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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 1 -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 1 -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,β-meATP (10 µM), a P2X1R desensitising agent, reduced the amplitude of these responses by 65% and prazosin (100 nM), an α 1 -adrenoceptor antagonist, decreased mean contraction amplitude by 69%. Stimulation of α 1 -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 α 1 -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., (© 2024. The Author(s).)

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. EFS responses were inhibited by the L-type Ca 2+ channel blocker nifedipine, but not by the SERCA inhibitor CPA or the SOCE blocker GSK7975A. The Kv7 channel opener retigabine and BK channel activator compound X inhibited purinergic responses, while blockade of Kv7 or BK channels with XE991 or iberiotoxin, respectively, augmented these responses. Application of the EPAC activator 007-AM or PKA activator 6-MB-cAMP inhibited EFS responses. These effects were unaffected by iberiotoxin; however, XE991 reduced the effects of 007-AM, but not 6-MB-cAMP. Kv7.5 was the only Kv7 transcript detected in isolated detrusor myocytes. These data suggest that purinergic contractions of the detrusor are regulated by BK and Kv7 channels and the latter may also play a role in EPAC-dependent inhibition of this activity., (© 2023 The Authors. Basic & Clinical Pharmacology & Toxicology published by John Wiley & Sons Ltd on behalf of Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).)

Beta-adrenoceptor (β-AR) agonists inhibit cholinergic contractions of airway smooth muscle (ASM), but the underlying mechanisms are unclear. ASM cells express M3 and M2 muscarinic receptors, but the bronchoconstrictor effects of acetylcholine are believed to result from activation of M3Rs, while the role of the M2Rs is confined to offsetting β-AR-dependent relaxations. However, a profound M2R-mediated hypersensitization of M3R-dependent contractions of ASM was recently reported, indicating an important role for M2Rs in cholinergic contractions of ASM. Here, we investigated if M2R-dependent contractions of murine bronchial rings were inhibited by activation of β-ARs. M2R-dependent contractions were apparent at low frequency (2Hz) electric field stimulation (EFS) and short (10s) stimulus  intervals. The β1-AR agonist, denopamine inhibited EFS-evoked contractions of ASM induced by reduction in stimulus interval from 100 to 10 s and was more effective at inhibiting contractions evoked by EFS at 2 than 20 Hz. Denopamine also abolished carbachol-evoked contractions that were resistant to the M3R antagonist 4-DAMP, similar to the effects of the M2R antagonists, methoctramine and AFDX-116. The inhibitory effects of denopamine on EFS-evoked contractions of ASM were smaller in preparations taken from M2R -/- mice, compared to wild-type (WT) controls. In contrast, inhibitory effects of the β3-AR agonist, BRL37344, on EFS-evoked contractions of detrusor strips taken from M2R -/- mice were greater than WT controls. These data suggest that M2R-dependent contractions of ASM were inhibited by activation of β1-ARs and that genetic ablation of M2Rs decreased the efficacy of β-AR agonists on cholinergic contractions., (© The Author(s) 2022. Published by Oxford University Press on behalf of American Physiological Society.)

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.. Contractions induced by 2Hz EFS were augmented by > 2.5-fold when the stimulus interval was reduced from 100 to 10 s. This effect was reversed by the M2R antagonists, methoctramine, and AFDX116, and was absent in M2R null mice. The M3R antagonist 4-DAMP abolished the entire response in both WT and M2R KO mice. The M2R-mediated potentiation of EFS-induced contractions was not observed when the stimulus frequency was increased to 20 Hz. A subthreshold concentration of carbachol enhanced the amplitude of EFS-evoked contractions in WT, but not M2R null mice. These data highlight a significant M2R-mediated potentiation of M3R-dependent contractions of ASM at low frequency stimulation that could be relevant in diseases such as asthma and COPD., (© The Author(s) 2021. Published by Oxford University Press on behalf of American Physiological Society.)

β 3 -Adrenoceptor (β 3 -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 3 -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 3 -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 β 3 -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,β-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 N 6 -monobutyryl-cAMP and the EPAC activator 8-pCPT-2'- O -AR agonists reduce postjunctional purinergic responses in the detrusor via a pathway involving activation of the cAMP effector EPAC.3 -AR agonists reduce postjunctional purinergic responses in the detrusor via a pathway involving activation of the cAMP effector EPAC.

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 Ca 2+ 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.

Acetylcholine contracts the bladder by binding to muscarinic M3 receptors on the detrusor, leading to Ca 2+ influx via voltage-gated Ca 2+ channels. The cellular mechanisms linking these events are poorly understood, but studies have suggested that activation of TRPC4 channels could be involved. The purpose of this study was to investigate if spontaneous and cholinergic-mediated contractions of the detrusor were impaired in TRPC4 deficient (TRPC4 -/- ) mice. Isometric tension recordings were made from strips of wild-type (WT) and TRPC4 -/- detrusor. Spontaneous phasic detrusor contractions were significantly smaller in TRPC4 -/- mice compared to wild-type, however no difference in response to exogenous application of 60 mM KCl was observed. Cholinergic responses, induced by electric-field stimulation (EFS), bath application of the cholinergic agonist carbachol, or the acetylcholinesterase inhibitor neostigmine were all significantly smaller in TRPC4 -/- detrusor strips than wild-type. Surprisingly, the TRPC4/5 inhibitor ML204 reduced EFS and CCh-evoked contractions in TRPC4 -/- detrusor strips. However, TRPC5 expression was up-regulated in these preparations and, in contrast to wild-type, EFS responses were reduced in amplitude by the TRPC5 channel inhibitor clemizole hydrochloride. This study demonstrates that TRPC4 channels are involved in spontaneous and cholinergic-mediated contractions of the murine detrusor. TRPC5 expression is up-regulated in TRPC4 -/- detrusor strips, and may partially compensate for loss of TRPC4 channels.

Purpose: Muscarinic receptor mediated contractions of the detrusor rely on Ca 2+ influx through voltage-gated Ca 2+ channels but to our knowledge the mechanism linking stimulation of M3Rs to the activation of voltage dependent Ca 2+ channels has not been established. TRPC4 channels are receptor operated cation channels that couple muscarinic receptor activation to depolarization of intestinal smooth muscle cells, voltage-activated Ca 2+ influx and contraction. We investigated whether TRPC4 channels are involved in cholinergic mediated contractions of the detrusor., Materials and Methods: Isometric tension recordings were made on strips of murine detrusor and intracellular Ca 2+ measurements were made on isolated detrusor myocytes using confocal microscopy. Transcriptional expression of TRPC and IP 3 R subtypes in intact detrusor strips and isolated detrusor myocytes was assessed using reverse transcriptase-polymerase chain reaction., Results: Cholinergic stimulation of the detrusor induced by electrical field stimulation or exogenous application of carbachol or neostigmine evoked contractions consisting of a transient plus a tonic response, which was blocked by ML204, an inhibitor of TRPC4 channels. A phasic oscillatory component was blocked by the IP 3 R inhibitor 2-APB. Carbachol evoked reproducible Ca 2+ responses in isolated detrusor myocytes, consisting of an initial Ca 2+ transient followed by Ca 2+ oscillations. ML204 inhibited the initial Ca 2+ transient whereas 2-APB inhibited the Ca 2+ oscillations. Reverse transcriptase-polymerase chain reaction experiments showed that TRPC4β, TRPC6 and IP 3 R1 were selectively expressed in isolated detrusor myocytes. Control experiments demonstrated that ML204 did not affect L-type Ca 2+ or BK current amplitude, caffeine induced Ca 2+ transients or KCl induced contractions of the detrusor., Conclusions: Muscarinic receptor mediated contractions of the detrusor involve the activation of TRPC4β channels., (Copyright © 2016 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Purpose: Adenosine triphosphate is capable of relaxing and contracting urethral smooth muscle. The mechanisms responsible for the relaxing effects of adenosine triphosphate have been well studied but those involved in the contractile response are still unclear. We investigated the contributions of interstitial cells of Cajal and smooth muscle cells to nerve mediated, adenosine triphosphate dependent contractions of urethral smooth muscle., Materials and Methods: Tension recordings were made from strips of rabbit urethral smooth muscle. Recordings were made of membrane potential and ionic currents from freshly isolated smooth muscle cells and interstitial cells of Cajal using the patch clamp technique., Results: Stimulating intramural nerves in urethral smooth muscle yielded contractions that were inhibited by the broad spectrum P2 receptor inhibitor pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate and the P2X receptor agonist α,β-methylene adenosine triphosphate but not by the P2Y receptor antagonist MRS2500. When studied under voltage clamp at a holding potential of -60 mV, interstitial cells of Cajal showed spontaneous transient inward currents that were increased in frequency by adenosine triphosphate but not by α,β-methylene adenosine triphosphate. In contrast, smooth muscle cells were quiescent but responded to adenosine triphosphate and α,β-methylene adenosine triphosphate by producing a single transient inward current. Currents evoked by adenosine triphosphate in smooth muscle cells were inhibited by α,β-methylene adenosine triphosphate, pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate and suramin, and by a decrease in extracellular Na+ from 130 to 13 mM., Conclusions: Stimulating purinergic nerves in rabbit urethral smooth muscle induces contractions via the activation of P2X receptors on smooth muscle cells., (Copyright © 2011 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Purpose: Adenosine triphosphate is thought to be an important neurotransmitter in urethral smooth muscle but its physiological role is still unclear. We characterized the effects of adenosine triphosphate on contractile and pacemaker activity in rabbit urethral smooth muscle., Materials and Methods: Tension recordings were made from strips of rabbit proximal urethral smooth muscle. Membrane currents from freshly isolated smooth muscle cells and interstitial cells of Cajal were recorded using the patch clamp technique. Intracellular Ca(2+) was measured using confocal microscopy., Results: Exogenous application of adenosine triphosphate (10 microM) evoked robust contractions that were inhibited by the type 2 purinergic receptor blocker suramin (100 microM) and the selective type 2 purinergic Y1 receptor antagonist MRS2500 (Tocris Bioscience, Ellisville, Missouri) (100 nM). Application of the type 2 purinergic Y receptor agonist 2-MeSADP (1 microM) mimicked the effects of adenosine triphosphate. When smooth muscle cells were studied under voltage clamp at -60 mV, adenosine triphosphate evoked a large single inward current (greater than 1.2 nA) but 2-MeSADP produced a small current (about 16 pA). In contrast, when interstitial cells of Cajal were held at -60 mV, they showed spontaneous transient inward currents that were increased in frequency by adenosine triphosphate and 2-MeSADP. These excitatory effects were inhibited by suramin and MRS2500. Interstitial cells of Cajal showed spontaneous Ca(2+) waves that were increased in frequency by adenosine triphosphate and 2-MeSADP. These effects were also inhibited by suramin and MRS2500., Conclusions: Contractile effects of adenosine triphosphate in urethral smooth muscle are mediated by the activation of type 2 purinergic Y receptors on interstitial cells of Cajal., (Copyright 2010 American Urological Association. Published by Elsevier Inc. All rights reserved.)

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 μ m) and to ATP (10 μ m). Ca2+ waves were inhibited by the nitric oxide donor DEA NONOate (10 μ m). 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 μ m) and xestospongin C (1 μ m) reduced Ca2+ waves. Ca2+ waves were insensitive to L‐type Ca2+ channel modulators nifedipine (1 μ m), nicardipine (1 μ m), isradipine (1 μ m) and FPL 64176 (1 μ m), and were unaffected by the T‐type Ca2+ channel antagonists NNC‐550396 (1 μ m) and TTA‐A2 (1 μ m). Ca2+ waves were reduced by the store operated Ca2+ entry blocker SKF 96365 (10 μ m) and by an Orai antagonist, GSK‐7975A (1 μ m). 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. [ABSTRACT FROM AUTHOR]

The involvement of Rho-associated kinase (ROK) in activation of rabbit urethral smooth muscle contraction was investigated by examining the effects of two structurally distinct inhibitors of ROK, Y27632 and H1152, on the contractile response to electric field stimulation, membrane depolarization with KCl, and a1-adrenoceptor stimulation with phenylephrine. Both compounds inhibited contractions elicited by all three stimuli. The protein kinase C inhibitor GF109203X, on the other hand, had no effect. Urethral smooth muscle strips were analyzed for phosphorylation of three potential direct or indirect substrates of ROK: 1) myosin regulatory light chains (LC20) at S19, 2) the myosin-targeting subunit of myosin light chain phosphatase (MYPT1) at T697 and T855, and 3) cofilin at S3. The following results were obtained: 1) under resting tension, LC20 was phosphorylated to 0.65 ± 0.02 mol Pi/mol LC20 (n = 21) at S19; 2) LC20 phosphorylation did not change in response to KCl or phenylephrine; 3) ROK inhibition had no effect on LC20 phosphorylation in the absence or presence of contractile stimuli; 4) under resting conditions, MYPT1 was partially phosphorylated at T697 and T855 and cofilin at S3; 5) phosphorylation of MYPT1 and cofilin was unaffected by KCl or phenylephrine; and 6) KCl- and phenylephrine-induced contraction-relaxation cycles did not correlate with actin polymerization-depolymerization. We conclude that ROK plays an important role in urethral smooth muscle contraction, but not via inhibition of MLCP or polymerization of actin. [ABSTRACT FROM AUTHOR]

Rhythmic electrical activity is a feature of most smooth muscles but the mechanical consequences can vary from regular rapid phasic contractions to sustained contracture. For many years it was thought that spontaneous electrical activity originated in smooth muscle cells but recently it has become apparent that there are specialized pacemaker cells in many organs that are morphologically and functionally distinct from smooth muscle and that the former cells are the source of spontaneous electrical activity. Such a pacemaker function is well documented for the ICC of the gastrointestinal tract but evidence is accumulating that ICC-like cells play a similar role in other types of smooth muscle. We have recently shown that there are specialized pacemaking cells in the rabbit urethra which are spontaneously active when freshly isolated, readily distinguishable from smooth muscle cells under bright field illumination and relatively easy to study using patch-clamp and confocal imaging techniques. Recent results suggest that calcium oscillations in isolated rabbit urethral interstitial cells are initiated by calcium release from ryanodine sensitive intracellular stores, that oscillation frequency is very sensitive to the external calcium concentration and that conversion of the primary oscillation to a propagated calcium wave depends upon IP3-induced calcium release. [ABSTRACT FROM AUTHOR]

We investigated effects of TMEM16A blockers benzbromarone, MONNA, CaCC inh A01 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. Benzbromarone (1 μM) markedly reduced the contractions with a more pronounced effect on their sustained component (at 10 min) compared to their initial component (at 2 min). Iberiotoxin (0.3 μM) enhanced the contractions, but they were still blocked by benzbromarone. MONNA (3 μM) and CaCC inh A01 (10 μM) had similar effects to benzbromarone, but were less potent. In contrast, Ani9 (10 μM) had no effect on carbachol-induced contractions. Confocal imaging revealed that benzbromarone (0.3 μM), MONNA (1 μM) and CaCC inh A01 (10 μM) increased intracellular calcium in isolated myocytes loaded with Fluo-4AM. In contrast, Ani9 (10 μM) had no effect on intracellular calcium. Benzbromarone and MONNA also increased calcium in calcium-free extracellular solution, but failed to do so when intracellular stores were discharged with caffeine (10 mM). Caffeine was unable to cause further discharge of the store when applied in the presence of benzbromarone. Ryanodine (100 μM) blocked the ability of benzbromarone (0.3 μM) to increase calcium, while tetracaine (100 μM) reversibly reduced the rise in calcium induced by benzbromarone. We conclude that benzbromarone and MONNA caused intracellular calcium release, probably by opening ryanodine receptors. Their ability to block carbachol contractions was likely due to this off-target effect. [ABSTRACT FROM AUTHOR]