Effects of sodium nitroprusside on cytosolic calcium level in vascular smooth muscle

The inhibitory effect of sodium nitroprusside on the cytosolic Ca2+ level ([Ca2+]cyt), measured simultaneously with contraction by means of a fluorescence dye, fura-2, and on the 45Ca2+ uptake was tested in the isolated rat aortic smooth muscle. Norepinephrine increased muscle tension, 45Ca2+ uptake and [Ca2+]cyt. In a Ca2+-deficient solution, norepinephrine transiently increased muscle tension and [Ca2+]cyt. Sodium nitroprusside inhibited all changes induced by norepinephrine although the inhibition of [Ca2+]cyt was less than that of muscle contraction. Sodium nitroprusside also inhibited the high K+-induced contraction at concentrations higher than those needed to inhibit norepinephrine-induced contraction. Inhibition of the high K+induced contraction was accompanied by a small decrease in [Ca2+]cyt and a smaller decrease in 45Ca2+ uptake. Methylene blue antagonized, and M&B 22,948 potentiated the inhibitory effect of sodium nitroprusside. These results suggest that sodium nitroprusside has multiple sites of action. At relatively low concentrations, sodium nitroprusside could inhibit the Ca2+ influx and Ca2+ release. At relatively high concentrations, this inhibitor could also augment Ca2+ sequestration and decrease the sensitivity of contractile elements to Ca2+.     

小檗胺对ROCC介导的血管平滑肌细胞内游离钙的影响

目的　研究小檗胺 (BA)对受体调控性Ca2 +通道介导的家兔胸主动脉血管平滑肌细胞内游离钙 ([Ca2 +]i)的影响。方法　家兔主动脉血管平滑肌以Fluo 3/AM负载 ,通过激光扫描共聚焦显微镜 (LSCM )测定 [Ca2 +]i。结果　在细胞外Ca2 +存在的条件下 ,BA 30 μmol·L-1不影响静息[Ca2 +]i;但对去甲肾上腺素 (NE) 30mmol·L-1、5 羟色胺 (5 HT) 1μmol·L-1诱导的 [Ca2 +]i 升高有明显的抑制作用。在无胞外钙时 ,对咖啡因 40mmol·L-1诱导的 [Ca2 +]i 升高没有作用。结论　BA对ROCC激活后的外钙内流有明显的抑制作用 ,对内钙释放没有影响。其作用与维拉帕米相似     

Effects of cyclopiazonic acid and ryanodine on cytosolic calcium and contraction in vascular smooth muscle.

1. In smooth muscle, both Ca2+ release from the sarcoplasmic reticulum (SR) and Ca2+ influx across the plasma membrane are responsible for the increase in the cytosolic Ca2+ level ([Ca2+]i). To understand further the role of SR on smooth muscle contraction, the effects of an inhibitor of the SR Ca2+ pump, cyclopiazonic acid (CPA 10 microM), an inhibitor of the Ca(2+) -induced Ca2+ release, ryanodine, (10 microM), and an activator of the Ca(2+) -induced Ca2+ release, caffeine (20 mM), on [Ca2+]i and contractile force were examined in the ferret portal vein loaded with a photoprotein, aequorin. 2. CPA induced a small increase in the aequorin signal reaching a maximum at 7 min. Several minutes after the increase in the aequorin signal, muscle tension increased reaching a maximum at 21.5 min. In contrast, ryanodine changed neither the aequorin signal nor contraction. In the presence of ryanodine, caffeine induced a sustained increase in the aequorin signal and transient contraction. After washing ryanodine and caffeine, the aequorin signal and muscle tone returned to their respective control levels. After treatment with ryanodine and caffeine, the second addition of caffeine was almost ineffective whereas CPA still increased the aequorin signal and muscle tension. 3. In the presence of external Ca2+, noradrenaline (NA, 10 microM) induced a transient increase followed by a sustained increase in the aequorin signal and sustained contraction. In contrast, KCl (70 mM) induced sustained increases in the aequorin signal and sustained contraction. In Ca(2+) -free solution, NA induced a small transient increase in the aequorin signal and a small transient contraction. These changes were inhibited in the presence of CPA or on pretreatment of the muscle with ryanodine and caffeine. These results suggest that CPA or ryanodine and caffeine depleted Ca2+ in SR. High K+ was ineffective in the absence of external Ca2+. 4. In the presence of external Ca2+ and CPA, NA and high K+ induced larger aequorin signals than in the absence of CPA, whereas the magnitude and shape of the contractions did not change. In contrast, pretreatment with ryanodine and caffeine did not have such an effect. In the muscle pretreated with ryanodine and caffeine, CPA changed the responses to high K+ and NA in a similar manner to that in the muscle without the pretreatment with ryanodine and caffeine. 5. Dissociation of contraction from [Ca2+]i as measured with aequorin suggests that NA and high K+ increase Ca2+ in two compartments: a compartment containing contractile elements (contractile compartment) and another compartment unrelated to contractile elements (non-contractile compartment). Because CPA augmented the stimulant-induced increase in aequorin signal without changing contraction, the non-contractile compartment may be located near the SR and the CPA-sensitive SR Ca2+ pump may regulate the Ca2+ level in this compartment. However, because CPA changed neither the magnitude nor shape of the contractions in the presence of external Ca2+, the SR Ca2+ pump may have little effect on regulation of Ca2+ level in the contractile compartment. Furthermore, the release of Ca2+ from SR seems to have little effect on the increase in the contractile Ca2+ because ryanodine and caffeine changed neither the aequorin signals nor contractions induced by NA and high K+ in the presence of external Ca2+ in the ferret portal vein.     

Protamine augments stretch induced calcium increase in vascular endothelium

1. Human umbilical vein endothelial cells cultured on a transparent silicone chamber were subjected to a short stretch pulse (ca. 1 s, 5-25% stretch) of their substrate and following increases in intracellular Ca(2+) concentration ([Ca(2+)](i)) were measured by fluorescence intensity ratiometry using fura-2. 2. In response to mechanical stretch, the cells in HEPES buffered saline exhibited a Ca(2+) transient in a dose dependent way. The response was completely dependent on external Ca(2+) and inhibited by gadolinium (Gd(3+)), suggesting that it was mediated by the activation of a stretch activated cation channel (SACatC). 3. Interestingly, the stretch induced Ca(2+) transient was significantly augmented in the presence of basic polypeptide, protamine. This augmented Ca(2+) response was inhibited neither by Gd(3+) nor by the deprivation of external Ca(2+), indicating that the SACatC is not responsible for this phenomenon. 4. In contrast, this augmentation was inhibited by depletion of intracellular Ca(2+) stores with thapsigargin or by the pretreatment with phospholipase inhibitors such as U73122 and manoalide. 5. These results suggest the presence of a metabotropic mechanoreceptor distinct from the SACatC in vascular endothelium. This augmented [Ca(2+)](i) increase may contribute to the vasodilating response induced by protamine during heparin neutralization in cardiac surgery.     

钾通道开放剂降低血管平滑肌细胞内游离钙浓度(英文)

目的:研究PCO—Pin,Nic,Lem及RP对VMSC内[Ca~(2 )]_i的改变及其可能机制。方法:VSMC加入Fura-2 AM 2.5μmol·L~(-1)37℃下孵育50min,[Ca~(2 )]_i用荧光分光光度计检测。结果:4种PCO能较弱地抑制K~ 30 mmol·L~(-1)诱导的[Ca~(2 )]_i增加,但明显抑制ATP 0.1mmol·L~(-1)诱导的[Ca~(2 )]_i峰相及持续相增加,且呈剂量依赖性。格列苯脲完全阻断Pin,Lem及RP的作用,只部分抑制Nic的作用。无钙液中先给4种PCO,能显著抑制ATP诱导的[Ca~(2 )]_i峰相增加。结论:4种PCO均抑制ATP诱导的[Ca~(2 )]_i增加,此作用与减少细胞外钙内流及细胞内钙释放有关。     

Store-operated calcium entry in vascular smooth muscle

In non-excitable cells, activation of G-protein-coupled phospholipase C (PLC)-linked receptors causes the release of Ca(2+) from intracellular stores, which is followed by transmembrane Ca(2+) entry. This Ca(2+) entry underlies a small and sustained phase of the cellular [Ca(2+)](i) increases and is important for several cellular functions including gene expression, secretion and cell proliferation. This form of transmembrane Ca(2+) entry is supported by agonist-activated Ca(2+)-permeable ion channels that are activated by store depletion and is referred to as store-operated Ca(2+) entry (SOCE) and represents a major pathway for agonist-induced Ca(2+) entry. In excitable cells such as smooth muscle cells, Ca(2+) entry mechanisms responsible for sustained cellular activation are normally considered to be mediated via either voltage-operated or receptor-operated Ca(2+) channels. Although SOCE occurs following agonist activation of smooth muscle, this was thought to be more important in replenishing Ca(2+) stores rather than acting as a source of activator Ca(2+) for the contractile process. This review summarizes our current knowledge of SOCE as a regulator of vascular smooth muscle tone and discusses its possible role in the cardiovascular function and disease. We propose a possible hypothesis for its activation and suggest that SOCE may represent a novel target for pharmacological therapeutic intervention.     

Alternative splicing modulates diltiazem sensitivity of cardiac and vascular smooth muscle Cav1.2 calcium channels

Background and purpose:

As a calcium channel blocker, diltiazem acts mainly on the voltage-gated calcium channels, Ca_v1.2, for its beneficial effects in cardiovascular diseases such as hypertension, angina and/or supraventricular arrhythmias. However, the effects of diltiazem on different isoforms of Ca_v1.2 channels expressed in heart and vascular smooth muscles remain to be investigated. Here, we characterized the effects of diltiazem on the splice variants of Ca_v1.2 channels, predominant in cardiac and vascular smooth muscles.

Experimental approach:

Cardiac and smooth muscle isoforms of Ca_v1.2 channels were expressed in human embryonic kidney cells and their electrophysiological properties were characterized using whole-cell patch-clamp techniques.

Key results:

Under closed-channel and use-dependent block (0.03 Hz), cardiac splice variant Ca_v1.2CM was less sensitive to diltiazem than two major smooth muscle splice variants, Ca_v1.2SM and Ca_v1.2b. Ca_v1.2CM has a more positive half-inactivation potential than the smooth muscle channels, and diltiazem shifted it less to negative potential. Additionally, the current decay was slower in Ca_v1.2CM channels. When we modified alternatively spliced exons of cardiac Ca_v1.2CM channels into smooth muscle exons, we found that all three loci contribute to the different diltiazem sensitivity between cardiac and smooth muscle splice isoforms.

Conclusions and implications:

Alternative splicing of Ca_v1.2 channels modifies diltiazem sensitivity in the heart and blood vessels. Gating properties altered by diltiazem are different in the three channels.     

Ca2+ localization and sensitivity in vascular smooth muscle

An increase in cytosolic Ca2+ level ([Ca2+]i) is a prerequisite for smooth muscle contraction. Simultaneous measurements of [Ca2+]i and muscle tension give direct information on the Ca2+ regulation of smooth muscle. The photoprotein aequorin and the fluorescent Ca2+ indicator fura-2 are widely used for this purpose. Although there are some inconsistencies between the results obtained with these two indicators, comparison between [Ca2+]i and muscle tension in vascular smooth muscle indicates that stimulation of alpha-adrenoceptors increases, whereas stimulation of beta-adrenoceptors decreases, both the Ca2+ sensitivity of contractile elements and [Ca2+]i. Thus, as Hideaki Karaki explains, contractility of vascular smooth muscle may be regulated not only by [Ca2+]i but also by the Ca2+ sensitivity of the contractile elements.     

Fluvastatin enhances apoptosis in cytokine-stimulated vascular smooth muscle cells.

Hydroxymethylglutaryl coenzyme A reductase inhibitors (statins) have been shown to attenuate proliferation of vascular smooth muscle cells (VSMCs) by mechanisms independent of lipid reduction. In the current study, we investigated the effect of lipophilic and hydrophilic statins (fluvastatin and pravastatin) on apoptosis in unstimulated or cytokine-stimulated VSMCs. The presence of apoptosis in rat VSMCs was evaluated by electrophoresis of DNA fragments and 4'6'-diamidine-2'-phenylindole staining and quantified by flow cytometry. Fluvastatin but not pravastatin enhanced apoptosis in interleukin-1beta-stimulated VSMCs. The proapoptotic effect of fluvastatin was fully reversed by mevalonate and geranylgeranyl-pyrophosphate, and partially by farnesyl-pyrophosphate, but not by squalene. Inhibition of the extracellular signal-regulated protein kinase (ERK1/2) pathway significantly increased fluvastatin-enhanced apoptosis, whereas inhibition of the p38-mitogen-activated protein kinase (MAPK) pathway significantly prevented this increase. However, fluvastatin showed no effect on the activity of ERK1/2 and p38-MAPK. Furthermore, fluvastatin-induced apoptosis was inhibited by YVAD-FMK (a caspase-1/interleukin-1beta-converting enzyme-like protease inhibitor) and DEVD-FMK (a caspase-3/CPP32 inhibitor), indicating involvement of an important segment in the apoptosis signaling pathway. These findings suggest that fluvastatin enhances apoptosis in cytokine-stimulated VSMCs and that protein prenylation, MAPK (ERK1/2 and p38-MAPK), and caspases are critically involved in the pathways of fluvastatin-enhanced apoptosis.     

Chronic nicotine treatment enhances vascular smooth muscle relaxation in rats

Aim:

To investigate the effect of chronic nicotine treatment on vascular function and to identify the underlying mechanisms.

Methods:

Adult rats were treated with nicotine (3 mg·kg⁻¹·d⁻¹, sc) for 6 weeks. After the rats were sacrificed, aortic rings were prepared for detecting vascular reactivity, and thoracic aorta and periaortic fat samples were collected for histological and molecular biology studies.

Results:

Chronic nicotine treatment significantly reduced periaortic fat, and specifically enhanced smooth muscle relaxation without altering the aortic adventitial fat and endothelium function. Pretreatment with the soluble guanylyl cyclase inhibitor ODQ (3 μmol/L) or PKG inhibitor Rp-8-Br-PET-cGMP (30 μmol/L) abolished the nicotine-induced enhancement of smooth muscle relaxation, whereas the cGMP analogue 8-Br-cGMP could mimic the nicotine-induced enhancement of smooth muscle relaxation. However, the chronic nicotine treatment did not alter PKG protein expression and activity in aortic media.

Conclusion:

Chronic nicotine treatment enhances vascular smooth muscle relaxation of rats via activation of PKG pathway.     

Metabolism of vasoactive peptides by vascular endothelium and smooth muscle aminopeptidase M

The cellular localization of vascular plasma membrane aminopeptidase M (AmM; EC3.4.11.2) was examined in cultured porcine aorta endothelium and smooth muscle cells. AmM was 14-fold higher on smooth muscle (117 +/- 16 units/mg) than on endothelium (8.4 +/- 0.2). Proportional to its cellular distribution, AmM hydrolyzed the N-terminus of kallidin to produce bradykinin, and degraded des(Asp1)angiotensin I, angiotensin III, hepta(5-11)substance P and Met5-enkephalin. In contrast, bradykinin, angiotensin II and substance P were resistant to AmM-mediated hydrolysis. Peptide metabolism was optimal at pH 7.0 and was inhibited by o-phenanthroline, bestatin (Ki = 2.2 +/- 0.1 microM) and amastatin (Ki = 25 +/- 5 nM). Des(Asp1)angiotensin I and angiotensin III had the highest affinity (lowest Km) for AmM (Km = 2.2 +/- 0.5 and 2.0 +/- 0.4 microM respectively), followed by hepta(5-11)substance P (53.9 +/- 1.7 microM) and Met5-enkephalin (75.7 +/- 3.5 microM). In contrast, maximal velocities of hydrolysis were higher for Met5-enkephalin (313 +/- 2 nmol/min/mg) than for hepta(5-11)substance P (109 +/- 18 nmol/min/mg) or angiotensin III (26.5 +/- 1.0 nmol/min/mg). As expected for hydrolysis by a common enzyme, AmM-mediated enkephalin degradation was inhibited competitively by angiotensin III (Ki = 0.34 +/- 0.04 microM), hepta(5-11)substance P (43.7 +/- 6.3 microM) and kallidin (62 microM). These data suggest that vascular AmM may modulate vasoactive peptide levels in vivo, particularly within the microenvironment of endothelial and smooth muscle cell surface receptors.     

眼镜蛇心脏毒素对血管平滑肌和内皮细胞的双向作用

AIM: To assess the cytotoxic effects of cobra cardiotoxin (CTX) on rat aorta. METHODS: Measure of contractility of aortic rings with or without endothelium. RESULTS: In endothelium-intact rings, CTX 10 mumol.L-1 induced a transient relaxation followed by a sustained contraction. Removal of the endothelium or pre-incubation of the rings with NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) abolished the transient relaxation but did not affect the magnitude of the contractile response induced by CTX. CTX itself induced contraction of vascular smooth muscle but also reduced contractions induced by phenylephrine (PhE) or KCl stimulation in a concentration-dependent manner. Contraction induced by CTX was dependent on the external Ca2+ concentration. Maximal contractile response to CTX was obtained in medium containing Ca2+ 1 mmol.L-1. This response decreased with higher Ca2+ concentration and disappeared when Ca2+ 7 mmol.L-1, organic and inorganic calcium channel blockers were present in the external solution before CTX addition. In preparations with the endothelium intact and incubated with CTX, relaxation by acetylcholine (ACh) stimulation of the tension induced by PhE was decreased. Endothelium-dependent relaxation to ACh was preserved when Ca2+ 5 mmol.L-1 was added to the medium prior to CTX. CONCLUSION: CTX first triggers the release of NO from the endothelium which results in muscle relaxation, and then causes smooth muscle contraction, Ca2+ and Ca2+ channel blockers prevented the effect of CTX.     

左旋千金藤定碱对培养牛主动脉血管平滑肌细胞胞内游离Ca~(2+)的影响

目的：研究左旋千金藤定碱（ｌ－ｓｔｅｐｈｏｌｉｄｉｎｅ，ＳＰＤ）对血管平滑肌的作用。方法：采用Ｆｕｒａ－２和ＡＲ－ＣＭ－ＭＩＣ阳离子测定系统测定培养牛主动脉血管平滑肌细胞内游离钙。结果：ＳＰＤ１～１００μｍｏｌ·Ｌ－１不影响静息［Ｃａ２＋］ｉ，但可剂量依赖地抑制高Ｋ＋引起的［Ｃａ２＋］ｉ增高，其ＩＣ５０为３９．６（９５％可信限２３．４～６７．１）μｍｏｌ·Ｌ－１，但其作用弱于尼群地平；ＳＰＤ１～１００μｍｏｌ·Ｌ－１对去甲肾上腺素、血管紧张素Ⅱ、５－ＨＴ、ＡＴＰ引起的［Ｃａ２＋］ｉ增高也有明显的抑制作用；高浓度ＳＰＤ对无外钙时去甲肾上腺素引起的［Ｃａ２＋］ｉ增高也有一定的抑制作用。结论：左旋千金藤定碱对培养血管平滑肌细胞电压依赖性钙通道和受体调控性钙通道均有抑制作用；其对电压依赖性钙通道的抑制作用弱于尼群地平。     

IL-13 enhances agonist-evoked calcium signals and contractile responses in airway smooth muscle

Growing evidence suggests that interleukin (IL)-13, a Th2-type cytokine, plays a critical role in the development of bronchial hyper-responsiveness (BHR), an essential feature of asthma, although the underlying mechanisms remain unknown. In the present study, we investigated whether IL-13 directly affects airway smooth muscle (ASM) function. In murine tracheal rings, IL-13 (100 ng ml-1, 24 h) significantly increased both the carbachol- and KCl-induced maximal force generation without affecting ASM sensitivity. In cultured human ASM cells, IL-13 (50 ng ml-1, 24 h) also augmented cytosolic calcium levels to bradykinin, histamine and carbachol by 60, 35 and 26%, respectively. The present study demonstrates that IL-13 may promote BHR by directly modulating ASM contractility, an effect that may be due to enhanced G protein-coupled receptor (GPCR)-associated calcium signaling.     

Endothelin dissociates muscle tension from cytosolic Ca2+ in vascular smooth muscle of rat carotid artery

Endothelin induced rapid increase followed by a decrease in cytosolic Ca2+ [( Ca2+]i) and a slow increase in muscle tension in the vascular smooth muscle strip of rat carotid artery. Thus, the endothelin-induced contraction was smaller, and it became gradually greater than high K-induced contraction at a given [Ca2+]i. In Ca2+-free solution, endothelin induced a transient increase in [Ca2+]i and a sustained contraction. These results suggest that endothelin-induced contraction is due to the increase in [Ca2+]i, the time-dependent change in Ca2+-sensitivity of contractile elements, and the mechanism which is independent of the increment in [Ca2+]i.     

Cholera toxin treatment of vascular smooth muscle cells decreases smooth muscle alpha-actin content and abolishes the platelet-derived growth factor-BB-stimulated DNA synthesis

The second messenger cyclic AMP regulates diverse biological processes such as cell morphology and cell growth. We examined the role of the second messenger cyclic AMP on rat aortic vascular smooth muscle cell (VSMC) morphology and the intracellular transduction pathway mediated by platelet-derived growth factor beta-receptor (PDGF-Rbeta). The effect of PDGF-BB on VSMCs growth was assessed by [(3)H]-thymidine incorporation. Tyrosine phosphorylation of PDGF-Rbeta, PLC-gamma1, ERK1 and ERK2, p125(FAK) and paxillin as well as Sm alpha-actin was examined by the chemiluminescence Western blotting method. Actin mRNA level was quantitated by Northern blotting. Visualization of Sm alpha-actin filaments, paxillin and PDGF-Rbeta was performed by immunfluorescence microscopy. Cholera toxin (CTX; 10 nM) treatment lead to a large and sustained increase in the cyclic AMP concentration after 2 h which correlated with change of VSMC morphology including complete disruption of the Sm alpha-actin filament array and loss of focal adhesions. Treatment of VSMCs with CTX did not influence tyrosine phosphorylation of p125(FAK) and paxillin but decreased the content of a Sm alpha-actin protein. Maximal decrease of 70% was observed after 24 h of treatment. CTX also caused a 90% decrease of the actin mRNA level. CTX treatment completely abolished PDGF-BB stimulated DNA-synthesis although PDGF-Rbeta level and subcellular distribution and translocation was not altered. Furthermore CTX attenuated the PDGF-BB-induced tyrosine phosphorylation of the PDGF-Rbeta, PI 3'-K, PLC-gamma1 and ERK1/2 indicating an action of cyclic AMP on PDGF-beta receptor. We conclude that although cyclic AMP attenuates the PDGF-Rbeta mediated intracellular transduction pathway, an intact actin filament may be required for the PDGF-BB-induced DNA synthesis in VSMCs.     

Different effects of verapamil on cytosolic Ca2+ and contraction in norepinephrine-stimulated vascular smooth muscle

Effects of verapamil on cytosolic Ca2+ levels ([Ca2+]cyt) and contraction in fura-2-loaded rat aorta were examined. Norepinephrine (NE) induced a greater contraction than KCl for a given increase in [Ca2+]cyt. Cumulative addition of verapamil decreased the NE-stimulated [Ca2+]cyt more strongly than contraction whereas verapamil decreased high K(+)-stimulated [Ca2+]cyt and contraction in parallel. In the presence of verapamil at a concentration needed to completely inhibit the high K(+)-induced increments, NE induced a transient increase, followed by a small sustained increase in [Ca2+]cyt which averaged 25% of that in the absence of verapamil. These changes were followed by a sustained contraction which averaged 60% of that in the absence of verapamil. In Ca2(+)-free solution, NE induced only a transient increase in [Ca2+]cyt whereas it induced a transient contraction, followed by a small sustained contraction. The second application of NE induced a small sustained contraction (10% of that in the presence of Ca2+) without increasing [Ca2+]cyt. These changes were not affected by verapamil. These results suggest that verapamil inhibits NE-induced increase in [Ca2+]cyt, but not the Ca2(+)-sensitization or Ca2(+)-independent contraction, and this may be the reason why the NE-induced contraction is less sensitive to verapamil than that induced by high K+.     

Bitter tasting compounds dilate airways by inhibiting airway smooth muscle calcium oscillations and calcium sensitivity

Background and Purpose

While selective, bitter tasting, TAS2R agonists can relax agonist-contracted airway smooth muscle (ASM), their mechanism of action is unclear. However, ASM contraction is regulated by Ca²⁺ signalling and Ca²⁺ sensitivity. We have therefore investigated how the TAS2R10 agonists chloroquine, quinine and denotonium regulate contractile agonist-induced Ca²⁺ signalling and sensitivity.

Experimental Approach

Airways in mouse lung slices were contracted with either methacholine (MCh) or 5HT and bronchodilation assessed using phase-contrast microscopy. Ca²⁺ signalling was measured with 2-photon fluorescence microscopy of ASM cells loaded with Oregon Green, a Ca²⁺-sensitive indicator (with or without caged-IP₃). Effects on Ca²⁺ sensitivity were assessed on lung slices treated with caffeine and ryanodine to permeabilize ASM cells to Ca²⁺.

Key Results

The TAS2R10 agonists dilated airways constricted by either MCh or 5HT, accompanied by inhibition of agonist-induced Ca²⁺ oscillations. However, in non-contracted airways, TAS2R10 agonists, at concentrations that maximally dilated constricted airways, did not evoke Ca²⁺ signals in ASM cells. Ca²⁺ increases mediated by the photolysis of caged-IP₃ were also attenuated by chloroquine, quinine and denotonium. In Ca²⁺-permeabilized ASM cells, the TAS2R10 agonists dilated MCh- and 5HT-constricted airways.

Conclusions and Implications

TAS2R10 agonists reversed bronchoconstriction by inhibiting agonist-induced Ca²⁺ oscillations while simultaneously reducing the Ca²⁺ sensitivity of ASM cells. Reduction of Ca²⁺ oscillations may be due to inhibition of Ca²⁺ release through IP₃ receptors. Further characterization of bronchodilatory TAS2R agonists may lead to the development of novel therapies for the treatment of bronchoconstrictive conditions.