Smooth Muscle Tone Research Articles

Novel imaging of the prostate reveals spontaneous gland contraction and excretory duct quiescence together with different drug effects.

Prostate carcinoma and benign prostate hyperplasia (BPH) with associated lower urinary tract symptoms (LUTS) are among the most prevalent and clinically relevant diseases in men. BPH is characterized by an enlargement of prostate tissue associated with increased tone of smooth muscle cells (SMCs) which surround the single glands composing the prostate. Secretions of the glands leave the prostate through local excretory ducts during the emission phase of ejaculation. Pharmacological treatment of BPH suggests different local drug targets based on reduction of prostate smooth muscle tone as the main effect and disturbed ejaculation as a common side effect. This highlights the need for detailed investigation of single prostate glands and ducts. We combined structural and functional imaging techniques-notably, clear lipid-exchanged, acrylamide-hybridized rigid imaging/immunostaining/ in situ hybridization-compatible tissue-hydrogel (CLARITY) and time-lapse imaging-and defined glands and ducts as distinct SMC compartments in human and rat prostate tissue. The single glands of the prostate (comprising the secretory part) are characterized by spontaneous contractions mediated by the surrounding SMCs, whereas the ducts (excretory part) are quiescent. In both SMC compartments, phosphodiesterase (PDE)-5 is expressed. PDE5 inhibitors have recently emerged as alternative treatment options for BPH. We directly visualized that the PDE5 inhibitors sildenafil and tadalafil act by reducing spontaneous contractility of the glands, thereby reducing the muscle tone of the organ. In contrast, the ductal (excretory) system and thus the prostate's contribution to ejaculation is unaffected by PDE5 inhibitors. Our differentiated imaging approach reveals new details about prostate function and local drug actions and thus may support clinical management of BPH.-Kügler, R., Mietens, A., Seidensticker, M., Tasch, S., Wagenlehner, F. M., Kaschtanow, A., Tjahjono, Y., Tomczyk, C. U., Beyer, D., Risbridger, G. P., Exintaris, B., Ellem, S. J., Middendorff, R. Novel imaging of the prostate reveals spontaneous gland contraction and excretory duct quiescence together with different drug effects.

Possible mechanisms of diverse spaceflight effects on endothelial function in different vascular beds

Possible mechanisms of diverse spaceflight effects on endothelial function in different vascular beds

The influence of pulmonary vascular pressures on lung diffusing capacity during incremental exercise in healthy aging.

Alveolar‐capillary surface area for pulmonary gas exchange falls with aging, causing a reduction in lung diffusing capacity for carbon monoxide (DLCO). However, during exercise additional factors may influence DLCO, including pulmonary blood flow and pulmonary vascular pressures. First, we sought to determine the age‐dependent effect of incremental exercise on pulmonary vascular pressures and DLCO. We also aimed to investigate the dependence of DLCO on pulmonary vascular pressures during exercise via sildenafil administration to reduce pulmonary smooth muscle tone. Nine younger (27 ± 4 years) and nine older (70 ± 3 years) healthy subjects performed seven 5‐min exercise stages at rest, 0 (unloaded), 10, 15, 30, 50, and 70% of peak workload before and after sildenafil. DLCO, cardiac output (Q), and pulmonary artery and wedge pressure (mPAP and mPCWP; subset of participants) were collected at each stage. mPAP was higher (P = 0.029) and DLCO was lower (P = 0.009) throughout exercise in older adults; however, the rate of rise in mPAP and DLCO with increasing Q was not different. A reduction in pulmonary smooth muscle tone via sildenafil administration reduced mPAP, mPCWP, and the transpulmonary gradient (TPG = mPAP–mPCWP) in younger and older subjects (P < 0.001). DLCO was reduced following the reduction in mPAP and TPG, regardless of age (P < 0.001). In conclusion, older adults successfully adapt to age‐dependent alterations in mPAP and DLCO. Furthermore, DLCO is dependent on pulmonary vascular pressures, likely to maintain adequate pulmonary capillary recruitment. The rise in pulmonary artery pressure with aging may be required to combat pulmonary vascular remodeling and maintain lung diffusing capacity, particularly during exercise.

The Control of Vascular Smooth Muscle Tone: Concepts Coming from Ca2+ and cAMP Signalling

The Control of Vascular Smooth Muscle Tone: Concepts Coming from Ca2+ and cAMP Signalling

A role for the Rho-kinase pathway in mediating smooth muscle tone of the porcine prostate and corpus cavernosum

A role for the Rho-kinase pathway in mediating smooth muscle tone of the porcine prostate and corpus cavernosum

KV channels and the regulation of vascular smooth muscle tone.

VSMCs in resistance arteries and arterioles express a diverse array of KV channels with members of the KV 1, KV 2 and KV 7 families being particularly important. Members of the KV channel family: (i) are highly expressed in VSMCs; (ii) are active at the resting membrane potential of VSMCs in vivo (-45 to -30 mV); (iii) contribute to the negative feedback regulation of VSMC membrane potential and myogenic tone; (iv) are activated by cAMP-related vasodilators, hydrogen sulfide and hydrogen peroxide; (v) are inhibited by increases in intracellular Ca2+ and vasoconstrictors that signal through Gq -coupled receptors; (vi) are involved in the proliferative phenotype of VSMCs; and (vii) are modulated by diseases such as hypertension, obesity, the metabolic syndrome and diabetes. Thus, KV channels participate in every aspect of the regulation of VSMC function in both health and disease.

TRAF6 regulates proliferation of stromal cells in the transition and peripheral zones of benign prostatic hyperplasia via Akt/mTOR signaling.

Increased prostatic smooth muscle tone and hyperplastic growth contribute to urethral obstruction and voiding symptoms in benign prostatic hyperplasia (BPH). It has been suggested that different proliferative potential of stromal cells between transition zone (TZ) and adjoining regions of the prostate plays a significant role in the development of BPH. However, the molecular mechanisms of this hyperplastic process remain unclear. We found tumor necrosis factor receptor-associated factor 6 (TRAF6) highly expressed in TZ stromal cells compared to peripheral zone (PZ) stromal cells by gene array analyzes. Therefore, we aim to study the potential mechanisms of stromal TRAF6 in promoting BPH progression. Stromal cells obtained from BPH-derived primary cultures. The TRAF6-siRNA vector were constructed and transfected into cultured human BPH primary TZ stromal cells, and TRAF6-overexpressing vector were constructed and transfected into cultured human BPH primary PZ stromal cells. Stromal cells were recombined with BPH-1 cells then subcutaneously inoculated into the kidney capsule of male nude mice. Cell proliferation was evaluated by CCK-8 assay. Multiple proteins in the Akt/mTOR pathway were assessed using western blot. TRAF6 levels were increased in TZ stroma compared with PZ stroma of BPH. The in vitro cell culture and in vivo cell recombination revealed that selective downregulation of TRAF6 in TZ stromal cells led to suppression of the proliferation, while upregulation of TRAF6 in PZ stromal cells enhanced the proliferation. We found that the Phosphorylation and Ubiquitination of Akt as well as the Phosphorylation of mTOR, P70S6K were decreased when TRAF6 was downregulated in primary cultured TZ stromal cells of BPH. TRAF6 can promote the proliferation of stromal cells of BPH via Akt/mTOR signaling. Our results may make stromal TRAF6 responsible for zonal characteristic of BPH and as a promising therapeutic strategy for BPH treatment.

Evidence for a Role of Vascular Endothelium in the Control of Arterial Wall Viscosity in Humans.

URL: https://eudract.ema.europa.eu. Unique identifier: RCB2007-A001-10-53.

Residual NO modulates contractile responses and membrane potential in isolated rat mesenteric arteries

Shear stress or vasocontriction causes endothelial nitric oxide (NO) release resulting in the regulation of vascular smooth muscle tone in small resistance arteries. Generation of NO is inhibited by nitric oxide synthase (NOS) inhibitors. In this study, we investigated the effect of residual NO, released even in the presence of NOS inhibitors, on the membrane depolarization and phenylephrine-induced contractions of smooth muscle. For this purpose, we used hydroxocobalamin (HC), an NO scavenger, in the presence of NOS inhibitiors, Nω-nitro- L-arginine (L-NA) or Nω-nitro-L-arginine methyl ester (L-NAME) in mesenteric arteries isolated from rats. Phenylephrine (0,01–10 μM), an α1-adrenoceptor agonist, led to depolarisation and concentration-dependent contraction in mesenteric arteries. The depolarisation and contractile responses were augmented by L-NA or L-NAME. Hydroxocobalamine (HC) or carboxy-PTIO (c-PTIO) also caused to further increase the membrane depolarization and contractions induced by phenylephrine in the presence of NOS inhibitors. Chemical removal of endothelium by saponin, tyrosin kinase inhibitor erbstatin A, but not calmodulin inhibitor calmidazolium inhibited the additional membrane depolarisation and contractile responses induced by L-NA or L-NAME and L-NA or L-NAME plus HC. These findings show that residual NO modulates the contractile responses in isolated rat mesenteric arteries by exerting a tonic inhibitor effect on the depolarization and vasoconstriction induced by phenylephrine.

The expression and functional activities of smooth muscle myosin and non-muscle myosin isoforms in rat prostate.

Benign prostatic hyperplasia (BPH) is mainly caused by increased prostatic smooth muscle (SM) tone and volume. SM myosin (SMM) and non‐muscle myosin (NMM) play important roles in mediating SM tone and cell proliferation, but these molecules have been less studied in the prostate. Rat prostate and cultured primary human prostate SM and epithelial cells were utilized. In vitro organ bath studies were performed to explore contractility of rat prostate. SMM isoforms, including SM myosin heavy chain (MHC) isoforms (SM1/2 and SM‐A/B) and myosin light chain 17 isoforms (LC 17a/b), and isoform ratios were determined via competitive RT‐PCR. SM MHC and NM MHC isoforms (NMMHC‐A, NMMHC‐B and NMMHC‐C) were further analysed via Western blotting and immunofluorescence microscopy. Prostatic SM generated significant force induced by phenylephrine with an intermediate tonicity between phasic bladder and tonic aorta type contractility. Correlating with this kind of intermediate tonicity, rat prostate mainly expressed LC 17a and SM1 but with relatively equal expression of SM‐A/SM‐B at the mRNA level. Meanwhile, isoforms of NMMHC‐A, B, C were also abundantly present in rat prostate with SMM present only in the stroma, while NMMHC‐A, B, C were present both in the stroma and endothelial. Additionally, the SMM selective inhibitor blebbistatin could potently relax phenylephrine pre‐contracted prostate SM. In conclusion, our novel data demonstrated the expression and functional activities of SMM and NMM isoforms in the rat prostate. It is suggested that the isoforms of SMM and NMM could play important roles in BPH development and bladder outlet obstruction.

Sex differences in the role of phospholipase A2 -dependent arachidonic acid pathway in the perivascular adipose tissue function in pigs.

The fat surrounding blood vessels (perivascular adipose tissue or PVAT) releases vasoactive compounds that regulate vascular smooth muscle tone. There are sex differences in the regulation of vascular tone, but, to date, no study has investigated whether there are sex differences in the regulation of blood vessel tone by PVAT. This study has identified that the cyclooxygenase products thromboxane and PGF2α are released from coronary artery PVAT from pigs. Thromboxane appears to mediate the PVAT-induced contraction in arteries from females, whereas PGF2α appears to mediate the contraction in arteries from males. These sex differences in the role of these prostanoids in the PVAT-induced contraction can be explained by a greater release of thromboxane from PVAT from female animals and greater sensitivity to PGF2α in the porcine coronary artery from males. Previous studies have demonstrated that perivascular adipose tissue (PVAT) causes vasoconstriction. In this present study, we determined the role of cyclooxygenase-derived prostanoids in this contractile response and determined whether there were any sex differences in the regulation of vascular tone by PVAT. Contractions in isolated segments of coronary arteries were determined using isolated tissue baths and isometric tension recording. Segments were initially cleaned of PVAT, which was then re-added to the tissue bath and changes in tone measured over 1h. Levels of PGF2α and thromboxane B2 (TXB2 ) were quantified by ELISA, and PGF2α (FP) and thromboxane A2 (TP) receptor expression determined by Western blotting. In arteries from both male and female pigs, re-addition of PVAT caused a contraction, which was partially inhibited by the cyclooxygenase inhibitors indomethacin and flurbiprofen. The FP receptor antagonist AL8810 attenuated the PVAT-induced contraction in arteries from males, whereas the TP receptor antagonist GR32191B inhibited the PVAT-induced contraction in arteries from females. Although there was no difference in PGF2α levels in PVAT between females and males, PGF2α produced a larger contraction in arteries from males, correlating with a higher FP receptor expression. In contrast, release of TXB2 from PVAT from females was greater than from males, but there was no difference in the contraction by the TXA2 agonist U46619, or TP receptor expression in arteries from different sexes. These findings demonstrate clear sex differences in PVAT function in which PGF2α and TXA2 antagonists can inhibit the PVAT-induced vasoconstriction in male and female PCAs, respectively.

Enhancement of S1P-induced contractile response in detrusor smooth muscle of rats having cystitis

Interstitial cystitis is a chronic disease characterized by lower abdominal pain and some nonspecific symptoms including an increase in urinary frequency and urgency. Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid that controls smooth muscle tone via G-protein coupled receptors (S1P1–3 receptors). S1P production is known to take place both in physiological states and some pathological situations, such as in overactive bladder syndrome. The intracellular mechanism of S1P-induced contractile response was investigated in β-escin permeabilized detrusor smooth muscle of rats having cyclophosphamide-induced cystitis. The bladder was isolated from rats and detrusor smooth muscle strips were permeabilized with β-escin. S1P (50µM)-induced contraction and calcium sensitization response were significantly increased in cystitis. S1P-induced augmented contractile response was inhibited by S1P2 receptor antagonist JTE-013 and S1P3 receptor antagonist suramin. S1P2 receptor protein expressions were increased in cystitis, where no change was observed in S1P3 expressions between control and cystitis groups. S1P-induced contraction was reduced by Rho kinase (ROCK) inhibitor Y-27632 and protein kinase C (PKC) inhibitor GF-109203X in both control and cystitis group. S1P-induced increased calcium sensitization response was decreased by ROCK inhibitor and PKC inhibitor in cystitis. Our findings provide the first evidence that interstitial cystitis triggers S1P-induced increase in intracellular calcium in permeabilized detrusor smooth muscle of female rats. Both S1P2 and S1P3 receptors are involved in S1P mediated enhanced contractile response. The augmentation in S1P-induced contraction in interstitial cystitis involves both PKC and ROCK pathways of calcium sensitization.

Age Related Differences in Responsiveness to Sildenafil and Tamsulosin are due to Myogenic Smooth Muscle Tone in the Human Prostate

Lower urinary tract symptoms (LUTS) due to Benign Prostatic Hyperplasia (BPH) are highly prevalent in older men, having a profound impact on patient quality of life. Current therapeutics for BPH/LUTS target neurogenic smooth muscle tone, but response is unpredictable and many patients fail to respond. Spontaneous myogenic tone is another component of smooth muscle contractility that is uncharacterized in human prostate. To better understand and improve the predictability of patient response, we defined myogenic contractility using human prostate specimens and examined the effect of existing therapeutics. We show that myogenic activity is present in the human prostate with the frequency of contractions in transition zone (TZ) specimens from BPH diagnosed patients approximately 160% greater than matched controls. α1-adrenoreceptor antagonists (Tamsulosin) and PDE5 inhibitors (Sildenafil) both significantly reduced myogenic contractile parameters, including frequency, with notable interpatient variability. Tamsulosin was more effective in older patients (R2 = 0.36, p < 0.01) and men with larger prostate volumes (R2 = 0.41, p < 0.05), while Sildenafil was more effective in younger men (R2 = 0.45, p < 0.05). As myogenic tone is significantly increased in BPH, therapeutics targeting this mechanism used with reference to patient characteristics could improve clinical outcomes and better predict patient response.

P6273CX3CR1 positive myeloid cells regulate vascular smooth muscle tone by inducing calcium oscillations via activation of IP3 receptors

P6273CX3CR1 positive myeloid cells regulate vascular smooth muscle tone by inducing calcium oscillations via activation of IP3 receptors

Interactions between glycopyrronium and indacaterol on cholinergic neurotransmission and contractile response in bovine trachealis

BackgroundMuscarinic-receptor antagonists and β-adrenoceptor agonists are used, alone or in combination, as first-line treatment for chronic obstructive pulmonary disease. Both drugs decrease airway smooth muscle tone by post-junctional mechanisms but they may have opposing effects on pre-junctional acetylcholine (ACh)-release.MethodsWe studied the effects of the muscarinic-receptor antagonist glycopyrronium (GLY), the β-adrenoceptor agonist indacaterol (IND) and their combination on electrically-induced ACh-release and contractile response in isolated bovine trachealis. Data were analyzed by paired t-test and analysis of variance for repeated or independent measures with Newmann-Keuls post-hoc test when appropriate.ResultsGLY 10−8 M decreased contractile response by 19 ± 6% (p = 0.010) without altering ACh-release. GLY 10−7 M and 10−6 M almost abolished contractile responses even if the ACh-release was increased by 27 ± 19% (p < 0.001) and 20 ± 8% (p = 0.004), respectively. IND 10−7 M had no significant effects on contractile response and ACh-release, whereas IND 10−6 M reduced contractile response by 24 ± 12% (p = 0.002) without altering ACh-release. IND 10−5 M decreased contractile response by 51 ± 17% (p < 0.001) and ACh-release by 22 ± 11% (p = 0.004). Co-incubation with GLY 10−8 M and IND 10−7 M did not alter ACh-release but inhibited contractile response by 41 ± 8% (p < 0.001). The latter effect was greater than with GLY 10−8 M, or IND 10−7 M, or IND 10−6 M given separately (p < 0.001 for all). The increment of ACh-release caused by GLY was attenuated by IND 10−5 M, though this did not affect contractile response.ConclusionsAt equimolar concentration, GLY alone attenuates airway smooth muscle contraction more than IND, despite an increased ACh-release. Combination of GLY with IND at submaximal concentrations has more than additive effect suggesting a synergistic post-junctional effect. Adding GLY to IND provides a greater inhibitory effect on airway smooth muscle contraction than increasing IND concentration.

Expression and function of phosphodiesterases (PDEs) in the rat urinary bladder

BackgroundIt has been shown that hosphodiesterases (PDEs) play an important role in mediating the smooth muscle tone of rat urinary bladder. However, the gene expression profiles of them were still unknown.MethodsUrinary bladder Strips were obtained from both neonatal and adult Sprague-Dawley rats. RT-PCR/western blot and organ bath were used to measure the expression and function of PDEs.ResultsAdult rat urinary bladder expressed various PDE mRNA with the following rank order: PDE5A ≈ PDE9A ≈ PDE10A > PDE2A ≈ PDE4A ≈ PDE4D > PDE4B ≈ PDE3B ≈ PDE8B ≈ PDE7A ≈ PDE7B > PDE1A. PDE1B, PDE1C, PDE3A, PDE4C, PDE8A, and PDE11A were not detected. Of interest, the mRNA and protein of PDE3A were significantly decreased in adult rat urinary bladder compared to neonatal rat urinary bladder. Cilostamide, a specific inhibitor for PDE3, significantly inhibited the amplitude and frequency of carbachol-enhanced phasic contractions of neonatal rat bladder strips by 38.8% and 12.1%, respectively. Compared to the neonatal rat bladder, the effect of cilostamide was significantly blunted in adult rat urinary bladder: the amplitude and frequency of carbachol-enhanced phasic contractions were decreased by 13.4% (P < 0.01 vs neonatal rat bladder) and 4.4%, respectively. However, the mRNA and the protein levels of PDE3B were similar between neonatal and adult rat bladder.ConclusionWe found that several PDE isoforms were expressed in the rat urinary bladder with distinct levels. Moreover, we showed that the function of PDE3 was blunted in adult rat bladder likely due to the decreased expression of PDE3A.

Role of differentially expressed microRNA-139-5p in the regulation of phenotypic internal anal sphincter smooth muscle tone

The present study focused on the role of microRNA-139-5p (miRNA-139-5p) in the regulation of basal tone in internal anal sphincter (IAS). Applying genome-wide miRNA microarrays on the phenotypically distinct smooth muscle cells (SMCs) within the rat anorectrum, we identified miRNA-139-5p as differentially expressed RNA repressor with highest expression in the purely phasic smooth muscle of anococcygeus (ASM) vs. the truly tonic smooth muscle of IAS. This pattern of miRNA-139-5p expression, previously shown to target ROCK2, was validated by target prediction using ingenuity pathway (IPA) and by qPCR analyses. Immunoblotting, immunocytochemistry (ICC), and functional assays using IAS tissues and cells subjected to overexpression/knockdown of miRNA-139-5p confirmed the inverse relationship between miRNA-139-5p and ROCK2 expressions/IAS tone. Overexpression of miRNA-139-5p caused a decrease, while knockdown by anti-miRNA-139-5p caused an increase in the IAS tone; these tissue contractile responses were confirmed by single-cell contraction using magnetic twisting cytometry (MTC). These findings suggest miRNA-139-5p is capable of significantly influencing the phenotypic tonicity in smooth muscle via ROCK2: a lack of tone in ASM may be associated with the suppression of ROCK2 by high expression of miRNA-139-5p, whereas basal IAS tone may be associated with the persistence of ROCK2 due to low expression of miRNA-139-5p.

The activity of cGMP-dependent protein kinase Iα is not directly regulated by oxidation-induced disulfide formation at cysteine 43

The type I cGMP-dependent protein kinases (PKGs) are key regulators of smooth muscle tone, cardiac hypertrophy, and other physiological processes. The two isoforms PKGIα and PKGIβ are thought to have unique functions because of their tissue-specific expression, different cGMP affinities, and isoform-specific protein-protein interactions. Recently, a non-canonical pathway of PKGIα activation has been proposed, in which PKGIα is activated in a cGMP-independent fashion via oxidation of Cys43, resulting in disulfide formation within the PKGIα N-terminal dimerization domain. A "redox-dead" knock-in mouse containing a C43S mutation exhibits phenotypes consistent with decreased PKGIα signaling, but the detailed mechanism of oxidation-induced PKGIα activation is unknown. Therefore, we examined oxidation-induced activation of PKGIα, and in contrast to previous findings, we observed that disulfide formation at Cys43 does not directly activate PKGIα in vitro or in intact cells. In transfected cells, phosphorylation of Ras homolog gene family member A (RhoA) and vasodilator-stimulated phosphoprotein was increased in response to 8-CPT-cGMP treatment, but not when disulfide formation in PKGIα was induced by H2O2 Using purified enzymes, we found that the Cys43 oxidation had no effect on basal kinase activity or Km and Vmax values; however, PKGIα containing the C43S mutation was less responsive to cGMP-induced activation. This reduction in cGMP affinity may in part explain the PKGIα loss-of-function phenotype of the C43S knock-in mouse. In conclusion, disulfide formation at Cys43 does not directly activate PKGIα, and the C43S-mutant PKGIα has a higher Ka for cGMP. Our results highlight that mutant enzymes should be carefully biochemically characterized before making in vivo inferences.

The impact of bilateral vagotomy on the physostigmine-induced airway constriction in ferrets

Vagal innervations have a great role in the respiratory function and are the main route of signal transmission from respiratory neural centers into the trachea and others conducting airways.We have investigated the role of central mechanisms related to vagal neural pathways and the cholinergic outflow in tracheobronchial smooth muscle tone and lung mechanics parameters.Parameters of lung mechanics such as lung resistance (RL), dynamic compliance (Cdyn) and pressure in bypassed tracheal segment (Ptseg) were measured before and after vagotomy and asphyxia test. Before vagotomy (BV), the control measurements were obtained and physostigmine was administered systemically, in increasing dose 10, 40 and 100μg/kg body weight (bw) with 15min interval between doses. After vagotomy (AV), administration of physostigmine with the same doses as BV has been done and the asphyxia challenge was conducted as per study protocol. The values of Ptseg and RL after physostigmine administration, BV vs. AV, respectively, at maximal dose of 100μg/kg bw were 32.5±3.3cm H2O, and 10.6±1.5cm H2O (p<0.0001); 0.16±0.04cm H2O/mL/s, and 0.067±0.006cm H2O/mL/s AV (P<0.05). The Cydn values were affected after physostigmine administration only at the lowest dose of 10μg/kg bw, and BV was 0.75±0.05mL/cm H2O vs. 0.53±0.04mL/cm H2O AV (P<0.004).Cholinergic outflow produced increases in tracheal tone, lung resistance and a decrease in dynamic compliance before, but not after vagotomy. Our results show the high impact of central neuronal mechanism in parameters of lung mechanics and respiration.This study indicates that vagal nerves have a crucial role, in the transmission of impulses initiated from central nervous system, in regulating the respiration by contraction or relaxation of airway smooth muscle tone.

Role of transient receptor potential vanilloid 1 in the modulation of airway smooth muscle tone and calcium handling

Asthma is a common disorder characterized, in part, by airway smooth muscle (ASM) hyperresponsiveness. Transient receptor potential vanilloid 1 (TRPV1) is a nonselective cation channel expressed on airway nerve fibers that modulates afferent signals, resulting in cough, and potentially bronchoconstriction. In the present study, the TRPV1 transcript was detected by RT-PCR in primary cultured human ASM cells, and the TRPV1 protein was detected in ASM of human trachea by immunohistochemistry. Proximity ligation assays suggest that TRPV1 is expressed in the sarcoplasmic reticulum membrane of human ASM cells in close association with sarco/endoplasmic reticulum Ca2+-ATPase-2. In guinea pig tracheal ring organ bath experiments, the TRPV1 agonist capsaicin led to ASM contraction, but this contraction was significantly attenuated by the sodium channel inhibitor bupivacaine (n = 4, P < 0.05) and the neurokinin-2 receptor antagonist GR-159897 (n = 4, P < 0.05), suggesting that this contraction is neutrally mediated. However, pretreatment of guinea pig and human ASM in organ bath experiments with the TRPV1 antagonist capsazepine inhibited the maintenance phase of an acetylcholine-induced contraction (n = 4, P < 0.01 for both species). Similarly, capsazepine inhibited methacholine-induced contraction of peripheral airways in mouse precision-cut lung slice (PCLS) experiments (n = 4-5, P < 0.05). Although capsazepine did not inhibit store-operated calcium entry in mouse ASM cells in PCLS (n = 4-7, P = nonsignificant), it did inhibit calcium oscillations (n = 3, P < 0.001). These studies suggest that TRPV1 is expressed on ASM, including the SR, but that ASM TRPV1 activation does not play a significant role in initiation of ASM contraction. However, capsazepine does inhibit maintenance of contraction, likely by inhibiting calcium oscillations.