RESUMEN
Airway smooth muscle (ASM) cells from mouse bronchus express a fast sodium current mediated by NaV1.7. We present evidence that this current is regulated by cAMP. ASM cells were isolated by enzymatic dispersal and studied using the whole cell patch clamp technique at room temperature. A fast sodium current, INa, was observed on holding cells under voltage clamp at -100 mV and stepping to -20 mV. This current was reduced in a concentration-dependent manner by denopamine (10 and 30 µM), a ß-adrenergic agonist. Forskolin (1 µM), an activator of adenylate cyclase, reduced the current by 35%, but 6-MB-cAMP (300 µM), an activator of protein kinase A (PKA), had no effect. In contrast, 8-pCPT-2-O-Me-cAMP-AM (007-AM, 10 µM), an activator of exchange protein directly activated by cAMP (Epac), reduced the current by 48%. The inhibitory effect of 007-AM was still observed in the presence of dantrolene (10 µM), an inhibitor of ryanodine receptors, and when cytosolic [Ca2+] was buffered by inclusion of 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, Sigma (BAPTA) (50 µM) in the pipette solution, suggesting that the inhibition of INa was not due to Ca2+-release from intracellular stores. When 007-AM was tested on the current-voltage relationship, it reduced the current at potentials from -30 to 0 mV, but had no effect on the steady-state activation curve. However, the steady-state inactivation V1/2, the voltage causing inactivation of 50% of the current, was shifted in the negative direction from -76.6 mV to -89.7 mV. These findings suggest that cAMP regulates INa in mouse ASM via Epac, but not PKA.NEW & NOTEWORTHY ß-adrenergic agonists are commonly used in inhalers to treat asthma and chronic obstructive pulmonary disease. These work by causing bronchodilation and reducing inflammation. The present study provides evidence that these drugs have an additional action, namely, to reduce sodium influx into airway smooth muscle cells via fast voltage-dependent channels. This may have the dual effect of promoting bronchodilation and reducing remodeling of the airways, which has a detrimental effect in these diseases.
Asunto(s)
AMP Cíclico , Sodio , Ratones , Animales , Sodio/metabolismo , AMP Cíclico/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Miocitos del Músculo Liso/metabolismo , Agonistas Adrenérgicos betaRESUMEN
Ca2+ and voltage-activated K+ (BK) channels are ubiquitous ion channels that can be modulated by accessory proteins, including ß, γ, and LINGO1 BK subunits. In this study, we utilized a combination of site-directed mutagenesis, patch clamp electrophysiology, and molecular modeling to investigate if the biophysical properties of BK currents were affected by coexpression of LINGO2 and to examine how they are regulated by oxidation. We demonstrate that LINGO2 is a regulator of BK channels, since its coexpression with BK channels yields rapid inactivating currents, the activation of which is shifted â¼-30 mV compared to that of BKα currents. Furthermore, we show the oxidation of BK:LINGO2 currents (by exposure to epifluorescence illumination or chloramine-T) abolished inactivation. The effect of illumination depended on the presence of GFP, suggesting that it released free radicals which oxidized cysteine or methionine residues. In addition, the oxidation effects were resistant to treatment with the cysteine-specific reducing agent DTT, suggesting that methionine rather than cysteine residues may be involved. Our data with synthetic LINGO2 tail peptides further demonstrate that the rate of inactivation was slowed when residues M603 or M605 were oxidized, and practically abolished when both were oxidized. Taken together, these data demonstrate that both methionine residues in the LINGO2 tail mediate the effect of oxidation on BK:LINGO2 channels. Our molecular modeling suggests that methionine oxidation reduces the lipophilicity of the tail, thus preventing it from occluding the pore of the BK channel.
Asunto(s)
Cisteína , Canales de Potasio de Gran Conductancia Activados por el Calcio , Canales de Potasio de Gran Conductancia Activados por el Calcio/genética , Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Cisteína/metabolismo , Oxidación-Reducción , Péptidos/metabolismo , Metionina/metabolismo , Calcio/metabolismoRESUMEN
BACKGROUND: Evidence suggests that the corpus cavernosum smooth muscle (CCSM) cells of several species, including humans, express purinergic P2X receptors, but it is not known if the corpus cavernosum has an excitatory purinergic innervation. AIM: In this study we aimed to determine if the mouse CCSM has a functional purinergic innervation. METHODS: Mouse CCSM myocytes were enzymatically isolated and studied using the perforated patch configuration of the patch clamp technique. Isometric tension was measured in whole cavernosum tissue subjected to electrical field stimulation (EFS) to evoke nerve-mediated responses. OUTCOMES: The mouse CCSM myocytes expressed P2X1 receptors, and adenosine triphosphate (ATP) evoked inward currents in these cells. In addition, P2X1-mediated contractions were recorded in whole tissue in response to EFS. RESULTS: In cells held under a voltage clamp at -60 mV, ATP (1 µm) evoked large inward currents (mean approximately 900 pA). This current rapidly declined but was repeatable at 8-minute intervals. α,ß-methylene ATP (10 µM), an agonist of P2X1 and P2X3 receptors, caused a similar current that also rapidly declined. Desensitization to α,ß-methylene ATP negated the effect of ATP, but the ATP effect was restored 8 minutes after washout of α,ß-methylene ATP. The effect of ATP was reversibly blocked by NF449 (1 µm), a selective antagonist of P2X1 receptors. In isometric tension experiments electrical field stimulation (EFS) at 0.5-8 Hz evoked frequency-dependent contractions in the presence of l-nitro arginine (l-NO-Arg) (100 µm). When phentolamine (3 µm) and atropine (1 µm) were applied, there remained a nonadrenergic, noncholinergic component of the response to EFS, consisting mainly of a transient contraction. This was significantly reduced by NF449 (1 µm). Finally, in immunocytochemistry experiments, isolated CCSM myocytes stained positively when exposed to an antibody raised against P2X1 receptors. CLINICAL IMPLICATIONS: Previous studies have shown that P2X1 receptors in CCSM are upregulated in diabetes. These findings, taken together with the functional evidence presented here, indicate that P2X1 receptors may provide an alternative therapeutic target for treatment of erectile dysfunction in patients with diabetes, which is known to be relatively resistant to treatment with phosphodiesterase 5 inhibitors. STRENGTHS AND LIMITATIONS: Strengths of this study are the use of a combination of functional experiments (patch clamp) and immunocytochemical analyses to show expression of P2X1 receptors on CCSM myocytes while also performing functional experiments to show that stimulation these receptors results in contraction of CCSM. A limitation of this study was the use of animal rather than human tissue. CONCLUSION: This investigation provides evidence that mouse corpus cavernosum smooth muscle cells express P2X1 receptors and that these receptors are involved in mediating part of the contractile response to nerve stimulation evoked by EFS.
RESUMEN
Stimulation of sympathetic nerves in the vas deferens yields biphasic contractions consisting of a rapid transient component resulting from activation of P2X1 receptors by ATP and a secondary sustained component mediated by activation of α1-adrenoceptors by noradrenaline. Noradrenaline can also potentiate the ATP-dependent contractions of the vas deferens, but the mechanisms underlying this effect are unclear. The purpose of the present study was to investigate the mechanisms underlying potentiation of transient contractions of the vas deferens induced by activation of α1-adrenoceptors. Contractions of the mouse vas deferens were induced by electric field stimulation (EFS). Delivery of brief (1s duration) pulses (4 Hz) yielded transient contractions that were inhibited tetrodotoxin (100 nM) and guanethidine (10 µM). α,ß-meATP (10 µM), a P2X1R desensitising agent, reduced the amplitude of these responses by 65% and prazosin (100 nM), an α1-adrenoceptor antagonist, decreased mean contraction amplitude by 69%. Stimulation of α1-adrenoceptors with phenylephrine (3 µM) enhanced EFS and ATP-induced contractions and these effects were mimicked by the phorbol ester PDBu (1 µM), which activates PKC. The PKC inhibitor GF109203X (1 µM) prevented the stimulatory effects of PDBu on ATP-induced contractions of the vas deferens but only reduced the stimulatory effects of phenylephrine by 40%. PDBu increased the amplitude of ATP-induced currents recorded from freshly isolated vas deferens myocytes and HEK-293 cells expressing human P2X1Rs by 93%. This study indicates that: (1) potentiation of ATP-evoked contractions of the mouse vas deferens by α1-adrenoceptor activation were not fully blocked by the PKC inhibitor GF109203X and (2) that the stimulatory effect of PKC on ATP-induced contractions of the vas deferens is associated with enhanced P2X1R currents in vas deferens myocytes.
Asunto(s)
Contracción Muscular , Conducto Deferente , Conducto Deferente/efectos de los fármacos , Conducto Deferente/fisiología , Animales , Masculino , Ratones , Contracción Muscular/efectos de los fármacos , Contracción Muscular/fisiología , Receptores Adrenérgicos alfa 1/metabolismo , Estimulación Eléctrica , Receptores Purinérgicos P2X1/metabolismo , Adenosina Trifosfato/farmacología , Ratones Endogámicos C57BL , HumanosRESUMEN
P2X1 receptors are adenosine triphosphate (ATP)-gated cation channels that are functionally important for male fertility, bladder contraction, and platelet aggregation. The activity of P2X1 receptors is modulated by lipids and intracellular messengers such as cAMP, which can stimulate protein kinase A (PKA). Exchange protein activated by cAMP (EPAC) is another cAMP effector; however, its effect on P2X1 receptors has not yet been determined. Here, we demonstrate that P2X1 currents, recorded from human embryonic kidney (HEK) cells transiently transfected with P2X1 cDNA, were inhibited by the highly selective EPAC activator 007-AM. In contrast, EPAC activation enhanced P2X2 current amplitude. The PKA activator 6-MB-cAMP did not affect P2X1 currents, but inhibited P2X2 currents. The inhibitory effects of EPAC on P2X1 were prevented by triple mutation of residues 21 to 23 on the amino terminus of P2X1 subunits to the equivalent amino acids on P2X2 receptors. Double mutation of residues 21 and 22 and single mutation of residue 23 also protected P2X1 receptors from inhibition by EPAC activation. Finally, the inhibitory effects of EPAC on P2X1 were also prevented by NSC23766, an inhibitor of Rac1, a member of the Rho family of small GTPases. These data suggest that EPAC is an important regulator of P2X1 and P2X2 receptors.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/farmacología , AMP Cíclico/metabolismo , Regulación de la Expresión Génica , Factores de Intercambio de Guanina Nucleótido/farmacología , Riñón/metabolismo , Receptores Purinérgicos P2X1/metabolismo , Receptores Purinérgicos P2X2/metabolismo , Adenosina Trifosfato , Aminoquinolinas/farmacología , Factores de Intercambio de Guanina Nucleótido/antagonistas & inhibidores , Células HEK293 , Humanos , Riñón/efectos de los fármacos , Pirimidinas/farmacología , Receptores Purinérgicos P2X1/genética , Receptores Purinérgicos P2X2/genética , Proteína de Unión al GTP rac1/antagonistas & inhibidoresRESUMEN
LINGO1 is a transmembrane protein that is up-regulated in the cerebellum of patients with Parkinson's disease (PD) and Essential Tremor (ET). Patients with additional copies of the LINGO1 gene also present with tremor. Pharmacological or genetic ablation of large conductance Ca2+-activated K+ (BK) channels also result in tremor and motor disorders. We hypothesized that LINGO1 is a regulatory BK channel subunit. We show that 1) LINGO1 coimmunoprecipitated with BK channels in human brain, 2) coexpression of LINGO1 and BK channels resulted in rapidly inactivating BK currents, and 3) LINGO1 reduced the membrane surface expression of BK channels. These results suggest that LINGO1 is a regulator of BK channels, which causes a "functional knockdown" of these currents and may contribute to the tremor associated with increased LINGO1 levels.
Asunto(s)
Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Secuencia de Aminoácidos , Línea Celular , Cerebelo/metabolismo , Humanos , Canales de Potasio de Gran Conductancia Activados por el Calcio/genética , Proteínas de la Membrana/genética , Proteínas del Tejido Nervioso/genética , Unión ProteicaRESUMEN
Isolated smooth muscle cells (SMCs) from mouse bronchus were studied using the whole cell patch-clamp technique at â¼21°C. Stepping from -100 mV to -20 mV evoked inward currents of mean amplitude -275 pA. These inactivated (tau = 1.1 ms) and were abolished when external Na+ was substituted with N-Methyl-d-glucamine. In current-voltage protocols, current peaked at -10 mV and reversed between +20 and +30 mV. The V1/2s of activation and inactivation were -25 and -86 mV, respectively. The current was highly sensitive to tetrodotoxin (IC50 = 1.5 nM) and the NaV1.7 subtype-selective blocker, PF-05089771 (IC50 = 8.6 nM), consistent with NaV1.7 as the underlying pore-forming α subunit. Two NaV1.7-selective antibodies caused membrane-delineated staining of isolated SMC, as did a nonselective pan-NaV antibody. RT-PCR, performed on groups of â¼15 isolated SMCs, revealed transcripts for NaV1.7 in 7/8 samples. Veratridine (30 µM), a nonselective NaV channel activator, reduced peak current evoked by depolarization but induced a sustained current of 40 pA. Both effects were reversed by tetrodotoxin (100 nM). In tension experiments, veratridine (10 µM) induced contractions that were entirely blocked by atropine (1 µM). However, in the presence of atropine, veratridine was able to modulate the pattern of activity induced by a combination of U-46619 (a thromboxane A2 mimetic) and PGE2 (prostaglandin E2), by eliminating bursts in favor of sustained phasic contractions. These effects were readily reversed to control-like activity by tetrodotoxin (100 nM). In conclusion, mouse bronchial SMCs functionally express NaV1.7 channels that are capable of modulating contractile activity, at least under experimental conditions.
Asunto(s)
Bronquios , Miocitos del Músculo Liso , Animales , Derivados de Atropina/metabolismo , Derivados de Atropina/farmacología , Bronquios/metabolismo , Ratones , Miocitos del Músculo Liso/metabolismo , Sodio/metabolismo , Tetrodotoxina/metabolismo , Tetrodotoxina/farmacología , Veratridina/metabolismo , Veratridina/farmacologíaRESUMEN
Urinary continence is maintained in the lower urinary tract by the contracture of urethral sphincters, including smooth muscle of the internal urethral sphincter. These contractions occlude the urethral lumen, preventing urine leakage from the bladder to the exterior. Over the past 20 years, research on the ionic conductances that contribute to urethral smooth muscle contractility has greatly accelerated. A debate has emerged over the role of interstitial cell of Cajal (ICC)-like cells in the urethra and their expression of Ca2+-activated Cl- channels encoded by anoctamin-1 [Ano1; transmembrane member 16 A (Tmem16a) gene]. It has been proposed that Ano1 channels expressed in urethral ICC serve as a source of depolarization for smooth muscle cells, increasing their excitability and contributing to tone. Although a clear role for Ano1 channels expressed in ICC is evident in other smooth muscle organs, such as the gastrointestinal tract, the role of these channels in the urethra is unclear, owing to differences in the species (rabbit, rat, guinea pig, sheep, and mouse) examined and experimental approaches by different groups. The importance of clarifying this situation is evident as effective targeting of Ano1 channels may lead to new treatments for urinary incontinence. In this review, we summarize the key findings from different species on the role of ICC and Ano1 channels in urethral contractility. Finally, we outline proposals for clarifying this controversial and important topic by addressing how cell-specific optogenetic and inducible cell-specific genetic deletion strategies coupled with advances in Ano1 channel pharmacology may clarify this area in future studies.NEW & NOTEWORTHY Studies from the rabbit have shown that anoctamin-1 (Ano1) channels expressed in urethral interstitial cells of Cajal (ICC) serve as a source of depolarization for smooth muscle cells, increasing excitability and tone. However, the role of urethral Ano1 channels is unclear, owing to differences in the species examined and experimental approaches. We summarize findings from different species on the role of urethral ICC and Ano1 channels in urethral contractility and outline proposals for clarifying this topic using cell-specific optogenetic approaches.
Asunto(s)
Anoctamina-1/metabolismo , Calcio/metabolismo , Músculo Liso/metabolismo , Miocitos del Músculo Liso/metabolismo , Animales , Señalización del Calcio/fisiología , Humanos , Células Intersticiales de Cajal/metabolismoRESUMEN
Haemophilus influenzae is the most common cause of bacterial infection in the lungs of chronic obstructive pulmonary disease (COPD) patients and contributes to episodes of acute exacerbation which are associated with increased hospitalization and mortality. Due to the ability of H. influenzae to adhere to host epithelial cells, initial colonization of the lower airways can progress to a persistent infection and biofilm formation. This is characterized by changes in bacterial behaviour such as reduced cellular metabolism and the production of an obstructive extracellular matrix (ECM). Herein we discuss the multiple mechanisms by which H. influenzae contributes to the pathogenesis of COPD. In particular, mechanisms that facilitate bacterial adherence to host airway epithelial cells, biofilm formation, and microbial persistence through immune system evasion and antibiotic tolerance will be discussed.
Asunto(s)
Infecciones por Haemophilus/microbiología , Haemophilus influenzae/crecimiento & desarrollo , Enfermedad Pulmonar Obstructiva Crónica/microbiología , Animales , Adhesión Bacteriana , Haemophilus influenzae/genética , Haemophilus influenzae/aislamiento & purificación , Haemophilus influenzae/fisiología , Humanos , Pulmón/microbiologíaRESUMEN
Potassium channels are the most diverse and ubiquitous family of ion channels found in cells. The Ca2+ and voltage gated members form a subfamily that play a variety of roles in both excitable and non-excitable cells and are further classified on the basis of their single channel conductance to form the small conductance (SK), intermediate conductance (IK) and big conductance (BK) K+ channels.In this chapter, we will focus on the mechanisms underlying the gating of BK channels, whose function is modified in different tissues by different splice variants as well as the expanding array of regulatory accessory subunits including ß, γ and LINGO subunits. We will examine how BK channels are modified by these regulatory subunits and describe how the channel gating is altered by voltage and Ca2+ whilst setting this in context with the recently published structures of the BK channel. Finally, we will discuss how BK and other calcium-activated channels are modulated by novel ion channel modulators and describe some of the challenges associated with trying to develop compounds with sufficient efficacy, potency and selectivity to be of therapeutic benefit.
Asunto(s)
Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio , Canales de Potasio de Gran Conductancia Activados por el Calcio , Calcio/metabolismo , Cinética , Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismoRESUMEN
Chronic obstructive pulmonary disease (COPD) is a debilitating heterogeneous disease characterised by unregulated proteolytic destruction of lung tissue mediated via a protease-antiprotease imbalance. In COPD, the relationship between the neutrophil serine protease, neutrophil elastase, and its endogenous inhibitor, alpha-1-antitrypsin (AAT) is the best characterised. AAT belongs to a superfamily of serine protease inhibitors known as serpins. Advances in screening technologies have, however, resulted in many members of the serpin superfamily being identified as having differential expression across a multitude of chronic lung diseases compared to healthy individuals. Serpins exhibit a unique suicide-substrate mechanism of inhibition during which they undergo a dramatic conformational change to a more stable form. A limitation is that this also renders them susceptible to disease-causing mutations. Identification of the extent of their physiological/pathological role in the airways would allow further expansion of knowledge regarding the complexity of protease regulation in the lung and may provide wider opportunity for their use as therapeutics to aid the management of COPD and other chronic airways diseases.
Asunto(s)
Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Serina Proteasas/metabolismo , Serpinas/metabolismo , Animales , Modelos Animales de Enfermedad , Humanos , Enfermedad Pulmonar Obstructiva Crónica/patología , Enfermedad Pulmonar Obstructiva Crónica/fisiopatología , Serpinas/química , Serpinas/uso terapéuticoRESUMEN
ß3-Adrenoceptor (ß3-AR) agonists are used to treat overactive bladder syndrome; however, their mechanism of action has not been determined. The aims of this study were to compare the effects of ß3-AR agonists on cholinergic versus purinergic receptor-mediated contractions of the detrusor and to examine the mechanisms underlying inhibition of the purinergic responses by ß3-AR agonists. Isometric tension recordings were made from strips of murine detrusor and whole cell current recordings were made from freshly isolated detrusor myocytes using the patch-clamp technique. Transcriptional expression of exchange protein directly activated by cAMP (EPAC) subtypes in detrusor strips was assessed using RT-PCR and real-time quantitative PCR. The ß3-AR agonists BRL37344 and CL316243 (100 nM) inhibited cholinergic nerve-mediated contractions of the detrusor by 19 and 23%, respectively, but did not reduce contractions induced by the cholinergic agonist carbachol (300 nM). In contrast, BRL37344 and CL316243 inhibited purinergic nerve-mediated responses by 55 and 56%, respectively, and decreased the amplitude of contractions induced by the P2X receptor agonist α,ß-methylene ATP by 40 and 45%, respectively. The adenylate cyclase activator forskolin inhibited purinergic responses, and these effects were mimicked by a combination of the PKA activator N6-monobutyryl-cAMP and the EPAC activator 8-pCPT-2'-O-methyl-cAMP-AM (007-AM). Application of ATP (1 µM) evoked reproducible P2X currents in isolated detrusor myocytes voltage-clamped at -60 mV. These responses were reduced in amplitude in the presence of BRL37344 and also by 007-AM. This study demonstrates that ß3-AR agonists reduce postjunctional purinergic responses in the detrusor via a pathway involving activation of the cAMP effector EPAC.
Asunto(s)
Agonistas de Receptores Adrenérgicos beta 3/farmacología , Contracción Muscular/efectos de los fármacos , Agonistas del Receptor Purinérgico P2X/farmacología , Receptores Adrenérgicos beta 3/efectos de los fármacos , Receptores Purinérgicos P2X/efectos de los fármacos , Vejiga Urinaria/efectos de los fármacos , Urodinámica/efectos de los fármacos , Animales , Agonistas Colinérgicos/farmacología , AMP Cíclico/metabolismo , Femenino , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Masculino , Ratones Endogámicos C57BL , Receptores Adrenérgicos beta 3/metabolismo , Receptores Purinérgicos P2X/metabolismo , Transducción de Señal , Vejiga Urinaria/inervación , Vejiga Urinaria/metabolismoRESUMEN
The urethra is a muscular tube that extends from the bladder neck and is composed of an inner layer of smooth muscle referred to as the internal urethral sphincter and an outer layer of striated muscle which forms the external urethral sphincter. The smooth muscle layer can be separated into an inner layer of longitudinally orientated smooth muscle and an outer, relatively thinner, layer of circular muscle. Tonic contraction of both the smooth and striated muscle components of the urethra generates a urethral closure pressure which exceeds intravesical pressure in the bladder to maintain urinary continence. It is likely that contraction of urethral smooth muscle is involved in the long-term maintenance of tone, since it can achieve this at relatively low energy cost, whereas the striated muscle contributes more to the rise in urethral tone that accompanies increases in bladder pressure secondary to coughing or other sudden increases in intra-abdominal pressure. The level of urethral smooth muscle tone is regulated by several autonomic neurotransmitters, including noradrenaline, acetylcholine, ATP and nitric oxide. However, it is also clear that urethral smooth muscle is capable of generating significant tone in the absence of neural input. In this chapter we will discuss the mechanisms responsible for contraction of urethral smooth muscle, with specific focus on the role of ion channels and Ca2+ handling proteins to this process. The mechanisms underlying spontaneous activity in urethral interstitial cells (UICs), putative pacemaker cells of the urethra, will also be examined along with the modulation of these mechanisms by key excitatory and inhibitory neurotransmitters.
Asunto(s)
Contracción Muscular , Músculo Esquelético/fisiología , Músculo Liso/fisiología , Uretra/fisiología , Humanos , Células Intersticiales de Cajal/fisiología , Vejiga Urinaria/fisiologíaRESUMEN
The corpus cavernosum smooth muscle is important for both erection of the penis and for maintaining penile flaccidity. Most of the time, the smooth muscle cells are in a contracted state, which limits filling of the corpus sinuses with blood. Occasionally, however, they relax in a co-ordinated manner, allowing filling to occur. This results in an erection. When contractions of the corpus cavernosum are measured, it can be deduced that the muscle cells work together in a syncytium, for not only do they spontaneously contract in a co-ordinated manner, but they also synchronously relax. It is challenging to understand how they achieve this.In this review we will attempt to explain the activity of the corpus cavernosum, firstly by summarising current knowledge regarding the role of ion channels and how they influence tone, and secondly by presenting data on the intracellular Ca2+ signals that interact with the ion channels. We propose that spontaneous Ca2+ waves act as a primary event, driving transient depolarisation by activating Ca2+-activated Cl- channels. Depolarisation then facilitates Ca2+ influx via L-type voltage-dependent Ca2+ channels. We propose that the spontaneous Ca2+ oscillations depend on Ca2+ release from both ryanodine- and inositol trisphosphate (IP3)-sensitive stores and that modulation by signalling molecules is achieved mainly by interactions with the IP3-sensitive mechanism. This pacemaker mechanism is inhibited by nitric oxide (acting through cyclic GMP) and enhanced by noradrenaline. By understanding these mechanisms better, it might be possible to design new treatments for erectile dysfunction.
Asunto(s)
Señalización del Calcio , Canales Iónicos/fisiología , Músculo Liso/fisiología , Pene/fisiología , Calcio/fisiología , Humanos , Masculino , Erección PenianaRESUMEN
Airway smooth muscle expresses abundant BKCa channels, but their role in regulating contractions remains controversial. This study examines the effects of two potent BKCa channel openers on agonist-induced phasic contractions in rabbit and mouse bronchi. First, we demonstrated the ability of 10 µM GoSlo-SR5-130 to activate BKCa channels in inside-out patches from rabbit bronchial myocytes, where it shifted the activation V1/2 by -88 ± 11 mV (100 nM Ca2+, n = 7). In mouse airway smooth muscle cells, GoSlo-SR5-130 dose dependently shifted V1/2 by 12-83 mV over a concentration range of 1-30 µM. Compound X, a racemic mixture of two enantiomers, reported to be potent BKCa channel openers, shifted V1/2 by 20-79 mV over a concentration range of 0.3-3 µM. In rabbit bronchial rings, exposure to histamine (1 µM) induced phasic contractions after a delay of ~35 min. These were abolished by GoSlo-SR5-130 (30 µM). Nifedipine (100 nM) and CaCCinhA01 (10 µM), a TMEM16A blocker, also abolished histamine-induced phasic contractions. In mouse bronchi, similar phasic contractions were evoked by exposure to U46619 (100 nM) and carbachol (100 nM). In each case, these were inhibited by concentrations of GoSlo-SR5-130 and compound X that shifted the activation V1/2 of BKCa channels in the order of -80 mV. In conclusion, membrane potential-dependent regulation of L-type Ca2+ channels appears to be important for histamine-, U46619-, and carbachol-induced phasic contractions in airway smooth muscle. Contractions can be abolished by BKCa channel openers, suggesting that these channels are potential targets for treating some causes of airway obstruction.
Asunto(s)
Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/genética , Potenciales de la Membrana/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Animales , Antraquinonas/farmacología , Bronquios/efectos de los fármacos , Bronquios/crecimiento & desarrollo , Bronquios/metabolismo , Relación Dosis-Respuesta a Droga , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/agonistas , Potenciales de la Membrana/genética , Ratones , Contracción Muscular/efectos de los fármacos , Miocitos del Músculo Liso/efectos de los fármacos , Técnicas de Placa-Clamp , Conejos , Ácidos Sulfónicos/farmacologíaRESUMEN
KEY POINTS: Contraction of urethral smooth muscle cells (USMCs) contributes to urinary continence. Ca2+ signalling in USMCs was investigated in intact urethral muscles using a genetically encoded Ca2+ sensor, GCaMP3, expressed selectively in USMCs. USMCs were spontaneously active in situ, firing intracellular Ca2+ waves that were asynchronous at different sites within cells and between adjacent cells. Spontaneous Ca2+ waves in USMCs were myogenic but enhanced by adrenergic or purinergic agonists and decreased by nitric oxide. Ca2+ waves arose from inositol trisphosphate type 1 receptors and ryanodine receptors, and Ca2+ influx by store-operated calcium entry was required to maintain Ca2+ release events. Ca2+ release and development of Ca2+ waves appear to be the primary source of Ca2+ for excitation-contraction coupling in the mouse urethra, and no evidence was found that voltage-dependent Ca2+ entry via L-type or T-type channels was required for responses to α adrenergic responses. ABSTRACT: Urethral smooth muscle cells (USMCs) generate myogenic tone and contribute to urinary continence. Currently, little is known about Ca2+ signalling in USMCs in situ, and therefore little is known about the source(s) of Ca2+ required for excitation-contraction coupling. We characterized Ca2+ signalling in USMCs within intact urethral muscles using a genetically encoded Ca2+ sensor, GCaMP3, expressed selectively in USMCs. USMCs fired spontaneous intracellular Ca2+ waves that did not propagate cell-to-cell across muscle bundles. Ca2+ waves increased dramatically in response to the α1 adrenoceptor agonist phenylephrine (10 µm) and to ATP (10 µm). Ca2+ waves were inhibited by the nitric oxide donor DEA NONOate (10 µm). Ca2+ influx and release from sarcoplasmic reticulum stores contributed to Ca2+ waves, as Ca2+ free bathing solution and blocking the sarcoplasmic Ca2+ -ATPase abolished activity. Intracellular Ca2+ release involved cooperation between ryanadine receptors and inositol trisphosphate receptors, as tetracaine and ryanodine (100 µm) and xestospongin C (1 µm) reduced Ca2+ waves. Ca2+ waves were insensitive to L-type Ca2+ channel modulators nifedipine (1 µm), nicardipine (1 µm), isradipine (1 µm) and FPL 64176 (1 µm), and were unaffected by the T-type Ca2+ channel antagonists NNC-550396 (1 µm) and TTA-A2 (1 µm). Ca2+ waves were reduced by the store operated Ca2+ entry blocker SKF 96365 (10 µm) and by an Orai antagonist, GSK-7975A (1 µm). The latter also reduced urethral contractions induced by phenylephrine, suggesting that Orai can function effectively as a receptor-operated channel. In conclusion, Ca2+ waves in mouse USMCs are a source of Ca2+ for excitation-contraction coupling in urethral muscles.
Asunto(s)
Canales de Calcio/metabolismo , Señalización del Calcio , Miocitos del Músculo Liso/metabolismo , Uretra/metabolismo , Agonistas Adrenérgicos/farmacología , Animales , Células Cultivadas , Acoplamiento Excitación-Contracción , Masculino , Ratones , Ratones Endogámicos C57BL , Contracción Muscular , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/fisiología , Óxido Nítrico/farmacología , Agonistas Purinérgicos/farmacología , Uretra/citología , Uretra/fisiologíaRESUMEN
OBJECTIVE: To test if carbachol (CCh)-evoked Ca2+ oscillations in freshly isolated murine detrusor myocytes are affected by ß3-adrenoceptor (ß-AR) modulators. MATERIALS AND METHODS: Isometric tension recordings were made from strips of murine detrusor, and intracellular Ca2+ measurements were made from isolated detrusor myocytes using confocal microscopy. Transcriptional expression of ß-AR sub-types in detrusor strips and isolated detrusor myocytes was assessed using reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time quantitative PCR (qPCR). Immunocytochemistry experiments, using a ß3-AR selective antibody, were performed to confirm that ß3-ARs were present on detrusor myocytes. RESULTS: The RT-PCR and qPCR experiments showed that ß1-, ß2- and ß3-AR were expressed in murine detrusor, but that ß3-ARs were the most abundant sub-type. The selective ß3-AR agonist BRL37344 reduced the amplitude of CCh-induced contractions of detrusor smooth muscle. These responses were unaffected by addition of the BK channel blocker iberiotoxin. BRL37344 also reduced the amplitude of CCh-induced Ca2+ oscillations in freshly isolated murine detrusor myocytes. This effect was mimicked by CL316,243, another ß3-AR agonist, and inhibited by the ß3-AR antagonist L748,337, but not by propranolol, an antagonist of ß1- and ß2-ARs. BRL37344 did not affect caffeine-evoked Ca2+ transients or L-type Ca2+ current in isolated detrusor myocytes. CONCLUSION: Inhibition of cholinergic-mediated contractions of the detrusor by ß3-AR agonists was associated with a reduction in Ca2+ oscillations in detrusor myocytes.
Asunto(s)
Agonistas de Receptores Adrenérgicos beta 3/farmacología , Carbacol/farmacología , Agonistas Colinérgicos/farmacología , Células Musculares/efectos de los fármacos , Agonistas Adrenérgicos beta/farmacología , Animales , Calcio/metabolismo , Etanolaminas/farmacología , Femenino , Masculino , Ratones Endogámicos C57BL , Contracción Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Péptidos/farmacología , Vejiga Urinaria/efectos de los fármacosRESUMEN
GoSlo-SR-5-6 is a novel large-conductance Ca(2+)-activated K(+) (BK) channel agonist that shifts the activation V1/2 of these channels in excess of -100 mV when applied at a concentration of 10 µM. Although the structure-activity relationship of this family of molecules has been established, little is known about how they open BK channels. To help address this, we used a combination of electrophysiology, mutagenesis, and mathematical modeling to investigate the molecular mechanisms underlying the effect of GoSlo-SR-5-6. Our data demonstrate that the effects of this agonist are practically abolished when three point mutations are made: L227A in the S4/S5 linker in combination with S317R and I326A in the S6C region. Our data suggest that GoSlo-SR-5-6 interacts with the transmembrane domain of the channel to enhance pore opening. The Horrigan-Aldrich model suggests that GoSlo-SR-5-6 works by stabilizing the open conformation of the channel and the activated state of the voltage sensors, yet decouples the voltage sensors from the pore gate.
Asunto(s)
Canales de Potasio de Gran Conductancia Activados por el Calcio/agonistas , Canales de Potasio de Gran Conductancia Activados por el Calcio/genética , Canales de Potasio de Gran Conductancia Activados por el Calcio/fisiología , MutagénesisRESUMEN
Rabbit corpus cavernosum smooth muscle (RCCSM) cells express ion channels that produce Ca2+-activated Cl- (IClCa) current, but low sensitivity to conventional antagonists has made its role in tone generation difficult to evaluate. We have reexamined this question using two new generation IClCa blockers, T16Ainh-A01 and CaCCinh-A01. Isolated RCCSM cells were studied using the perforated patch method. Current-voltage protocols revealed that both L-type Ca2+ current and IClCa T16Ainh-A01 and CaCCinh-A01 (10 µM) reduced IClCa by ~85%, while 30 µM abolished it. L-type Ca2+ current was unaffected by 10 µM CaCCinh-A01 but was reduced by 50% at 30 µM CaCCinh-A01, 46% at 10 µM T16Ainh-A01, and 78% at 30 µM T16Ainh-A01. Both drugs reduced spontaneous isometric tension in RCCSM strips, by 60-70% at 10 µM and >90% at 30 µM. Phenylephrine (PE)-enhanced tension was also reduced (ED50 = 3 µM, CaCCinh-A01; 14 µM, T16Ainh-A01). CaCCinh-A01 at 10 µM had little effect on 60 mM KCl contractures, though they were reduced by 30 µM CaCCinh-A01 and T16Ainh-A01 (10 µM and 30 µM) consistent with their effects on L-type Ca2+ current. Both drugs also reversed the stimulatory effect of PE on intracellular Ca2+ waves, studied with laser scanning confocal microscopy in isolated RCCSM cells. In conclusion, although both drugs were effective blockers of IClCa, the effect of T16Ainh-A01 on L-type Ca2+ current precludes its use for evaluating the role of IClCa in tone generation. However, 10 µM CaCCinh-A01 selectively blocked IClCa versus L-type Ca2+ current and reduced spontaneous and PE-induced tone, suggesting that IClCa is important in maintaining penile detumescence.