSMC, where GPR30 expression is significantly reduced, estradiol inhibits ERK phosphorylation. Transfer of the genes encoding GPR30 led to estradiol stimulation of ERK phosphorylation, which is opposite the effects of estradiol in the primary culture of VSMCs. Transduction of the mineralocorticoid receptor (MR) had no effect on estradiol effects on ERK. Estradiol-mediated stimulation of ERK subsequent to heterologous GPR30 expression was pertussis toxin sensitive and phosphoinositide 3-kinase (PI3 kinase) dependent; under these conditions, estradiol also inhibited protein kinase A (PKA). In contrast, in the absence of GPR30 expression in cultured VSMC, estradiol stimulated PKA activity and inhibited ERK phosphorylation. To determine the functional effect of GPR30 (vs. estrogen receptor expression), we assessed estradiol-mediated apoptosis. In the absence of GPR30 expression, estradiol inhibited apoptosis. This effect was enhanced with ERalpha expression. In contrast, with GPR30 expression, estradiol stimulated apoptosis in an ERK-dependent manner. Thus the effect of estradiol on vascular smooth muscle cell apoptosis is likely dependent on the balance between ER-mediated PKA activation and GPR30-mediated PKA inhibition and PI3 kinase activation. Taken together, we postulate that modulation of GPR30 expression or activity may be an important determinant of the effects of estradiol in the vasculature.

Phosphorylation-independent regulation of metabotropic glutamate receptor 5 desensitization and internalization by G protein-coupled receptor kinase 2 in neurons.

The uncoupling of metabotropic glutamate receptors (mGluRs) from heterotrimeric G proteins represents an essential feedback mechanism that protects neurons against receptor overstimulation that may ultimately result in damage. The desensitization of mGluR signaling is mediated by both second messenger-dependent protein kinases and G protein-coupled receptor kinases (GRKs). Unlike mGluR1, the attenuation of mGluR5 signaling in HEK 293 cells is reported to be mediated by a phosphorylation-dependent mechanism. However, the mechanisms regulating mGluR5 signaling and endocytosis in neurons have not been investigated. Here we show that a 2-fold overexpression of GRK2 leads to the attenuation of endogenous mGluR5-mediated inositol phosphate (InsP) formation in striatal neurons and siRNA knockdown of GRK2 expression leads to enhanced mGluR5-mediated InsP formation. Expression of a catalytically inactive GRK2-K220R mutant also effectively attenuates mGluR5 signaling, but the expression of a GRK2-D110A mutant devoid in Galpha(q/11) binding increases mGluR5 signaling in response to agonist stimulation. Taken together, these results indicate that the attenuation of mGluR5 responses in striatal neurons is phosphorylation-independent. In addition, we find that mGluR5 does not internalize in response to agonist treatment in striatal neuron, but is efficiently internalized in cortical neurons that have higher levels of endogenous GRK2 protein expression. When overexpressed in striatal neurons, GRK2 promotes agonist-stimulated mGluR5 internalization. Moreover, GRK2-mediated promotion of mGluR5 endocytosis does not require GRK2 catalytic activity. Thus, we provide evidence that GRK2 mediates phosphorylation-independent mGluR5 desensitization and internalization in neurons.

Increased enzyme activity and beta-adrenergic mediated vasodilation in subjects expressing a single-nucleotide variant of human adenylyl cyclase 6.

OBJECTIVE: cAMP is a critical regulator of metabolic and cardiovascular function. However, the role of genetic variability in the regulation of cAMP-mediated effects is unclear. Therefore, we assessed the effect of the expression of a recently identified missense genetic variant of adenylyl cyclase isoform 6 (ADCY6 S674). METHODS AND RESULTS: In rat vascular smooth muscle cells, gene transfer of ADCY6 S674 increased adenylyl cyclase activity and arborization to a greater extent than gene transfer of ADCY6 A674. Similarly, in adherent mononuclear leukocyte cells isolated from ADCY6 S674-expressing human subjects, both adenylyl cyclase activity and adenylyl cyclase-mediated cell retraction were significantly increased. Additionally, in dorsal hand vein LVDT studies, subjects expressing the hyper-functional ADCY6 S674 variant had significantly greater vascular sensitivity to the beta-adrenergic agonist isoproterenol as assessed by both a greater potency and greater maximal effect than subjects expressing the ADCY6 A674 enzyme. CONCLUSIONS: These data indicate that the expression of a novel, relatively common variant of ADCY6 parallels an increase in adenylyl cyclase activity and adenylyl cyclase-mediated function in humans.

Rapid effects of aldosterone on clonal human vascular smooth muscle cells.

It has been increasingly appreciated that aldosterone elicits acute vascular effects through nongenomic signaling pathways. Our previous studies demonstrated that aldosterone attenuated phenylephrine-mediated constriction in intact vessels [via phosphatidylinositol 3-kinase-dependent nitric oxide synthase activation] but enhanced vasoconstrictor responses in endothelium-denuded arteries. To determine the mechanism of this vasoconstrictor response, we assessed the effect of aldosterone on myosin light-chain phosphorylation and contraction in clonal adult human vascular smooth muscle cells. Acute aldosterone exposure mediated dose-dependent myosin light-chain phosphorylation, inhibited by spironolactone and phosphatidylinositol 3-kinase inhibition. These rapid effects of aldosterone were mimicked by estradiol and hydrocortisone and were also inhibitable by both spironolactone and eplerenone. In parallel to its effects on myosin light-chain phosphorylation, aldosterone mediated dose-dependent contraction responses that were inhibited by spironolactone. Comparable contractile responses were seen with both 17beta-estradiol and hydrocortisone. In total, these data are consistent with a mechanism of acute aldosterone-mediated contraction common to both glucocorticoids and estrogen. Steroid-mediated vasoconstriction may represent an important pathobiological mechanism of vascular disease, especially in the setting of preexisting endothelial dysfunction.

Adenylyl cyclase isoform-selective regulation of vascular smooth muscle proliferation and cytoskeletal reorganization.

Compartmentation of cAMP signaling been demonstrated to be attributable to the structural association of protein kinase A (PKA) (via association with A-kinase anchoring proteins [AKAPs]) with phosphodiesterase and AKAP-dependent effector molecules. However, other mechanisms contributing to compartmentalization have not been rigorously explored, including the possibility that different isoforms of adenylyl cyclase (AC) may be functionally "compartmentalized" because of differential association with tethering or signaling molecules. To this end, we examined the effect of adenoviral transduction of representative AC isoforms (AC1, AC2, AC5, and AC6) on cellular cAMP production, PKA activation, extracellular signal-regulated kinase (ERK) activation, cell doubling and proliferation, as well as arborization responses (an index of cAMP-mediated cytoskeletal re-organization) in vascular smooth muscle cells. When isoforms were expressed at levels to achieve comparable forskolin-stimulated AC activity, only gene transfer of AC6 significantly enhanced PKA-dependent vasodilator-stimulated phosphoprotein (VASP) phosphorylation and arborization responses. Treatment of control cells, which express AC6 endogenously, as well as vascular smooth overexpressing the AC6 isoform with small interfering RNA directed against AC6, significantly suppressed both isoproterenol-stimulated cAMP accumulation and arborization. Notably, the selective effects of AC6 expression were abrogated in the presence of phosphodiesterase suppression. In contrast, only the expression of AC1 enhanced forskolin-stimulated association of ERK with AC, demonstrated by coimmuno-isolation of ERK with Flag-tagged AC1, but not with Flag-tagged AC6. To determine whether these isoform-selective effects of AC were unique to differentiated and morphologically compartmentalized vascular smooth muscle cells or were a general property of these isoforms, we examined the consequence of expression of these various isoforms in human embryonic kidney (HEK) cells. Indeed, we observed similar isoform-dependent association of AC1 with ERK, activation of ERK by stimulation of AC1 with forskolin, and AC1-dependent lengthening of doubling time, indicating that these properties of AC1 are cell autologous and likely result from AC1-dependent protein-protein interactions. In aggregate, these findings suggest that isoform-selective signaling complexes likely contribute to various functional consequences of cAMP elevation in vascular smooth muscle cells.

The impact of blunted beta-adrenergic responsiveness on growth regulatory pathways in hypertension.

The effects of vasodilator hormones acting through receptors linked to adenylyl cyclase are impaired in the hypertensive state. This has been ascribed to impaired receptor-G protein coupling. However, these receptors also act via effectors not linked to adenylyl cyclase activation. These "alternate" mechanisms may be especially important in growth regulation and might be unaffected (or enhanced) with G protein-coupled receptor-G protein uncoupling. Therefore, we assessed the effects of beta-adrenergic activation on 1) regulation of phosphatidylinositol 3-kinase (PI3 kinase) and extracellular signal-regulated kinase (ERK) activation-two tyrosine kinase-dependent enzymes linked to cell growth-and 2) microarray analysis in vascular smooth muscle cells from spontaneously hypertensive rats (SHR). Isoproterenol-stimulated phosphorylation of ERK1/2 was impaired in SHR. The effect of forskolin was unaltered. In contrast, both vasopressin and angiotensin 2-mediated stimulation of ERK activation was enhanced in SHR. In addition, beta-adrenergic-mediated inhibition of PI3 kinase activity was attenuated in SHR (whereas the effect of forskolin remained intact). In microarray studies, the effect of isoproterenol to regulate transcription was significantly impaired in SHR (as was the effect of forskolin). Together, these data support the hypothesis that the blunted vasodilator effects of hormones linked to adenylyl cyclase activation are an index of a more generalized impairment in modulating growth regulatory pathways. Furthermore, this study supports the hypothesis that the blunting of beta-adrenergic responses relating to increased G protein-coupled receptor kinase 2 expression reflects a "generalized uncoupling" of beta-adrenergic-mediated responses and do not support the concept of "enhanced coupling" of "alternate" pathways of beta-adrenergic growth regulatory pathways in the hypertensive state.