Relaxation Of Muscle Cells Research Articles

The Appendix as a Viable Source of Neural Progenitor Cells to Functionally Innervate Bioengineered Gastrointestinal Smooth Muscle Tissues

Appendix-derived neural progenitor cells (NPCs) have both neurogenic and gliogenic potential, but use of these cells for enteric neural cell therapy has not been addressed. The objective of this study was to determine whether NPCs obtained from the appendix would differentiate into enteric neural subsets capable of inducing neurotransmitter-mediated smooth muscle cell (SMC) contraction and relaxation. NPCs were isolated from the appendix and small intestine (SI) of rabbits. Bioengineered internal anal sphincter constructs were developed using the same source of smooth muscle and innervated with NPCs derived from either the appendix or SI. Innervated constructs were assessed for neuronal differentiation markers through Western blots and immunohistochemistry, and functionality was assessed through force-generation studies. Expression of neural and glial differentiation markers was observed in constructs containing appendix- and SI-derived NPCs. The addition of acetylcholine to both appendix and SI constructs caused a robust contraction that was decreased by pretreatment with the neural inhibitor tetrodotoxin (TTX). Electrical field stimulation caused relaxation of constructs that was completely abolished in the presence of TTX and significantly reduced on pretreatment with nitric oxide synthase inhibitor (Nω-nitro-l-arginine methyl ester hydrochloride [l-NAME]). These data indicate that in the presence of identical soluble factors arising from intestinal SMCs, enteric NPCs derived from the appendix and SI differentiate in a similar manner and are capable of responding to physiological stimuli. This coculture paradigm could be used to explore the nature of the soluble factors derived from SMCs and NPCs in generating specific functional innervations. This study demonstrates the ability of neural stem cells isolated from the appendix to differentiate into mature functional enteric neurons. The differentiation of neural stem cells from the appendix is similar to differentiation of neural stem cells derived from the gastrointestinal tract. The appendix is a vestigial organ that can be removed with minimal clinical consequence through laparoscopy. Results presented in this paper indicate that the appendix is a potential source of autologous neural stem cells required for cell therapy for the gastrointestinal tract.

Modulating Calcium Entry into Microvascular Endothelium by Controlling Membrane Potential During Submaximal Muscarinic Receptor Activation

Vascular smooth muscle cell relaxation and vasodilation are coupled to a rise in endothelial cell [Ca2+]i and hyperpolarization. However, the role of membrane potential (Vm) in regulating endothelial cell [Ca2+]i remains controversial. To test the hypothesis that Vm can govern Ca2+ entry into endothelial cells in accord with its electrochemical gradient, we evaluated [Ca2+]i using Fura-2 photometry (F340/F380) while controlling Vm using intracellular current injection. Intact endothelial tubes (width: 60 μm, length: ≤ 1 mm) were freshly isolated from superior epigastric arteries of C57BL/6 mice (n≥7; 32°C, pH 7.4). Endothelial cells were electrically coupled to each other through gap junctions and lacked inherent voltage-sensitive ion channel activity as indicated by linearity (R2 = 0.990±0.002) of the current-voltage relationship throughout the Vm range of ≈-80 mV to +10 mV during current injection (±0.5 to 5 nA). In the absence of receptor activation, Vm was −37±1 mV and [Ca2+]i did not change with hyperpolarization or depolarization. In contrast, during submaximal activation of muscarinic (Gq protein-coupled) receptors (GPCRs) with acetylcholine (100 nM; ∼EC50), [Ca2+]i increased with hyperpolarization to −60±1 mV and decreased with depolarization to −28±2 mV. The effect of membrane hyperpolarization on [Ca2+]i was abolished following removal of [Ca2+]o (control [Ca2+]o = 2 mM) and reduced by half in endothelium isolated from TRPV4-/- mice during Vm > −50 mV. Thus, submaximal stimulation of GPCRs enables physiological changes in Vm to modulate Ca entry through voltage-insensitive ion channels in the plasma membrane of native microvascular endothelial cells. (Support: NIH grants R37-HL041026, 5T32-AR048523, 1K99-AG047198)

Epoxyeicosatrienoic acids act through TRPV4–TRPC1–KCa1.1 complex to induce smooth muscle membrane hyperpolarization and relaxation in human internal mammary arteries

Human left internal mammary arteries (LIMAs) are commonly used as donor grafts for coronary bypass surgery. Previous reports suggested that 11,12-epoxyeicosatrienoic acid (11,12-EET) is an important endothelial-derived hyperpolarizing factor (EDHF) in human LIMAs and that EETs act through large conductance Ca2+-activated K+ channels (KCa1.1) to induce smooth muscle cell hyperpolarization and relaxation in these tissues. In this study, we aimed to explore the role of vanilloid transient receptor potential channel 4 (TRPV4) and canonical transient receptor potential channel 1 (TRPC1) channels in the EET-induced smooth muscle hyperpolarization and vascular relaxation in human LIMAs. Co-immunoprecipitation studies demonstrated that TRPV4, TRPC1, and KCa1.1 physically interacted with each other to form a complex. Sharp microelectrode and vascular tension studies demonstrated that 11,12-EET (300nmol/L) and 4α-phorbol 12,13-didecanoate (5μmol/L) were able to induce smooth muscle membrane hyperpolarization and vascular relaxation in isolated human LIMA segments. The hyperpolarizing and relaxant effects were markedly reduced by treatments that could suppress the expression/activity of TRPV4, TRPC1, or KCa1.1. With the use of human embryonic kidney 293 cells that over-expressed with TRPV4, TRPC1 and KCa1.1, we found that TRPC1 is the linker through which TRPV4 and KCa1.1(α) can interact. The present study revealed that 11,12-EET targets the TRPV4–TRPC1–KCa1.1 complex to induce smooth muscle cell hyperpolarization and vascular relaxation in human LIMAs. This finding provides novel mechanistic insights for the EET action in human LIMAs.

Potential of garlic (Allium sativum) in lowering high blood pressure: mechanisms of action and clinical relevance.

Garlic supplements have shown promise in the treatment of uncontrolled hypertension, lowering blood pressure (BP) by about 10 mmHg systolic and 8 mmHg diastolic, similar to standard BP medication. Aged garlic extract, which contains S-allylcysteine as the bioactive sulfur compound, in particular is standardizable and highly tolerable, with little or no known harmful interaction when taken with other BP-reducing or blood-thinning medication. Here we describe biologically plausible mechanisms of garlic’s BP-lowering effect. Garlic-derived polysulfides stimulate the production of the vascular gasotransmitter hydrogen sulfide (H2S) and enhance the regulation of endothelial nitric oxide (NO), which induce smooth muscle cell relaxation, vasodilation, and BP reduction. Several dietary and genetic factors influence the efficiency of the H2S and NO signaling pathways and may contribute to the development of hypertension. Sulfur deficiency might play a part in the etiology of hypertension, and could be alleviated with supplementation of organosulfur compounds derived from garlic.

Resolvin D1 reverses reactivity and Ca2+ sensitivity induced by ET-1, TNF-α, and IL-6 in the human pulmonary artery.

Pulmonary hypertension (PH) is a rare and progressive disease characterized by an inflammatory status and vessel wall remodeling, resulting in increased pulmonary artery resistance. During the last decade, treatments have been proposed; most of them target the endothelial pathways that stimulate smooth muscle cell relaxation. However, PH remains associated with significant morbidity. We hypothesized that inflammation plays a crucial role in the severity of the abnormal vasoconstriction in PH. The goal of this study was to assess the effects of resolvin D1 (RvD1), a potent anti-inflammatory agent, on the pharmacological reactivity of human pulmonary arteries (HPAs) via an in vitro model of induced hyperreactivity. The effects of RvD1 and monoacylglyceride compounds were measured on contractile activity and Ca(2+) sensitivity developed by HPAs that had been pretreated (or not) under proinflammatory conditions with either 10 ng/ml TNF-α or 10 ng/ml IL-6 or under hyperreactive conditions with 5 nM endothelin-1. The results demonstrated that, compared with controls, 24-h pretreatment with TNF-α, IL-6, or endothelin-1 increased reactivity and Ca(2+) sensitivity of HPAs as revealed by agonist challenges with 80 mM KCl, 1 μM serotonin (5-hydroxytryptamine), 30 nM U-46619, and 1 μM phorbol 12,13-dibutyrate. However, 300 nM RvD1 as well as 1 μM monoacylglyceride-docosapentaenoic acid monoglyceride strongly reversed the overresponsiveness induced by both proinflammatory and hyperreactive treatments. In pretreated pulmonary artery smooth muscle cells, Western blot analyses revealed that RvD1 treatment decreased the phosphorylation level of CPI-17 and expression of transmembrane protein member 16A while increasing the detection of G protein-coupled receptor 32. The present data demonstrate that RvD1, a trihydroxylated docosahexaenoic acid derivative, decreases induced overreactivity in HPAs via a reduction in CPI-17 phosphorylation and transmembrane protein member 16A expression.

NMR Assignment and Secondary Structure of Coiled Coil Domain of C-terminal Myosin Binding Subunit of Myosin Phosphatase

Protein-protein interactions between the C-terminal domain of Myosin Binding Subunit (MBS) of MLC Phosphatase (MBS(CT180); C-terminal 180 aa) and the N-terminal coiled coil (CC) leucine zipper (LZ) domain of PKGIα, PKG-Iα(1-159) play an important role in the process of Smooth Muscle Cell relaxation. The paucity of three-dimensional structural information for MBS(CT180) prevents an atomic level understanding of the MBS-PKG contractile complex. MBS(CT180) is comprised of three structurally different sub-domains including a non-canonical CC, a CC, and a LZ. Recently we reported polypeptide purification and biophysical characterization of the CC domain and the LZ domain of MBS(CT180) (Sharma et al, Prot Expr Purif 2012). Here we report (1)H, (13)C, (15)N chemical shift assignments of homodimeric CC MBS domain encompassing amino acid residues Asp931-Leu980 using 2D and 3D heteronuclear NMR spectroscopy. Secondary structure analyses deduced from these NMR chemical shift data have identified a contiguous stretch of 36 residues from Phe932 to Ala967 that is involved in the formation of coiled coil α-helical region within CC MBS domain. The N-terminal residue Asp931 and the C-terminally positioned residues Thr968-Ala975, Arg977, and Ser978 adopt nonhelical loop conformations.

Characterization of vascular endothelial and smooth muscle cell pathways and their contributions to myogenic and agonist‐mediated control (677.16)

Vascular smooth muscle cell contraction and relaxation is central to the local regulation of blood flow to tissue. At the arteriolar level, interactions between vascular endothelial cells and smooth muscel cells play critical role in determining the level of vascular tone. During mechanical and/or agonist stimulations, vascular Ca2+ dynamics are finely adjusted via a number of signaling pathways and the Ca2+ levels in the smooth muscle cells determine the level of contraction. To quantify how cellular Ca2+ dynamics are transformed into changes in vascular tone, we have constructed a cellular‐based model of isolated resistance vessel by integrating models of vessel wall mechanics, smooth muscle force generation, smooth muscle Ca2+ handling and electrophysiology with an endothelial electrophysiology and Ca2+ handling model. The dose‐response vasodilation and vasoconstriction data of different sizes of vessels in response to pharmacological vasoconstrictors and vasodilators, measured using a novel isovolumic myograph developed by Lu and Kassab are analyzed. The model simulates how changes in intraluminal pressure are used to maintain a constant vessel diameter during application of phenylephrine and acetylcholine. In order to simulate these sets of experimental data the model captures integrated vessel behavior including ionic (Ca2+, K+, Na+, etc.), IP3 and nitric oxide myoendothelial transport. Model analysis shows that α‐adrenergic pathways in smooth muscle and NO pathways in endothelium are sufficient to capture the experimentally observed phenylephrine and acetylcholine mediated responses.Grant Funding Source: NIH U01 HL118738, NSF CBET 1133260 and NIH P50‐GM094503

Protease‐activated receptor ‐3 signals independent of protease‐activated receptor‐1 to regulate endothelial nitric oxide synthase in human endothelial cells (1097.1)

Protease‐activated receptor‐3 (PAR‐3) is widely recognized as a non‐signaling elusive member of a G protein‐coupled receptor family known to regulate the endothelial nitric oxide synthase (eNOS)/nitric oxide (NO) signaling pathway via phosphorylation of its regulatory sites Thr495 and Ser1177. The eNOS/NO pathway is responsible for vascular smooth muscle cell relaxation and disruption of this pathway leads to endothelial dysfunction, laminar shear stress and vascular inflammation, a precursor to cellular induced hypertension. Using human coronary artery endothelial cells (HCAECs) as a model, we demonstrated that PAR‐3 is highly co‐expressed with PAR‐1 and ‐2 and can be activated using a synthetic peptide ligand, TFRGAP. We hypothesize that PAR‐3 can signal independently of PAR‐1 and ‐2 to regulate eNOS via site‐ specific phosphorylation and induce pro‐inflammation via the NFkB pathway. Analysis using eNOS specific antibodies showed that 500uM of TFRGAP lead to the phosphorylation of the positive site of eNOS‐Ser1177 and the negative site ‐Thr495, 30 seconds and 15 minutes respectively. Pretreatment of HCAECs with 10uM SCH79797, a selective PAR‐1 inhibitor, did not block the increase in phosphorylation at both eNOS regulatory sites stimulated by TFRGAP. Similar results where found in a stable PAR‐1 deficient cell line of HCAECs which confirmed the signaling potential of PAR‐3 using thrombin and the PAR‐3 activating peptide. Our data suggests that PAR‐3 is able to regulate eNOS/NO signaling via site‐specific phosphorylation that is independent of PAR‐1 activation. In additional studies cytokine production was measured using an ELISA after PAR‐3 activation. Data from these studies will lead to a better understanding of endothelial dysfunction and vascular inflammation while identifying potential drug targets for cardiovascular diseases.Grant Funding Source: The project [poster] described was supported by CTSA award No. UL1TR000445

Modulating Ca2+ entry into endothelial tubes by controlling membrane potential during muscarinic receptor activation (664.7)

Coupling of intracellular Ca2+ [Ca2+]i to hyperpolarization of endothelial cells underlies smooth muscle cell relaxation in resistance vessels. Whether membrane potential (Vm) influences [Ca2+]i levels in the endothelium remains controversial. To resolve the impact of Vm on Ca2+ influx, Fura‐2 photometry was evaluated during intracellular current injection (‐5 to +5 nA; 20 s pulses) to control Vm of endothelial tubes (width, 60 µm; length, 蠄 1 mm) freshly isolated from superior epigastric arteries of adult (4‐6 mo) male C57BL/6 mice (n蠅4). Relative to resting Vm (‐40±2 mV), current injection hyperpolarized (Vm in mV; ‐1 nA: ‐52±2, ‐3 nA: ‐64±2, ‐5 nA: ‐79±2) and depolarized (+1 nA: ‐33±1, +3 nA: ‐13±2, +5 nA: 8±3) cells. Under resting conditions, changing Vm did not affect [Ca2+]i (e.g., F340/F380 = 0.68±0.03 at rest and ‐5 nA). However, during exposure to acetylcholine (ACh; 100 nM, ~EC50), the plateau phase of the [Ca2+]i response was either enhanced [∆F340/F380 & ∆Vm (mV); ‐1 nA: 0.07±0.02 & ‐10±1; ‐3 nA: 0.16±0.02 & ‐23±2; ‐5 nA: 0.21±0.02 & ‐38±1] or reduced (+1 nA: ‐0.08±0.02 & 11±2; +3 nA: ‐0.18±0.03 & 28±3; +5 nA: ‐0.34±0.03 & 49±4) according to voltage amplitude and polarity (P<0.05). These effects of ACh and ∆Vm were abolished in the absence of extracellular Ca2+ (2 mM). Thus Vm can modulate Ca2+ entry during submaximal activation of ion channels that enable Ca2+ influx in accord with the electrochemical driving force.Grant Funding Source: Supported by R37‐HL041026 & 5T32‐AR048523

Emerging Trabecular Outflow Drugs

Emerging Trabecular Outflow Drugs

Isolation of Microvascular Endothelial Tubes from Mouse Resistance Arteries

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic by-products, as exemplified by exercising skeletal muscle. Endothelial cells (ECs) line the intima of all resistance vessels and serve a key role in controlling diameter (e.g. endothelium-dependent vasodilation) and, thereby, the magnitude and distribution of tissue blood flow. The regulation of vascular resistance by ECs is effected by intracellular Ca2+ signaling, which leads to production of diffusible autacoids (e.g. nitric oxide and arachidonic acid metabolites)1-3 and hyperpolarization4,5 that elicit smooth muscle cell relaxation. Thus understanding the dynamics of endothelial Ca2+ signaling is a key step towards understanding mechanisms governing blood flow control. Isolating endothelial tubes eliminates confounding variables associated with blood in the vessel lumen and with surrounding smooth muscle cells and perivascular nerves, which otherwise influence EC structure and function. Here we present the isolation of endothelial tubes from the superior epigastric artery (SEA) using a protocol optimized for this vessel. To isolate endothelial tubes from an anesthetized mouse, the SEA is ligated in situ to maintain blood within the vessel lumen (to facilitate visualizing it during dissection), and the entire sheet of abdominal muscle is excised. The SEA is dissected free from surrounding skeletal muscle fibers and connective tissue, blood is flushed from the lumen, and mild enzymatic digestion is performed to enable removal of adventitia, nerves and smooth muscle cells using gentle trituration. These freshly-isolated preparations of intact endothelium retain their native morphology, with individual ECs remaining functionally coupled to one another, able to transfer chemical and electrical signals intercellularly through gap junctions6,7. In addition to providing new insight into calcium signaling and membrane biophysics, these preparations enable molecular studies of gene expression and protein localization within native microvascular endothelium.

Isolation of Microvascular Endothelial Tubes from Mouse Resistance Arteries

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic by-products, as exemplified by exercising skeletal muscle. Endothelial cells (ECs) line the intima of all resistance vessels and serve a key role in controlling diameter (e.g. endothelium-dependent vasodilation) and, thereby, the magnitude and distribution of tissue blood flow. The regulation of vascular resistance by ECs is effected by intracellular Ca2+ signaling, which leads to production of diffusible autacoids (e.g. nitric oxide and arachidonic acid metabolites)1-3 and hyperpolarization4,5 that elicit smooth muscle cell relaxation. Thus understanding the dynamics of endothelial Ca2+ signaling is a key step towards understanding mechanisms governing blood flow control. Isolating endothelial tubes eliminates confounding variables associated with blood in the vessel lumen and with surrounding smooth muscle cells and perivascular nerves, which otherwise influence EC structure and function. Here we present the isolation of endothelial tubes from the superior epigastric artery (SEA) using a protocol optimized for this vessel.To isolate endothelial tubes from an anesthetized mouse, the SEA is ligated in situ to maintain blood within the vessel lumen (to facilitate visualizing it during dissection), and the entire sheet of abdominal muscle is excised. The SEA is dissected free from surrounding skeletal muscle fibers and connective tissue, blood is flushed from the lumen, and mild enzymatic digestion is performed to enable removal of adventitia, nerves and smooth muscle cells using gentle trituration. These freshly-isolated preparations of intact endothelium retain their native morphology, with individual ECs remaining functionally coupled to one another, able to transfer chemical and electrical signals intercellularly through gap junctions6,7. In addition to providing new insight into calcium signaling and membrane biophysics, these preparations enable molecular studies of gene expression and protein localization within native microvascular endothelium.

Vasodilation by GPER in mesenteric arteries involves both endothelial nitric oxide and smooth muscle cAMP signaling

Our previous work showed that chronic activation of the membrane-bound estrogen receptor GPR30/GPER significantly lowers blood pressure in ovariectomized hypertensive mRen2.Lewis female rats which may, in part, reflect direct vasodilatory actions. The current study assessed the hypothesis that cyclic adenosine monophosphate (cAMP) signaling contributes to GPER-mediated vasorelaxation. In mesenteric resistance arteries from intact Lewis females, relaxation to 17-β-estradiol (E2; 47±3% of phenylephrine contraction vs. vehicle 89±2%, P<0.001) or G-1 (44±8%, P<0.001) was blunted to a similar extent by denuding (P<0.001) or the nitric oxide synthase inhibitor l-NAME (P<0.001). In contrast, the cyclooxygenase inhibitor indomethacin did not alter vasodilation (P>0.05). The cAMP analog Rp-cAMPS partially attenuated vasodilation (65±7%, P<0.001), while the combination of l-NAME and Rp-cAMPS exhibited additive effects to effectively abolish vasorelaxation (P>0.05 vs. vehicle). Pretreatment of endothelium-intact vessels with the adenylyl cyclase inhibitor SQ (63±6%) or the guanylyl cyclase inhibitor ODQ (62±9%) both partially inhibited the response to G-1 (P<0.01), while pretreatment with the both inhibitors completely abolished vasorelaxation (P>0.05 vs. vehicle). In denuded vessels only SQ reduced the response (88±3%, P<0.001). Moreover, G-1 significantly increased intracellular cAMP levels in cultured mesenteric smooth muscle cells (P<0.05). We conclude that GPER-dependent vasorelaxation apparently involves both endothelial release of nitric oxide which activates guanylyl cyclase and smooth muscle cell activation of adenylyl cyclase. Downstream production of cyclic nucleotides and stimulation of protein kinases may phosphorylate proteins to promote vascular smooth muscle cell relaxation. The ability of GPER to initiate these signaling pathways may contribute to the beneficial vascular effects of estrogen.

Steroid-sensitive Gene 1 Is a Novel Cyclic GMP-dependent Protein Kinase I Substrate in Vascular Smooth Muscle Cells

NO, via its second messenger cGMP, activates protein kinase GI (PKGI) to induce vascular smooth muscle cell relaxation. The mechanisms by which PKGI kinase activity regulates cardiovascular function remain incompletely understood. Therefore, to identify novel protein kinase G substrates in vascular cells, a λ phage coronary artery smooth muscle cell library was constructed and screened for phosphorylation by PKGI. The screen identified steroid-sensitive gene 1 (SSG1), which harbors several predicted PKGI phosphorylation sites. We observed direct and cGMP-regulated interaction between PKGI and SSG1. In cultured vascular smooth muscle cells, both the NO donor S-nitrosocysteine and atrial natriuretic peptide induced SSG1 phosphorylation, and mutation of SSG1 at each of the two predicted PKGI phosphorylation sites completely abolished its basal phosphorylation by PKGI. We detected high SSG1 expression in cardiovascular tissues. Finally, we found that activation of PKGI with cGMP regulated SSG1 intracellular distribution.

ChemInform Abstract: Crystal Structures of the Calcium Pump and Sarcolipin in the Mg2+‐Bound E1 State

AbstractReview: 42 refs.

Emerging role of angiotensin type 2 receptor (AT2R)/Akt/NO pathway in vascular smooth muscle cell in the hyperthyroidism.

Hyperthyroidism is characterized by increased vascular relaxation and decreased vascular contraction and is associated with augmented levels of triiodothyronine (T3) that contribute to the diminished systemic vascular resistance found in this condition. T3 leads to augmented NO production via PI3K/Akt signaling pathway, which in turn causes vascular smooth muscle cell (VSMC) relaxation; however, the underlying mechanisms involved remain largely unknown. Evidence from human and animal studies demonstrates that the renin-angiotensin system (RAS) plays a crucial role in vascular function and also mediates some of cardiovascular effects found during hyperthyroidism. Thus, in this study, we hypothesized that type 2 angiotensin II receptor (AT2R), a key component of RAS vasodilatory actions, mediates T3 induced-decreased vascular contraction. Marked induction of AT2R expression was observed in aortas from T3-induced hyperthyroid rats (Hyper). These vessels showed decreased protein levels of the contractile apparatus: α-actin, calponin and phosphorylated myosin light chain (p-MLC). Vascular reactivity studies showed that denuded aortic rings from Hyper rats exhibited decreased maximal contractile response to angiotensin II (AngII), which was attenuated in aortic rings pre-incubated with an AT2R blocker. Further study showed that cultured VSMC stimulated with T3 (0.1 µmol/L) for 24 hours had increased AT2R gene and protein expression. Augmented NO levels and decreased p-MLC levels were found in VSMC stimulated with T3, both of which were reversed by a PI3K/Akt inhibitor and AT2R blocker. These findings indicate for the first time that the AT2R/Akt/NO pathway contributes to decreased contractile responses in rat aorta, promoted by T3, and this mechanism is independent from the endothelium.

Altered electrical reactivity of endothelial tubes with aging: Role of mitochondria and Ca2+‐activated K+channels

Hyperpolarization of endothelial cells (ECs) is integral to coordinating smooth muscle cell relaxation throughout resistance networks and endothelial function is compromised with aging. Aberrations in mitochondrial function may contribute to endothelial dysfunction however little is known of such effects with aging in the microcirculation. We tested (P&lt;0.05) whether disrupting the proton gradient across the inner mitochondrial membrane (IMM) altered plasma membrane potential (Vm) in endothelial tubes (width, 60 μm; length, 1–3 mm) freshly isolated (bath pH, 7.4; 32°C) from superior epigastric arteries of Young (4–6 mo) and Old (24–26 mo) male C57BL/6 mice. The IMM proton ionophore FCCP (10 nM‐10 μM) depolarized Young ECs progressively (from resting Vm of −33±2 mV to −17±2 mV; n=5). In contrast, FCCP (10 nM‐10 μM) hyperpolarized Old ECs progressively (from −42±4 to −67±3 mV; n=5) which was abolished by SKCa/IKCa channel blockade with apamin (Ap, 300 nM) + charybdotoxin (ChTx, 100 nM). In Old, 300 nM FCCP hyperpolarized ECs by −12±1 mV and this reversed to 9±1 mV depolarization with Ap+ChTx (n=4). Despite no effect on Vm of Young, BKCa channel activation (NS1619, 30 μM) hyperpolarized Old ECs by −13±1 mV (n=6). We hypothesize that changes in endothelial function with aging reflects mitochondrial dysregulation of SKCa/IKCa channels with acquired BKCa channel activity. (R01HL086483, R37HL041026, F32HL110701).

How Bitter Medicine Could Clear Up Asthma

​AirwayAirway obstructive diseases, such as asthma and chronic obstructive pulmonary disease, cause the airways to narrow and make it difficult to breathe. Broncodilators are used to treat these conditions, but they often don't work in severe cases and can cause serious side effects, such as abnormal heart rhythms and increased blood pressure. Recently, scientists discovered a potential alternative to currently available bronchodilators. These compounds act on bitter taste receptors in the airways called TAS2Rs, a class of proteins long thought to be restricted to taste buds on the tongue for the purpose of detecting and avoiding harmful toxins. Bitter substances are more effective than existing bronchodilators at causing the relaxation of smooth muscle cells—which control the diameter of the airways—and opening up the airways in a mouse model of asthma. But the mechanism of action of these promising compounds has been under dispute, limiting the potential of developing them into a new class of drugs for patients. Bitter tasting compounds, synthesized or natively present in plants such as bitter melon, act on bitter taste receptors (TAS2R) in airway smooth muscle cells, which can inhibit L-type calcium channels and lead to muscle cell relaxation. In this issue of PLOS Biology, a team led by Ronghua ZhuGe of the University of Massachusetts Medical School has uncovered how bitter compounds open up the airways. They found that these substances stimulate TAS2Rs to affect calcium signaling and contraction in smooth muscle cells. The study reveals a new cell-based screening method for quickly identifying bitter compounds that have the potential of becoming powerful broncodilators for the treatment of airway obstructive diseases. ZhuGe and his team used freshly dissected smooth muscle cells and tissues, which should more accurately reflect calcium signaling and cell contraction than the cultured cells used in another recent study. Similar to the previous study, ZhuGe and his collaborators found that bitter compounds produced a counterintuitive effect: they stimulated TAS2Rs to cause a rise in calcium levels in smooth muscle cells, which might be expected to cause these cells to contract because broncoconstrictors also increase calcium levels in these cells. But unlike the previous study, the researchers discovered that the rise in calcium was not sufficient to cause the cells to contract. Moreover, bitter compounds actually reversed the effects of broncoconstrictors by inhibiting L-type voltage-dependent calcium channels, thereby blocking calcium influx into smooth muscle cells and causing them to relax. Thus, bitter substances stimulate TAS2Rs to activate two opposing calcium signaling pathways, depending on the circumstances. Under normal conditions, the compounds can modestly raise calcium levels in smooth muscle cells without causing contraction, but they reverse the rise in calcium levels caused by broncoconstrictors. Together, these findings resolve the paradox about how bitter substances can cause airway dilation even though they raise calcium levels in smooth muscle cells. By revealing the mechanisms by which bitter compounds open up the airways, the study paves the way for developing safer and more effective therapies for airway obstructive diseases. For one, the results suggest that bitter compounds should be explored as a more effective asthma treatment than currently available L-type calcium channel blockers, which don't work very well. And by simultaneously measuring calcium signaling and cell contraction, scientists will be able to efficiently identify the most promising bitter compounds. Because there are thousands of available bitter substances, some of which can stimulate TAS2Rs at extremely low concentrations, it is likely that very strong bronchodilators can be identified in future studies. Zhang C-H, Lifshitz LM, Uy KF, Ikebe M, Fogarty KE, et al. (2013) The Cellular and Molecular Basis of Bitter Tastant-Induced Bronchodilation. doi:10.1371/journal.pbio.1001501

The sarcolipin-bound calcium pump stabilizes calcium sites exposed to the cytoplasm

The contraction and relaxation of muscle cells is controlled by the successive rise and fall of cytosolic Ca(2+), initiated by the release of Ca(2+) from the sarcoplasmic reticulum and terminated by re-sequestration of Ca(2+) into the sarcoplasmic reticulum as the main mechanism of Ca(2+) removal. Re-sequestration requires active transport and is catalysed by the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA), which has a key role in defining the contractile properties of skeletal and heart muscle tissue. The activity of SERCA is regulated by two small, homologous membrane proteins called phospholamban (PLB, also known as PLN) and sarcolipin (SLN). Detailed structural information explaining this regulatory mechanism has been lacking, and the structural features defining the pathway through which cytoplasmic Ca(2+) enters the intramembranous binding sites of SERCA have remained unknown. Here we report the crystal structure of rabbit SERCA1a (also known as ATP2A1) in complex with SLN at 3.1 Å resolution. The regulatory SLN traps the Ca(2+)-ATPase in a previously undescribed E1 state, with exposure of the Ca(2+) sites through an open cytoplasmic pathway stabilized by Mg(2+). The structure suggests a mechanism for selective Ca(2+) loading and activation of SERCA, and provides new insight into how SLN and PLB inhibition arises from stabilization of this E1 intermediate state without bound Ca(2+). These findings may prove useful in studying how autoinhibitory domains of other ion pumps modulate transport across biological membranes.

Urinary Tract Symptoms (LUTS) Secondary to Benign Prostatic Hyperplasia (BPH) and LUTS/BPH with Erectile Dysfunction in Asian Men: A Systematic Review Focusing on Tadalafil

This review assesses lower urinary tract symptoms (LUTS) due to benign prostatic hyperplasia (BPH) with or without erectile dysfunction (ED) and related therapies focusing on tadalafil. A literature search was obtained and reviewed for the epidemiology, treatment therapies, pathophysiology, and efficacy and safety of phosphodiesterase type 5 inhibitor (PDE5i) tadalafil in patients with LUTS/BPH. Approximately 42% of men aged 51 to 60 years have BPH. Approximately 90% of men aged 45 to 80 years have LUTS. Occurrence of LUTS increases with age for almost all racial/ethnic groups (range, 32% to 56%) with prevalence of LUTS highest among Hispanic men, then Blacks, Caucasians, and Asians. There is an independent relationship with LUTS/BPH and ED, with approximately 70% of men with LUTS/BPH having ED with severity of one disease often correlating with the other. The European Urological Association guidelines include the use of the PDE5i tadalafil. Tadalafil is the only therapy recommended for treatment of co-existing BPH and ED, while other therapies have unwanted ED side effects. The mode of action of tadalafil may involve different areas of the lower urinary tract such as smooth muscle cell relaxation in the bladder neck, prostate, and urethra, but there may also be resulting modulation of the afferent nerve activity. Tadalafil (5 mg) in Asian men with LUTS/BPH, similar to global studies, is efficacious and safe. Tadalafil (5 mg) improves co-existing LUTS/BPH and ED, independently. Men with LUTS/BPH likely also have ED. Asian men with LUTS/BPH have similar incidence rates, co-existing ED, comorbid diseases, and risks as non-Asian men. Tadalafil can improve co-existing LUTS/BPH and ED.