Evidence of an excitatory purinergic innervation in mouse corpus cavernosum smooth muscle.

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.

Interstitial cell of Cajal-like cells (ICC-LC) exhibit dynamic spontaneous activity but are not functionally innervated in mouse urethra.

Urethral smooth muscle cells (USMC) contract to occlude the internal urethral sphincter during bladder filling. Interstitial cells also exist in urethral smooth muscles and are hypothesized to influence USMC behaviours and neural responses. These cells are similar to Kit interstitial cells of Cajal (ICC), which are gastrointestinal pacemakers and neuroeffectors.

Regulation of nerve-evoked contractions of the murine vas deferens.

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 α-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 α-adrenoceptors.

Modulation of fast sodium current in airway smooth muscle cells by exchange protein directly activated by cAMP.

Airway smooth muscle (ASM) cells from mouse bronchus express a fast sodium current mediated by Na1.7. We present evidence that this current is regulated by cAMP.

BK channel modulation by positively charged peptides and auxiliary γ subunits mediated by the Ca2+-bowl site.

The large-conductance, Ca2+-, and voltage-activated K+ (BK) channel consists of the pore-forming α (BKα) subunit and regulatory β and γ subunits. The γ1-3 subunits facilitate BK channel activation by shifting the voltage-dependence of channel activation toward the hyperpolarization direction by about 50-150 mV in the absence of Ca2+. We previously found that the intracellular C-terminal positively charged regions of the γ subunits play important roles in BK channel modulation.

Blockade of Kv7 channels reverses the inhibitory effects of exchange protein directly activated by cAMP activation on purinergic contractions of the murine detrusor.

Purinergic contractions of the detrusor are reduced by cAMP, but the underlying mechanisms are unclear. We examined the effects of BK and Kv7 channel modulators on purinergic contractions of the detrusor and tested if the inhibitory effects of activators of the cAMP effectors, PKA and EPAC, were reduced by blockade of BK or Kv7 channels. Purinergic contractions of the murine detrusor were induced by electric field stimulation (EFS) or application of the P2X receptor agonist α,β-MeATP.

The TMEM16A blockers benzbromarone and MONNA cause intracellular Ca-release in mouse bronchial smooth muscle cells.

We investigated effects of TMEM16A blockers benzbromarone, MONNA, CaCCA01 and Ani9 on isometric contractions in mouse bronchial rings and on intracellular calcium in isolated bronchial myocytes. Separate concentrations of carbachol (0.1-10 μM) were applied for 10 min periods to bronchial rings, producing concentration-dependent contractions that were well maintained throughout each application period.

Oxidation modulates LINGO2-induced inactivation of large conductance, Ca-activated potassium channels.

Ca and voltage-activated K (BK) channels are ubiquitous ion channels that can be modulated by accessory proteins, including β, 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 ∼-30 mV compared to that of BKα currents.

Ca -activated Cl channels (TMEM16A) underlie spontaneous electrical activity in isolated mouse corpus cavernosum smooth muscle cells.

Penile detumescence is maintained by tonic contraction of corpus cavernosum smooth muscle cells (CCSMC), but the underlying mechanisms have not been fully elucidated. The purpose of this study was to characterize the mechanisms underlying activation of TMEM16A Ca -activated Cl channels in freshly isolated murine CCSMC. Male C57BL/6 mice aged 10-18 weeks were euthanized via intraperitoneal injection of sodium pentobarbital (100 mg.

Functional expression of Na1.7 channels in freshly dispersed mouse bronchial smooth muscle cells.

Isolated smooth muscle cells (SMCs) from mouse bronchus were studied using the whole cell patch-clamp technique at ∼21°C. Stepping from -100 mV to -20 mV evoked inward currents of mean amplitude -275 pA. These inactivated (tau = 1.

Contribution of Postjunctional M2 Muscarinic Receptors to Cholinergic Nerve-Mediated Contractions of Murine Airway Smooth Muscle.

Postjunctional M2Rs on airway smooth muscle (ASM) outnumber M3Rs by a ratio of 4:1 in most species, however, it is the M3Rs that are thought to mediate the bronchoconstrictor effects of acetylcholine. In this study, we describe a novel and profound M2R-mediated hypersensitization of M3R-dependent contractions of ASM at low stimulus frequencies..

Modulation of carbachol-induced Ca oscillations in airway smooth muscle cells by PGE.

PGE is a potent bronchodilator, but the mechanisms underlying this effect have not been fully elucidated. Acetylcholine-induced contractions of airway smooth muscle (ASM) are associated with the generation of repetitive Ca oscillations in airway smooth muscle cells (ASMC) and the force of contraction is positively correlated with the frequency of the underlying Ca oscillations. The purpose of the present study was to examine if carbachol-evoked Ca oscillations in isolated ASMC were inhibited by PGE.

Fast voltage-dependent sodium (Na ) currents are functionally expressed in mouse corpus cavernosum smooth muscle cells.

Background And Purpose: Corpus cavernosum smooth muscle (CCSM) exhibits phasic contractions that are coordinated by ion channels. Mouse models are commonly used to study erectile dysfunction, but there are few published electrophysiological studies of mouse CCSM. We describe the voltage-dependent sodium (Na ) currents in mouse CCSM and investigate their function.

Regulation of P2X1 receptors by modulators of the cAMP effectors PKA and EPAC.

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.

Trypsin-Like Proteases and Their Role in Muco-Obstructive Lung Diseases.

Trypsin-like proteases (TLPs) belong to a family of serine enzymes with primary substrate specificities for the basic residues, lysine and arginine, in the P1 position. Whilst initially perceived as soluble enzymes that are extracellularly secreted, a number of novel TLPs that are anchored in the cell membrane have since been discovered. Muco-obstructive lung diseases (MucOLDs) are characterised by the accumulation of hyper-concentrated mucus in the small airways, leading to persistent inflammation, infection and dysregulated protease activity.

Calcium-Activated K Channels (K) and Therapeutic Implications.

Potassium channels are the most diverse and ubiquitous family of ion channels found in cells. The Ca 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 β, γ and LINGO subunits.

Role of Ano1 Ca-activated Cl channels in generating urethral tone.

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.

LINGO1 is a regulatory subunit of large conductance, Ca-activated potassium channels.

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 Ca-activated K (BK) channels also result in tremor and motor disorders.

Vasodilation of rat skeletal muscle arteries by the novel BK channel opener GoSlo is mediated by the simultaneous activation of BK and K 7 channels.

Background And Purpose: BK channels play important roles in various physiological and pathophysiological processes and thus have been the target of several drug development programmes focused on creating new efficacious BK channel openers, such as the GoSlo-SR compounds. However, the effect of GoSlo-SR compounds on vascular smooth muscle has not been studied. Therefore, we tested the hypothesis that GoSlo-SR compounds dilate arteries exclusively by activating BK channels.

Ion Channels and Intracellular Calcium Signalling in Corpus Cavernosum.

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.

Spontaneous Activity in Urethral Smooth Muscle.

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.

β-Adrenoceptor agonists inhibit purinergic receptor-mediated contractions of the murine detrusor.

β-Adrenoceptor (β-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 β-AR agonists on cholinergic versus purinergic receptor-mediated contractions of the detrusor and to examine the mechanisms underlying inhibition of the purinergic responses by β-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.