TRPC5 controls the adrenaline-mediated counter regulation of hypoglycemia.

Hypoglycemia triggers autonomic and endocrine counter-regulatory responses to restore glucose homeostasis, a response that is impaired in patients with diabetes and its long-term complication hypoglycemia-associated autonomic failure (HAAF). We show that insulin-evoked hypoglycemia is severely aggravated in mice lacking the cation channel proteins TRPC1, TRPC4, TRPC5, and TRPC6, which cannot be explained by alterations in glucagon or glucocorticoid action. By using various TRPC compound knockout mouse lines, we pinpointed the failure in sympathetic counter-regulation to the lack of the TRPC5 channel subtype in adrenal chromaffin cells, which prevents proper adrenaline rise in blood plasma. Using electrophysiological analyses, we delineate a previously unknown signaling pathway in which stimulation of PAC1 or muscarinic receptors activates TRPC5 channels in a phospholipase-C-dependent manner to induce sustained adrenaline secretion as a crucial step in the sympathetic counter response to insulin-induced hypoglycemia. By comparing metabolites in the plasma, we identified reduced taurine levels after hypoglycemia induction as a commonality in TRPC5-deficient mice and HAAF patients.

Saraf-dependent activation of mTORC1 regulates cardiac growth.

Pathological cardiac hypertrophy is an independent risk for heart failure (HF) and sudden death. Deciphering signaling pathways regulating intracellular Ca2+ homeostasis that control adaptive and pathological cardiac growth may enable identification of novel therapeutic targets. The objective of the present study is to determine the role of the store-operated calcium entry-associated regulatory factor (Saraf), encoded by the Tmem66 gene, on cardiac growth control in vitro and in vivo. Saraf is a single-pass membrane protein located at the sarco/endoplasmic reticulum and regulates intracellular calcium homeostasis. We found that Saraf expression was upregulated in the hypertrophied myocardium and was sufficient for cell growth in response to neurohumoral stimulation. Increased Saraf expression caused cell growth, which was associated with dysregulation of calcium-dependent signaling and sarcoplasmic reticulum calcium content. In vivo, Saraf augmented cardiac myocyte growth in response to angiotensin II and resulted in increased cardiac remodeling together with worsened cardiac function. Mechanistically, Saraf activated mTORC1 (mechanistic target of rapamycin complex 1) and increased protein synthesis, while mTORC1 inhibition blunted Saraf-dependent cell growth. In contrast, the hearts of Saraf knockout mice and Saraf-deficient myocytes did not show any morphological or functional alterations after neurohumoral stimulation, but Saraf depletion resulted in worsened cardiac function after acute pressure overload. SARAF knockout blunted transverse aortic constriction cardiac myocyte hypertrophy and impaired cardiac function, demonstrating a role for SARAF in compensatory myocyte growth. Collectively, these results reveal a novel link between sarcoplasmic reticulum calcium homeostasis and mTORC1 activation that is regulated by Saraf.

Connexin45 provides optimal atrioventricular nodal conduction in the adult mouse heart.

RATIONALE: The gap junctional protein connexin (Cx) 45 is strongly expressed in the early embryonic myocardium. In the adult hearts of mice and humans, the expression mainly is restricted to the cardiac conduction system. Cx45 plays an essential role for development and function of the embryonic heart because general and cardiomyocyte-directed deficiencies of Cx45 in mice lead to embryonic lethality attributable to morphological and functional cardiovascular defects. The function of Cx45 in the adult mouse has not yet been cleared. OBJECTIVE: To clarify the function of Cx45 in the adult mouse heart. METHODS AND RESULTS: To circumvent the embryonic lethality resulting from Cx45 deficiency, mice were generated in which deletion of Cx45 specifically was induced in cardiomyocytes of adult mice. These Cx45-deficient mice were viable but showed a decrease in atrioventricular nodal conductivity. In addition, the Cx30.2 protein that is coexpressed with Cx45 in the cardiac conduction system was posttranscriptionally reduced by 70% in mutant hearts. Furthermore, deletion of both Cx45 and Cx30.2 resulted in viable mice that, however, showed stronger impairment of atrioventricular nodal conduction than the single Cx45-deficient mice. CONCLUSIONS: Cx45 is required for optimal impulse propagation in the atrioventricular node and stabilizes the level of the coexpressed Cx30.2 protein in the adult mouse heart. In contrast to the embryo, Cx45 is not essential for the viability of adult mice.

OCaR1 endows exocytic vesicles with autoregulatory competence by preventing uncontrolled Ca2+ release, exocytosis, and pancreatic tissue damage.

Regulated exocytosis is initiated by increased Ca2+ concentrations in close spatial proximity to secretory granules, which is effectively prevented when the cell is at rest. Here we showed that exocytosis of zymogen granules in acinar cells was driven by Ca2+ directly released from acidic Ca2+ stores including secretory granules through NAADP-activated two-pore channels (TPCs). We identified OCaR1 (encoded by Tmem63a) as an organellar Ca2+ regulator protein integral to the membrane of secretory granules that controlled Ca2+ release via inhibition of TPC1 and TPC2 currents. Deletion of OCaR1 led to extensive Ca2+ release from NAADP-responsive granules under basal conditions as well as upon stimulation of GPCR receptors. Moreover, OCaR1 deletion exacerbated the disease phenotype in murine models of severe and chronic pancreatitis. Our findings showed OCaR1 as a gatekeeper of Ca2+ release that endows NAADP-sensitive secretory granules with an autoregulatory mechanism preventing uncontrolled exocytosis and pancreatic tissue damage.

G(13)-mediated signaling pathway is required for pressure overload-induced cardiac remodeling and heart failure.

BACKGROUND: Cardiac remodeling in response to pressure or volume overload plays an important role in the pathogenesis of heart failure. Various mechanisms have been suggested to translate mechanical stress into structural changes, one of them being the release of humoral factors such as angiotensin II and endothelin-1, which in turn promote cardiac hypertrophy and fibrosis. A large body of evidence suggests that the prohypertrophic effects of these factors are mediated by receptors coupled to the G(q/11) family of heterotrimeric G proteins. Most G(q/11)-coupled receptors, however, can also activate G proteins of the G(12/13) family, but the role of G(12/13) in cardiac remodeling is not understood. METHODS AND RESULTS: We use siRNA-mediated knockdown in vitro and conditional gene inactivation in vivo to study the role of the G(12/13) family in pressure overload-induced cardiac remodeling. We show in detail that inducible cardiomyocyte-specific inactivation of the α subunit of G(13), Gα(13), does not affect basal heart function but protects mice from pressure overload-induced hypertrophy and fibrosis as efficiently as inactivation of Gα(q/11). Furthermore, inactivation of Gα(13) prevents the development of heart failure up to 1 year after overloading. On the molecular level, we show that Gα(13), but not Gα(q/11), controls agonist-induced expression of hypertrophy-specific genes through activation of the small GTPase RhoA and consecutive activation of myocardin-related transcription factors. CONCLUSION: Our data show that the G(12/13) family of heterotrimeric G proteins is centrally involved in pressure overload-induced cardiac remodeling and plays a central role in the transition to heart failure.

Deletion of the last five C-terminal amino acid residues of connexin43 leads to lethal ventricular arrhythmias in mice without affecting coupling via gap junction channels.

The cardiac intercalated disc harbors mechanical and electrical junctions as well as ion channel complexes mediating propagation of electrical impulses. Cardiac connexin43 (Cx43) co-localizes and interacts with several of the proteins located at intercalated discs in the ventricular myocardium. We have generated conditional Cx43D378stop mice lacking the last five C-terminal amino acid residues, representing a binding motif for zonula occludens protein-1 (ZO-1), and investigated the functional consequences of this mutation on cardiac physiology and morphology. Newborn and adult homozygous Cx43D378stop mice displayed markedly impaired and heterogeneous cardiac electrical activation properties and died from severe ventricular arrhythmias. Cx43 and ZO-1 were co-localized at intercalated discs in Cx43D378stop hearts, and the Cx43D378stop gap junction channels showed normal coupling properties. Patch clamp analyses of isolated adult Cx43D378stop cardiomyocytes revealed a significant decrease in sodium and potassium current densities. Furthermore, we also observed a significant loss of Nav1.5 protein from intercalated discs in Cx43D378stop hearts. The phenotypic lethality of the Cx43D378stop mutation was very similar to the one previously reported for adult Cx43 deficient (Cx43KO) mice. Yet, in contrast to Cx43KO mice, the Cx43 gap junction channel was still functional in the Cx43D378stop mutant. We conclude that the lethality of Cx43D378stop mice is independent of the loss of gap junctional intercellular communication, but most likely results from impaired cardiac sodium and potassium currents. The Cx43D378stop mice reveal for the first time that Cx43 dependent arrhythmias can develop by mechanisms other than impairment of gap junction channel function.

Membrane-bound Interleukin-1α mediates leukocyte adhesion during atherogenesis.

Introduction: The interleukin-1 (IL-1) family and the NLR family pyrin domain-containing 3 (NLRP3) inflammasome contribute to atherogenesis but the underlying mechanisms are incompletely understood. Unlike IL-1ß, IL-1α is not dependent on the NLRP3 inflammasome to exert its pro-inflammatory effects. Here, a non-genetic model was applied to characterize the role of IL-1α, IL-1ß, and NLRP3 for the pathogenesis of atherosclerosis. Methods: Atherogenesis was induced by gain-of-function PCSK9-AAV8 mutant viruses and feeding of a high-fat western diet (WTD) for 12 weeks in C57Bl6/J wildtype mice (WT) and in Il1a-/-, Nlrp3-/-, and Il1b-/- mice. Results: PCSK9-Il1a-/- mice showed reduced atherosclerotic plaque area in the aortic root with lower lipid accumulation, while no difference was observed between PCSK9-WT, PCSK9-Nlrp3-/- and PCSK9-Il1b-/- mice. Serum proteomic analysis showed a reduction of pro-inflammatory cytokines (e.g., IL-1ß, IL-6) in PCSK9-Il1a-/- as well as in PCSK9-Nlrp3-/- and PCSK9-Il1b-/- mice. Bone marrow dendritic cells (BMDC) of PCSK9-WT, PCSK9-Nlrp3-/-, and PCSK9-Il1b-/- mice and primary human monocytes showed translocation of IL-1α to the plasma membrane (csIL-1α) upon stimulation with LPS. The translocation of IL-1α to the cell surface was regulated by myristoylation and increased in mice with hypercholesterolemia. CsIL-1α and IL1R1 protein-protein interaction on endothelial cells induced VCAM1 expression and monocyte adhesion, which was abrogated by the administration of neutralizing antibodies against IL-1α and IL1R1. Conclusion: The results highlight the importance of IL-1α on the cell surface of circulating leucocytes for the development of atherosclerosis. PCSK9-Il1a-/- mice, but not PCSK9-Nlrp3-/- or PCSK9-Il1b-/- mice, are protected from atherosclerosis after induction of hypercholesterolemia independent of circulating cytokines. Myristoylation and translocation of IL-1α to the cell surface in myeloid cells facilitates leukocyte adhesion and contributes to the development of atherosclerosis.

The activity of glyoxylase 1 is regulated by glucose-responsive phosphorylation on Tyr136.

OBJECTIVE: Methylglyoxal (MG) is a highly reactive α-oxoaldehyde that glycates proteins. MG has been linked to the development of diabetic complications: MG is the major precursor of advanced glycation end products (AGEs), a risk marker for diabetic complications in humans. Furthermore, flies and fish with elevated MG develop insulin resistance, obesity, and hyperglycemia. MG is detoxified in large part through the glyoxalase system, whose rate-limiting enzyme is glyoxalase I (Glo1). Hence, we aimed to study how Glo1 activity is regulated. METHODS: We studied the regulation and effect of post-translational modifications of Glo1 in tissue culture and in mouse models of diabetes. RESULTS: We show that Glo1 activity is promoted by phosphorylation on Tyrosine 136 via multiple kinases. We find that Glo1 Y136 phosphorylation responds in a bimodal fashion to glucose levels, increasing in cell culture from 0 mM to 5 mM (physiological) glucose, and then decreasing at higher glucose concentrations, both in cell culture and in mouse models of hyperglycemia. CONCLUSIONS: These data, together with published findings that elevated MG leads to hyperglycemia, suggest the existence of a deleterious positive feedback loop whereby hyperglycemia leads to reduced Glo1 activity, contributing to elevated MG levels, which in turn promote hyperglycemia. Hence, perturbations elevating either glucose or MG have the potential to start an auto-amplifying feedback loop contributing to diabetic complications.

Deep Metabolic Profiling Assessment of Tissue Extraction Protocols for Three Model Organisms.

Metabolic profiling harbors the potential to better understand various disease entities such as cancer, diabetes, Alzheimer's, Parkinson's disease or COVID-19. To better understand such diseases and their intricate metabolic pathways in human studies, model animals are regularly used. There, standardized rearing conditions and uniform sampling strategies are prerequisites towards a successful metabolomic study that can be achieved through model organisms. Although metabolomic approaches have been employed on model organisms before, no systematic assessment of different conditions to optimize metabolite extraction across several organisms and sample types has been conducted. We address this issue using a highly standardized metabolic profiling assay analyzing 630 metabolites across three commonly used model organisms (Drosophila, mouse, and zebrafish) to find an optimal extraction protocol for various matrices. Focusing on parameters such as metabolite coverage, concentration and variance between replicates we compared seven extraction protocols. We found that the application of a combination of 75% ethanol and methyl tertiary-butyl ether (MTBE), while not producing the broadest coverage and highest concentrations, was the most reproducible extraction protocol. We were able to determine up to 530 metabolites in mouse kidney samples, 509 in mouse liver, 422 in zebrafish and 388 in Drosophila and discovered a core overlap of 261 metabolites in these four matrices. To enable other scientists to search for the most suitable extraction protocol in their experimental context and interact with this comprehensive data, we have integrated our data set in the open-source shiny app "MetaboExtract". Hereby, scientists can search for metabolites or compound classes of interest, compare them across the different tested extraction protocols and sample types as well as find reference concentration values.

Novel Nongenetic Murine Model of Hyperglycemia and Hyperlipidemia-Associated Aggravated Atherosclerosis.

Objective: Atherosclerosis, the main pathology underlying cardiovascular diseases is accelerated in diabetic patients. Genetic mouse models require breeding efforts which are time-consuming and costly. Our aim was to establish a new nongenetic model of inducible metabolic risk factors that mimics hyperlipidemia, hyperglycemia, or both and allows the detection of phenotypic differences dependent on the metabolic stressor(s). Methods and Results: Wild-type mice were injected with gain-of-function PCSK9D377Y (proprotein convertase subtilisin/kexin type 9) mutant adeno-associated viral particles (AAV) and streptozotocin and fed either a high-fat diet (HFD) for 12 or 20 weeks or a high-cholesterol/high-fat diet (Paigen diet, PD) for 8 weeks. To evaluate atherosclerosis, two different vascular sites (aortic sinus and the truncus of the brachiocephalic artery) were examined in the mice. Combined hyperlipidemic and hyperglycemic (HGHCi) mice fed a HFD or PD displayed characteristic features of aggravated atherosclerosis when compared to hyperlipidemia (HCi HFD or PD) mice alone. Atherosclerotic plaques of HGHCi HFD animals were larger, showed a less stable phenotype (measured by the increased necrotic core area, reduced fibrous cap thickness, and less α-SMA-positive area) and had more inflammation (increased plasma IL-1ß level, aortic pro-inflammatory gene expression, and MOMA-2-positive cells in the BCA) after 20 weeks of HFD. Differences between the HGHCi and HCi HFD models were confirmed using RNA-seq analysis of aortic tissue, revealing that significantly more genes were dysregulated in mice with combined hyperlipidemia and hyperglycemia than in the hyperlipidemia-only group. The HGHCi-associated genes were related to pathways regulating inflammation (increased Cd68, iNos, and Tnfa expression) and extracellular matrix degradation (Adamts4 and Mmp14). When comparing HFD with PD, the PD aggravated atherosclerosis to a greater extent in mice and showed plaque formation after 8 weeks. Hyperlipidemic and hyperglycemic mice fed a PD (HGHCi PD) showed less collagen (Sirius red) and increased inflammation (CD68-positive cells) within aortic plaques than hyperlipidemic mice (HCi PD). HGHCi-PD mice represent a directly inducible hyperglycemic atherosclerosis model compared with HFD-fed mice, in which atherosclerosis is severe by 8 weeks. Conclusion: We established a nongenetically inducible mouse model allowing comparative analyses of atherosclerosis in HCi and HGHCi conditions and its modification by diet, allowing analyses of multiple metabolic hits in mice.

Rho kinase and hypertension.

Arterial hypertension is a multifactorial disease that is characterised by increased peripheral vascular resistance often accompanied by smooth muscle cell hypertrophy and proliferation. Rho kinases (ROCKs) are the most extensively studied effectors of the small G-protein RhoA and abnormalities in RhoA/ROCK signalling have been observed in various cardiovascular disease including hypertension. The RhoA/ROCK-pathway is a key player in different smooth muscle cell functions including contractility, proliferation and migration. Furthermore, there is extensive crosstalk between RhoA/ROCK- and NO-signalling. Therefore, not only ROCK inhibitors but also NO-donators or pleiotropic agents like statins exert their beneficial effects on the cardiovascular system at least in part via Rho/Rho-kinase.

Smooth muscle-specific deletion of nitric oxide-sensitive guanylyl cyclase is sufficient to induce hypertension in mice.

BACKGROUND: Arterial hypertension is one of the major diseases in industrial countries and a major cause of mortality. One of the main vascular factors responsible for the relaxation of blood vessels and regulation of blood pressure is nitric oxide (NO). NO acts predominantly via NO-sensitive guanylyl cyclase (NO-GC), which is made up of 2 different subunits (alpha and beta). Deletion of the beta(1) subunit leads to a global NO-GC knockout, and these mice are hypertensive. However, global deletion of NO-GC in mice does not allow identification of the cell/tissue type responsible for the elevated blood pressure. METHODS AND RESULTS: To determine the relative contribution of smooth muscle cells to the hypertension seen in NO-GC knockout mice, we generated smooth muscle-specific knockout mice for the beta(1) subunit of NO-GC using a tamoxifen-inducible system. Male mice were investigated because the Cre transgene used is located on the Y chromosome. Tamoxifen injection led to a rapid reduction of NO-GC expression in smooth muscle but did not affect that in other tissues. Parallel to a reduction in NO-induced cGMP accumulation, NO-induced relaxation of aortic smooth muscle was gradually lost after induction by tamoxifen. Concomitantly, these animals developed hypertension within 3 to 4 weeks. CONCLUSIONS: We generated a model in which the development of hypertension can be visualized over time by deletion of a single gene in smooth muscle cells. In sum, our data provide evidence that deletion of NO-GC solely in smooth muscle is sufficient to cause hypertension.

Myogenic vasoconstriction requires G12/G13 and LARG to maintain local and systemic vascular resistance.

Myogenic vasoconstriction is an autoregulatory function of small arteries. Recently, G-protein-coupled receptors have been involved in myogenic vasoconstriction, but the downstream signalling mechanisms and the in-vivo-function of this myogenic autoregulation are poorly understood. Here, we show that small arteries from mice with smooth muscle-specific loss of G12/G13 or the Rho guanine nucleotide exchange factor ARHGEF12 have lost myogenic vasoconstriction. This defect was accompanied by loss of RhoA activation, while vessels showed normal increases in intracellular [Ca2+]. In the absence of myogenic vasoconstriction, perfusion of peripheral organs was increased, systemic vascular resistance was reduced and cardiac output and left ventricular mass were increased. In addition, animals with defective myogenic vasoconstriction showed aggravated hypotension in response to endotoxin. We conclude that G12/G13- and Rho-mediated signaling plays a key role in myogenic vasoconstriction and that myogenic tone is required to maintain local and systemic vascular resistance under physiological and pathological condition.

Helix 8 is the essential structural motif of mechanosensitive GPCRs.

G-protein coupled receptors (GPCRs) are versatile cellular sensors for chemical stimuli, but also serve as mechanosensors involved in various (patho)physiological settings like vascular regulation, cardiac hypertrophy and preeclampsia. However, the molecular mechanisms underlying mechanically induced GPCR activation have remained elusive. Here we show that mechanosensitive histamine H1 receptors (H1Rs) are endothelial sensors of fluid shear stress and contribute to flow-induced vasodilation. At the molecular level, we observe that H1Rs undergo stimulus-specific patterns of conformational changes suggesting that mechanical forces and agonists induce distinct active receptor conformations. GPCRs lacking C-terminal helix 8 (H8) are not mechanosensitive, and transfer of H8 to non-responsive GPCRs confers, while removal of H8 precludes, mechanosensitivity. Moreover, disrupting H8 structural integrity by amino acid exchanges impairs mechanosensitivity. Altogether, H8 is the essential structural motif endowing GPCRs with mechanosensitivity. These findings provide a mechanistic basis for a better understanding of the roles of mechanosensitive GPCRs in (patho)physiology.

Age-dependent blood pressure elevation is due to increased vascular smooth muscle tone mediated by G-protein signalling.

AIMS: Arterial hypertension is a major risk factor for cardiovascular diseases. The kidney and its natriuretic function are in the centre of the prevailing models to explain the pathogenesis of hypertension; however, the mechanisms underlying blood pressure elevation remain unclear in most patients. Development of hypertension is strongly correlated with age, and this blood pressure increase typically accelerates in the fourth decade of life. The cause of age-dependent blood pressure elevation is poorly understood. This study aims to understand the role of procontractile G-protein-mediated signalling pathways in vascular smooth muscle in age-dependent hypertension. METHODS AND RESULTS: Similar to humans at mid-life, we observed in 1-year-old mice elevated blood pressure levels without any evidence for increased vessel stiffness, impaired renal function, or endocrine abnormalities. Hypertensive aged mice showed signs of endothelial dysfunction and had an increased vascular formation of reactive oxygen species (ROS) and elevated endothelial ET-1 expression. Age-dependent hypertension could be normalized by ETA receptor blockade, smooth muscle-specific inactivation of the gene encoding the ETA receptor, as well as by acute disruption of downstream signalling via induction of smooth muscle-specific Gα12/Gα13, Gαq/Gα11, or LARG deficiency using tamoxifen-inducible smooth muscle-specific conditional mouse knock-out models. Induction of smooth muscle-specific ETA receptor deficiency normalized the blood pressure in aged mice despite the continuous presence of signs of endothelial dysfunction. CONCLUSION: Age-dependent blood pressure elevation is due to a highly reversible activation of procontractile signalling in vascular smooth muscle cells indicating that increased vascular tone can be a primary factor in the development of hypertension.

Procontractile G protein-mediated signaling pathways antagonistically regulate smooth muscle differentiation in vascular remodeling.

Vascular smooth muscle (Sm) cells (VSMCs) are highly plastic. Their differentiation state can be regulated by serum response factor (SRF), which activates genes involved in Sm differentiation and proliferation by recruiting cofactors, such as members of the myocardin family and ternary complex factors (TCFs), respectively. However, the extracellular cues and upstream signaling mechanisms regulating SRF-dependent VSMC differentiation under in vivo conditions are poorly understood. In this study, we show that the procontractile signaling pathways mediated by the G proteins G(12)/G(13) and G(q)/G(11) antagonistically regulate VSMC plasticity in different models of vascular remodeling. In mice lacking Gα(12)/Gα(13) or their effector, the RhoGEF protein LARG, RhoA-dependent SRF-regulation was blocked and down-regulation of VSMC differentiation marker genes was enhanced. This was accompanied by an excessive vascular remodeling and exacerbation of atherosclerosis. In contrast, Sm-specific Gα(q)/Gα(11) deficiency blocked activation of extracellular signal-regulated kinase 1/2 and the TCF Elk-1, resulting in a reduced VSMC dedifferentiation in response to flow cessation or vascular injury. These data show that the balanced activity of both G protein-mediated pathways in VSMCs is required for an appropriate vessel remodeling response in vascular diseases and suggest new approaches to modulate Sm differentiation in vascular pathologies.

Nicotinic acid- and monomethyl fumarate-induced flushing involves GPR109A expressed by keratinocytes and COX-2-dependent prostanoid formation in mice.

The antidyslipidemic drug nicotinic acid and the antipsoriatic drug monomethyl fumarate induce cutaneous flushing through activation of G protein-coupled receptor 109A (GPR109A). Flushing is a troublesome side effect of nicotinic acid, but may be a direct reflection of the wanted effects of monomethyl fumarate. Here we analyzed the mechanisms underlying GPR109A-mediated flushing and show that both Langerhans cells and keratinocytes express GPR109A in mice. Using cell ablation approaches and transgenic cell type-specific GPR109A expression in Gpr109a-/- mice, we have provided evidence that the early phase of flushing depends on GPR109A expressed on Langerhans cells, whereas the late phase is mediated by GPR109A expressed on keratinocytes. Interestingly, the first phase of flushing was blocked by a selective cyclooxygenase-1 (COX-1) inhibitor, and the late phase was sensitive to a selective COX-2 inhibitor. Both monomethyl fumarate and nicotinic acid induced PGE2 formation in isolated keratinocytes through activation of GPR109A and COX-2. Thus, the early and late phases of the GPR109A-mediated cutaneous flushing reaction involve different epidermal cell types and prostanoid-forming enzymes. These data will help to guide new efficient approaches to mitigate nicotinic acid-induced flushing and may help to exploit the potential antipsoriatic effects of GPR109A agonists in the skin.

Anaphylactic shock depends on endothelial Gq/G11.

Anaphylactic shock is a severe allergic reaction involving multiple organs including the bronchial and cardiovascular system. Most anaphylactic mediators, like platelet-activating factor (PAF), histamine, and others, act through G protein-coupled receptors, which are linked to the heterotrimeric G proteins G(q)/G(11), G(12)/G(13), and G(i). The role of downstream signaling pathways activated by anaphylactic mediators in defined organs during anaphylactic reactions is largely unknown. Using genetic mouse models that allow for the conditional abrogation of G(q)/G(11)- and G(12)/G(13)-mediated signaling pathways by inducible Cre/loxP-mediated mutagenesis in endothelial cells (ECs), we show that G(q)/G(11)-mediated signaling in ECs is required for the opening of the endothelial barrier and the stimulation of nitric oxide formation by various inflammatory mediators as well as by local anaphylaxis. The systemic effects of anaphylactic mediators like histamine and PAF, but not of bacterial lipopolysaccharide (LPS), are blunted in mice with endothelial G alpha(q)/G alpha(11) deficiency. Mice with endothelium-specific G alpha(q)/G alpha(11) deficiency, but not with G alpha(12)/G alpha(13) deficiency, are protected against the fatal consequences of passive and active systemic anaphylaxis. This identifies endothelial G(q)/G(11)-mediated signaling as a critical mediator of fatal systemic anaphylaxis and, hence, as a potential new target to prevent or treat anaphylactic reactions.

Inducible knockout mutagenesis reveals compensatory mechanisms elicited by constitutive BK channel deficiency in overactive murine bladder.

The large-conductance, voltage-dependent and Ca(2+)-dependent K(+) (BK) channel links membrane depolarization and local increases in cytosolic free Ca(2+) to hyperpolarizing K(+) outward currents, thereby controlling smooth muscle contractility. Constitutive deletion of the BK channel in mice (BK(-/-)) leads to an overactive bladder associated with increased intravesical pressure and frequent micturition, which has been revealed to be a result of detrusor muscle hyperexcitability. Interestingly, time-dependent and smooth muscle-specific deletion of the BK channel (SM-BK(-/-)) caused a more severe phenotype than displayed by constitutive BK(-/-) mice, suggesting that compensatory pathways are active in the latter. In detrusor muscle of BK(-/-) but not SM-BK(-/-) mice, we found reduced L-type Ca(2+) current density and increased expression of cAMP kinase (protein kinase A; PKA), as compared with control mice. Increased expression of PKA in BK(-/-) mice was accompanied by enhanced beta-adrenoceptor/cAMP-mediated suppression of contractions by isoproterenol. This effect was attenuated by about 60-70% in SM-BK(-/-) mice. However, the Rp isomer of adenosine-3',5'-cyclic monophosphorothioate, a blocker of PKA, only partially inhibited enhanced cAMP signaling in BK(-/-) detrusor muscle, suggesting the existence of additional compensatory pathways. To this end, proteome analysis of BK(-/-) urinary bladder tissue was performed, and revealed additional compensatory regulated proteins. Thus, constitutive and inducible deletion of BK channel activity unmasks compensatory mechanisms that are relevant for urinary bladder relaxation.

G12-G13-LARG-mediated signaling in vascular smooth muscle is required for salt-induced hypertension.

The tone of vascular smooth muscle cells is a primary determinant of the total peripheral vascular resistance and hence the arterial blood pressure. Most forms of hypertension ultimately result from an increased vascular tone that leads to an elevated total peripheral resistance. Regulation of vascular resistance under normotensive and hypertensive conditions involves multiple mediators, many of which act through G protein-coupled receptors on vascular smooth muscle cells. Receptors that mediate vasoconstriction couple with the G-proteins G(q)-G11 and G12-G13 to stimulate phosphorylation of myosin light chain (MLC) via the Ca2+/MLC kinase- and Rho/Rho kinase-mediated signaling pathways, respectively. Using genetically altered mouse models that allow for the acute abrogation of both signaling pathways by inducible Cre/loxP-mediated mutagenesis in smooth muscle cells, we show that G(q)-G11-mediated signaling in smooth muscle cells is required for maintenance of basal blood pressure and for the development of salt-induced hypertension. In contrast, lack of G12-G13, as well as of their major effector, the leukemia-associated Rho guanine nucleotide exchange factor (LARG), did not alter normal blood pressure regulation but did block the development of salt-induced hypertension. This identifies the G12-G13-LARG-mediated signaling pathway as a new target for antihypertensive therapies that would be expected to leave normal blood pressure regulation unaffected.