Receptors In Smooth Muscle Research Articles

Abstract 13832: GPR39 is a High Affinity Vascular Smooth Muscle Receptor for P450 Eicosanoids

Epoxyeicosatrienoic (EETs) and hydroxyeicosatetraenoic acids (HETEs) are endogenous vasoactive P450 eicosanoids that play important roles in cardiovascular physiology and disease, but their mechanisms of action are not fully understood. We developed a clickable photo-crosslinking probe (EET-P) based on EETs regio-isomer 14,15-EET, which allowed us to purify and enrich 14,15-EET binding proteins in mouse heart vascular smooth muscle cells (VSMCs). Proteomic analysis generated a list of proteins bound by EET-P that included the orphan G-protein coupled receptor 39 (GPR39). Consistent with the molecular weight of GPR39, sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) analysis revealed a prominent ~50 kD protein crosslinked to EET-P, and pretreatment with either 14,15-EET or its antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE) completely displaced EET-P binding. Heterologous expression of GPR39 but not a control plasmid in HEK293 cells conferred responsiveness to both 14,15-EET and 15-HETE, as assayed by extracellular-regulated kinase (ERK) activation. Immuno-histochemical analysis localized GPR39 expression in both mouse heart micro-vessels and primary cultured VSMCs. Live-cell imaging of VSMC intracellular calcium ([Ca 2+ ]i) revealed a dual and antagonistic regulatory mechanism in which 15-HETE induces and 14,15-EET suppresses VSMC calcium transients. Short hairpin RNA (shRNA) knockdown of GPR39 in VSMCs was sufficient to abolish effects of both 15-HETE and 14,15-EET on VSMC calcium. Our results identify GPR39 as a high-affinity, lipid-sensing GPCR antagonistically regulated by two endogenous P450 eicosanoids with opposing actions on VSMC calcium, and offer a unique window into multi-ligand GPCRs and their role in integrating the multitude of hormonal signals to regulate local tissue function and response to disease state.

Calcitonin Gene-Related Peptide Rescues Proximity Associations of Its Receptor Components, Calcitonin Receptor-Like Receptor and Receptor Activity-Modifying Protein 1, in Rat Uterine Artery Smooth Muscle Cells Exposed to Tumor Necrosis Factor Alpha.

Calcitonin gene-related peptide (CALCB), adrenomedullin (ADM), and ADM2/intermedin play critical roles in vascular adaptation during pregnancy through calcitonin receptor-like receptor (CALCRL) and receptor activity-modifying proteins (RAMPs). This study was designed to assess the predominant RAMP that associates with CALCRL to form a functional receptor in the rat uterine artery smooth muscle (RUASM). We also determined if these receptor component associations are decreased by tumor necrosis factor (TNF) alpha and if CALCB, ADM, or ADM2 can rescue CALCRL/RAMP associations. Using proximity ligation assay in RUASM cells, this study shows that CALCRL predominantly associates with RAMP1 forming a CALCB receptor, and minimally with RAMP2 and RAMP3 that confer specificity for ADM and ADM2. However, knockdown of RAMP1 mRNA increases the interaction between CALCRL and RAMP3 without affecting the association of CALCRL and RAMP2. Furthermore, CALCB, ADM, and ADM2 have no effects on the associations of CALCRL with any of the RAMPs in RUASM cells. Interestingly, CALCB reverses the TNFalpha-induced decreases in CALCRL/RAMP1 associations. Furthermore, CALCB increases ERK1/2 phosphorylation in a time-dependent manner in RUASM, and the protective effect of CALCB on TNFalpha-induced inhibition of CALCRL/RAMP1 associations was significantly blocked in presence of ERK inhibitor (PD98059). In conclusion, this study demonstrates that CALCRL predominantly associates with RAMP1 forming a CALCB-specific receptor complex in RUASM cells, which is dissociated by TNFalpha. Rescue of TNFalpha-induced dissociation of CALCRL/RAMP1 complex by CALCB in RUASM cells suggests a potential use of CALCB in developing therapeutic strategies for pregnancy-related complications that are vulnerable to abnormal levels of TNFalpha, such as fetal growth restriction and preeclampsia.

Co-Administration of Soy Isoflavones and Vitamin D in Management of Irritable Bowel Disease.

Background and AimsThe substantial characteristics of Irritable Bowel Syndrome (IBS) are associated with estrogens in women. Both soy isoflavones and vitamin D can modulate estrogen receptors in the colonic smooth muscles. The aim of this study was to investigate the effects of soy isoflavones, vitamin D and their probable interactions in women with IBS.MethodsIn a factorial blinded randomized clinical trial, 100 women with IBS (age:18-75yr, were randomly assigned in 4 arms to receive either placebo of vitamin D and placebo of soy isoflavones (P+P), or placebo of vitamin D and soy isoflavones (P+S), or vitamin D and placebo of soy isoflavones (D+P), or vitamin D and soy isoflavones (D+S) for 6 weeks. Dosage of soy isoflavone was 2 capsules of 20 mg soy isoflavones per day, and dosage of vitamin D was one pearl of 50’000 IU biweekly. The clinical outcomes were IBS symptoms severity scores (IBS-SSS), disease- specific quality of life (IBS-QOL) and total score (IBS-TS) that evaluated at weeks 0, 6, and 10, and compared to each other.ResultsIBS-TS improved significantly in both S+P and D+P groups (p- value = 0.004, 0.015). The interaction effect of soy isoflavones and vitamin D on IBS-TS was significant (p<0.05). The interaction effect of soy isoflavones with vitamin D and the main effect of vitamin D on IBS-SSS were not statistically significant, whereas IBS-SSS decreased significantly in S+P and D+P groups (p-value = 0.001, 0.047 respectively).ConclusionOur results indicate that co-administration of soy isoflavones with vitamin D did not improve the IBS- SSS and IBS- QOL; however, it improved the IBS-TS.Trial RegistrationClinical Trials.gov NCT02026518

ANGIOTENSIN II RECEPTOR TYPE 1 EXPRESSION IN PATIENTS WITH MULTIFOCAL ATHEROSCLEROSIS

Local expression of rennin-angiotensin system (RAS) components significantly increases in patients with arterial hypertension and atherosclerosis with or without elevation of RAS activity in the blood. Our objectives was to assessment and compare the expression level of angiotensin II type 1 receptor in the smooth muscle cells in arteries of patients with multifocal atherosclerosis and the role of local angiotensin II receptor type 1 (AT1R) expression in the disease progression. The study results suggest that the tissue RAS activity increases inhomogeneously among patients. It was interestly that AT1R expression levels in intact arteries does not differ of those in atherosclerotic arteries. Most of patients had expression of angiotensin II type 1 receptor in smooth muscle of arteries; strong elevation had 43,8% — 50,0%. Our study suggest of role of local RAS activity, but we proposed existence of another than only angiotensin II type 1 receptor mechanisms of low or high susceptibility of arteries to atherosclerosis in patient with multifocal atherosclerosis.

Transient Receptor Potential Melastatin 4 Channel Regulation by Inositol Trisphosphate Receptor in Human Urinary Bladder Smooth Muscle

Overactive bladder (OAB) is a highly prevalent and debilitating condition that lacks an effective therapeutic treatment. The contraction and relaxation of urinary bladder smooth muscle (UBSM) facilitates the voiding and storage of urine, the two main functions of the urinary bladder. Some forms of OAB have been directly linked to UBSM dysfunction. Thus, efforts to uncover unknown regulatory mechanisms in UBSM function are urgently needed. Among the potential novel targets for OAB, emerging evidence points to key roles for transient receptor potential melastatin 4 (TRPM4) channels in UBSM excitability and contractility. However, the cellular mechanisms regulating TRPM4 channel activity in human UBSM has been virtually unexplored. As a potential cellular mechanism regulating TRPM4 channel activity in UBSM, we have directed our attention to the inositol trisphosphate (IP3) receptors (IP3Rs), which facilitate the release of Ca2+ from the sarcoplasmic reticulum (SR). Since the TRPM4 channels are activated by Ca2+, IP3R‐mediated Ca2+ release of the SR represents a potential Ca2+ source for TRPM4 channel activation. In accordance with the approved IRB Pro00045232 of the Medical University of South Carolina, we used clinically‐characterized human UBSM tissues from 6 donor patients to investigate the molecular and functional interactions of the TRPM4 channels and IP3Rs in human UBSM. Using in situ proximity ligation assay (PLA) and amphotericin‐B perforated patch‐clamp electrophysiology, we sought to test the hypothesis that TRPM4 channels are tightly associated with the IP3Rs and are activated by IP3R‐mediated Ca2+ release of the SR in human UBSM. As determined by in situ PLA, we demonstrated colocalization of the TRPM4 channels and IP3Rs in human UBSM cells. As the TRPM4 channels and IP3Rs must be located within close apposition to functionally interact, these findings support the concept of a TRPM4‐IP3R regulatory mechanism in human UBSM function. To investigate IP3R regulation of TRPM4 channel activity, we sought to determine the consequences of IP3R pharmacological inhibition on TRPM4 channel‐mediated transient inward cation currents (TICCs). In freshly‐isolated human UBSM cells, the selective IP3R inhibitor xestospongin C significantly decreased TICCs. These findings suggest that the SR IP3Rs have a key role in mediating the Ca2+‐dependent activation of TRPM4 channels in human UBSM. In conclusion, this study provides novel insight into the cellular mechanisms regulating TRPM4 channel activity in human UBSM function. Our study reveals that the TRPM4 channels and IP3Rs are spatially and functionally coupled in human UBSM, information that is critical for further evaluating the potential role of the TRPM4 channels as novel therapeutic targets for OAB.Support or Funding InformationSupported by NIH grant R01‐DK106964 to Georgi V. Petkov and F31‐DK104528 to Aaron Provence.

Association between Polymorphisms in Bitter Taste Receptor Genes and Clinical Features in Korean Asthmatics

Background: Bitter taste receptors (TAS2R) in human airway smooth muscle have recently been shown to have an important role in bronchodilation, together with β₂-adrenergic receptors. Object: To evaluate the association between genetic variations in TAS2R and clinical features, including bronchodilator response and asthma control. Method: We analyzed the association between single nucleotide polymorphisms (SNPs) of TAS2R10 and TAS2R14 and variables such as demographic data, atopy, duration of disease, and asthma control status, including variables such as asthma control test (ACT) score, percent predicted value of forced expiratory volume in 1 s (FEV₁), forced vital capacity (FVC), and FEV₁/FVC ratio, as well as bronchodilator response (BDR), in 721 asthma patients in Korea. Result: Three novel SNPs of 633G>A, 645C>A, and -79G>A in TAS2R10 and 3 known SNPs of -815T>C, -1267G>A, and -1897T>C in TAS2R14 were analyzed. Increased BDR was significantly associated with SNPs of -815T>C [OR (95% CI) = 1.88 (1.01-3.49), p = 0.04 ] [J Gen Physiol 2005;125:535-553; Am J Respir Cell Mol Biol 2010;42:373-3812], -1267A>G [OR (95% CI) = 2.07 (1.03-4.15), p = 0.04] and -1897T>C [OR (95% CI) = 3.05 (1.01-9.23), p = 0.04, in a dominant model, and OR = 1.91 (1.08−3.36), p = 0.02, in a codominant model] of the TAS2R14 gene. There was a significant association between -815T>C and a low mean ACT score [OR (95% CI) = 5.84 (1.94-17.61), p = 0.001]. In haplotype analysis, TAC, CAT, and TGT, or TG and CA haplotypes on TAS2R14 were significantly associated with increased BDR; CAT and CA haplotypes were significantly associated with a low ACT score. Conclusion: Genetic variations in TAS2Rs may be valuable genetic markers to predict therapeutic response and outcomes in asthma. Further research in an independent cohort is needed.

Portulaca olerace Affects Muscarinic Receptors of Guinea Pig Tracheal Smooth Muscle

The relaxant effect of Portulaca oleracea L. (P. oleracea) on tracheal smooth muscles has been shown. The effect of the plant on muscarinic receptors of tracheal smooth muscles as one possible mechanism responsible for its relaxant effect was examined. Concentration response curves to methacholine were performed in the presence of three concentrations of aqueous-ethanol extract from aerial part of the plant (0.25, 0.50 and 1.00 mg/ml), 10 nM atropine, and saline. The values of EC50 (effective concentration causing 50% of maximum response) and CR-1 ((EC50 of test solutions/EC50 of saline)-1) were measured. The study was done using three designs as non-incubated (group 1), incubated tissues with propranolol and chlorpheniramine (group 2) and incubated tissues with propranolol (group 3). Concentration-response curves to methacholine were shifted to the right and EC50 methacholine were higher in the presence of atropine, medium and highest extract concentrations in all groups and its low concentration in group 3 compared to saline (P<0.05 to P<0.001). In addition, EC50 methacholine in the presence of high extract concentration (27.00±2.12) in group 2 was greater than its low (10.33±1.54) and medium (16.83±2.52) concentrations (P<0.001 and P<0.05, respectively). The values of the CR-1 obtained in the presence of all extract concentrations in all groups were lower than that of atropine (P<0.05 to P<0.001). An inhibitory effect for the extract of P. oleracea muscarinic receptors of tracheal smooth muscles was shown and a histamine (H1) receptor blockade was also suggested.

Simultaneous Spectrophotometric Estimation of Candesartan Cilexetil and Hydrochlorothiazidein Tablet Dosage Form

A new simple derivative spectrophotometric method has been developed for the simultaneous determination of Candesartan Cilexetil and Hydrochlorothiazide in tablet dosage forms. Candesartan antagonizes the effect of angiotensin II (vasoconstriction and aldosterone secretion) by blocking the angiotensin II receptor in vascular smooth muscle and the adrenal gland and decrease the blood pressure whereas Hydrochlorothiazide increases chloride, sodium and water excretion by interfering with transport of sodium ions across renal tubular epithelium. And there by show the diuretic action. The first derivative method is based on the measurement of absorbance of one drug at the zero crossing point of another drug. Candesartan Cilexetil and Hydrochlorothiazide were determined at two different wavelengths 222.69 (zero crossing point of Hydrochlorothiazide) and 254.63 nm (zero crossing point of Candesartan Cilexetil) from the derivative spectra respectively. The methods hows linearity over the concentration range 0.5-50 and 0.1-50 µg/ml for Candesartan Cilexetil and Hydrochlorothiazide respectively in phosphate buffer. The proposed method was validated and can be used for routine analysis of combined tablet dosage forms containing Candesartan Cilexetil and Hydrochlorothiazide. Key words: Candesartan Cilexetil, Derivative spectroscopy, Hydrochlorothiazide, Simultaneous determination.

The ERK1/2 pathway participates in the upregulation of the expression of mesenteric artery α1 receptors by intravenous tail injections of mmLDL in mice

Minimally modified low density lipoprotein (mmLDL) is a risk factor for cardiovascular diseases. However, no studies examining the effect of mmLDL on vascular smooth muscle receptors have been released. The current study investigated the effect of mmLDL on the mesenteric artery α1 adrenoceptor and the molecular mechanisms. Mice were divided into the normal saline (NS), mmLDL, and mmLDL+U0126 groups. In the mmLDL+U0126 group, the animals were subjected to an intravenous tail injection of mmLDL and an intraperitoneal injection of U0126. Vascular tension caused by noradrenaline (NA) in mesenteric arteries was measured with a sensitive myograph system. The serum levels of oxLDL, TNF-α, and IL-1β were detected using enzyme-linked immunosorbent assays. The expressions of the α1 adrenoceptor, the α2 adrenoceptor, TNF-α, IL-1β, and pERK1/2 were detected using real-time polymerase chain reactions and Western blot analysis. Compared with the NS group, the mmLDL group exhibited a noticeably enhanced NA shrinkage dose–response curve and a significantly increased Emax value (P<0.01). Prazosin (α1 adrenoceptor antagonist) caused a noticeable right shift of the dose–response curve. U0126 inhibited the increases in the serum levels and vessel wall expression of IL-1β and TNF-α and enhanced the NA shrinkage dose–response curve caused by mmLDL, as observed by a significantly decreased Emax value (P<0.01). It inhibited the increased α1 adrenoceptor expression caused by mmLDL. The serum levels of IL-1β and TNF-α demonstrated a positive correlation with the NA-induced maximum shrinkage percentage. U0126 inhibited the mmLDL-induced increase in the pERK1/2 protein level in the vessel wall. In conclusion, mmLDL increased the serum levels of IL-1β and TNF-α in vivo by activating the ERK1/2 pathway, which resulted in α1 receptor-mediated vasoconstriction and an increase in the expression of α1 adrenoceptor. The results of this study may provide new ideas for the prevention and cure of cardiovascular diseases in the future.

Novel identification of the free fatty acid receptor FFAR1 that promotes contraction in airway smooth muscle.

Obesity is one of the major risk factors for asthma. Previous studies have demonstrated that free fatty acid levels are elevated in the plasma of obese individuals. Medium- and long-chain free fatty acids act as endogenous ligands for the free fatty acid receptors FFAR1/GPR40 and FFAR4/GPR120, which couple to Gq proteins. We investigated whether FFAR1 and FFAR4 are expressed on airway smooth muscle and whether they activate Gq-coupled signaling and modulate airway smooth muscle tone. We detected the protein expression of FFAR1 and FFAR4 in freshly dissected native human and guinea pig airway smooth muscle and cultured human airway smooth muscle (HASM) cells by immunoblotting and immunohistochemistry. The long-chain free fatty acids (oleic acid and linoleic acid) and GW9508 (FFAR1/FFAR4 dual agonist) dose-dependently stimulated transient intracellular Ca(2+) concentration ([Ca(2+)]i) increases and inositol phosphate synthesis in HASM cells. Downregulation of FFAR1 or FFAR4 in HASM cells by small interfering RNA led to a significant inhibition of the long-chain free fatty acids-induced transient [Ca(2+)]i increases. Oleic acid, linoleic acid, or GW9508 stimulated stress fiber formation in HASM cells, potentiated acetylcholine-contracted guinea pig tracheal rings, and attenuated the relaxant effect of isoproterenol after an acetylcholine-induced contraction. In contrast, TUG-891 (FFAR4 agonist) did not induce the stress fiber formation or potentiate acetylcholine-induced contraction. These results suggest that FFAR1 is the functionally dominant free fatty acid receptor in both human and guinea pig airway smooth muscle. The free fatty acid sensors expressed on airway smooth muscle could be an important modulator of airway smooth muscle tone.

The Inhibitory Effects of Ferula assafoetida on Muscarinic Receptors of Guinea-Pig Tracheal Smooth Muscle

Background: In traditional medicine, asafoetida obtained from the roots of different Ferula assafoetida (F. asafoetida) is used for various therapeutic purposes such as whooping cough, pneumonia and bronchitis in children, as well as asthma. Objectives: In this study, the effect of asafoetida on muscarinic receptors of tracheal smooth muscle, as one possible mechanism responsible for the relaxant effect seen for the plant, was evaluated. Materials and Methods: The effects of three cumulative concentrations of aqueous extract of F. asafoetida (2.5, 5 and 10 mg/mL), 10 nM atropine, and saline on muscarinic receptors were tested in tracheal smooth muscle samples by performing cumulative concentration response curve to methacholine (a muscarinic receptor agonist) and assessing the shift in concentration response curves due to different concentrations of the extract and atropine. Results: The EC 50 obtained in the presence of higher concentration of the extract muscle was significantly higher compared to saline (P < 0.01). The maximum responses to methacholine in the presence of higher concentration of the extract (10 mg/mL) was significantly lower than that of saline (P < 0.05). The values of CR-1, obtained in the presence of all concentrations of the extract, were significantly lower compared to atropine in the experimental group (P < 0.01 to P < 0.001). Conclusions: These results showed an inhibitory effect for the extract of asafetida on muscarinic receptors of tracheal smooth muscle.

The impact of prolapse mesh on vaginal smooth muscle structure and function.

To evaluate the impact of prolapse meshes on vaginal smooth muscle structure (VaSM) and function, and to evaluate these outcomes in the context of the mechanical and textile properties of the mesh. Three months following the implantation of three polypropylene prolapse meshes with distinct textile and mechanical properties, mesh tissue explants were evaluated for smooth muscle contraction, innervation, receptor function, and innervation density. Magee-Womens Research Institute at the University of Pittsburgh. Thirty-four parous rhesus macaques of similar age, parity, and pelvic organ prolapse quantification (POP-Q) scores. Macaques were implanted with mesh via sacrocolpopexy. The impact of Gynemesh(™) PS (Ethicon; n=7), Restorelle(®) (Coloplast; n=7), UltraPro(™) parallel and UltraPro(™) perpendicular (Ethicon; n=6 and 7, respectively) were compared with sham-operated controls (n=7). Outcomes were analysed by Kruskal-Wallis ANOVA, Mann-Whitney U-tests and multiple regression analysis (P<0.05). Vaginal tissue explants were evaluatedfor the maximum contractile force generated following muscle, nerve, and receptor stimulation, and for peripheral nerve density. Muscle myofibre, nerve, and receptor-mediated contractions were negatively affected by mesh only in the grafted region (P<0.001, P=0.002, and P=0.008, respectively), whereas cholinergic and adrenergic nerve densities were affected in the grafted (P=0.090 and P=0.008, respectively) and non-grafted (P=0.009 and P=0.005, respectively) regions. The impact varied by mesh property, as mesh stiffness was a significant predictor of the negative affect on muscle function and nerve density (P<0.001 and P=0.013, respectively), whereas mesh and weight was a predictor of receptor function (P<0.001). Mesh has an overall negative impact on VaSM, and the effects are a function of mesh properties, most notably, mesh stiffness. Prolapse mesh affects vaginal smooth muscle.

Purinergic transmission in blood vessels.

There are nineteen different receptor proteins for adenosine, adenine and uridine nucleotides, and nucleotide sugars, belonging to three families of G protein-coupled adenosine and P2Y receptors, and ionotropic P2X receptors. The majority are functionally expressed in blood vessels, as purinergic receptors in perivascular nerves, smooth muscle and endothelial cells, and roles in regulation of vascular contractility, immune function and growth have been identified. The endogenous ligands for purine receptors, ATP, ADP, UTP, UDP and adenosine, can be released from different cell types within the vasculature, as well as from circulating blood cells, including erythrocytes and platelets. Many purine receptors can be activated by two or more of the endogenous ligands. Further complexity arises because of interconversion between ligands, notably adenosine formation from the metabolism of ATP, leading to complex integrated responses through activation of different subtypes of purine receptors. The enzymes responsible for this conversion, ectonucleotidases, are present on the surface of smooth muscle and endothelial cells, and may be coreleased with neurotransmitters from nerves. What selectivity there is for the actions of purines/pyrimidines comes from differential expression of their receptors within the vasculature. P2X1 receptors mediate the vasocontractile actions of ATP released as a neurotransmitter with noradrenaline (NA) from sympathetic perivascular nerves, and are located on the vascular smooth muscle adjacent to the nerve varicosities, the sites of neurotransmitter release. The relative contribution of ATP and NA as functional cotransmitters varies with species, type and size of blood vessel, neuronal firing pattern, the tone/pressure of the blood vessel, and in ageing and disease. ATP is also a neurotransmitter in non-adrenergic non-cholinergic perivascular nerves and mediates vasorelaxation via smooth muscle P2Y-like receptors. ATP and adenosine can act as neuromodulators, with the most robust evidence being for prejunctional inhibition of neurotransmission via A1 adenosine receptors, but also prejunctional excitation and inhibition of neurotransmission via P2X and P2Y receptors, respectively. P2Y2, P2Y4 and P2Y6 receptors expressed on the vascular smooth muscle are coupled to vasocontraction, and may have a role in pathophysiological conditions, when purines are released from damaged cells, or when there is damage to the protective barrier that is the endothelium. Adenosine is released during hypoxia to increase blood flow via vasodilator A2A and A2B receptors expressed on the endothelium and smooth muscle. ATP is released from endothelial cells during hypoxia and shear stress and can act at P2Y and P2X4 receptors expressed on the endothelium to increase local blood flow. Activation of endothelial purine receptors leads to the release of nitric oxide, hyperpolarising factors and prostacyclin, which inhibits platelet aggregation and thus ensures patent blood flow. Vascular purine receptors also regulate endothelial and smooth muscle growth, and inflammation, and thus are involved in the underlying processes of a number of cardiovascular diseases.

Heteromerization of chemokine (C-X-C motif) receptor 4 with α1A/B-adrenergic receptors controls α1-adrenergic receptor function

Recent evidence suggests that chemokine (C-X-C motif) receptor 4 (CXCR4) contributes to the regulation of blood pressure through interactions with α1-adrenergic receptors (ARs) in vascular smooth muscle. The underlying molecular mechanisms, however, are unknown. Using proximity ligation assays to visualize single-molecule interactions, we detected that α1A/B-ARs associate with CXCR4 on the cell surface of rat and human vascular smooth muscle cells (VSMC). Furthermore, α1A/B-AR could be coimmunoprecipitated with CXCR4 in a HeLa expression system and in human VSMC. A peptide derived from the second transmembrane helix of CXCR4 induced chemical shift changes in the NMR spectrum of CXCR4 in membranes, disturbed the association between α1A/B-AR and CXCR4, and inhibited Ca(2+) mobilization, myosin light chain (MLC) 2 phosphorylation, and contraction of VSMC upon α1-AR activation. CXCR4 silencing reduced α1A/B-AR:CXCR4 heteromeric complexes in VSMC and abolished phenylephrine-induced Ca(2+) fluxes and MLC2 phosphorylation. Treatment of rats with CXCR4 agonists (CXCL12, ubiquitin) reduced the EC50 of the phenylephrine-induced blood pressure response three- to fourfold. These observations suggest that disruption of the quaternary structure of α1A/B-AR:CXCR4 heteromeric complexes by targeting transmembrane helix 2 of CXCR4 and depletion of the heteromeric receptor complexes by CXCR4 knockdown inhibit α1-AR-mediated function in VSMC and that activation of CXCR4 enhances the potency of α1-AR agonists. Our findings extend the current understanding of the molecular mechanisms regulating α1-AR and provide an example of the importance of G protein-coupled receptor (GPCR) heteromerization for GPCR function. Compounds targeting the α1A/B-AR:CXCR4 interaction could provide an alternative pharmacological approach to modulate blood pressure.

Cyclosporin A upregulates ETB receptor in vascular smooth muscle via activation of mitogen-activating protein kinases and NF-κB pathways

Hypertension is one of the most frequent complications of solid organ transplantation, and cyclosporin A (CsA) plays a predominant role in the pathophysiology of post-transplant hypertension. However, the exact molecular mechanisms of CsA-induced hypertension remain obscure. We previously showed that CsA increased the mRNA expression and contractile function of endothelin B (ETB) receptor in vascular smooth muscle cells. The present study was designed to investigate the underlying mechanisms of CsA-induced upregulation of ETB receptor in vasculature. Rat mesenteric arteries were incubated with CsA in an organ culture system, and results showed that CsA enhanced ETB receptor mRNA in the time- and dose-dependent manner, and increased protein expression levels of ETB receptor after treatment with CsA 10−5M for 6h. Furthermore, CsA induced phosphorylation of extracellular regulated protein kinases 1 and 2 (ERK1/2), p38, and translocation of nuclear factor-kappaB (NF-κB) p65 in vasculature. Blocking ERK1/2, p38, or NF-κB activation with their specific inhibitors markedly attenuated CsA-induced upregulation of ETB receptor mRNA expression and protein levels, and ETB receptor-mediated contraction. In summary, this study showed that mitogen-activating protein kinases (ERK1/2 and p38) and the downstream transcriptional factor NF-κB pathways were involved in CsA-induced upregulation of ETB receptor in arterial smooth muscle cells.

Potassium channelopathy-like defect underlies early-stage cerebrovascular dysfunction in a genetic model of small vessel disease

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), caused by dominant mutations in the NOTCH3 receptor in vascular smooth muscle, is a genetic paradigm of small vessel disease (SVD) of the brain. Recent studies using transgenic (Tg)Notch3(R169C) mice, a genetic model of CADASIL, revealed functional defects in cerebral (pial) arteries on the surface of the brain at an early stage of disease progression. Here, using parenchymal arterioles (PAs) from within the brain, we determined the molecular mechanism underlying the early functional deficits associated with this Notch3 mutation. At physiological pressure (40 mmHg), smooth muscle membrane potential depolarization and constriction to pressure (myogenic tone) were blunted in PAs from TgNotch3(R169C) mice. This effect was associated with an ∼ 60% increase in the number of voltage-gated potassium (KV) channels, which oppose pressure-induced depolarization. Inhibition of KV1 channels with 4-aminopyridine (4-AP) or treatment with the epidermal growth factor receptor agonist heparin-binding EGF (HB-EGF), which promotes KV1 channel endocytosis, reduced KV current density and restored myogenic responses in PAs from TgNotch3(R169C) mice, whereas pharmacological inhibition of other major vasodilatory influences had no effect. KV1 currents and myogenic responses were similarly altered in pial arteries from TgNotch3(R169C) mice, but not in mesenteric arteries. Interestingly, HB-EGF had no effect on mesenteric arteries, suggesting a possible mechanistic basis for the exclusive cerebrovascular manifestation of CADASIL. Collectively, our results indicate that increasing the number of KV1 channels in cerebral smooth muscle produces a mutant vascular phenotype akin to a channelopathy in a genetic model of SVD.

Current optimization of combined therapy for chronic obstructive pulmonary disease

Testing the new combined bronchodilator Anoro Ellipta in different clinical trials gives to its high clinical efficacy and safety in chronic obstructive pulmonary disease. The drug contains the molecules of sustained-release selective β2-adrenergic receptor agonist (vilanterol) and a muscarinic cholinergic receptor antagonist (umeclidinium bromide). The bronchodilating mechanisms of umeclidinium bromide are in the competitive inhibition of the binding of acetylcholine with muscarinic acetylcholine receptors of airway smooth muscles whereas in those of vilanterol are in that with the stimulation of intracellular adenylate cyclase. On days 1 and 24 after inhalation of the first dose of vilanterol and umeclidinium bromide, there was a significant increase in the forced expiratory volume in one second as compared to placebo. No clinical effects on QT interval on an electrocardiogram and cardiac rhythm were found. The benefits of an inhalation device (Ellipta) are its innovation design ensuring the effective delivery of an aerosol dose into the airway, convenience, and simplicity.

Thyroid hormone affects both endothelial and vascular smooth muscle cells in rat arteries

Hypothyroidism impairs endothelium-dependent dilatations, while hyperthyroidism augments the production of endothelial nitric oxide. Thus, experiments were designed to determine if thyroid hormone causes endothelium-dependent responses, or alleviates diabetic endothelial dysfunction. Isometric tension was measured in rings with or without endothelium of arteries from normal and diabetic Sprague-Dawley rats. Release of 6-keto prostaglandin F1α and thromboxane B2 were measured by enzyme linked immunosorbent assay and protein levels [endothelial nitric oxide synthase (eNOS), cyclooxygenases (COX)] by immunoblotting. Triiodothyronine (T3) caused concentration-dependent (3×10−6–3×10−5M) relaxations in mesenteric (pEC50, 4.96±0.19) and femoral (pEC50, 4.57±0.35) arteries without endothelium. In femoral arteries of rats with diabetes, 5-methylamino-2-[[(2S,3R,5R,8S,9S)-3,5,9-trimethyl-2-(1-oxo-(1H-pyrrol-2- -yl)propan-2-yl)-1,7-dioxaspiro-(5,5)undecan-8-yl]methyl]benzooxazole-4-carboxylic acid (A23187, 3×10−7 to 10−6M) caused partly endothelium-dependent contractions. After chronic T3-treatment with (10μg/kg/day; four weeks), the contractions to A23187 of preparations with and without endothelium were comparable, the thromboxane B2-release was reduced (by 38.1±9.2%). The pEC50 of 9, 11-dideoxy-11α, 9α-epoxymethanoprostaglandin F2α (U46619, TP-receptor agonist) was increased in T3-treated diabetic rats compared with controls (8.53±0.06 vs 7.94±0.09). The protein expression of eNOS increased (by 228%) but that of COX-1 decreased (by 35%) after chronic T3 treatment. In human umbilical vein endothelial cells incubated for one week with T3 (10−10–10−7M) in the presence but not in the absence of interleukin-1β (1ng/ml), the expression of eNOS was increased compared to control. In conclusion, thyroid hormone acutely relaxes mesenteric and femoral vascular smooth muscle, but given chronically reduces the release of endothelium-derived vasoconstrictor prostanoids while enhancing the responsiveness of TP receptors of vascular smooth muscle.

Hydrogen sulfide is involved in dexamethasone-induced hypertension in rat

Glucocorticoid (GC)-induced hypertension is a common clinical problem still poorly understood.The presence of GC receptor (GR) in vascular smooth muscle and endothelial cells suggests a direct role for GC in vasculature. In response to hemodynamic shear stress, endothelium tonically releases nitric oxide (NO), endothelial-derived hyperpolarizing factor (EDHF) and prostacyclin contributing to vascular homeostasis. Recently, hydrogen sulfide (H2S) has been proposed as a candidate for EDHF. H2S is endogenously mainly formed from L-cysteine by the action of cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE). It plays many physiological roles and contributes to cardiovascular function. Here we have evaluated the role played by H2S in mesenteric arterial bed and in carotid artery harvested from rats treated with vehicle or dexamethasone (DEX; 1.5 mg/kg/day) for 8 days. During treatments systolic blood pressure was significantly increased in conscious rats. EDHF contribution was evaluated in ex-vivo by performing a concentration–response curve induced by acetylcholine (Ach) in presence of a combination of indomethacin and L-NG-Nitroarginine methyl ester in both vascular districts. EDHF-mediated relaxation was significantly reduced in DEX-treated group in both mesenteric bed and carotid artery. EDHF-mediated relaxation was abolished by pre-treatment with both apamin and charybdotoxin, inhibitors of small and big calcium-dependent potassium channels respectively, or with propargylglycine, inhibitor of CSE.Western blot analysis revealed a marked reduction in CBS and CSE expression as well as H2S production in homogenates of mesenteric arterial bed and carotid artery from DEX-treated rats. In parallel, H2S plasma levels were significantly reduced in DEX group compared with vehicle.In conclusion, an impairment in EDHF/H2S signaling occurs in earlier state of GC-induced hypertension in rats suggesting that counteracting this dysfunction may be beneficial to manage DEX-associated increase in blood pressure.

New ideas in urology: 2014 update

Pharmacists practicing in a variety of settings should be aware of the most recent findings on medications used in urology. Hot topics include safety concerns about testosterone replacement therapy (TRT), novel treatments for overactive bladder, and daily dosing of phosphodiesterase-5 enzyme (PDE) inhibitors for erectile dysfunction (ED).