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The Ras-related GTP-binding protein D (RRAGD) gene plays a crucial role in cellular processes. Recently, RRAGD variants found in patients have been implicated in a novel disorder with kidney tubulopathy and dilated cardiomyopathy. Currently, the consequences of RRAGD variants at the organismal level are unknown. Therefore, this study investigated the impact of RRAGD variants on cardiac function using a zebrafish embryo model. Furthermore, the potential usage of rapamycin, an mTOR inhibitor, as a therapy was assessed in this model. Zebrafish embryos were injected with RRAGD p.S76L and p.P119R cRNA and the resulting heart phenotypes were studied. Our findings reveal that overexpression of RRAGD mutants resulted in decreased ventricular fractional shortening, ejection fraction, and pericardial swelling. In RRAGD S76L-injected embryos, lower survival and heartbeat were observed, whereas survival was unaffected in RRAGD P119R embryos. These observations were reversible following therapy with the mTOR inhibitor rapamycin. Moreover, no effects on electrolyte homeostasis were observed. Together, these findings indicate a crucial role of RRAGD in cardiac function. In the future, the molecular mechanisms by which RRAGD variants result in cardiac dysfunction and if the effects of rapamycin are specific for RRAGD-dependent cardiomyopathy should be studied in clinical studies. NEW & NOTEWORTHY: The resultant heart-associated phenotypes in the zebrafish embryos of this study serve as a valuable experimental model for this rare cardiomyopathy. Moreover, the potential therapeutic property of rapamycin in cardiac dysfunctions was highlighted, making this study a pivotal step toward prospective clinical applications. [ABSTRACT FROM AUTHOR]

G protein-coupled receptors (GPCRs) are ubiquitously expressed cell surface receptors that mediate numerous physiological responses and are highly druggable. Upon activation, GPCRs rapidly couple to heterotrimeric G proteins and are then phosphorylated and internalized from the cell surface. Recent studies indicate that GPCRs not only localize at the plasma membrane but also exist in intracellular compartments where they are competent to signal. Intracellular signaling by GPCRs is best described to occur at endosomes. Several studies have elegantly documented endosomal GPCR-G protein and GPCR-β-arrestin signaling. Besides phosphorylation, GPCRs are also posttranslationally modified with ubiquitin. GPCR ubiquitination has been studied mainly in the context of receptor endosomal-lysosomal trafficking. However, new studies indicate that ubiquitination of endogenous GPCRs expressed in endothelial cells initiates the assembly of an intracellular p38 mitogen-activated kinase signaling complex that promotes inflammatory responses from endosomes. In this mini-review, we discuss emerging discoveries that provide critical insights into the function of ubiquitination in regulating GPCR inflammatory signaling at endosomes. [ABSTRACT FROM AUTHOR]

We recently demonstrated that acute oral ketone monoester intake induces a stimulation of postprandial myofibrillar protein synthesis rates comparable to that elicited following the ingestion of 10 g whey protein or their coingestion. The present investigation aimed to determine the acute effects of ingesting a ketone monoester, whey protein, or their coingestion on mechanistic target of rapamycin (mTOR)-related protein-protein colocalization and intracellular trafficking in human skeletal muscle. In a randomized, double-blind, parallel group design, 36 healthy recreationally active young males (age: 24.2 ± 4.1 yr) ingested either: 1) 0.36 g·kg−1 bodyweight of the ketone monoester (R)-3-hydroxybutyl (R)-3-hydroxybutyrate (KET), 2) 10 g whey protein (PRO), or 3) the combination of both (KET + PRO). Muscle biopsies were obtained in the overnight postabsorptive state (basal conditions), and at 120 and 300 min in the postprandial period for immunofluorescence assessment of protein translocation and colocalization of mTOR-related signaling molecules. All treatments resulted in a significant (Interaction: P < 0.0001) decrease in tuberous sclerosis complex 2 (TSC2)-Ras homolog enriched in brain (Rheb) colocalization at 120 min versus basal; however, the decrease was sustained at 300 min versus basal (P < 0.0001) only in KET + PRO. PRO and KET + PRO increased (Interaction: P < 0.0001) mTOR-Rheb colocalization at 120 min versus basal; however, KET + PRO resulted in a sustained increase in mTOR-Rheb colocalization at 300 min that was greater than KET and PRO. Treatment intake increased mTOR-wheat germ agglutinin (WGA) colocalization at 120 and 300 min (Time: P = 0.0031), suggesting translocation toward the fiber periphery. These findings demonstrate that ketone monoester intake can influence the spatial mechanisms involved in the regulation of mTORC1 in human skeletal muscle. NEW & NOTEWORTHY: We explored the effects of a ketone monoester (KET), whey protein (PRO), or their coingestion (KET + PRO) on mTOR-related protein-protein colocalization and intracellular trafficking in human muscle. All treatments decreased TSC2-Rheb colocalization at 120 minutes; however, KET + PRO sustained the decrease at 300 min. Only PRO and KET + PRO increased mTOR-Rheb colocalization; however, the increase at 300 min was greater in KET + PRO. Treatment intake increased mTOR-WGA colocalization, suggesting translocation to the fiber periphery. Ketone bodies influence the spatial regulation of mTOR. [ABSTRACT FROM AUTHOR]

Acetylcholine plays an essential role in the regulation of detrusor muscle contractions, and antimuscarinics are widely used in the management of overactive bladder syndrome. However, several adverse effects limit their application and patients' compliance. Thus, this study aimed to further analyze the signal transduction of M2 and M3 receptors in the murine urinary bladder to eventually find more specific therapeutic targets. Experiments were performed on adult male wild-type, M2, M3, M2/M3, or Gαq/11 knockout (KO), and pertussis toxin (PTX)-treated mice. Contraction force and RhoA activity were measured in the urinary bladder smooth muscle (UBSM). Our results indicate that carbamoylcholine (CCh)-induced contractions were associated with increased activity of RhoA and were reduced in the presence of the Rho-associated kinase (ROCK) inhibitor Y-27632 in UBSM. CCh-evoked contractile responses and RhoA activation were markedly reduced in detrusor strips lacking either M2 or M3 receptors and abolished in M2/M3 KO mice. Inhibition of Gai-coupled signaling by PTX treatment shifted the concentration-response curve of CCh to the right and diminished RhoA activation. CCh-induced contractile responses were markedly decreased in Gαq/11 KO mice; however, RhoA activation was unaffected. In conclusion, cholinergic detrusor contraction and RhoA activation are mediated by both M2 and M3 receptors. Furthermore, whereas both Gai and Gαq/11 proteins mediate UBSM contraction, the activation at the RhoA-ROCK pathway appears to be linked specifically to Gαi. These findings may aid the identification of more specific therapeutic targets for bladder dysfunctions. [ABSTRACT FROM AUTHOR]

This article, titled "Advances in GPCRs: structure, mechanisms, disease, and pharmacology," provides an overview of the current state of research on G protein-coupled receptors (GPCRs). GPCRs are a large family of cell membrane receptors that play a crucial role in regulating physiological processes by responding to various ligands. The collection of articles in this journal highlights recent advances in understanding GPCR structure, mechanisms of signal transduction, and their role in diseases. The articles also discuss the development of targeted therapies and interventions based on GPCR research. The authors emphasize the importance of studying GPCR signaling in different contexts and using relevant assays and technologies. The collection covers a wide range of topics, including the role of GPCRs in cardiovascular diseases, inflammation, and neurological diseases. Overall, this collection provides valuable references for researchers and industry professionals interested in GPCRs and their implications for drug development. [Extracted from the article]

We tested the hypothesis that, compared with normothermia, the increase in glomerular filtration rate (GFR) after an oral protein load (defined as the GFR reserve) is attenuated during moderate passive heat stress in young healthy adults. Sixteen participants (5 women; 26 ± 2 yr) completed two experimental visits, heat stress or a normothermic time-control, assigned in a block-randomized crossover design. During the heat stress trial, core temperature was increased by 0.6°C in the first hour before commencing a 2-min cold pressor test (CPT) to assess renal vasoconstrictor responses. One-hour post-CPT, subjects ingested a whey protein shake (1.2 g of protein/kg body wt), and measurements were taken pre-, 75, and 150 min postprotein. Segmental artery vascular resistance was calculated as the quotient of Doppler ultrasound-derived segmental artery blood velocity and mean arterial pressure and provided an estimate of renal vascular tone. GFR was estimated from creatinine clearance. The increase in segmental artery vascular resistance during the CPT was attenuated during heat stress (end CPT: 5.6 ± 0.9 vs. 4.7 ± 1.1 mmHg/cm/s, P = 0.024). However, the reduction in segmental artery vascular resistance in response to an oral protein load did not differ between heat stress (at 150 min: 1.9 ± 0.4 mmHg/cm/s) and normothermia (at 150 min: 1.8 ± 0.5 mmHg/cm/s; P = 0.979). The peak increase in creatinine clearance postprotein, independent of time, was attenuated during heat stress (+26 ± 19 vs. +16 ± 20 mL/ min, P = 0.013, n = 13). GFR reserve is diminished by mild passive heat stress. Moreover, renal vasoconstrictor responses are attenuated by mild passive heat stress, but renal vasodilator responses are maintained. [ABSTRACT FROM AUTHOR]

Wilms' tumor interacting protein (Wtip) has been implicated in cell junction assembly and cell differentiation and interacts with proteins in the podocyte slit diaphragm, where it regulates podocyte phenotype. To define Wtip expression and function in the kidney, we created a Wtip-deleted mouse model using b-galactosidase-neomycin (β-geo) gene trap technology. Wtip gene trap mice were embryonic lethal, suggesting additional developmental roles outside kidney function. Using β-geo heterozygous and normal mice, Wtip expression was identified in the developing kidneys, heart, and eyes. In the kidney, expression was restricted to podocytes, which appeared initially at the capillary loop stage coinciding with terminal podocyte differentiation. Heterozygous mice had an expected lifespan and showed no evidence of proteinuria or glomerular pathology. However, heterozygous mice were more susceptible to glomerular injury than wild-type littermates and developed more significant and prolonged proteinuria in response to lipopolysaccharide or adriamycin. In normal human kidneys, WTIP expression patterns were consistent with observations in mice and were lost in glomeruli concurrent with loss of synaptopodin expression in disease. Mechanistically, we identified the Rho guanine nucleotide exchange factor 12 (ARHGEF12) as a binding partner for WTIP. ARHGEF12 was expressed in human podocytes and formed high-affinity interactions through their LIM- and PDZ-binding domains. Our findings suggest that Wtip is essential for early murine embryonic development and maintaining normal glomerular filtration barrier function, potentially regulating slit diaphragm and foot process function through Rho effector proteins. NEW & NOTEWORTHY This study characterized dynamic expression patterns of Wilms' tumor interacting protein (Wtip) and demonstrates the novel role of Wtip in murine development and maintenance of the glomerular filtration barrier. [ABSTRACT FROM AUTHOR]

The canonical model for G protein-coupled receptors (GPCRs) activation assumes that stimulation of heterotrimeric G protein signaling upon ligand binding occurs solely at the cell surface and that duration of the stimulation is transient to prevent overstimulation. In this model, GPCR signaling is turned-off by receptor phosphorylation via GPCR kinases (GRKs) and subsequent recruitment of β-arrestins, resulting in receptor internalization into endosomes. Internalized receptors can then recycle back to the cell surface or be trafficked to lysosomes for degradation. However, over the last decade, this model has been extended by discovering that some internalized GPCRs continue to signal via G proteins from endosomes. This is the case for the parathyroid hormone (PTH) type 1 receptor (PTHR), which engages on sustained cAMP signaling from endosomes upon PTH stimulation. Accumulative evidence shows that the location of signaling has an impact on the physiological effects of GPCR signaling. This mini-review discusses recent insights into the mechanisms of PTHR endosomal signaling and its physiological impact. [ABSTRACT FROM AUTHOR]

G protein-coupled receptors (GPCRs) are of considerable interest due to their importance in a wide range of physiological functions and in a large number of Food and Drug Administration (FDA)-approved drugs as therapeutic entities. With continued study of their function and mechanism of action, there is a greater understanding of how effector molecules interact with a receptor to initiate downstream effector signaling. This review aims to explore the signaling pathways, dynamic structures, and physiological relevance in the cardiovascular system of the three most important GPCR signaling effectors: heterotrimeric G proteins, GPCR kinases (GRKs), and β-arrestins. We will first summarize their prominent roles in GPCR pharmacology before transitioning into less well-explored areas. As new technologies are developed and applied to studying GPCR structure and their downstream effectors, there is increasing appreciation for the elegance of the regulatory mechanisms that mediate intracellular signaling and function. [ABSTRACT FROM AUTHOR]

The role of different G protein-coupled receptors (GPCRs) in the cardiovascular system is well understood in cardiomyocytes and vascular smooth muscle cells (VSMCs). In the former, stimulation of Gs-coupled receptors leads to increases in contractility, whereas stimulation of Gq-coupled receptors modulates cellular survival and hypertrophic responses. In VSMCs, stimulation of GPCRs also modulates contractile and cell growth phenotypes. Here, we will focus on the relatively less well-studied effects of GPCRs in cardiac fibroblasts, focusing on key signaling events involved in the activation and differentiation of these cells. We also review the hierarchy of signaling events driving the fibrotic response and the communications between fibroblasts and other cells in the heart. We discuss how such events may be distinct depending on where the GPCRs and their associated signaling machinery are localized in these cells with an emphasis on nuclear membrane-localized receptors. Finally, we explore what such connections between the cell surface and nuclear GPCR signaling might mean for cardiac fibrosis. [ABSTRACT FROM AUTHOR]

G protein-gated inwardly rectifying K+ (GIRK/Kir3) channels exert a critical inhibitory influence on neurons. Neuronal GIRK channels mediate the G protein-dependent, direct/postsynaptic inhibitory effect of many neurotransmitters including γ-aminobutyric acid (GABA), serotonin, dopamine, adenosine, somatostatin, and enkephalin. In addition to their complex regulation by G proteins, neuronal GIRK channel activity is sensitive to phosphatidylinositol 4,5-bisphosphate (PIP2), phosphorylation, regulator of G protein signaling (RGS) proteins, intracellular Na+ and Ca2+, and cholesterol. The application of genetic and viral manipulations in rodent models, together with recent progress in the development of GIRK channel modulators, has increased our understanding of the physiological and behavioral impact of neuronal GIRK channels. Work in rodent models has also revealed that neuronal GIRK channel activity is modified, transiently or persistently, by various stimuli including exposure drugs of abuse, changes in neuronal activity patterns, and aversive experience. A growing body of preclinical and clinical evidence suggests that dysregulation of GIRK channel activity contributes to neurological diseases and disorders. The primary goals of this review are to highlight fundamental principles of neuronal GIRK channel biology, mechanisms of GIRK channel regulation and plasticity, the nascent landscape of GIRK channel pharmacology, and the potential relevance of GIRK channels to the pathophysiology and treatment of neurological diseases and disorders. [ABSTRACT FROM AUTHOR]

In this study, we elucidate factors that regulate the trafficking and activity of a well-conserved olfactory receptor (OR), olfactory receptor 558 (Olfr558), and its human ortholog olfactory receptor 51E1 (OR51E1). Results indicate that butyrate activates Olfr558/OR51E1 leading to the production of cAMP, and evokes Ca2+ influx. We also find olfactory G protein (Golf) increases cAMP production induced by Olfr558/OR51E1 activation but does not affect trafficking. Given the 93% sequence identity between OR51E1 and Olfr558, it is surprising to note that OR51E1 has significantly more surface expression yet similar total protein expression. We find that replacing the Olfr558 N-terminus with that of OR51E1 significantly increases trafficking; in contrast, there is no change in surface expression conferred by the OR51E1 TM2, TM3, or TM4 domains. A previous analysis of human OR51E1 single nucleotide polymorphisms (SNPs) identified an A156T mutant primarily found in South Asia as the most abundant (albeit still rare). We find that the OR51E1 A156T mutant has reduced surface expression and cAMP production without a change in total protein expression. In sum, this study of a well-conserved olfactory receptor identifies both protein regions and specific amino acid residues that play key roles in protein trafficking and also elucidates common effects of Golf on the regulation of both the human and murine OR. [ABSTRACT FROM AUTHOR]

G protein-coupled chemoattractant receptors are class A GPCRs that couple primarily to the Gi class of heterotrimeric G proteins. Initially identified for their abilities to mediate leukocyte chemotaxis, chemoattractant GPCRs such as the formyl peptide receptors (FPRs) have been known for their diverse cellular functions in response to a variety of agonists. Stimulation of FPR2, in particular, leads to ligand-dependent activation of proinflammatory signaling as well as anti-inflammatory and proresolving signaling. Recently, the structures of FPR2-Gi protein complexed with ligands of different compositions have been solved by crystallization and cryo-electron microscopy. Analysis of the structural data as well as molecular simulation has led to the findings that the FPR2 binding pocket is sufficiently large for accommodation of several different types of ligands but in different poses. This mini-review focuses on the structural and conformational aspects of FPR2 for mechanisms underlying its biased agonism. [ABSTRACT FROM AUTHOR]

G protein-coupled receptors (GPCRs) are the largest family of transmembrane receptors and are the target of approximately one-third of all Food and Drug Administration (FDA)-approved pharmaceutical drugs. GPCRs interact with many transducers, such as heterotrimeric G proteins, GPCR kinases (GRKs), and β-arrestins. Recent experiments have demonstrated that some ligands can activate distinct effector proteins over others, a phenomenon termed "biased agonism." These discoveries have raised the potential of developing drugs which preferentially activate therapeutic signaling pathways over those that lead to deleterious side effects. However, to date, only one biased GPCR therapeutic has received FDA approval and many others have either failed to meet their specified primary end points and or demonstrate superiority over currently available treatments. In addition, there is a lack of understanding regarding how biased agonism measured at a GPCR leads to specific downstream physiological responses. Here, we briefly summarize the history and current status of biased agonism at GPCRs and suggest adoption of a "systems pharmacology" approach upon which to develop GPCR-targeted drugs that demonstrate heightened therapeutic efficacy with improved side effect profiles. [ABSTRACT FROM AUTHOR]

GNAO1 encodes Gαo, a heterotrimeric G protein α subunit in the Gi/o family. In this report, we used a Gnao1 mouse model "G203R" previously described as a "gain-of-function" Gnao1 mutant with movement abnormalities and enhanced seizure susceptibility. Here, we report an unexpected second mutation resulting in a loss-of-function Gαo protein, and describe alterations in central synaptic transmission. Whole cell patch clamp recordings from Purkinje cells (PCs) in acute cerebellar slices from Gnao1 mutant mice showed significantly lower frequencies of spontaneous and miniature inhibitory postsynaptic currents (sIPSCs and mIPSCs) compared with WT mice. There was no significant change in sEPSCs or mEPSCs. Whereas mIPSC frequency was reduced, mIPSC amplitudes were not affected, suggesting a presynaptic mechanism of action. A modest decrease in the number of molecular layer interneurons was insufficient to explain the magnitude of IPSC suppression. Paradoxically, Gi/o inhibitors (pertussis toxin) enhanced the mutant-suppressed mIPSC frequency and eliminated the difference between WT and Gnao1 mice. Although GABAB receptor regulates mIPSCs, neither agonists nor antagonists of this receptor altered function in the mutant mouse PCs. This study is an electrophysiological investigation of the role of Gi/o protein in cerebellar synaptic transmission using an animal model with a loss-of-function Gi/o protein. [ABSTRACT FROM AUTHOR]

Rho GTPases are a family of small G proteins that regulate a wide array of cellular processes related to their key roles controlling the cytoskeleton. Cancer is a multistep disease caused by the accumulation of genetic mutations and epigenetic alterations, from the initial stages of cancer development when cells in normal tissues undergo transformation, to the acquisition of invasive and metastatic traits, responsible for a large number of cancer related deaths. In this review, we discuss the role of Rho GTPase signaling in cancer in every step of disease progression. Rho GTPases contribute to tumor initiation and progression, by regulating proliferation and apoptosis, but also metabolism, senescence, and cancer cell stemness. Rho GTPases play a major role in cell migration and in the metastatic process. They are also involved in interactions with the tumor microenvironment and regulate inflammation, contributing to cancer progression. After years of intensive research, we highlight the importance of relevant models in the Rho GTPase field, and we reflect on the therapeutic opportunities arising for cancer patients. [ABSTRACT FROM AUTHOR]

Chronic metabolic acidosis stimulates cell-mediated net Ca2+ efflux from bone mediated by increased osteoblastic cyclooxygenase 2, leading to prostaglandin E2-induced stimulation of receptor activator of NF-κB ligand-induced osteoclastic bone resorption. Ovarian cancer G protein-coupled receptor-1 (OGR1), an osteoblastic H+-sensing G protein-coupled receptor, is activated by acidosis and leads to increased bone resorption. As regulator of G protein signaling (RGS) proteins limit GPCR signaling, we tested whether RGS proteins themselves are regulated by metabolic acidosis. Primary osteoblasts were isolated from neonatal mouse calvariae and incubated in physiological neutral or acidic (MET) medium. Cells were collected, and RNA was extracted for real-time PCR analysis with mRNA levels normalized to ribosomal protein L13a. RGS1, RGS2, RGS3, RGS4, RGS10, RGS11, and RGS18 mRNA did not differ between MET and neutral medium; however, by 30 min, MET decreased RGS16, which persisted for 60 min and 3 h. Incubation of osteoblasts with the OGR1 inhibitor CuCl2 inhibited the MET-induced increase in RGS16 mRNA. Gallein, a specific inhibitor of Gβγ signaling, was used to determine if downstream signaling by the βγ-subunit was critical for the response to acidosis. Gallein decreased net Ca2+ efflux from calvariae and cyclooxygenase 2 and receptor activator of NF-κB ligand gene expression from isolated osteoblasts. These results indicate that regulation of RGS16 plays an important role in modulating the response of the osteoblastic GPCR OGR1 to metabolic acidosis and subsequent stimulation of osteoclastic bone resorption. [ABSTRACT FROM AUTHOR]

G protein-coupled receptors (GPCRs) regulate many cellular and physiological processes, responding to a diverse range of extracellular stimuli including hormones, neurotransmitters, odorants, and light. Decades of biochemical and pharmacological studies have provided fundamental insights into the mechanisms of GPCR signaling. Thanks to recent advances in structural biology, we now possess an atomistic understanding of receptor activation and G protein coupling. However, how GPCRs and G proteins interact in living cells to confer signaling efficiency and specificity remains insufficiently understood. The development of advanced optical methods, including single-molecule microscopy, has provided the means to study receptors and G proteins in living cells with unprecedented spatio-temporal resolution. The results of these studies reveal an unexpected level of complexity, whereby GPCRs undergo transient interactions among themselves as well as with G proteins and structural elements of the plasma membrane to form short-lived signaling nanodomains that likely confer both rapidity and specificity to GPCR signaling. These findings may provide new strategies to pharmaceutically modulate GPCR function, which might eventually pave the way to innovative drugs for common diseases such as diabetes or heart failure. [ABSTRACT FROM AUTHOR]

Idiopathic pulmonary arterial hypertension (IPAH) is a rapidly progressive disease with several treatment options. Long-term mortality remains high with great heterogeneity in treatment response. Even though most of the pathology of IPAH is observed in the lung, there is systemic involvement. Platelets from patients with IPAH have characteristic metabolic shifts and defects in activation; therefore, we investigated whether they could be used to identify other disease-specific abnormalities. We used proteomics to investigate protein expression changes in platelets from patients with IPAH compared with healthy controls. Key abnormalities of nitric oxide pathway were tested in platelets from a larger cohort of unique patients with IPAH. Platelets showed abnormalities in the prostacyclin and nitric oxide pathways, which are dysregulated in IPAH and hence targets of therapy. We detected reduced expression of G protein αs and increased expression of the regulatory subunits of the cAMP-dependent protein kinase (PKA) type II isoforms, supporting an overall decrease in the activation of the prostacyclin pathway. We noted reduced levels of the soluble guanylate cyclase (sGC) subunits and increased expression of the phosphodiesterase type 5 A (PDE5A), conditions that affect the response to nitric oxide. Ensuing analysis of 38 unique patients with IPAH demonstrated considerable variation in the levels and specific activity of sGC, a finding with novel implications for personalized therapy. Platelets have some of the characteristic vasoactive signal abnormalities seen in IPAH and may provide comprehensive ex vivo mechanistic information to direct therapeutic decisions. [ABSTRACT FROM AUTHOR]

This document is an editorial published in the American Journal of Physiology: Renal Physiology. It discusses the role of G protein-coupled receptors (GPCRs) in renal physiology. GPCRs are plasmamembrane receptors that respond to various stimuli and regulate physiological responses. The kidney contains multiple GPCRs with important roles, and recent studies have highlighted the complexity of GPCR signaling in the kidney. The editorial invites submissions related to GPCR biology and function for further research in this area. [Extracted from the article]

Renal ischemia-reperfusion (I/R) injury is associated with markedly reduced protein expression of aquaporins (AQPs). Membrane G protein-coupled bile acid receptor-1 (TGR5) has shown protective roles in some kidney diseases. The purpose of the current study was to investigate whether activation of TGR5 prevented the decreased protein expression of AQPs in rodents with renal I/R injury and potential mechanisms. TGR5 agonist lithocholic acid (LCA) treatment reduced polyuria after renal I/R injury in rats. LCA prevented the decreased abundance of AQP2 protein and upregulated hypoxia-inducible factor (HIF)-1α protein expression, which were associated with decreased protein abundance of NF-κB p65 and IL-1β. After renal I/R, mice with tgr5 gene deficiency exhibited further decreases in AQP2 and HIF-1α protein abundance and increases of IL-1β and NF-κB p65 protein expression compared with wild-type mice. In primary cultured inner medullary collecting duct cells with hypoxia/reoxygenation, LCA induced markedly increased protein expression of AQP2 and HIF-1α, which were partially prevented by the PKA inhibitor H89. FG4592, a prolyl-4-hydroxylase domain-containing protein inhibitor, increased HIF-1α and AQP2 protein abundance in association with decreased NF-κB p65 protein expression in inner medullary collecting duct cells with hypoxia/reoxygenation. In conclusion, TGR5 stimulation by LCA prevented downregulation of renal AQPs in kidney with I/R injury, likely through activating HIF-1α signaling and suppressing inflammatory responses. NEW & NOTEWORTHY Stimulation of the membrane G protein-coupled bile acid receptor TGR5 by lithocholic acid (LCA) reduced polyuria in rats with renal ischemia-reperfusion (I/R) injury. LCA increased abundance of aquaporin-2 (AQP2) protein and upregulated hypoxia-inducible factor (HIF)-1α protein expression in association with decreased NF-κB p65 and IL-1β. After I/R, mice with tgr5 gene deficiency exhibited more severe decreases in AQP2 and HIF-1α protein abundance and inflammatory responses. TGR5 activation exhibits a protective role in acute renal injury induced by I/R. [ABSTRACT FROM AUTHOR]

The G-protein subunits Gqα and Gnα (Gq/nα) couple receptors to phospholipase C, leading to increased intracellular calcium. In this study we investigated the consequences of Gq/11a deficiency in the dorsomedial hypothalamus (DMH), a critical site for the control of energy homeostasis. Mice with DMH-specific deletion of (Gq/nα)(DMHGq/11KO) were generated by stereotaxic injection of adeno-associated virus (AAV)-Cre-green fluorescent protein (GFP) into the DMH of Gqaflox/floxG amice. Compared with control mice that received DMH injection of AAV-GFP, mice developed obesity associated with reduced energy expenditure without significant changes in food intake or physical activity. DMHGq/11KO mice showed no defects in the ability of the melanocortin agonist melanotan II to acutely stimulate energy expenditure or to inhibit food intake. At room temperature (22C), DMHGq/11KO mice showed reduced sympathetic nervous system activity in brown adipose tissue (BAT) and heart, accompanied with decreased basal BAT uncoupling protein 1 (Ucp1) gene expression and lower heart rates. These mice were cold intolerant when acutely exposed to cold (6C for 5 h) and had decreased cold-stimulated BAT Ucp1 gene expression. DMHGq/11KO mice also failed to adapt to gradually declining ambient temperatures and to develop adipocyte browning in inguinal white adipose tissue although their BAT Ucp1 was proportionally stimulated. Consistent with impaired cold-induced thermogenesis, the onset of obesity in DMHGq/11KO mice was significantly delayed when housed under thermoneutral conditions (30C). Thus our results show that Gqa and G11a in the DMH are required for the control of energy homeostasis by stimulating energy expenditure and thermoregulation. [ABSTRACT FROM AUTHOR]

Testosterone supplementation during energy deficit promotes whole body lean mass accretion, but the mechanisms underlying that effect remain unclear. To elucidate those mechanisms, skeletal muscle molecular adaptations were assessed from muscle biopsies collected before, 1 h, and 6 h after exercise and a mixed meal (40 g protein, 1 h postexercise) following 14 days of weight maintenance (WM) and 28 days of an exercise- and diet-induced 55% energy deficit (ED) in 50 physically active nonobese men treated with 200 mg testosterone enanthate/wk (TEST) or placebo (PLA) during the ED. Participants (n = 10/group) exhibiting substantial increases in leg lean mass and total testosterone (TEST) were compared with those exhibiting decreases in both of these measures (PLA). Resting androgen receptor (AR) protein content was higher and fibroblast growth factor-inducible 14 (Fn14), IL-6 receptor (IL-6R), and muscle ring-finger protein-1 gene expression was lower in TEST vs. PLA during ED relative to WM (P < 0.05). Changes in inflammatory, myogenic, and proteolytic gene expression did not differ between groups after exercise and recovery feeding. Mechanistic target of rapamycin signaling (i.e., translational efficiency) was also similar between groups at rest and after exercise and the mixed meal. Muscle total RNA content (i.e., translational capacity) increased more during ED in TEST than PLA (P < 0.05). These findings indicate that attenuated proteolysis at rest, possibly downstream of AR, Fn14, and IL-6R signaling, and increased translational capacity, not efficiency, may drive lean mass accretion with testosterone administration during energy deficit. [ABSTRACT FROM AUTHOR]

Respiratory depression is a potentially fatal side effect of opioid analgesics and a major limitation to their use. G protein-biased opioid agonists have been proposed as "safer" analgesics with less respiratory depression. These agonists are biased to activate G proteins rather than β-arrestin signaling. Respiratory depression has been shown to correlate with both G protein bias and intrinsic efficacy, and recent work has refuted the role of β-arrestin signaling in opioid-induced respiratory depression. In addition, there is substantial evidence that G proteins do, in fact, mediate respiratory depression by actions in respiratory-controlling brainstem neurons. Based on these studies, we provide the perspective that protection from respiratory depression displayed by newly developed G protein-biased agonists is due to factors other than G protein versus arrestin bias. [ABSTRACT FROM AUTHOR]

Older adults have shown an attenuated postexercise increase in muscle protein synthesis rates following ingestion of smaller amounts of protein compared with younger adults. Consequently, it has been suggested that older adults require the ingestion of more protein to increase postexercise muscle protein synthesis rates compared with younger adults. We investigated whether coingestion of 1.5 g of free leucine with a single 15-g bolus of protein further augments the postprandial muscle protein synthetic response during recovery from resistance-type exercise in older men. Twenty-four healthy older men (67 ± 1 yr) were randomly assigned to ingest 15 g of milk protein concentrate (MPC80) with (15G+LEU; n = 12) or without (15G; n = 12) 1.5 g of free leucine after performing a single bout of resistancetype exercise. Postprandial protein digestion and amino acid absorption kinetics, whole body protein metabolism, and postprandial myofibrillar protein synthesis rates were assessed using primed, continuous infusions with L-[ring-2H5]phenylalanine, L-[ring-2H2]tyrosine, and L-[1-13C]leucine combined with ingestion of intrinsically L-[1-13C]phenylalanine-labeled milk protein. A total of 70 ± 1% (10.5 ± 0.2 g) and 75 ± 2% (11.2 ± 0.3 g) of the protein-derived amino acids were released in the circulation during the 6-h postexercise recovery phase in 15G+LEU and 15G, respectively (P < 0.05). Postexercise myofibrillar protein synthesis rates were 16% (0.058 ± 0.003 vs. 0.049 ± 0.002%/h, P < 0.05; based on L-[ring-2H5]phenylalanine) and 19% (0.071 ± 0.003 vs. 0.060 ± 0.003%/h, P < 0.05; based on L-[1-13C]leucine) greater in 15G+LEU compared with 15G. Leucine coingestion further augments the postexercise muscle protein synthetic response to the ingestion of a single 15-g bolus of protein in older men. [ABSTRACT FROM AUTHOR]

Endocytosis is a major mechanism through which cellular signaling by G protein-coupled receptors (GPCRs) is terminated. However, recent studies demonstrate that GPCRs are internalized in an active state and continue to signal from within endosomes, resulting in effects on cellular function that are distinct to those arising at the cell surface. Endocytosis inhibitors are commonly used to define the importance of GPCR internalization for physiological and pathophysiological processes. Here, we provide the first detailed examination of the effects of these inhibitors on neurogenic contractions of gastrointestinal smooth muscle, a key preliminary step to evaluate the importance of GPCR endocytosis for gut function. Inhibitors of clathrin-mediated endocytosis (Pitstop2, PS2) or G protein-coupled receptor kinase-2/3-dependent phosphorylation (Takeda compound 101, Cmpd101), signifi- cantly reduced GPCR internalization. However, they also attenuated cholinergic contractions through different mechanisms. PS2 abolished contractile responses by colonic muscle to SNC80 and morphine, which strongly and weakly internalize δ-opioid and μ-opioid receptors, respectively. PS2 did not affect the increased myogenic contractile activity following removal of an inhibitory neural influence (tetrodotoxin) but suppressed electrically evoked neurogenic contractions. Ca2+ signaling by myenteric neurons in response to exogenous ATP was unaffected by PS2, suggesting inhibitory actions on neurotransmitter release rather than neurotransmission. In contrast, Cmpd101 attenuated contractions to the cholinergic agonist carbachol, indicating direct effects on smooth muscle. We conclude that, although PS2 and Cmpd101 are effective blockers of GPCR endocytosis in enteric neurons, these inhibitors are unsuitable for the study of neurally mediated gut function due to their inhibitory effects on neuromuscular transmission and smooth muscle contractility. [ABSTRACT FROM AUTHOR]

Ovarian cancer G protein-coupled receptor 1 (OGR1) is a recently deorphanized G protein-coupled receptor shown to signal in response to low extracellular pH (↓pHo) or certain benzodiazepines. The pleiotropic nature of OGR1 signaling in human airway smooth muscle (HASM) cells suggests that OGR1 is a potential therapeutic target for the management of obstructive lung diseases. However, the basic pharmacological and regulatory features of OGR1 remain poorly understood. We employed model systems of heterologously expressed [human embryonic kidney 293 (HEK293) cells] or endogenous (HASM) OGR1 to assess changes in expression, subcellular localization, and signaling capabilities following acute or chronic treatment with ↓pHo or the benzodiazepines lorazepam and sulazepam. In HEK293 cells expressing OGR1, treatment with ↓pHo and/or lorazepam, but not sulazepam, caused rapid OGR1 internalization. In HASM cells, acute treatment with ↓pHo or benzodiazepines did not alter abundance of OGR1 mRNA; however, significant downregulation was observed following chronic treatment. Acute and chronic pretreatment of HASM cells with sulazepam or lorazepam resulted in receptor desensitization as demonstrated by reduced phosphorylation of vasodilator-stimulated phosphoprotein (VASP) or p42/p44 upon rechallenge. Acid (acute but not chronic) pretreatment of HASM cells induced desensitization of OGR1-mediated VASP (but not p42/p44) phosphorylation. In contrast to a recent study reporting OGR1 upregulation and sensitization in cardiac tissue subject to ischemic/acidic insult, chronic OGR1 activation in multiple model systems did not increase OGR1 expression or signaling capacity. The ability to induce OGR1 internalization and desensitization was activator dependent, reflecting the ability of different activators to induce specific receptor confirmations and engagement of specific heterotrimeric G proteins. [ABSTRACT FROM AUTHOR]

High-fat diet (HFD) feeding or leptin-deficient mice are extensively used as models resembling features of human nonalcoholic fatty liver disease (NAFLD). The concurrence of experimental factors as fat content and source or total caloric intake leads to prominent differences in the development of the hepatic steatosis and related disturbances. In this work, we characterized the hepatic lipid accumulation induced by HFD in wild-type (WT) and ob/ob mice with the purpose of differentiating adaptations to HFD from those specific of increased overfeeding due to leptin deficiency-associated hyperphagia. Given that most published works have been done in male models, we used female mice with the aim of increasing the body of evidence regarding NAFLD in female subjects. HFD promoted liver lipid accumulation only in the hyperphagic strain. Nevertheless, a decrease of lipid droplet-associated cholesteryl ester (CE) in both WT and obese animals was observed. These changes were accompanied by an improvement in the profile of lipoproteins that transport cholesterol and liver function markers in plasma from ob/ob mice and a lower hepatic index. Using primary hepatocytes from female mice, overaccumulation of CE induced by 0.4 mM oleic acid reversed in the presence of a specific Takeda G protein-coupled bile acid receptor agonist. Nevertheless, hepatocytes from male mice were not responsive. This study suggests that enterohepatic circulation of bile acids might be one of the factors that can affect sex dimorphism in NAFLD development, which underlines the importance of including female models in the NAFLD research field. NEW & NOTEWORTHY This work provides new insight into the use of high-fat diet as a model to induce nonalcoholic fatty liver disease in wild-type and ob/ob female mice. We show that high-fat diet induces steatosis only in ob/ob mice while, surprisingly, several health indicators improve. Noteworthy, experiments with primary hepatocytes from male and female mice show that they express Takeda G protein-coupled bile acid receptor and that it and bile acid enterohepatic circulation might be accountable for sex dimorphism in nonalcoholic fatty liver disease development. [ABSTRACT FROM AUTHOR]

G protein pathway suppressor 2 (GPS2) is a multifunctional protein and transcriptional regulation factor that is involved in the G protein MAPK signaling pathway. It has been shown that the MAPK signaling pathway plays an important role in the regulation of renal large-conductance Ca2+- activated potassium (BK) channels. In this study, we investigated the effects of GPS2 on BK channel activity and protein expression. In human embryonic kidney (HEK) BK stably expressing cells transfected with either GPS2 or its vector control, a single-cell recording showed that GPS2 significantly increased BK channel activity (NPo), increasing BK open probability (Po), and channel number (N) compared with the control. In Cos-7 cells and HEK 293 T cells, GPS2 overexpression significantly enhanced the total protein expression of BK in a dose-dependent manner. Knockdown of GPS2 expression significantly decreased BK protein expression, while increasing ERK1/2 phosphorylation. Knockdown of ERK1/2 expression reversed the GPS2 siRNA-mediated inhibition of BK protein expression in Cos-7 cells. Pretreatments of Cos-7 cells with either the lysosomal inhibitor bafilomycin A1 or the proteasomal inhibitor MG132 partially reversed the inhibitory effects of GPS2 siRNA on BK protein expression. In addition, feeding a high-potassium diet significantly increased both GPS2 and BK protein abundance in mice. These data suggest that GPS2 enhances BK channel activity and its protein expression by reducing ERK1/2 signaling-mediated degradation of the channel. [ABSTRACT FROM AUTHOR]

G protein-coupled receptor signaling mechanisms are implicated in many aspects of cardiovascular control, and dysfunction of such signaling mechanisms is commonly associated with disease states. Investigators have identified a large number of regulator of G protein signaling (RGS) proteins that variously contribute to the modulation of intracellular secondmessenger signaling kinetics. These many RGS proteins each interact with a specific set of second-messenger cascades and receptor types and exhibit tissue specific expression patterns. Increasing evidence supports the contribution of RGS proteins, or their loss, in the pathogenesis of cardiovascular dysfunctions. This review summarizes the current understanding of the functional contributions of RGS proteins, particularly within the B/R4 family, in cardiovascular disorders of pregnancy including gestational hypertension, uterine artery dysfunction, and preeclampsia. [ABSTRACT FROM AUTHOR]

Olfactory receptors (ORs) are not exclusively expressed in the olfactory sensory neurons; they are also observed outside of the olfactory system in all other human tissues tested to date, including the testis, lung, intestine, skin, heart, and blood. Within these tissues, certain ORs have been determined to be exclusively expressed in only one tissue, whereas other ORs are more widely distributed in many different tissues throughout the human body. For most of the ectopically expressed ORs, limited data are available for their functional roles. They have been shown to be involved in the modulation of cell-cell recognition, migration, proliferation, the apoptotic cycle, exocytosis, and pathfinding processes. Additionally, there is a growing body of evidence that they have the potential to serve as diagnostic and therapeutic tools, as ORs are highly expressed in different cancer tissues. Interestingly, in addition to the canonical signaling pathways activated by ORs in olfactory sensory neurons, alternative pathways have been demonstrated in nonolfactory tissues. In this review, the existing data concerning the expression, as well as the physiological and pathophysiological functions, of ORs outside of the nose are highlighted to provide insights into future lines of research. [ABSTRACT FROM AUTHOR]

Previously, we showed that receptor for activated C kinase 1 (Rack1) regulates growth of colon cells in vitro, partly by suppressing Src kinase activity at key cell cycle checkpoints, in apoptotic and cell survival pathways and at cell-cell adhesions. Here, we generated mouse models of Rack1 deficiency to assess Rack1's function in intestinal epithelia in vivo. Intestinal Rack1 deficiency resulted in proliferation of crypt cells, diminished differentiation of crypt cells into enterocyte, goblet, and enteroendocrine cell lineages, and expansion of Paneth cell populations. Following radiation injury, the morphology of Rack1-deleted small bowel was strikingly abnormal with development of large polypoid structures that contained many partly formed villi, numerous back-to-back elongated and regenerating crypts, and high-grade dysplasia in surface epithelia. These abnormalities were not observed in Rack1-expressing areas of intestine or in control mice. Following irradiation, apoptosis of enterocytes was strikingly reduced in Rack1-deleted epithelia. These novel findings reveal key functions for Rack1 in regulating growth of intestinal epithelia: suppressing crypt cell proliferation and regeneration, promoting differentiation and apoptosis, and repressing development of neoplasia. NEW & NOTEWORTHY Our findings reveal novel functions for receptor for activated C kinase 1 (Rack1) in regulating growth of intestinal epithelia: suppressing crypt cell proliferation and regeneration, promoting differentiation and apoptosis, and repressing development of neoplasia. [ABSTRACT FROM AUTHOR]

Estrogens have historically been associated with female reproduction, but work over the last two decades established that estrogens and their main nuclear receptors (ESR1 and ESR2) and G protein-coupled estrogen receptor (GPER) also regulate male reproductive and nonreproductive organs. 17β-Estradiol (E2) is measureable in blood of men and males of other species, but in rete testis fluids, E2 reaches concentrations normally found only in females and in some species nanomolar concentrations of estrone sulfate are found in semen. Aromatase, which converts androgens to estrogens, is expressed in Leydig cells, seminiferous epithelium, and other male organs. Early studies showed E2 binding in numerous male tissues, and ESR1 and ESR2 each show unique distributions and actions in males. Exogenous estrogen treatment produced male reproductive pathologies in laboratory animals and men, especially during development, and studies with transgenic mice with compromised estrogen signaling demonstrated an E2 role in normal male physiology. Efferent ductules and epididymal functions are dependent on estrogen signaling through ESR1, whose loss impaired ion transport and water reabsorption, resulting in abnormal sperm. Loss of ESR1 or aromatase also produces effects on nonreproductive targets such as brain, adipose, skeletal muscle, bone, cardiovascular, and immune tissues. Expression of GPER is extensive in male tracts, suggesting a possible role for E2 signaling through this receptor in male reproduction. Recent evidence also indicates that membrane ESR1 has critical roles in male reproduction. Thus estrogens are important physiological regulators in males, and future studies may reveal additional roles for estrogen signaling in various target tissues. [ABSTRACT FROM AUTHOR]

The neuromodulator-gated current (IMI) found in the crab stomatogastric ganglion is activated by neuromodulators that are essential to induce the rhythmic activity of the pyloric network in this system. One of these neuromodulators is also known to control the correlated expression of voltage-gated ionic currents in pyloric neurons, as well as synaptic plasticity and strength. Thus understanding the mechanism by which neuromodulator receptors activate IMI should provide insights not only into how oscillations are initiated but also into how other processes, and currents not directly activated by them, are regulated. To determine what specific signaling molecules are implicated in this process, we used a battery of agonists and antagonists of common signal transduction pathways. We found that the G protein inhibitor GDPβS and the G protein activator GTPγS significantly affect IMI amplitude, suggesting that its activation is mediated by G proteins. Interestingly, when using the more specific G protein blocker pertussis toxin, we observed the expected inhibition of IMI amplitude but, unexpectedly, in a calcium-dependent fashion. We also found that antagonists of calcium- and calmodulin-associated signaling significantly reduce IMI amplitude. In contrast, we found little evidence for the role of cyclic nucleotide signaling, phospholipase C (PLC), or kinases and phosphatases, except two calmodulin-dependent kinases. In sum, these results suggest that proctolin-induced IMI is mediated by a G protein whose pertussis toxin sensitivity is altered by external calcium concentration and appears to depend on intracellular calcium, calmodulin, and calmodulin-activated kinases. In contrast, we found no support for IMI being mediated by PLC signaling or cyclic nucleotides. NEW & NOTEWORTHY Neuronal rhythmic activity is generated by either network-based or cell-autonomous mechanisms. In the pyloric network of decapod crustaceans, the activation of a neuromodulator-gated pacemaker current is crucial for the generation of rhythmic activity. This current is activated by several neuromodulators, including peptides and acetylcholine, presumably via metabotropic receptors. We have previously demonstrated a novel extracellular calcium-sensitive voltage-dependence mechanism of this current. We presently report that the activation mechanism depends on intracellular and extracellular calcium-sensitive components. [ABSTRACT FROM AUTHOR]

The preglomerular microcirculation of spontaneously hypertensive rats (SHR) is hypersensitive to angiotensin (ANG) II, and studies have shown that this is likely due to enhanced coincident signaling between G protein subunits αq (Gαq; released by ANG II) and βγ (Gβγ released by Gi-coupled receptors) to active phospholipase C (PLC). Here we investigated the molecular basis for the enhanced coincident signaling between Gβγ and Gαq in SHR preglomerular vascular smooth muscle cells (PGVSMCs). Because receptor for activated C kinase 1 (RACK1; a scaffolding protein) organizes interactions between Gβγ, Gαq, and PLC, we included RACK1 in this investigation. Cell fractionation studies demonstrated increased levels of membrane (but not cytosolic) Gγ, Gαq, PLCγ3, and RACK1 in SHR PGVSMCs compared with Wistar-Kyoto rat PGVSMCs. In SHR PGVSMCs, coimmunoprecipitation demonstrated RACK1 binding to Gγ and PLCγ3, but only at cell membranes. Pertussis toxin (which blocks Gβγ) and U73122 (which blocks PLC) reduced membrane RACK1; however, RACK1 knockdown (shRNA) did not affect membrane levels of Gγ, Gαq, or PLCγ3. In a novel gel contraction assay, RACK1 knockdown in SHR PGVSMCs attenuated contractions to ANG II and abrogated the ability of neuropeptide Y (which signals via Gβγ) to enhance ANG II-induced contractions. We conclude that in SHR PGVSMCs the enlarged pool of Gβγ and PLCγ3 recruits RACK1 to membranes and RACK1 then organizes signaling. Consequently, knockdown of RACK1 prevents coincident signaling between ANG II and the Gi pathway. This is the first study to implicate RACK1 in vascular smooth muscle cell contraction and suggests that RACK1 inhibitors could be effective cardiovascular drugs. [ABSTRACT FROM AUTHOR]

Binding of angiotensin II (ANG II) to the AT1 receptor (AT1R) in the proximal tubule stimulates Na+/H+ exchanger isoform 3 (NHE3) activity through multiple signaling pathways. However, the effects of ANG II/AT1R-induced inihibitory G protein (Gi) activation and subsequent decrease in cAMP accumulation on NHE3 regulation are not well established. We therefore tested the hypothesis that ANG II reduces cAMP/PKA-mediated phosphorylation of NHE3 on serine 552 and, in doing so, stimulates NHE3 activity. Under basal conditions, ANG II stimulated NHE3 activity but did not affect PKA-mediated NHE3 phosphorylation at serine 552 in opossum kidney (OKP) cells. However, in the presence of the cAMP-elevating agent forskolin (FSK), ANG II blocked FSK-induced NHE3 inhibition, reduced intracellular cAMP concentrations, lowered PKA activity, and prevented the FSK-mediated increase in NHE3 serine 552 phosphorylation. All effects of ANG II were blocked by pretreating OKP cells with the AT1R antagonist losartan, highlighting the contribution of the AT1R/Gi pathway in ANG II-mediated NHE3 upregulation under cAMP-elevating conditions. Accordingly, Gi inhibition by pertussis toxin treatment decreased NHE3 activity both in vitro and in vivo and, more importantly, prevented the stimulatory effect of ANG II on NHE3 activity in rat proximal tubules. Collectively, our results suggest that ANG II counteracts the effects of cAMP/PKA on NHE3 phosphorylation and inhibition by activating the AT1R/Gi pathway. Moreover, these findings support the notion that NHE3 dephosphorylation at serine 552 may represent a key event in the regulation of renal proximal tubule sodium handling by ANG II in the presence of natriuretic hormones that promote cAMP accumulation and transporter phosphorylation. [ABSTRACT FROM AUTHOR]

Autosomal dominant polycystic kidney disease (ADPKD) is a signalopathy of renal tubular epithelial cells caused by naturally occurring mutations in two distinct genes, polycystic kidney disease 1 (PKD1) and 2 (PKD2). Genetic variants in PKD1, which encodes the polycystin-1 (PC-1) protein, remain the predominant factor associated with the pathogenesis of nearly two-thirds of all patients diagnosed with PKD. Although the relationship between defective PC-1 with renal cystic disease initiation and progression remains to be fully elucidated, there are numerous clinical studies that have focused upon the control of effector systems involving heterotrimeric G protein regulation. A major regulator in the activation state of heterotrimeric G proteins are G proteincoupled receptors (GPCRs), which are defined by their seven transmembranespanning regions. PC-1 has been considered to function as an unconventional GPCR, but the mechanisms by which PC-1 controls signal processing, magnitude, or trafficking through heterotrimeric G proteins remains to be fully known. The diversity of heterotrimeric G protein signaling in PKD is further complicated by the presence of non-GPCR proteins in the membrane or cytoplasm that also modulate the functional state of heterotrimeric G proteins within the cell. Moreover, PC-1 abnormalities promote changes in hormonal systems that ultimately interact with distinct GPCRs in the kidney to potentially amplify or antagonize signaling output from PC-1. This review will focus upon the canonical and noncanonical signaling pathways that have been described in PKD with specific emphasis on which heterotrimeric G proteins are involved in the pathological reorganization of the tubular epithelial cell architecture to exacerbate renal cystogenic pathways. [ABSTRACT FROM AUTHOR]

The thalamus acts as a conduit for sensory and other information traveling to the cortex. In response to continuous sensory stimulation in vivo, the firing rate of thalamocortical neurons initially increases, but then within a minute firing rate decreases and T-type Ca2+ channel-dependent action potential burst firing emerges. While neuromodulatory systems could play a role in this inhibitory response, we instead report a novel and cell-autonomous inhibitory mechanism intrinsic to the thalamic relay neuron. Direct intracellular stimulation of thalamocortical neuron firing initially triggered a continuous and high rate of action potential discharge, but within a minute membrane potential (Vm) was hyperpolarized and firing rate to the same stimulus was decreased. This self-inhibition was observed across a wide variety of thalamic nuclei, and in a subset firing mode switched from tonic to bursting. The self-inhibition resisted blockers of intracellular Ca2+ signaling, Na+-K+-ATPases, and G protein-regulated inward rectifier (GIRK) channels as implicated in other neuron subtypes, but instead was in part inhibited by an ATP-sensitive K+ channel blocker. The results identify a new homeostatic mechanism within the thalamus capable of gating excitatory signals at the single-cell level. [ABSTRACT FROM AUTHOR]

Interaction of polycystin-1 (PC1) and Gα12 is important for development of kidney cysts in autosomal dominant polycystic kidney disease (ADPKD). The integrity of cell polarity and cell-cell adhesions (mainly E-cadherin-mediated adherens junction) is altered in the renal epithelial cells of ADPKD. However, the key signaling pathway for this alteration is not fully understood. Madin-Darby canine kidney (MDCK) cells maintain the normal integrity of epithelial cell polarity and adherens junctions. Here, we found that deletion of Pkd1 increased activation of Gα12, which then promoted the cystogenesis of MDCK cells. The morphology of these cells was altered after the activation of Gα12. By using liquid chromatography-mass spectrometry, we found several proteins that could be related this change in the extracellular milieu. E-cadherin was one of the most abundant peptides after active Gα12 was induced. Gα12 activation or Pkd1 deletion increased the shedding of E-cadherin, which was mediated via increased ADAM10 activity. The increased shedding of E-cadherin was blocked by knockdown of ADAM10 or specific ADAM10 inhibitor GI254023X. Pkd1 deletion or Gα12 activation also changed the distribution of E-cadherin in kidney epithelial cells and caused β-catenin to shift from cell membrane to nucleus. Finally, ADAM10 inhibitor, GI254023X, blocked the cystogenesis induced by PC1 knockdown or Gα12 activation in renal epithelial cells. Our results demonstrate that the E-cadherin/β-catenin signaling pathway is regulated by PC1 and Gα12 via ADAM10. Specific inhibition of this pathway, especially ADAM10 activity, could be a novel therapeutic regimen for ADPKD. [ABSTRACT FROM AUTHOR]

During myocardial ischemia, upregulation of the hedgehog (Hh) pathway promotes neovascularization and increases cardiomyocyte survival. The canonical Hh pathway activates a transcriptional program through the Gli family of transcription factors by derepression of the seven-transmembrane protein smoothened (Smo). The mechanisms linking Smo to Gli are complex and, in some cell types, involve coupling of Smo to G¡ proteins. In the present study, we investigated, for the first time, the transcriptional response of cardiomyocytes to sonic hedgehog (Shh) and the role of G¡ protein utilization. Our results show that Shh strongly activates Gli1 expression by quantitative PCR in a Smodependent manner in neonatal rat ventricular cardiomyocytes. Microarray analysis of gene expression changes elicited by Shh and sensitive to a Smo inhibitor identified a small subset of 37 cardiomyocyte- specific genes regulated by Shh, including some in the PKA and purinergic signaling pathways. In addition, neonatal rat ventricular cardiomyocytes infected with an adenovirus encoding GiCT, a peptide that impairs receptor-Gi protein coupling, showed reduced activation of Hh targets. In vitro data were confirmed in transgenic mice with cardiomyocyte-inducible GiCT expression. Transgenic GiCT mice showed specific reduction of Gli1 expression in the heart under basal conditions and failed to upregulate the Hh pathway upon ischemia and reperfusion injury, unlike their littermate controls. This study characterizes, for the first time, the transcriptional response of cardiomyocytes to Shh and establishes a critical role for Smo coupling to G¡ in Hh signaling in the normal and ischemic myocardium. [ABSTRACT FROM AUTHOR]

Extracellular acidification has been observed in allergic inflammatory diseases. Recently, we demonstrated that the proton-sensing receptor G protein-coupled receptor 65 (GPR65) regulates eosinophil survival in an acidic environment in vitro and eosinophil accumulation in an allergic lung inflammation model. For mast cells, another inflammatory cell type critical for allergic responses, it remains unknown whether GPR65 is expressed and/or regulates mast cell viability. Thus, in the present study, we employed in vitro experiments and an intestinal anaphylaxis model in which both mastocytosis and eosinophilia can be observed, particularly in the gastrointestinal tract, to enable us to directly compare the effect of GPR65 expression on these two cell types. We identified GPR65 expression on mast cells; however, unlike eosinophil viability, mast cell viability in vitro is not affected by acidification or GPR65 expression. Mechanistically, we determined that mast cells do not respond to extracellular acidification with increased cAMP levels. Furthermore, in the intestinal anaphylaxis model, we observed a significant reduction of eosinophils (59.1 ± 9.2% decrease) in the jejunum of allergen-challenged GPR65-deficient mice compared with allergen-challenged wild-type mice, despite the degree of antigen sensitization and the expression levels of Th2 cytokines (Il4, Il13) and eosinophil chemokines (Ccl11, Ccl24) in the jejunum being comparable. In contrast, the accumulation of mast cells in allergen-challenged mice was not affected by GPR65 deficiency. In conclusion, our study demonstrates differential regulation of eosinophils and mast cells in inflammatory tissue, with mast cell viability and accumulation being independent of GPR65. [ABSTRACT FROM AUTHOR]

Acute intermittent hypoxia (AIH; three 5-min hypoxic episodes) causes a form of phrenic motor facilitation (pMF) known as phrenic long-term facilitation (pLTF); pLTF is initiated by spinal activation of Gq protein-coupled 5-HT2 receptors. Because α1 adrenergic receptors are expressed in the phrenic motor nucleus and are also Gq protein-coupled, we hypothesized that α1 receptors are sufficient, but not necessary for AIH-induced pLTF. In anesthetized, paralyzed, and ventilated rats, episodic spinal application of the ai receptor agonist phenylephrine (PE) elicited dose-dependent pMF (10 and 100 pM, P < 0.05; but not 1 µM). PE-induced pMF was blocked by the α1 receptor antagonist prazosin (1 mM; - 20 ± 20% at 60 min, - 5 ± 21% at 90 min; n = 6). Although α1 receptor activation is sufficient to induce pMF, it was not necessary for AIH-induced pLTF because intrathecal prazosin (1 mM) did not alter AIH-induced pLTF (56 ± 9% at 60 min, 78 ± 12% at 90 min; n = 9). Intravenous (iv) prazosin (150 µ/kg) appeared to reduce pLTF (21 ± 9% at 60 min, 26 ± 8% at 90 min), but this effect was not significant. Hypoglossal long-term facilitation was unaffected by intrathecal prazosin, but was blocked by iv prazosin (-4 ± 14% at 60 min, -13 ± 18% at 90 min), suggesting different LTF mechanisms in different motor neuron pools. In conclusion, Gq protein-coupled α1 adrenergic receptors evoke pMF, but they are not necessary for AIH-induced pLTF. [ABSTRACT FROM AUTHOR]

Skeletal muscle is the major tissue disposing of dietary glucose, a function regulated by insulin-elicited signals that impart mobilization of GLUT4 glucose transporters to the plasma membrane. This phenomenon, also central to adipocyte biology, has been the subject of intense and productive research for decades. We focus on muscle cell studies scrutinizing insulin signals and vesicle traffic in a spatiotemporal manner. Using the analogy of an integrated circuit to approach the intersection between signal transduction and vesicle mobilization, we identify signaling relays ("software") that engage structural/mechanical elements ("hardware") to enact the rapid mobilization and incorporation of GLUT4 into the cell surface. We emphasize how insulin signal transduction switches from tyrosine through lipid and serine phosphorylation down to activation of small G proteins of the Rab and Rho families, describe key negative regulation step of Rab GTPases through the GTPase-activating protein activity of the Akt substrate of 160 kDa (AS160), and focus on the mechanical effectors engaged by Rabs 8A and 10 (the molecular motor myosin Va), and the Rho GTPase Rac1 (actin filament branching and severing through Arp2/3 and cofilin). Finally, we illustrate how actin filaments interact with myosin 1c and ɑ-Actinin4 to promote vesicle tethering as preamble to fusion with the membrane. [ABSTRACT FROM AUTHOR]

Canonical transient receptor potential- 6 (TRPC6) channels have been implicated in the pathogenesis of kidney disease and in the regulation of vascular smooth muscle tone, podocyte function, and a variety of processes in other cell types. The question of whether their gating is intrinsically mechanosensitive has been controversial. In this study we have examined activation of two alleles of TRPC6 transiently expressed in CHO-K1 cells: the wild-type human TRPC6 channel, and TRPC6-N143S, an allele originally identified in a family with autosomal dominant familial focal and segmental glomerulosclerosis (FSGS). We observed that both channel variants carried robust cationic currents that could be evoked by application of membrane-permeable analogs of diacylglycerol (DAG) or by the P2Y receptor agonist ATP. The amplitudes and characteristics of currents evoked by the DAG analog or ATP were indistinguishable in cells expressing the two TRPC6 alleles. By contrast, hypoosmotic stretch evoked robust currents in wild-type TRPC6 channels but had no discernible effect on currents in cells expressing TRPC6-N143S, indicating that the mutant form lacks mechanosensitivity. Coexpression of TRPC6-N143S with wild-type TRPC6 or TRPC3 channels did not alter stretch-evoked responses compared with when TRPC3 channels were expressed by themselves, indicating that TRPC6-N143S does not function as a dominantnegative. These data indicate that mechanical activation and activation evoked by DAG or ATP occur through fundamentally distinct biophysical mechanisms, and they provide support for the hypothesis that protein complexes containing wild-type TRPC6 subunits can be intrinsically mechanosensitive. [ABSTRACT FROM AUTHOR]

Adenylyl cyclases (AC) catalyze formation of cAMP, a critical component of G protein-coupled receptor signaling. So far, nine distinct membrane-bound AC isoforms (AC1-9) and one soluble AC (sAC) have been identified and, except for AC8, all of them are expressed in the kidney. While the role of ACs in renal cAMP formation is well established, we are just beginning to understand the function of individual AC isoforms, particularly with regard to hormonal regulation of transporter and channel phosphorylation, membrane abundance, and trafficking. This review focuses on the role of different AC isoforms in regulating renal water and electrolyte transport in health as well as potential pathological implications of disordered AC isoform function. In particular, we focus on modulation of transporter and channel abundance, activity, and phosphorylation, with an emphasis on studies employing genetically modified animals. As will be described, it is now evident that specific AC isoforms can exert unique effects in the kidney that may have important implications in our understanding of normal physiology as well as disease pathogenesis. [ABSTRACT FROM AUTHOR]

The majority of the biological actions attributed to somatostatin (SST) are thought to be mediated by SST receptor 2 (sst2), the most ubiquitous sst, and, to a lesser extent, by sst5. However, a growing body of evidence suggests a relevant role of sst1 in mediating SST actions in (patho)physiological situations (i.e., endometriosis, type 2 diabetes). Moreover, sst1 together with sst2 and sst5 is involved in the well-known actions of SST on pituitary somatotropes in pig and primates. Here, we cloned the porcine sst1 (psst1) and performed a structural and functional characterization using both primary and heterologous models. The psst1 sequence presents the majority of signature motifs shared among G proteincoupled receptors and, specifically, among ssts and exhibits a high homology with other mammalian sst1, with only minor differences in the amino-terminal domain, reinforcing the idea of an early evolutive divergence between mammalian and nonmammalian sst1s. psst1 is functional in terms of decreasing cAMP levels in response to SST when transfected in heterologous models. The psst1 receptor is expressed in several tissues, and analyses of gene cis elements predict regulation by multiple transcription factors and metabolic stimuli. Finally, psst1 is coexpressed with other sst subtypes in various tissues, and in vitro data demonstrate that psst1 can interact with itself forming homodimers and with other ssts forming heterodimers. These data highlight the functional importance of sst1 on the SST-mediated effects and its functional interaction with different ssts, which point out the necessity of exploring the consequences of such interactions. [ABSTRACT FROM AUTHOR]

Blood flow acts parallel to the coronary luminal endothelial surface layer (LESL) and modulates multiple parenchymal functions via the release of paracrine agents. Evidence indicates that the LESL may be a flow-sensing organelle and that perhaps through flow-induced lectin (L)·oligosaccharide (O) complex formation (L·O) participates in this process. LESL integrins and selectins are both lectinic and flow sensitive, but the L properties of flow-sensitive G protein-coupled receptors (GPCRs) are unknown. Therefore, we investigated the presence of L in the LESL and hypothesized that if flow-sensitive GPCRs are L, flow and O will determine their response to receptor activation. The LESL protein fraction isolated from guinea pig hearts was passed through an affinity chromatography column made of three sugars, mannose, galactose, and N-acetylglucosamine, and the lectinic fraction was eluted. Immune dot blot was used to identify L proteins in the LESL fraction. Our results indicate the following. 1) Two-dimensional SDS-PAGE (2D-SDS-PAGE) of the LESL lectinic fraction revealed at least 167 Ls. 2) Among these Ls, we identified three selectins and the GPCRs: angiotensin II, bradykinin (B2-R), adenosine A1 and A2, prolactin, endothelin, α1-adrenergic (α1A-R), thromboxane A2, β1-adrenergic, β3-adrenergic, and insulin receptors; the first six GPCRs are known to be flow sensitive. 3) The amplitude of receptor-induced vascular responses by α1A-R and B2-R activation (phenylephrine or bradykinin, respectively) was a function of flow and O (hyaluronidate). Our results support a novel mechanism of GPCR-mediated responses to flow via L·O interaction. [ABSTRACT FROM AUTHOR]

In the porcine coronary artery, regenerated endothelium is dysfunctional as regards the responses to endothelium-dependent agonists. The current study aimed to determine the possible involvement of histamine in such dysfunction. Pigs were treated chronically with pyrilamine (H1 receptor inhibitor, 2 mg·kg-1·day-1) with part of their coronary endothelium and allowed to regenerate for 28 days after balloon denudation. The results showed a reduction in relaxation to bradykinin (Gq protein dependent) only in the pyrilamine-treated group (area under the curve, 269.7 ± 13.4 vs. 142.0 ± 31.0, native endothelium vs. regenerated endothelium) but not in the control group (253.0 ± 22.1 vs. 231.9 ± 29.5, native endothelium vs. regenerated endothelium). The differences in the relaxation to serotonin (Gi protein dependent) between native and regenerated endothelium were not affected by the pyrilamine treatment (control group, 106.3 ± 17.0 vs. 55.61 ± 12.7; and pyrilamine group, 106.0 ± 8.20 vs. 49.30 ± 6.31, native endothelium vs. regenerated endothelium). These findings indicate that during regeneration of the endothelium, the activation of H1 receptors by endogenous histamine may be required to maintain the endothelium-dependent Gq protein-mediated relaxation to bradykinin, suggesting a beneficial role of the monoamine in the process of endothelial regeneration. [ABSTRACT FROM AUTHOR]

Chemokines participate in various processes of monocyte recruitment including monocyte arrest and migration. Our group and others have demonstrated that growth-related oncogene (GRO)-[alpha] (CXCL1) can support monocyte arrest in models of inflammation. Here we employed a parallel plate-flow chamber and Transwell reconstitution assay to test whether GRO family chemokines were sufficient for Mono Mac 6 (a human monocytic cell line) and isolated human monocyte recruitment. Our study shows that 1) GRO-[alpha], -[beta] (CXCL2), and -[gamma] (CXCL3) all act as arrest chemokines for monocyte adhesion on vascular cell adhesion molecule (VCAM)-1 under flow in the presence of P-selectin; 2) CXCR2 is the functional receptor for GRO-family chemokines in monocyte arrest; however, CXCR2 is not an arrest chemokine receptor in general, since epithelial neutrophil-activating peptide ENA-78 failed to arrest monocytes; 3) GRO-[alpha], -[beta], and -[gamma] all fail to increase intracellular free [Ca.sup.2+] or mediate monocyte chemotaxis; and 4) signaling through G[[alpha].sub.i] protein, phosphoinositide 3-kinase, and actin polymerization but not [Ca.sup.2+] mobilization or the mitogen-activated kinases p38 and MAPK/extracellular signal-related kinase are necessary for GRO-[alpha]-mediated Mono Mac 6 cell arrest under flow. We conclude that the GRO-family chemokines are specialized monocyte-arrest chemokines. Their role in monocyte recruitment in inflammation can be inhibited by blocking CXCR2 function or downstream signaling events. growth-related oncogene; P-selectin; vascular cell adhesion molecule-l; phosphoinositide 3-kinase; G protein; signaling

Arrhythmia-prone subepicardial border zone (EBZ) tissue demonstrates decreased G protein receptor kinase 2 (GRK2) activity and increased sensitivity to isoproterenol 6-24 h after coronary artery ligation (CAL) in the dog. With the use of a semiquantitative immunofluorescence technique, the relative fluorescence intensity (RF) of GRK2 in EBZ decreased to 24% of that in a remote site (RS) (P < 0.01, n = 30 cells from 3 dogs), whereas GRK5 RF did not change. Confocal studies of cardiac tissue from transgenic mice overexpressing GRK2 validated the use of a semilogarithmic relationship between RF and GRK2 activity. As shown with the use of quantitative real-time RT-PCR, both GRK2 and GRK5 mRNA were not decreased at 24 h in EBZ (n = 6 dogs) relative to RS control, indicating that the decrease of GRK2 in the EBZ is likely due to posttranscriptional degradation following CAL. Pretreatment of six dogs with the selective proteasome inhibitor bortezomib provided 100% (EBZ) and 50% (infarct) protection against loss of GRK2 at 24 h. There was an absence of rapid (>300 beats/min) and very rapid (>360 beats/min) ventricular triplets that are highly predictive of sudden cardiac death during ECG monitoring in the bortezomib-pretreated animals in contrast to nonpretreated infarcted animals. We have demonstrated that the dramatic decrease in GRK2 in cardiac ischemic tissue can bc largely blocked by prior proteasome blockade and that this is associated with significant cardioprotection against malignant ventricular tachyarrhythmias. [beta]-adrenergic receptor; G protein receptor kinase 2; sudden cardiac death; ventricular arrhythmias; immunofluorescence microscopy