Hyperpolarization Of Smooth Muscle Cells Research Articles

Pulmonary Fibrosis Augments CGRP-Dependent Hyperpolarization and Vasodilation of Mouse Pulmonary Arteries

Calcitonin gene related peptide (CGRP) hyperpolarizes membrane potential (Vm) of pulmonary arterial smooth muscle cells (SMCs) and endothelial cells (ECs) through PKA-dependent activation of KATP channels. CGRP can diminish the severity of pulmonary fibrosis (PF), however, the effects on vascular signaling were poorly defined. We hypothesized that hyperpolarization to CGRP would be augmented in a mouse model of PF. PF was induced by intratracheal delivery of bleomycin (3 wk), and saline was used as a control (sham). Pulmonary arteries (PAs; 100-150 μm diameter) from male C57BL/6J mice (age, ~5 mo), were cannulated, pressurized to 16 cmH2O, and studied at 37°C. Alternatively, intact endothelial tubes were studied at 32°C following dissociation of SMCs. Vm was recorded continuously using intracellular microelectrodes. At rest, Vm was similar for SMCs (bleomycin = -43±3 mV, sham = -44±3 mV; n = 5/group) and ECs (bleomycin = -45±4 mV, sham = -44±3 mV; n=5/group). Increasing concentrations of CGRP (10−10-10−6 M) evoked hyperpolarization of Vm with greater potency in SMCs (EC50: bleomycin = 2 nM, sham = 15 nM) and ECs (EC50: bleomycin = 3 nM, sham = 8 nM) from PF mice, without changing effcacy (SMCs: bleomycin ΔVm = -24±4 mV, sham ΔVm = -21±3 mV; ECs: bleomycin ΔVm = -26±2 mV, sham ΔVm=23±4 mV). Consistently, vasodilation to CGRP in PAs preconstricted with uridine triphosphate (UTP, 5 μM; EC50) was enhanced in PF (92±6% maximum dilation;) vs. sham (83±4% maximum dilation) mice (n=5/group). Greater vasodilation and hyperpolarization of SMCs to CGRP persisted in endothelium-disrupted PAs and during treatment with L-NAME (10−4 M) indicating that ECs are not required for greater responsiveness to CGRP in SMCs. With no effect on resting Vm, inhibition of KATP channels with 1 μM glibenclamide or PKA with protein kinase inhibitor [PKI-(14-22)-amide, myristoylated; 1 μM] significantly attenuated (p<0.05) hyperpolarization of SMCs and ECs (ΔVm ≤5 mV for all groups). Both glibenclamide and PKI attenuated vasodilation to CGRP in PAs and eliminated differences between groups. In a mouse model of PF, CGRP-dependent hyperpolarization of pulmonary arterial SMCs and ECs is augmented through increased PKA-dependent activation of KATP channels leading to increased vasodilator sensitivity. AHA CDA#931652. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

PDGFRα+ Interstitial Cells are Effector Cells of PACAP Signaling in Mouse and Human Colon.

Background & AimsPlatelet-derived growth factor receptor α (PDGFRα)-positive interstitial cells (PIC) are interposed between enteric nerve fibers and smooth muscle cells (SMCs) in the tunica muscularis of the gastrointestinal tract. PIC have robust expression of small conductance Ca2+ activated K+ channels 3 (SK3 channels) and transduce inhibitory inputs from purinergic and sympathetic nerves in mouse and human colon. We investigated whether PIC also express pituitary adenylate cyclase-activating polypeptide (PACAP) receptors, PAC1 (PAC1R), and are involved in mediating inhibitory regulation of colonic contractions by PACAP in mouse and human colons.MethodsGene expression analysis, Ca2+ imaging, and contractile experiments were performed on mouse colonic muscles. Ca2+ imaging, intracellular electrical recordings, and contractile experiments were performed on human colonic muscles.ResultsAdcyap1r1 (encoding PAC1R) is highly expressed in mouse PIC. Interstitial cells of Cajal (ICC) and SMCs expressed far lower levels of Adcyap1r. Vipr1 and Vipr2 were expressed at low levels in PIC, ICC, and SMCs. PACAP elicited Ca2+ transients in mouse PIC and inhibited spontaneous phasic contractions via SK channels. In human colonic muscles, PAC1R agonists elicited Ca2+ transients in PIC, hyperpolarized SMCs through SK channels and inhibited spontaneous phasic contractions.ConclusionsPIC of mouse and human colon utilize PAC1R-SK channel signal pathway to inhibit colonic contractions in response to PACAP. Effects of PACAP are in addition to the previously described purinergic and sympathetic inputs to PIC. Thus, PIC integrate inhibitory inputs from at least 3 neurotransmitters and utilize several types of receptors to activate SK channels and regulate colonic contractile behaviors.

Endothelium-Independent Relaxation of Vascular Smooth Muscle Induced by Persimmon-Derived Polyphenol Phytocomplex in Rats.

The vasorelaxant effect of polyphenols is well known, and the mortality rate due to coronary artery disease is low in people who consume polyphenol-containing foods. We aimed to elucidate the mechanism by which polyphenols derived from persimmon juice (PJ) and persimmon leaves (PLs) induce vasorelaxation and suppress vasocontraction in the superior mesenteric arteries isolated from male Sprague Dawley rats. Vasocontraction was induced with 1 µM phenylephrine, and polyphenol-induced vasorelaxation was expressed as a percentage of the previous tone induced by phenylephrine. PJ powder (100 mg/L) induced higher levels of vasorelaxation (mean ± standard error of the mean, 88.6% ± 4.4%) than PLs powder (1 g/L; 72.0% ± 10.8%). Nitric oxide pathway inhibitors (NG-nitro-L-arginine methyl ester + carboxy-PTIO) did not affect persimmon-derived polyphenol-induced vasorelaxation, whereas potassium chloride, tetraethylammonium, and potassium-channel inhibitors did. Vasorelaxation was endothelium independent with both extracts. Phenylephrine-induced vasocontraction was suppressed by pretreatment with PJ and PLs powder, even when inositol triphosphate-mediated Ca2+ release and extracellular Ca2+ influx were inhibited. These results suggest that persimmon-derived polyphenol phytocomplex cause vasorelaxation and inhibit vasocontraction through hyperpolarization of smooth muscle cells. Persimmon-derived polyphenols may be able to prevent cardiovascular diseases caused by abnormal contraction of blood vessels.

Reduced Function of Endothelial Nitric Oxide and Hyperpolarization in Artery Grafts with Poor Runoff

BackgroundThe endothelium regulates vascular tonus by releasing nitric oxide (endothelium-derived nitric oxide, EDNO) and hyperpolarizing factor (endothelium-derived hyperpolarizing factor, EDHF). In vein grafts with poor runoff, lack of function of these factors causes severe intimal hyperplasia. This study evaluated how the functions of EDNO and EDHF are altered in artery grafts under poor runoff conditions. Materials and MethodsThe right common carotid arteries of rabbits were excised and implanted in their original positions as autogenous grafts under normal runoff conditions (“nonoccluded grafts”) or poor runoff conditions (“poor runoff grafts”). Histochemical changes, acetylcholine (ACh)-induced effects on endothelium-dependent relaxation and smooth muscle cell (SMC) hyperpolarization were examined. ResultsBoth artery graft types displayed negligible intimal hyperplasia. In the absence and presence of an EDNO synthase inhibitor, ACh-induced relaxation was lower in grafts with poor runoff than in nonoccluded grafts. Furthermore, ACh-induced but not nonreceptor agonist A23187-induced SMC hyperpolarization was lower in the poor runoff graft group than in the nonoccluded graft group. ConclusionsUnlike in those in vein grafts, the functions of EDNO and EDHF in autogenous carotid artery grafts under poor runoff conditions were reduced but partly maintained. In such artery grafts, intimal hyperplasia caused by surgical operation was not present. These results may explain some of the mechanisms underlying the improved patency of artery grafts compared with vein grafts.

Hybrids between H2S-donors and betamethasone 17-valerate or triamcinolone acetonide inhibit mast cell degranulation and promote hyperpolarization of bronchial smooth muscle cells

Glucocorticoids represent the standard gold treatment of inflammation in asthmatic patients. More recently, H2S has been described to exert positive effect on this disease. Bearing in mind that an improved pharmacological activity and a reduced toxicity can be obtained through hybridization of different molecules, simultaneously modulating multiple targets, we designed and synthesized novel betamethasone 17-valerate and triamcinolone acetonide hybrids with well-known H2S-donor moieties. Synthesized compounds have been evaluated for the potential H2S-releasing profile both in cell-free environment and into the cytosol of bronchial smooth muscle cells (BSMCs). The two hybrids 4b and 5b were investigated by molecular modelling studies and results indicated that the steric accessibility of the isothiocyanate carbon atom can account for their different H2S releasing properties. Furthermore, the most promising derivatives 4b and 5b have been tested for inhibitory effect on mast cell degranulation and for the ability to induce cell membrane hyperpolarization in BSMCs. Significant inhibitory effect on mast cell degranulation was assessed, resulting to reduce β-hexosaminidase release more efficiently than the corresponding native drugs. Both compounds determined a massive membrane hyperpolarization of BSMCs and proved to be 4-fold more effective compared to reference compound NS1619. These effects represent an enrichment of the pharmacological activity of the native drugs.

Role of Transient Receptor Potential Vanilloid 4 in Vascular Function.

Transient receptor potential vanilloid 4 (TRPV4) channels are widely expressed in systemic tissues and can be activated by many stimuli. TRPV4, a Ca2+-permeable cation channel, plays an important role in the vasculature and is implicated in the regulation of cardiovascular homeostasis processes such as blood pressure, vascular remodeling, and pulmonary hypertension and edema. Within the vasculature, TRPV4 channels are expressed in smooth muscle cells, endothelial cells, and perivascular nerves. The activation of endothelial TRPV4 contributes to vasodilation involving nitric oxide, prostacyclin, and endothelial-derived hyperpolarizing factor pathways. TRPV4 activation also can directly cause vascular smooth muscle cell hyperpolarization and vasodilation. In addition, TRPV4 activation can evoke constriction in some specific vascular beds or under some pathological conditions. TRPV4 participates in the control of vascular permeability and vascular damage, particularly in the lung capillary endothelial barrier and lung injury. It also participates in vascular remodeling regulation mainly by controlling vasculogenesis and arteriogenesis. This review examines the role of TRPV4 in vascular function, particularly in vascular dilation and constriction, vascular permeability, vascular remodeling, and vascular damage, along with possible mechanisms, and discusses the possibility of targeting TRPV4 for therapy.

Subcellular Injury of ASMEs Involves in the Genesis of Refractory Hypotension in Severe Shock

The Refractory Hypotension (RH) leads to severe hypoperfusion which results in vital organ failure and death. It has been shown that one of the main reasons for RH is Arteriolar Smooth Muscle Cell (ASMC) hyperpolarization, which results in the inhibition of L-type calcium channel and Ca2+ influx after vasoconstrictor stimulation, that finally leads to refractory hypotension during severe shock. The activation of KATP channels by depletion of intracellular ATP content causes ASMC hyperpolarization. It is usually recognized that the depletion of ATP content originates from microcirculatory disturbance and refractory hypotension is only a functional problem of ASMC with treatment using vasopressors, and no morphological changes of smooth muscle cell were reported in RH. This review shows that mitochondrial damage is another important reason for depletion of ATP level and that protection of mitochondrial dysfunction can increase the blood pressure and survival rate during severe shock, which indicated that subcellular injury of ASMCs is involved in the genesis of refractory hypotension. Protecting and repairing ASMC subcellular injury is a new approach to treatment of severe shock.

Aqueous Fraction from Hibiscus sabdariffa Relaxes Mesenteric Arteries of Normotensive and Hypertensive Rats through Calcium Current Reduction and Possibly Potassium Channels Modulation.

Background/Objectives: Hibiscus sabdariffa L. (H. sabdariffa (HS)) extract has a vascular relaxant effect on isolated rat thoracic aorta, but data on small resistance arteries, which play an important role on the development of hypertension, are still missing. The purposes of this study were (1) to assess the effect on isolated mesenteric arteries (MA) from normotensive (Wistar and Wistar-Kyoto (WKY)) and spontaneous hypertensive rats (SHR); (2) to elucidate the mechanism(s) of action underling the relaxant effect in light of bioactive components. Methods: Vascular effects of HS aqueous fraction (AF) on isolated MA rings, as well as its mechanisms of action, were assessed using the contractility and intracellular microelectrode technique. The patch clamp technique was used to evaluate the effect of HS AF on the L-type calcium current. Extraction and enrichment of AF were carried out using liquid–liquid extraction, and the yield was analyzed using HPLC. Results: The HS AF induced a concentration-dependent relaxant effect on MA rings of SHR (EC50 = 0.83 ± 0.08 mg/mL), WKY (EC50 = 0.46 ± 0.04 mg/mL), and Wistar rats (EC50 = 0.44 ± 0.08 mg/mL) pre-contracted with phenylephrine (10 µM). In Wistar rats, the HS AF maximum relaxant effect was not modified after endothelium removal or when a guanylate cyclase inhibitor (ODQ, 10 µM) and a selective β2-adrenergic receptor antagonist (ICI-118551, 1 µM) were incubated with the preparation. Otherwise, it was reduced by 34.57 ± 10.66% when vascular rings were pre-contracted with an 80 mM [K+] solution (p < 0.001), which suggests an effect on ionic channels. HS AF 2 mg/mL significantly decreased the peak of the L-type calcium current observed in cardiac myocytes by 24.4%. Moreover, though the vasorelaxant effect of HS, AF was reduced by 27% when the nonselective potassium channels blocker (tetraethylammonium (TEA) 20 mM) was added to the bath (p < 0.01). The extract did not induce a membrane hyperpolarization of smooth muscle cells, which might suggest an absence of a direct effect on background potassium current. Conclusion: These results highlight that the antihypertensive effect of HS probably involves a vasorelaxant effect on small resistance arteries, which is endothelium independent. L-type calcium current reduction contributes to this effect. The results could also provide a link between the vasorelaxant effect and the bioactive compounds, especially anthocyanins.

Using lectures to identify student misconceptions: a study on the paradoxical effects of hyperkalemia on vascular smooth muscle.

Medical students have difficulty understanding the mechanisms underlying hyperkalemia-mediated local control of blood flow. Such control mechanisms are crucial in the brain, kidney, and skeletal muscle vasculature. We aimed to identify medical students' misconceptions via assessment of students' in-class knowledge and, subsequently, improve future teaching of this concept. In-class polling was performed with the TurningPoint clicker response system (n = 860) to gauge students' understanding of three physiological concepts related to hyperkalemia: membrane potential (Vm), conductance, and smooth muscle response. Vm includes the concepts of equilibrium potential (Veq) for specific ions, as well as driving force (DF = Vm - Veq). Students understood the concept of DF (~70% answered correctly), suggesting their understanding of Vm. However, students misunderstood that hyperkalemia results in depolarization (~52% answered correctly) and leads to an increase in potassium conductance (~31% answered correctly). Clarification of the type of smooth muscle as vascular increased the percentage of correct responses (~51 to 73%). The data indicate that students lacked knowledge of specific potassium conductance in various muscle types, resulting in divergent responses, such as the canonical depolarization in skeletal muscle versus hyperpolarization in smooth muscle cells during hyperkalemia. Misunderstanding of this crucial concept of conductance is directly related to the students' performance. Furthermore, we connected the paradoxical effect of hyperkalemia to pathological acute and chronic hyperkalemia clinical scenarios.

Norepinephrine Has Dual Effects on Human Colonic Contractions Through Distinct Subtypes of Alpha 1 Adrenoceptors.

Background & AimsColonic musculature contain smooth muscle cells (SMC), interstitial cells of Cajal (ICC), and platelet-derived growth factor receptor α+ cells (PDGFRα+ cells), which are electrically coupled and operate together as the SIP syncytium. PDGFRα+ cells have enriched expression of small conductance Ca2+-activated K+ (SK) channels. Purinergic enteric neural input activates SK channels in PDGFRα+ cells, hyperpolarizes SMC, and inhibits colonic contractions. Recently we discovered that PDGFRα+ cells in mouse colon have enriched expression of α1A adrenoceptors (ARs), which coupled to activation of SK channels and inhibited colonic motility, and α1A ARs were principal targets for sympathetic regulation of colonic motility. Here we investigated whether PDGFRα+ cells in human colon express α1A ARs and share the roles as targets for sympathetic regulation of colonic motility.MethodsIsometric tension recording, intracellular recording, and Ca2+ imaging were performed on muscles of the human colon. Responses to α1 ARs agonists or electric field stimulation with AR antagonists and neuroleptic reagents were studied.ResultsExogenous or endogenous norepinephrine released from nerve fibers inhibited colonic contractions through binding to α1A ARs or enhanced colonic contractions by acting on α1D ARs. Inhibitory responses were blocked by apamin, an antagonist of SK channels. Phenylephrine, α1 AR agonists, or norepinephrine increased intracellular [Ca2+] in PDGFRα+ cells, but not in ICC, and hyperpolarized SMCs by binding to α1 ARs expressed by PDGFRα+ cells.ConclusionsHuman colonic contractions are inhibited by α1A ARs expressed in PDGFRα+ cells and activated by α1D ARs expressed in SMC.

Endothelium-Dependent Hyperpolarization (EDH) in Diabetes: Mechanistic Insights and Therapeutic Implications.

Diabetes mellitus is one of the major risk factors for cardiovascular disease and is an important health issue worldwide. Long-term diabetes causes endothelial dysfunction, which in turn leads to diabetic vascular complications. Endothelium-derived nitric oxide is a major vasodilator in large-size vessels, and the hyperpolarization of vascular smooth muscle cells mediated by the endothelium plays a central role in agonist-mediated and flow-mediated vasodilation in resistance-size vessels. Although the mechanisms underlying diabetic vascular complications are multifactorial and complex, impairment of endothelium-dependent hyperpolarization (EDH) of vascular smooth muscle cells would contribute at least partly to the initiation and progression of microvascular complications of diabetes. In this review, we present the current knowledge about the pathophysiology and underlying mechanisms of impaired EDH in diabetes in animals and humans. We also discuss potential therapeutic approaches aimed at the prevention and restoration of EDH in diabetes.

Calcitonin gene-related peptide hyperpolarizes mouse pulmonary artery endothelial tubes through KATP channel activation.

The sensory neurotransmitter calcitonin gene-related peptide (CGRP) is associated with vasodilation of systemic arteries through activation of ATP-sensitive K+ (KATP) channels in smooth muscle cells (SMCs); however, its effects on endothelial cell (EC) membrane potential ( Vm) are unresolved. In pulmonary arteries (PAs) of C57BL/6J mice, we questioned whether CGRP would hyperpolarize ECs as well as SMCs. Intact PAs were isolated and immunostained for CGRP to confirm sensory innervation; vessel segments (1-2 mm long, ∼150 µm diameter) with intact or denuded endothelium were cannulated and pressurized to 16 cmH2O at 37°C. Increasing concentrations (10-10-10-6 M) of CGRP progressively dilated PAs preconstricted with UTP (10-5 M); SMCs hyperpolarized similarly (Δ Vm ∼20 mV) before and after endothelial denudation. To study native intact PA ECs, SMCs were dissociated to isolate endothelial tubes, and their integrity was confirmed by vital dye uptake, nuclear staining, and reproducible electrical and intracellular Ca2+ responses to acetylcholine (10-5 M) over 2 h. Increasing [CGRP] hyperpolarized ECs in a manner similar to SMCs, with each cell layer demonstrating robust immunostaining for CGRP receptor proteins. Increasing concentrations (10-10-10-6 M) of pinacidil, a KATP channel agonist, resulted in progressive hyperpolarization of SMCs of intact PAs (Δ Vm ∼30 mV), which was blocked by glibenclamide (10-6 M), as was hyperpolarization of ECs and SMCs to CGRP. Inhibition of protein kinase A with protein kinase inhibitor (10-5 M) also inhibited hyperpolarization to CGRP. We demonstrate [CGRP]-dependent hyperpolarization of ECs for the first time while validating freshly isolated PA endothelial tubes as an experimental model. Redundant electrical signaling to CGRP in ECs and SMCs implies an integral role for KATP channels in PA dilation.

Adenosine Receptors As Drug Targets for Treatment of Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is a clinical condition characterized by pulmonary arterial remodeling and vasoconstriction, which promote chronic vessel obstruction and elevation of pulmonary vascular resistance. Long-term right ventricular (RV) overload leads to RV dysfunction and failure, which are the main determinants of life expectancy in PAH subjects. Therapeutic options for PAH remain limited, despite the introduction of prostacyclin analogs, endothelin receptor antagonists, phosphodiesterase type 5 inhibitors, and soluble guanylyl cyclase stimulators within the last 15 years. Through addressing the pulmonary endothelial and smooth muscle cell dysfunctions associated with PAH, these interventions delay disease progression but do not offer a cure. Emerging approaches to improve treatment efficacy have focused on beneficial actions to both the pulmonary vasculature and myocardium, and several new targets have been investigated and validated in experimental PAH models. Herein, we review the effects of adenosine and adenosine receptors (A1, A2A, A2B, and A3) on the cardiovascular system, focusing on the A2A receptor as a pharmacological target. This receptor induces pulmonary vascular and heart protection in experimental models, specifically models of PAH. Targeting the A2A receptor could potentially serve as a novel and efficient approach for treating PAH and concomitant RV failure. A2A receptor activation induces pulmonary endothelial nitric oxide synthesis, smooth muscle cell hyperpolarization, and vasodilation, with important antiproliferative activities through the inhibition of collagen deposition and vessel wall remodeling in the pulmonary arterioles. The pleiotropic potential of A2A receptor activation is highlighted by its additional expression in the heart tissue, where it participates in the regulation of intracellular calcium handling and maintenance of heart chamber structure and function. In this way, the activation of A2A receptor could prevent the production of a hypertrophic and dysfunctional phenotype in animal models of cardiovascular diseases.

Enhancement of Nitric Oxide Production Is Responsible for Minimal Intimal Hyperplasia of Autogenous Rabbit Arterial Grafts.

Vascular endothelium induces smooth muscle cell (SMC) relaxation mainly mediated by endothelium-derived nitric oxide (EDNO) and endothelium-derived hyperpolarizing factor (EDHF). It has previously been reported that functions of these endothelium factors have been greatly impaired in vein grafts. The present study was undertaken to determine whether the functions of EDNO and EDHF might be altered in artery graft.Methods and Results:In rabbits, the right carotid artery was excised and implanted in its original position as an autogenous graft ("artery graft") and the non-operated left carotid artery served as the "control artery". Histochemical changes, acetylcholine (ACh)-induced effects on the intracellular concentration of Ca2+([Ca2+]i) in endothelial cells, endothelium-dependent SMC hyperpolarization and relaxation, and tissue cGMP content were examined on post-operative day 28. "Artery graft" displayed a minimal amount of intimal hyperplasia. When compared with the "control artery", it exhibited greater ACh-induced, endothelium-dependent relaxation, but the reverse was true when EDNO production was blocked. In the "artery graft" (vs. the "control artery"), basal cGMP content was greater, whereas the [Ca2+]iincrease in endothelial cells and the endothelium-dependent SMC-hyperpolarization induced by ACh were less. It is suggested that the [Ca2+]i-independent EDNO production covers the loss of function of endothelium-dependent SMC hyperpolarization and minimizes intimal hyperplasia caused by surgical operation in autogenous carotid artery graft.

Endothelium-Dependent Hyperpolarization and Endothelial Dysfunction

The endothelium controls vascular tone not only by releasing various vasoactive substances but also by another pathway associated with the hyperpolarization of both endothelial and vascular smooth muscle cells and is termed endothelium-dependent hyperpolarization (EDH). These responses involve an increase in the endothelial intracellular Ca concentration by the activation of transient receptor potential channels (predominantly TRPV4) followed by the opening of Ca-activated K channels of small and intermediate conductance (SKCa and IKCa). These channels show a distinct subcellular distribution. SKCa are widely distributed over the plasma membrane but segregates at sites of homocellular endothelial junctions, whereas IKCa are preferentially expressed in the myoendothelial projections. Following KCa activation, smooth muscle hyperpolarization is evoked by electrical coupling through myoendothelial gap junctions and/or by the potassium efflux that subsequently activates smooth muscle Kir2.1 and/or Na/K-ATPase. Alteration of the EDH contributes to the endothelial dysfunctions observed in various pathologies or conversely compensates for the loss in NO bioavailability. A better characterization of EDH should allow determining whether new druggable targets can be identified for the treatment of cardiovascular diseases.

TRPML1‐Mediated Ca2+ Signaling in Cerebral Artery Smooth Muscle

Late endosomes and lysosomes (collectively referred to as endolysosomes) are small acidic organelles present in all cells that maintain a steep Ca2+ gradient compared with the cytosol. Therefore, activation of Ca2+ permeable ion channels within the endolysosomal membrane can transiently generate localized microdomains of elevated Ca2+ within the cytosol. The goal of the current study is to elucidate the importance of Ca2+ signals arising from acidic Ca2+ stores in cerebral artery smooth muscle cells (SMC). Members of the mucolipin transient receptor potential (TRPML) subfamily of cation channels are Ca2+ permeable and selectively localize to endolysosomes. Mutations in the human TRPML1 gene (MCOLN1) result in the heritable childhood neurodegenerative disease type IV mucolipidosis (ML‐IV). Quantitative RT‐PCR analysis showed the presence of TRPML1, TRPML2, and TRPML3 in mRNA extracted from cerebral pial arteries from C57Bl/6 mice, with TRPML1 being most abundant. Furthermore, freshly isolated cerebral pial artery SMC showed positive immunofluorescence labeling for TRPML1, which co‐localized with the endolysosomal marker LAMP‐1. Immunolabeling experiments also suggest colocalization of TRPML1 and NADPH oxidase isoform 2 (NOX2) in endolysosomes in pial artery SMC. Ca2+ signals arising from TRPML1‐mediated endolysosomal release were investigated using a TRPML1‐GCaMP3 fusion protein. This genetic construct is targeted to endolysosomes and selectively detects Ca2+ released into the cytosol from these organelles. In HEK293 cells expressing TRPML1‐GCaMP3, an increase in cytosolic fluorescence was detected in response to the selective TRPML1 activator ML‐SA1 (10 μM), signifying that Ca2+ release from acidic stores through TRPML1 can be detected using these methods. We also investigated the functional significance of TRPML1‐mediated intracellular Ca2+ signals. In cerebral SMC, large conductance Ca2+ activated K+ (BK) channels are activated by spatially and temporally limited release of Ca2+ from the sarcoplasmic reticulum via ryanodine receptors (RyR), which generate microdomains of high cytosolic [Ca2+] known as Ca2+ sparks. This pathway promotes smooth muscle cell hyperpolarization and vasodilation. The activity of RyR is elevated by localized increases in cytosolic [Ca2+] to cause Ca2+‐induced Ca2+ release. Therefore, we hypothesized that Ca2+ release from endolysosomes sensitizes RyR to increase Ca2+ spark and BK channel activity. Ca2+ sparks were recorded from cerebral SMC loaded with the fast Ca2+ indicator fluo‐4AM using high‐speed, high‐resolution confocal microscopy. We found that Ca2+ spark sites co‐localized with endolysosomes labeled with LysoTracker Red DND‐99. Furthermore, preliminary experiments show that Ca2+ spark frequency and BK channel activity are increased by ML‐SA1 (3 μM). These data suggest a novel pathway for cerebral blood flow regulation in which local Ca2+ release from endolysosomes through TRPML1 channels sensitizes RyR, stimulating Ca2+ spark and BK channel activity to promote vasodilation. It is possible that disrupted cerebral blood flow control resulting from loss of TRPML1 activity in SMC contributes to neurodegeneration associated with ML‐IV.Support or Funding InformationSupport: R01HL091905 (SE).

SGLT inhibitors attenuate NO-dependent vascular relaxation in the pulmonary artery but not in the coronary artery.

Inhibitors of sodium-glucose cotransporter (SGLT)2 are a new class of oral drugs for type 2 diabetic patients that reduce plasma glucose levels by inhibiting renal glucose reabsorption. There is increasing evidence showing the beneficial effect of SGLT2 inhibitors on glucose control; however, less information is available regarding the impact of SGLT2 inhibitors on cardiovascular outcomes. The present study was designed to determine whether SGLT inhibitors regulate vascular relaxation in mouse pulmonary and coronary arteries. Phlorizin (a nonspecific SGLT inhibitor) and canagliflozin (a SGLT2-specific inhibitor) relaxed pulmonary arteries in a dose-dependent manner, but they had little or no effect on coronary arteries. Pretreatment with phlorizin or canagliflozin significantly inhibited sodium nitroprusside (SNP; a nitric oxide donor)-induced vascular relaxation in pulmonary arteries but not in coronary arteries. Phlorizin had no effect on cGMP-dependent relaxation in pulmonary arteries. SNP induced membrane hyperpolarization in human pulmonary artery smooth muscle cells, and pretreatment of cells with phlorizin and canagliflozin attenuated SNP-induced membrane hyperpolarization by decreasing K(+) activities induced by SNP. Contrary to the result observed in ex vivo experiments with SGLT inhibitors, SNP-dependent relaxation in pulmonary arteries was not altered by chronic administration of canagliflozin. On the other hand, canagliflozin administration significantly enhanced SNP-dependent relaxation in coronary arteries in diabetic mice. These data suggest that SGLT inhibitors differentially regulate vascular relaxation depending on the type of arteries, duration of the treatment, and health condition, such as diabetes.

Pleiotropic Effects of Bitter Taste Receptors on [Ca2+]i Mobilization, Hyperpolarization, and Relaxation of Human Airway Smooth Muscle Cells.

Asthma is characterized by airway inflammation and airflow obstruction from human airway smooth muscle (HASM) constriction due to increased local bronchoconstrictive substances. We have recently found bitter taste receptors (TAS2Rs) on HASM, which increase [Ca2+]i and relax the muscle. We report here that some, but not all, TAS2R agonists decrease [Ca2+]i and relax HASM contracted by G-protein coupled receptors (GPCRs) that stimulate [Ca2+]i. This suggests both a second pathway by which TAS2Rs relax, and, a heterogeneity of the response phenotype. We utilized eight TAS2R agonists and five procontractile GPCR agonists in cultured HASM cells. We find that heterogeneity in the inhibitory response hinges on which procontractile GPCR is activated. For example, chloroquine inhibits [Ca2+]i increases from histamine, but failed to inhibit [Ca2+]i increases from endothelin-1. Conversely, aristolochic acid inhibited [Ca2+]i increases from endothelin-1 but not histamine. Other dichotomous responses were found when [Ca2+]i was stimulated by bradykinin, angiotensin, and acetylcholine. There was no association between [Ca2+]i inhibition and TAS2R subtype, nor whether [Ca2+]i was increased by Gq- or Gi-coupled GPCRs. Selected studies revealed a correlation between [Ca2+]i inhibition and HASM cell-membrane hyperpolarization. To demonstrate physiologic correlates, ferromagnetic beads were attached to HASM cells and cell stiffness measured by magnetic twisting cytometry. Consistent with the [Ca2+]i inhibition results, chloroquine abolished the cell stiffening response (contraction) evoked by histamine but not by endothelin-1, while aristolochic acid inhibited cell stiffening from endothelin-1, but not from histamine. In studies using intact human bronchi, these same differential responses were found. Those TAS2R agonists that decreased [Ca2+]i, promoted hyperpolarization, and decreased HASM stiffness, caused relaxation of human airways. Thus TAS2Rs relax HASM in two ways: a low-efficiency de novo [Ca2+]i stimulation, and, a high-efficiency inhibition of GPCR-stimulated [Ca2+]i. Furthermore, there is an interaction between TAS2Rs and some GPCRs that facilitates this [Ca2+]i inhibition limb.

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.

PDE and sGC hand in hand to see the light.

In PNAS, Sikka et al. (1) suggest that melanopsin, a non–image-forming opsin (opsin4, Opn4), may play a physiological role in the regulation of vascular tone by mediating photorelaxation. The authors conclude that the response to light is due to hyperpolarization of the vascular smooth muscle cells resulting from the activation of soluble guanylyl cyclase (sGC), but does not involve protein kinase G (PKG). This study not only provides an intriguing molecular explanation for a phenomenon (photorelaxation) that has puzzled vascular biologists for more than half a century (2), but also forces us to rethink the fate of the products generated by sGC.