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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 Ca2+ 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. [ABSTRACT FROM AUTHOR]

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 Ca2+‐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.kg−1). Whole‐cell patch clamp, pharmacological, and immunocytochemical experiments were performed on isolated CCSM. Tension measurements were performed in whole tissue. TMEM16A expression in murine corpus cavernosum was confirmed using immunocytochemistry. Isolated CCSMC developed spontaneous transient inward currents (STICs) under voltage clamp and spontaneous transient depolarizations (STDs) in current clamp mode of the whole cell, perforated patch clamp technique. STICs reversed close to the predicted Cl− equilibrium potential and both STICs and STDs were blocked by the TMEM16A channel blockers, Ani9 and CaCC(inh)‐A01. These events were also blocked by GSK7975A (ORAI inhibitor), cyclopiazonic acid (CPA, sarcoplasmic reticulum [SR] Ca2+‐ATPase blocker), tetracaine (RyR blocker), and 2APB (IP3R blocker), suggesting that they were dependent on Ca2+ release from intracellular Ca2+ stores. Nifedipine (L‐type Ca2+ channel blocker) did not affect STICs, but reduced the duration of STDs. Phenylephrine induced transient depolarizations and transient inward currents which were blocked by Ani9. Similarly, phenylephrine induced phasic contractions of intact corpus cavernosum muscle strips and these events were also inhibited by Ani9. This study suggests that contraction of CCSM is regulated by activation of TMEM16A channels and therefore inhibition of these channels could lead to penile erection. [ABSTRACT FROM AUTHOR]

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. Graphical Abstract [ABSTRACT FROM AUTHOR]

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 = 1.1 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 ~15 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. [ABSTRACT FROM AUTHOR]

The aim of the present study was to investigate the properties and role of capacitative Caz+ entry (CCE) in interstitial cells (IC) isolated from the rabbit urethra. [Ca.sup.2+] entry in IC was larger in cells with depleted intracellular [Ca.sup.2+] stores compared with controls, consistent with influx via a CCE pathway. The nonselective [Ca.sup.2+] entry blockers [Gd.sup.3+] (10 [micro]M), [La.sup.3+] (10 [micro]M), and [Ni.sup.2+] (100 [micro]M) reduced CCE by 67% (n = 14), 65% (n = 11), and 55% (n = 9), respectively. These agents did not inhibit [Ca.sup.2+] entry when stores were not depleted. Conversely, CCE in IC was resistant to SKF-96365 (10 [micro]M), wortmannin (10 [micro]M), and nifedipine (1 [micro]M). Spontaneous transient inward currents were recorded from IC voltage-clamped at -60 mV. These events were not significantly affected by [Gd.sup.3+] (10 [micro]M) or [La.sup.3+] (10 [micro]M) and were only slightly decreased in amplitude by 100 [micro]M [Ni.sup.2+]. The results from this study demonstrate that freshly dispersed IC from the rabbit urethra possess a CCE pathway. However, influx via this pathway does not appear to contribute to spontaneous activity in these cells. smooth muscle; patch clamp; spontaneous transient inward currents

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 (NaV) currents in mouse CCSM and investigate their function. Experimental Approach We used electrophysiological, pharmacological and immunocytochemical methods to study the NaV currents in isolated CCSM cells from C57BL/6 mice. Tension measurements were carried out using crural sections of the corpus cavernosum in whole tissue. Key Results: Fast, voltage‐dependent, sodium currents in mouse CCSM were induced by depolarising steps. Steady‐state activation and inactivation curves revealed a window current between −60 and −30 mV. Two populations of NaV currents, 'TTX‐sensitive' and 'TTX‐insensitive', were identified. TTX‐sensitive currents showed 48% block with the NaV channel subtype‐specific blockers ICA‐121431 (NaV1.1–1.3), PF‐05089771 (NaV1.7) and 4,9‐anhydro‐TTX (NaV1.6). TTX‐insensitive currents were resistant to blockade by A803467, specific for NaV1.8 channels. Immunocytochemistry confirmed expression of NaV1.5 and NaV1.4 in freshly dispersed CCSM cells. Veratridine, a NaV channel activator, reduced time‐dependent inactivation of NaV currents and increased duration of evoked action potentials. Veratridine induced phasic contractions in CCSM strips, reversible with TTX and nifedipine but not KB‐R7943. Conclusion and Implications: There are fast, voltage‐dependent, sodium currents in mouse CCSM. Stimulation of these currents increased contractility of CCSM in vitro, suggesting an involvement in detumescence and potentially providing a clinically relevant target in erectile dysfunction. Further work will be necessary to define its role. [ABSTRACT FROM AUTHOR]

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. Graphical Abstract [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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.Experimental Approach: Experiments were performed on rat Gracilis muscle, saphenous, mesenteric and tail arteries using isobaric and isometric myography, sharp microelectrodes, digital droplet PCR and the patch-clamp technique.Key Results: GoSlo-SR compounds dilated isobaric and relaxed and hyperpolarised isometric vessel preparations and their effects were abolished after (a) functionally eliminating K+ channels by pre-constriction with 50 mM KCl or (b) blocking all K+ channels known to be expressed in vascular smooth muscle. However, these effects were not blocked when BK channels were inhibited. Surprisingly, the Kv 7 channel inhibitor XE991 reduced their effects considerably, but neither Kv 1 nor Kv 2 channel blockers altered the inhibitory effects of GoSlo-SR. However, the combined blockade of BK and Kv 7 channels abolished the GoSlo-SR-induced relaxation. GoSlo-SR compounds also activated Kv 7.4 and Kv 7.5 channels expressed in HEK 293 cells.Conclusion and Implications: This study shows that GoSlo-SR compounds are effective relaxants in vascular smooth muscle and mediate their effects by a combined activation of BK and Kv 7.4/Kv 7.5 channels. Activation of Kv 1, Kv 2 or Kv 7.1 channels or other vasodilator pathways seems not to be involved. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

β3-Adrenoceptor (β3-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 β3-AR agonists on cholinergic versus purinergic receptor-mediated contractions of the detrusor and to examine the mechanisms underlying inhibition of the purinergic responses by β3-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 β3-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 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 β3-AR agonists reduce postjunctional purinergic responses in the detrusor via a pathway involving activation of the cAMP effector EPAC. [ABSTRACT FROM AUTHOR]

Objective: To test if carbachol (CCh)-evoked Ca2+ oscillations in freshly isolated murine detrusor myocytes are affected by β3-adrenoceptor (β-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 β-AR sub-types in detrusor strips and isolated detrusor myocytes was assessed using reverse transcriptase-polymerase chain reaction (RTPCR) and real-time quantitative PCR (qPCR). Immunocytochemistry experiments, using a b3-AR selective antibody, were performed to confirm that b3-ARs were present on detrusor myocytes. Results: The RT-PCR and qPCR experiments showed that β1-, β2- and β3-AR were expressed in murine detrusor, but that β3-ARs were the most abundant sub-type. The selective β3-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 CChinduced Ca2+ oscillations in freshly isolated murine detrusor myocytes. This effect was mimicked by CL316,243, another b3-AR agonist, and inhibited by the b3-AR antagonist L748,337, but not by propranolol, an antagonist of β1- and b2-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 β3-AR agonists was associated with a reduction in Ca2+ oscillations in detrusor myocytes. [ABSTRACT FROM AUTHOR]

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]

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. [ABSTRACT FROM AUTHOR]

BK channels are critical regulators of neuronal activity, controlling firing, neurotransmitter release, cerebellar function, and BK channel mutations have been linked to seizure disorders. Modulation of BK channel gating is well characterized, regulated by accessory subunit interactions, intracellular signaling pathways, and membrane potential. In contrast, the role of intracellular trafficking mechanisms in controlling BK channel function, especially in live cells, has been less studied. Fluorogen-activating peptides (FAPs) are well-suited for trafficking and physiological studies due to the binding of malachite green (MG)-based dyes with sub-nanomolar affinity to the FAP, resulting in bright, photostable, far-red fluorescence. Cell-excluded MG dyes enable the selective tagging of surface protein and tracking through endocytic pathways. We used CRISPR to insert the FAP at the extracellular N-terminus of BKα in the first exon of its native locus, enabling regulation by the native promoter elements and tag incorporation into multiple splice isoforms. Motor coordination was found to be normal; however, BK channel expression seems to be reduced in some locations. Alternate start site selection or post-translational proteolytic processing resulted in incomplete FAP tagging of the BKα proteins in brain tissues. In Purkinje cell somata, FAP revealed BK channel clustering previously only observed by electron microscopy. Measurement of these clusters in β4+/- and β4-/- mice showed that puncta number and cluster fluorescence intensity on the soma are reduced in β4-/- knockout animals. This novel mouse line provides a versatile fluorescent platform for studying endogenous BK channels in living and fixed tissues. Future studies could apply this line to ex vivo neuronal cultures to study live-cell channel trafficking. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Stimulation of intramural nerves in the vas deferens of many species yields a classical biphasic contraction comprised of an initial fast component, mediated by P2X receptors and a second slower component, mediated by α1-adrenoceptors. It is also recognized that sympathetic nerve-mediated contractions of the vas deferens can be modulated by acetylcholine (Ach), however there is considerable disagreement in the literature regarding the precise contribution of cholinergic nerves to contraction of the vas deferens. In this study we examined the effect of cholinergic modulators on electric field stimulation ( EFS)-evoked contractions of rabbit vas deferens and on cytosolic Ca2+ levels in isolated vas deferens smooth muscle cells ( VDSMC). The sustained component of EFS-evoked contractions was inhibited by atropine and by the selective M3R antagonist, 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (4- DAMP). EFS-evoked contractions were potentiated by Ach, carbachol (Cch), and neostigmine. The sustained phase of the EFS-evoked contraction was inhibited by prazosin, an α1-adrenoceptor antagonist and guanethidine, an inhibitor of noradrenaline release, even in the continued presence of Ach, Cch or neostigmine. The soluble guanylate cyclase (sGC) inhibitor, 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one enhanced the amplitude of EFS-evoked contractions and reduced the inhibitory effects of 4- DAMP. Isolated VDSMC displayed spontaneous Ca2+ oscillations, but did not respond to Cch. However, the α1-adrenoceptor agonist, phenylephrine, evoked a Ca2+ transient and contracted the cells. These data suggest that EFS-evoked contractions of the rabbit vas deferens are potentiated by activation of M3 receptors and reduced by activation of a sGC-dependent inhibitory pathway. [ABSTRACT FROM AUTHOR]

Key points Tonic contractions of rabbit urethra are associated with spontaneous electrical slow waves that are thought to originate in pacemaker cells termed interstitial cells of Cajal (ICC)., ICC pacemaker activity results from their ability to generate propagating Ca2+ waves, although the exact mechanisms of propagation are not understood., In this study, we have identified spontaneous localised Ca2+ events for the first time in urethral ICC; these were due to Ca2+ release from the endoplasmic reticulum (ER) via ryanodine receptors (RyRs) and, while they often remained localised, they sometimes initiated propagating Ca2+ waves., We show that propagation of Ca2+ waves in urethral ICC is critically dependent upon Ca2+ influx via reverse mode NCX., Our data provide a clearer understanding of the intracellular mechanisms involved in the generation of ICC pacemaker activity., Abstract Interstitial cells of Cajal (ICC) are putative pacemaker cells in the rabbit urethra. Pacemaker activity in ICC results from spontaneous propagating Ca2+ waves that are modulated by [Ca2+]o and whose propagation is inhibited by inositol tri-phosphate receptor (IP3R) blockers. The purpose of this study was to further examine the role of Ca2+ influx and Ca2+ release in the propagation of Ca2+ waves. Intracellular Ca2+ was measured in Fluo-4-loaded ICC using a Nipkow spinning disc confocal microscope at fast acquisition rates (50 fps). We identified previously undetected localised Ca2+ events originating from ryanodine receptors (RyRs). Inhibiting Ca2+ influx by removing [Ca2+]o or blocking reverse mode sodium-calcium exchange (NCX) with KB-R 7943 or SEA-0400 abolished Ca2+ waves, while localised Ca2+ events persisted. Stimulating RyRs with 1 m m caffeine restored propagation. Propagation was also inhibited when Ca2+ release sites were uncoupled by buffering intracellular Ca2+ with EGTA-AM. This was reversed when Ca2+ influx via NCX was increased by reducing [Na+]o to 13 m m. Low [Na+]o also increased the frequency of Ca2+ waves and this effect was blocked by tetracaine and ryanodine but not 2-aminoethoxydiphenyl borate (2-APB). RT-PCR revealed that isolated ICC expressed both RyR2 and RyR3 subtypes. We conclude: (i) RyRs are required for the initiation of Ca2+ waves, but wave propagation normally depends on activation of IP3Rs; (ii) under resting conditions, propagation by IP3Rs requires sensitisation by influx of Ca2+ via reverse mode NCX; (iii) propagation can be maintained by RyRs if they have been sensitised to Ca2+. [ABSTRACT FROM AUTHOR]

Interstitial cells of Cajal (ICC) serve as electrical pacemakers in the rabbit urethra. Pacemaking activity in ICC results from spontaneous intracellular Ca 2+ waves that rely on Ca 2+ release from endoplasmic reticulum (ER) stores. The purpose of this study was to investigate if the action of protein kinase A (PKA) affected the generation of Ca 2+ waves in ICC. Intracellular [Ca 2+ ] was measured in fluo-4 loaded ICC, freshly isolated from the rabbit urethra using a Nipkow spinning disc confocal microscope. Application of the PKA inhibitor H-89 (10 μM) significantly inhibited the generation of spontaneous Ca 2+ waves in ICC and this was associated with a significant decrease in the ER Ca 2+ load, measured with 10 mM caffeine responses. Ca 2+ waves could be rescued in the presence of H-89 by stimulating ryanodine receptors (RyRs) with 1 mM caffeine but not by activation of inositol 1,4,5 tri-phosphate receptors (IP 3 Rs) with 10 μM phenylephrine. Increasing intracellular PKA with the cAMP agonists forskolin and 8-bromo-cAMP failed to yield an increase in Ca 2+ wave activity. We conclude that PKA may be maximally active under basal conditions in ICC and that inhibition of PKA with H-89 leads to a decreased ER Ca 2+ load sufficient to inactivate IP 3 Rs but not RyRs. [ABSTRACT FROM AUTHOR]

• PGE 2 inhibited CCh-evoked Ca2± oscillations in freshly isolated ASMC. • The inhibitory effects of PGE 2 were mediated by activation of EP 2 Rs. • CCh-evoked Ca2± oscillations relied on Ca2±release via RyRs and IP 3 Rs. • Inhibition of PKA only partially reversed the inhibitory effects of PGE 2. • PKA and EPAC activation reduced the frequency of the CCh responses. PGE 2 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 Ca2+ oscillations in airway smooth muscle cells (ASMC) and the force of contraction is positively correlated with the frequency of the underlying Ca2+ oscillations. The purpose of the present study was to examine if carbachol-evoked Ca2+ oscillations in isolated ASMC were inhibited by PGE 2. Isolated murine ASMC loaded with fluo4-AM were imaged with a Nipkow spinning disk confocal microscope. Cells responded to application of CCh (1 μM) by generating an initial Ca2+ transient followed by a series of Ca2+ oscillations. This activity was abolished by PGE 2 (300 nM) and the EP 2 R agonist (R)-butaprost (3 μM) and the inhibitory effects of PGE 2 were reversed by application of the EP 2 R antagonist PF-04418948 (100 nM). Activation of adenylate cyclase using forskolin (1 μM) mimicked the effects of PGE 2. The PKA activator, 6-MB-cAMP (300 μM) reduced the frequency of CCh-induced Ca2+ oscillations by 33% and the PKA inhibitor Rp-8-CPT-cAMPs partially reversed the inhibitory effects of PGE 2. The EPAC activator 007-AM (10 μM) reduced the frequency of the oscillations by 60% and joint application of 007-AM and 6-MB-cAMP reduced oscillation frequency by ∼85%. CCh-induced Ca2+ oscillations were inhibited by 2-APB and tetracaine, but caffeine-evoked Ca2+ transients were resistant to PGE 2. These data suggest that PGE 2 inhibits CCh-induced Ca2+ oscillations in murine ASMC via stimulation of EP 2 Rs and a mechanism involving activation of PKA and EPAC. [Display omitted] [ABSTRACT FROM AUTHOR]

Keywords: PIP2; protein kinase C; SOCE; TRPC1; vasoconstriction EN PIP2 protein kinase C SOCE TRPC1 vasoconstriction 1037 1038 2 02/17/21 20210215 NES 210215 Store-operated-Ca SP 2+ sp -entry (SOCE) is increasingly recognized as an important signalling pathway mediating smooth muscle cell (SMC) contraction, proliferation, migration and growth (Trebak I et al i . 2013). However, in native contractile VSMC, SOCE is considered to be mediated by other store-operated channels, such as canonical transient receptor potential 1 (TRPC1) channels, independently of Orai1 (Baudel I et al i . 2020a). Furthermore, store-operated currents in VSMCs were reduced by a pan-PKC isoform inhibitor (GF109203X), demonstrating the importance not only of TRPC1 channels, but also PKC with respect to mediating SOCE. [Extracted from the article]

We used the perforated patch-clamp technique at 37°C to investigate the mechanisms underlying the activation of a transient large-conductance K+ (tBK) current in rabbit urethral smooth muscle cells. The tBK current required an elevation of intracellular Ca2+, resulting from ryanodine receptor (RyR) activation via Ca2+-induced Ca2+ release, triggered by Ca2+ influx through L-type Ca2+ (CaV) channels. Carbachol inhibited tBK current by reducing Ca2+ influx and Ca2+ release and altered the shape of spike complexes recorded under current-clamp conditions. The tBK currents were blocked by iberiotoxin and penitrem A (300 and 100 nM, respectively) and were also inhibited when external Ca2+ was removed or the CaV channel inhibitors nifedipine (10 μM) and Cd2+ (100 μM) were applied. The tBK current was inhibited by caffeine (10 mM), ryanodine (30 μM), and tetracaine (100 μM), suggesting that RyR-mediated Ca2+ release contributed to the activation of the tBK current. When IP3 receptors (IP3Rs) were blocked with 2-aminoethoxydiphenyl borate (2-APB, 100 μM), the amplitude of the tBK current was not reduced. However, when Ca2+ release via IP3Rs was evoked with phenylephrine (1 μM) or carbachol (1 μM), the tBK current was inhibited. The effect of carbachol was abolished when IP3Rs were blocked with 2-APB or by inhibition of muscarinic receptors with the M3 receptor antagonist 4-diphenylacetoxy- N-methylpiperidine methiodide (1 μM). Under current-clamp conditions, bursts of action potentials could be evoked with depolarizing current injection. Carbachol reduced the number and amplitude of spikes in each burst, and these effects were reduced in the presence of 2-APB. In the presence of ryanodine, the number and amplitude of spikes were also reduced, and carbachol was without further effect. These data suggest that IP3-generating agonists can modulate the electrical activity of rabbit urethral smooth muscle cells and may contribute to the effects of neurotransmitters on urethral tone. [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]

Interstitial cells of Cajal (ICC) isolated from the rabbit urethra exhibit pacemaker activity that results from spontaneous Ca2+ waves. The purpose of this study was to investigate if this activity was influenced by Ca2+ uptake into mitochondria. Spontaneous Ca2+ waves were recorded using a Nipkow spinning disk confocal microscope and spontaneous transient inward currents (STICs) were recorded using the whole-cell patch clamp technique. Disruption of the mitochondrial membrane potential with the electron transport chain inhibitors rotenone (10 μm) and antimycin A (5 μm) abolished Ca2+ waves and increased basal Ca2+ levels. Similar results were achieved when mitochondria membrane potential was collapsed using the protonophores FCCP (0.2 μm) and CCCP (1 μm). Spontaneous Ca2+ waves were not inhibited by the ATP synthase inhibitor oligomycin (1 μm), suggesting that these effects were not attributable to an effect on ATP levels. STICs recorded under voltage clamp at −60 mV were also inhibited by CCCP and antimycin A. Dialysis of cells with the mitochondrial uniporter inhibitor RU360 (10 μm) also inhibited STICS. Stimulation of Ca2+ uptake into mitochondria using the plant flavonoid kaempferol (10 μm) induced a series of propagating Ca2+ waves. The kaempferol-induced activity was inhibited by application of caffeine (10 mm) or removal of extracellular Ca2+, but was not significantly affected by the IP3 receptor blocker 2-APB (100 μm). These data suggest that spontaneous Ca2+ waves in urethral ICC are regulated by buffering of cytoplasmic Ca2+ by mitochondria. [ABSTRACT FROM AUTHOR]

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 μ m) evoked large inward currents (mean approximately 900 pA). This current rapidly declined but was repeatable at 8-minute intervals. α,β-methylene ATP (10 μM), an agonist of P2X1 and P2X3 receptors, caused a similar current that also rapidly declined. Desensitization to α,β-methylene ATP negated the effect of ATP, but the ATP effect was restored 8 minutes after washout of α,β-methylene ATP. The effect of ATP was reversibly blocked by NF449 (1 μ m), 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 μ m). When phentolamine (3 μ m) and atropine (1 μ m) 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 μ m). 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. [ABSTRACT FROM AUTHOR]