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Transient receptor potential vanilloid 1 (TRPV1) is a calcium-permeable ion channel that is gated by the pungent constituent of red chili pepper, capsaicin, and by related chemicals from the group of vanilloids, in addition to noxious heat. It is expressed mostly in sensory neurons to act as a detector of painful stimuli produced by pungent chemicals and high temperatures. Although TRPV1 is also found outside the sensory nervous system, its expression and function in the bladder detrusor smooth muscle (DSM) remain controversial. Here, by using Ca 2+ imaging and patch clamp on isolated rat DSM cells, in addition to tensiometry on multicellular DSM strips, we show that TRPV1 is expressed functionally in only a fraction of DSM cells, in which it acts as an endoplasmic reticulum Ca 2+ -release channel responsible for the capsaicin-activated [Ca 2+ ] i rise. Carbachol-stimulated contractions of multicellular DSM strips contain a TRPV1-dependent component, which is negligible in the circular DSM but reaches ≤50% in the longitudinal DSM. Activation of TRPV1 in rat DSM during muscarinic cholinergic stimulation is ensured by phospholipase A 2 -catalysed derivation of arachidonic acid and its conversion by lipoxygenases to eicosanoids, which act as endogenous TRPV1 agonists. Immunofluorescence detection of TRPV1 protein in bladder sections and isolated DSM cells confirmed both its preferential expression in the longitudinal DSM sublayer and its targeting to the endoplasmic reticulum. We conclude that TRPV1 is an essential contributor to the cholinergic contraction of bladder longitudinal DSM, which might be important for producing spatial and/or temporal anisotropy of bladder wall deformation in different regions during parasympathetic stimulation. KEY POINTS: The transient receptor potential vanilloid 1 (TRPV1) heat/capsaicin receptor/channel is localized in the endoplasmic reticulum membrane of detrusor smooth muscle (DSM) cells of the rat bladder, operating as a calcium-release channel. Isolated DSM cells are separated into two nearly equal groups, within which the cells either show or do not show TRPV1-dependent [Ca 2+ ] i rise. Carbachol-stimulated, muscarinic ACh receptor-mediated contractions of multicellular DSM strips contain a TRPV1-dependent component. This component is negligible in the circular DSM but reaches ≤50% in longitudinal DSM. Activation of TRPV1 in rat DSM during cholinergic stimulation involves phospholipase A 2 -catalysed derivation of arachidonic acid and its conversion by lipoxygenases to eicosanoids, which act as endogenous TRPV1 agonists., (© 2024 The Authors. The Journal of Physiology © 2024 The Physiological Society.)

Approximately 10% of US births deliver preterm before 37 weeks of completed gestation. Premature infants are at risk for life-long debilitating morbidities and death, and spontaneous preterm labour explains 50% of preterm births. In all cases existing treatments are ineffective, and none are FDA approved. The mechanisms that initiate preterm labour are not well understood but may result from dysfunctional regulation of quiescence mechanisms. Human pregnancy is accompanied by large increases in blood flow, and the uterus must enlarge by orders of magnitude to accommodate the growing fetus. This mechanical strain suggests that stretch-activated channels may constitute a mechanism to explain gestational quiescence. Here we identify for the first time that Piezo1, a mechanosensitive cation channel, is present in the uterine smooth muscle and microvascular endothelium of pregnant myometrium. Piezo is downregulated during preterm labour, and stimulation of myometrial Piezo1 in an organ bath with the agonist Yoda1 relaxes the tissue in a dose-dependent fashion. Further, stimulation of Piezo1 while inhibiting protein kinase A, AKT, or endothelial nitric oxide synthase mutes the negative inotropic effects of Piezo1 activation, intimating that actions on the myocyte and endothelial nitric oxide signalling contribute to Piezo1-mediated contractile dynamics. Taken together, these data highlight the importance of stretch-activated channels in pregnancy maintenance and parturition, and identify Piezo1 as a tocolytic target of interest. KEY POINTS: Spontaneous preterm labour is a serious obstetric dilemma without a known cause or effective treatments. Piezo1 is a stretch-activated channel important to muscle contractile dynamics. Piezo1 is present in the myometrium and is dysregulated in women who experience preterm labour. Activation of Piezo1 by the agonist Yoda1 relaxes the myometrium in a dose-dependent fashion, indicating that Piezo1 modulation may have therapeutic benefits to treat preterm labour., (© 2022 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Key Points: S100A4 is expressed in many tissues, including smooth muscle (SM), but its physiologic function is unknown. S100A4 regulates the motility of metastatic cancer cells by binding to non-muscle (NM) myosin II. Contractile stimulation causes the polymerization of NM myosin in airway SM, which is necessary for tension development. NM myosin regulates the assembly of adhesion junction signalling complexes (adhesomes) that catalyse actin polymerization. In airway SM, ACh (acetylcholine) stimulated the binding of S100A4 to the NM myosin heavy chain, which was catalysed by RhoA GTPase via the RhoA-binding protein, rhotekin. The binding of S100A4 to NM myosin was required for NM myosin polymerization, adhesome assembly and actin polymerization. S100A4 plays a critical function in the regulation of airway SM contraction by catalysing NM myosin filament assembly. The interaction of S100A4 with NM myosin may also play an important role in the physiologic function of other tissues., Abstract: S100A4 binds to the heavy chain of non-muscle (NM) myosin II and can regulate the motility of crawling cells. S100A4 is widely expressed in many tissues including smooth muscle (SM), although its role in the regulation of their physiologic function is not known. We hypothesized that S100A4 contributes to the regulation of contraction in airway SM by regulating a pool of NM myosin II at the cell cortex. NM myosin II undergoes polymerization in airway SM and regulates contraction by catalysing the assembly of integrin-associated adhesome complexes that activate pathways that catalyse actin polymerization. ACh stimulated the interaction of S100A4 with NM myosin II in airway SM at the cell cortex and catalysed NM myosin filament assembly. RhoA GTPase regulated the activation of S100A4 via rhotekin, which facilitated the formation of a complex between RhoA, S100A4 and NM myosin II. The depletion of S100A4, RhoA or rhotekin from airway SM tissues using short hairpin RNA or small interfering RNA prevented NM myosin II polymerization as well as the recruitment of vinculin and paxillin to adhesome signalling complexes in response to ACh, and inhibited actin polymerization and tension development. S100A4 depletion did not affect ACh-stimulated SM myosin regulatory light chain phosphorylation. The results show that S100A4 plays a critical role in tension development in airway SM tissue by catalysing NM myosin filament assembly, and that the interaction of S100A4 with NM myosin in response to contractile stimulation is activated by RhoA GTPase. These results may be broadly relevant to the physiologic function of S100A4 in other cell and tissue types., (© 2020 The Authors. The Journal of Physiology © 2020 The Physiological Society.)

Key Points: Colonic intramuscular interstitial cells of Cajal (ICC-IM) exhibit spontaneous Ca 2+ transients manifesting as stochastic events from multiple firing sites with propagating Ca 2+ waves occasionally observed. Firing of Ca 2+ transients in ICC-IM is not coordinated with adjacent ICC-IM in a field of view or even with events from other firing sites within a single cell. Ca 2+ transients, through activation of Ano1 channels and generation of inward current, cause net depolarization of colonic muscles. Ca 2+ transients in ICC-IM rely on Ca 2+ release from the endoplasmic reticulum via IP 3 receptors, spatial amplification from RyRs and ongoing refilling of ER via the sarcoplasmic/endoplasmic-reticulum-Ca 2+ -ATPase. ICC-IM are sustained by voltage-independent Ca 2+ influx via store-operated Ca 2+ entry. Some of the properties of Ca 2+ in ICC-IM in the colon are similar to the behaviour of ICC located in the deep muscular plexus region of the small intestine, suggesting there are functional similarities between these classes of ICC., Abstract: A component of the SIP syncytium that regulates smooth muscle excitability in the colon is the intramuscular class of interstitial cells of Cajal (ICC-IM). All classes of ICC (including ICC-IM) express Ca 2+ -activated Cl - channels, encoded by Ano1, and rely upon this conductance for physiological functions. Thus, Ca 2+ handling in ICC is fundamental to colonic motility. We examined Ca 2+ handling mechanisms in ICC-IM of murine proximal colon expressing GCaMP6f in ICC. Several Ca 2+ firing sites were detected in each cell. While individual sites displayed rhythmic Ca 2+ events, the overall pattern of Ca 2+ transients was stochastic. No correlation was found between discrete Ca 2+ firing sites in the same cell or in adjacent cells. Ca 2+ transients in some cells initiated Ca 2+ waves that spread along the cell at ∼100 µm s -1 . Ca 2+ transients were caused by release from intracellular stores, but depended strongly on store-operated Ca 2+ entry mechanisms. ICC Ca 2+ transient firing regulated the resting membrane potential of colonic tissues as a specific Ano1 antagonist hyperpolarized colonic muscles by ∼10 mV. Ca 2+ transient firing was independent of membrane potential and not affected by blockade of L- or T-type Ca 2+ channels. Mechanisms regulating Ca 2+ transients in the proximal colon displayed both similarities to and differences from the intramuscular type of ICC in the small intestine. Similarities and differences in Ca 2+ release patterns might determine how ICC respond to neurotransmission in these two regions of the gastrointestinal tract., (© 2019 The Authors. The Journal of Physiology © 2019 The Physiological Society.)

Key Points: Electrical pacemaking in gastrointestinal muscles is generated by specialized interstitial cells of Cajal that produce the patterns of contractions required for peristalsis and segmentation in the gut. The calcium-activated chloride conductance anoctamin-1 (Ano1) has been shown to be responsible for the generation of pacemaker activity in GI muscles, but this conclusion is established from studies of juvenile animals in which effects of reduced Ano1 on gastric emptying and motor patterns could not be evaluated. Knocking down Ano1 expression using Cre/LoxP technology caused dramatic changes in in gastric motor activity, with disrupted slow waves, abnormal phasic contractions and delayed gastric emptying; modest changes were noted in the small intestine. Comparison of the effects of Ano1 antagonists on muscles from juvenile and adult small intestinal muscles suggests that conductances in addition to Ano1 may develop with age and contribute to pacemaker activity., Abstract: Interstitial cells of Cajal (ICC) generate slow waves and transduce neurotransmitter signals in the gastrointestinal (GI) tract, facilitating normal motility patterns. ICC express a Ca 2+ -activated Cl - conductance (CaCC), and constitutive knockout of the channel protein anoctamin-1 leads to loss of slow waves in gastric and intestinal muscles. These knockout experiments were performed on juvenile mice. However, additional experiments demonstrated significant differences in the sensitivity of gastric and intestinal muscles to antagonists of anoctamin-1 channels. Furthermore, the significance of anoctamin-1 and the electrical and mechanical behaviours facilitated by this conductance have not been evaluated on the motor behaviours of adult animals. Cre/loxP technology was used to generate cell-specific knockdowns of anoctamin-1 in ICC (Kit CreERT2/+ ;Ano1 tm2jrr/+ ) in GI muscles. The recombination efficiency of Kit CreERT was evaluated with an eGFP reporter, molecular techniques and immunohistochemistry. Electrical and contractile experiments were used to examine the consequences of anoctamin-1 knockdown on pacemaker activity, mechanical responses, gastric motility patterns, gastric emptying and GI transit. Reduced anoctamin-1 caused loss of gastric, but not intestinal slow waves. Irregular spike complexes developed in gastric muscles, leading to uncoordinated antral contractions, delayed gastric emptying and increased total GI transit time. Slow waves in intestinal muscles of juvenile mice were more sensitive to anoctamin-1 antagonists than slow waves in adult muscles. The low susceptibility to anoctamin-1 knockdown and weak efficacy of anoctamin-1 antagonists in inhibiting slow waves in adult small intestinal muscles suggest that a conductance in addition to anoctamin-1 may develop in small intestinal ICC with ageing and contribute to pacemaker activity., (© 2019 The Authors. The Journal of Physiology © 2019 The Physiological Society.)

Key Points: Studies of urothelial cells, bladder sheets or lumens of filled bladders have suggested that mediators released from urothelium into suburothelium (SubU)/lamina propria (LP) activate mechanisms controlling detrusor excitability. None of these approaches, however, has enabled direct assessment of availability of mediators at SubU/LP during filling. We developed an ex vivo mouse bladder preparation with intact urothelium and SubU/LP but no detrusor, which allows direct access to the SubU/LP surface of urothelium during filling. Pressure-volume measurements during filling demonstrated that bladder compliance is governed primarily by the urothelium. Measurements of purine mediators in this preparation demonstrated asymmetrical availability of purines in lumen and SubU/LP, suggesting that interpretations based solely on intraluminal measurements of mediators may be inaccurate. The preparations are suitable for assessments of release, degradation and transport of mediators in SubU/LP during bladder filling, and are superior to experimental approaches previously used for urothelium research., Abstract: The purpose of this study was to develop a decentralized (ex vivo) detrusor smooth muscle (DSM)-denuded mouse bladder preparation, a novel model that enables studies on availability of urothelium-derived mediators at the luminal and anti-luminal aspects of the urothelium during filling. Urinary bladders were excised from C57BL6/J mice and the DSM was removed by fine-scissor dissection without touching the mucosa. Morphology and cell composition of the preparation wall, pressure-volume relationships during filling, and fluorescent dye permeability of control, protamine sulfate- and lipopolysaccharide-treated denuded bladders were characterized. The preparation wall contained intact urothelium and suburothelium (SubU)/lamina propria (LP) and lacked the DSM and the serosa. The utility of the model for physiological research was validated by measuring release, metabolism and transport of purine mediators at SubU/LP and in bladder lumen during filling. We determined asymmetrical availability of purines (e.g. ATP, ADP, AMP and adenosine) in lumen and at SubU/LP during filling, suggesting differential mechanisms of release, degradation and bilateral transurothelial transport of purines during filling. Some observations were validated in DSM-denuded bladder of the cynomolgus monkey (Macaca fascicularis). The novel model was superior to current models utilized to study properties of the urothelium (e.g. cultured urothelial cells, bladder mucosa sheets mounted in Ussing chambers or isolated bladder strips in organ baths) in that it enabled direct access to the vicinity of SubU/LP during authentic bladder filling. The model is particularly suitable for understanding local mechanisms of urothelium-DSM connectivity and for broad understanding of the role of urothelium in regulating continence and voiding., (© 2018 The Authors. The Journal of Physiology © 2018 The Physiological Society.)

Key Points: K V 7 channels are a family of voltage-dependent K + channels expressed in many cell types, which open in response to membrane depolarization to regulate cell excitability. Drugs that target K V 7 channels are used clinically to treat epilepsy. Interestingly, these drugs also cause urinary retention, but it was unclear how. In this study, we focused on two possible mechanisms by which retigabine could cause urinary retention: by decreasing smooth muscle excitability, or by decreasing sensory nerve outflow. Urinary bladder smooth muscle had no measurable K V 7 channel currents. However, the K V 7 channel agonist retigabine nearly abolished sensory nerve outflow from the urinary bladder during bladder filling. We conclude that K V 7 channel activation likely affects urinary bladder function by blocking afferent nerve outflow to the brain, which is key to sensing bladder fullness., Abstract: K V 7 channels are voltage-dependent K + channels that open in response to membrane depolarization to regulate cell excitability. K V 7 activators, such as retigabine, were used to treat epilepsy but caused urinary retention. Using electrophysiological recordings from freshly isolated mouse urinary bladder smooth muscle (UBSM) cells, isometric contractility of bladder strips, and ex vivo measurements of bladder afferent activity, we explored the role of K V 7 channels as regulators of murine urinary bladder function. The K V 7 activator retigabine (10 μM) had no effect on voltage-dependent K + currents or resting membrane potential of UBSM cells, suggesting that these cells lacked retigabine-sensitive K V 7 channels. The K V 7 inhibitor XE-991 (10 μM) inhibited UBSM K + currents; the properties of these currents, however, were typical of K V 2 channels and not K V 7 channels. Retigabine inhibited voltage-dependent Ca 2+ channel (VDCC) currents and reduced steady-state contractions to 60 mM KCl in bladder strips, suggesting that reduction in VDCC current was sufficient to directly affect UBSM function. To determine if retigabine altered ex vivo bladder sensory outflow, we measured afferent activity during simulated transient contractions (TCs) of the bladder wall. Simulated TCs caused bursts of afferent activity that were nearly abolished by retigabine. The effects of retigabine were blocked by co-incubation with XE-991, suggesting specific activation of K V 7 channels on afferent nerves. These results indicate that retigabine primarily affects urinary bladder function by inhibiting TC generation and afferent nerve activity, which are key to sensing bladder fullness. Any direct inhibition of UBSM contractility is likely to be from non-specific effects on VDCCs and K V 2 channels., (© 2018 The Authors. The Journal of Physiology © 2018 The Physiological Society.)

Key Points: Non-muscle (NM) and smooth muscle (SM) myosin II are both expressed in smooth muscle tissues, however the role of NM myosin in SM contraction is unknown. Contractile stimulation of tracheal smooth muscle tissues stimulates phosphorylation of the NM myosin heavy chain on Ser1943 and causes NM myosin filament assembly at the SM cell cortex. Expression of a non-phosphorylatable NM myosin mutant, NM myosin S1943A, in SM tissues inhibits ACh-induced NM myosin filament assembly and SM contraction, and also inhibits the assembly of membrane adhesome complexes during contractile stimulation. NM myosin regulatory light chain (RLC) phosphorylation but not SM myosin RLC phosphorylation is regulated by RhoA GTPase during ACh stimulation, and NM RLC phosphorylation is required for NM myosin filament assembly and SM contraction. NM myosin II plays a critical role in airway SM contraction that is independent and distinct from the function of SM myosin., Abstract: The molecular function of non-muscle (NM) isoforms of myosin II in smooth muscle (SM) tissues and their possible role in contraction are largely unknown. We evaluated the function of NM myosin during contractile stimulation of canine tracheal SM tissues. Stimulation with ACh caused NM myosin filament assembly, as assessed by a Triton solubility assay and a proximity ligation assay aiming to measure interactions between NM myosin monomers. ACh stimulated the phosphorylation of NM myosin heavy chain on Ser1943 in tracheal SM tissues, which can regulate NM myosin IIA filament assembly in vitro. Expression of the non-phosphorylatable mutant NM myosin S1943A in SM tissues inhibited ACh-induced endogenous NM myosin Ser1943 phosphorylation, NM myosin filament formation, the assembly of membrane adhesome complexes and tension development. The NM myosin cross-bridge cycling inhibitor blebbistatin suppressed adhesome complex assembly and SM contraction without inhibiting NM myosin Ser1943 phosphorylation or NM myosin filament assembly. RhoA inactivation selectively inhibited phosphorylation of the NM myosin regulatory light chain (RLC), NM myosin filament assembly and contraction, although it did not inhibit SM RLC phosphorylation. We conclude that the assembly and activation of NM myosin II is regulated during contractile stimulation of airway SM tissues by RhoA-mediated NM myosin RLC phosphorylation and by NM myosin heavy chain Ser1943 phosphorylation. NM myosin II actomyosin cross-bridge cycling regulates the assembly of membrane adhesome complexes that mediate the cytoskeletal processes required for tension generation. NM myosin II plays a critical role in airway SM contraction that is independent and distinct from the function of SM myosin., (© 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.)

Key Points: The internal anal sphincter develops tone important for maintaining high anal pressure and continence. Controversy exists regarding the mechanisms underlying tone development. We examined the hypothesis that tone depends upon electrical slow waves (SWs) initiated in intramuscular interstitial cells of Cajal (ICC-IM) by activation of Ca 2+ -activated Cl - channels (ANO1, encoded by Ano1) and voltage-dependent L-type Ca 2+ channels (Cav L , encoded by Cacna1c). Measurement of membrane potential and contraction indicated that ANO1 and Cav L have a central role in SW generation, phasic contractions and tone, independent of stretch. ANO1 expression was examined in wildtype and Ano1 /+egfp mice with immunohistochemical techniques. Ano1 and Cacna1c expression levels were examined by quantitative PCR in fluorescence-activated cell sorting. ICC-IM were the predominant cell type expressing ANO1 and the most likely candidate for SW generation. SWs in ICC-IM are proposed to conduct to smooth muscle where Ca 2+ entry via Cav L results in phasic activity that sums to produce tone., Abstract: The mechanism underlying tone generation in the internal anal sphincter (IAS) is controversial. We examined the hypothesis that tone depends upon generation of electrical slow waves (SWs) initiated in intramuscular interstitial cells of Cajal (ICC-IM) by activation of Ca 2+ -activated Cl - channels (encoded by Ano1) and voltage-dependent L-type Ca 2+ channels (encoded by Cacna1c). Phasic contractions and tone in the IAS were nearly abolished by ANO1 and Cav L antagonists. ANO1 antagonists also abolished SWs as well as transient depolarizations that persisted after addition of Cav L antagonists. Tone development in the IAS did not require stretch of muscles, and the sensitivity of contraction to ANO1 antagonists was the same in stretched versus un-stretched muscles. ANO1 expression was examined in wildtype and Ano1 /+egfp mice with immunohistochemical techniques. Dual labelling revealed that ANO1 expression could be resolved in ICC but not smooth muscle cells (SMCs) in the IAS and rectum. Ano1, Cacna1c and Kit gene expression were the same in extracts of IAS and rectum muscles. In IAS cells isolated with fluorescence-activated cell sorting, Ano1 expression was 26.5-fold greater in ICC than in SMCs while Cacna1c expression was only 2-fold greater in SMCs than in ICC. These data support a central role for ANO1 and Cav L in the generation of SWs and tone in the IAS. ICC-IM are the probable cellular candidate for ANO1 currents and SW generation. We propose that ANO1 and Cav L collaborate to generate SWs in ICC-IM followed by conduction to adjacent SMCs where phasic calcium entry through Cav L sums to produce tone., (© 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.)

Key Points: The extent of myosin regulatory light chain phosphorylation (RLC) necessary for smooth muscle contraction depends on the respective activities of Ca(2+) /calmodulin-dependent myosin light chain kinase and myosin light chain phosphatase (MLCP), which contains a regulatory subunit MYPT1 bound to the phosphatase catalytic subunit and myosin. MYPT1 showed significant constitutive T696 and T853 phosphorylation, which is predicted to inhibit MLCP activity in isolated ileal smooth muscle tissues, with additional phosphorylation upon pharmacological treatment with the muscarinic agonist carbachol. Electrical field stimulation (EFS), which releases ACh from nerves, increased force and RLC phosphorylation but not MYPT1 T696 or T853 phosphorylation. The conditional knockout of MYPT1 or the knockin mutation T853A in mice had no effect on the frequency-maximal force responses to EFS in isolated ileal tissues. Physiological RLC phosphorylation and force development in ileal smooth muscle depend on myosin light chain kinase and MLCP activities without changes in constitutive MYPT1 phosphorylation., Abstract: Smooth muscle contraction initiated by myosin regulatory light chain (RLC) phosphorylation is dependent on the relative activities of Ca(2+) /calmodulin-dependent myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP). We have investigated the physiological role of the MLCP regulatory subunit MYPT1 in ileal smooth muscle in adult mice with (1) smooth muscle-specific deletion of MYPT1; (2) non-phosphorylatable MYPT1 containing a T853A knockin mutation; and (3) measurements of force and protein phosphorylation responses to cholinergic neurostimulation initiated by electric field stimulation. Isolated MYPT1-deficient tissues from MYPT1(SM-/-) mice contracted and relaxed rapidly with moderate differences in sustained responses to KCl and carbachol treatments and washouts, respectively. Similarly, measurements of regulatory proteins responsible for RLC phosphorylation during contractions also revealed moderate changes. There were no differences in contractile or RLC phosphorylation responses to carbachol between tissues from normal mice vs. MYPT1 T853A knockin mice. Quantitatively, there was substantial MYPT1 T696 and T853 phosphorylation in wild-type tissues under resting conditions, predicting a high extent of MLCP phosphatase inhibition. Reduced PP1cδ activity in MYPT1-deficient tissues may be similar to attenuated MLCP activity in wild-type tissues resulting from constitutively phosphorylated MYPT1. Electric field stimulation increased RLC phosphorylation and force development in tissues from wild-type mice without an increase in MYPT1 phosphorylation. Thus, physiological RLC phosphorylation and force development in ileal smooth muscle appear to be dependent on MLCK and MLCP activities without changes in constitutive MYPT1 phosphorylation., (© 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.)

Key Points: Platelet derived growth factor receptor α (PDGFRα(+) ) cells in colonic muscles are innervated by enteric inhibitory motor neurons. PDGFRα(+) cells generate Ca(2+) transients in response to exogenous purines and these responses were blocked by MRS-2500. Stimulation of enteric neurons, with cholinergic and nitrergic components blocked, evoked Ca(2+) transients in PDGFRα(+) and smooth muscle cells (SMCs). Responses to nerve stimulation were abolished by MRS-2500 and not observed in muscles with genetic deactivation of P2Y1 receptors. Ca(2+) transients evoked by nerve stimulation in PDGFRα(+) cells showed the same temporal characteristics as electrophysiological responses. PDGFRα(+) cells express gap junction genes, and drugs that inhibit gap junctions blocked neural responses in SMCs, but not in nerve processes or PDGFRα(+) cells. PDGFRα(+) cells are directly innervated by inhibitory motor neurons and purinergic responses are conducted to SMCs via gap junctions., Abstract: Interstitial cells, known as platelet derived growth factor receptor α (PDGFRα(+) ) cells, are closely associated with varicosities of enteric motor neurons and suggested to mediate purinergic hyperpolarization responses in smooth muscles of the gastrointestinal tract (GI), but this concept has not been demonstrated directly in intact muscles. We used confocal microscopy to monitor Ca(2+) transients in neurons and post-junctional cells of the murine colon evoked by exogenous purines or electrical field stimulation (EFS) of enteric neurons. EFS (1-20 Hz) caused Ca(2+) transients in enteric motor nerve processes and then in PDGFRα(+) cells shortly after the onset of stimulation (latency from EFS was 280 ms at 10 Hz). Responses in smooth muscle cells (SMCs) were typically a small decrease in Ca(2+) fluorescence just after the initiation of Ca(2+) transients in PDGFRα(+) cells. Upon cessation of EFS, several fast Ca(2+) transients were noted in SMCs (rebound excitation). Strong correlation was noted in the temporal characteristics of Ca(2+) transients evoked in PDGFRα(+) cells by EFS and inhibitory junction potentials (IJPs) recorded with intracellular microelectrodes. Ca(2+) transients and IJPs elicited by EFS were blocked by MRS-2500, a P2Y1 antagonist, and absent in P2ry1((-/-)) mice. PDGFRα(+) cells expressed gap junction genes, and gap junction uncouplers, 18β-glycyrrhetinic acid (18β-GA) and octanol blocked Ca(2+) transients in SMCs but not in neurons or PDGFRα(+) cells. IJPs recorded from SMCs were also blocked. These findings demonstrate direct innervation of PDGFRα(+) cells by motor neurons. PDGFRα(+) cells are primary targets for purinergic neurotransmitter(s) in enteric inhibitory neurotransmission. Hyperpolarization responses are conducted to SMCs via gap junctions., (© 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.)

Protease-activated receptors (PARs) are G protein-coupled receptors activated by proteolytic cleavage at their amino termini by serine proteases. PAR activation contributes to the inflammatory response in the gastrointestinal (GI) tract and alters GI motility, but little is known about the specific cells within the tunica muscularis that express PARs and the mechanisms leading to contractile responses. Using real time PCR, we found PARs to be expressed in smooth muscle cells (SMCs), interstitial cells of Cajal (ICC) and platelet-derived growth factor receptor α positive (PDGFRα(+)) cells. The latter cell-type showed dominant expression of F2r (encodes PAR1) and F2rl1 (encodes PAR2). Contractile and intracellular electrical activities were measured to characterize the integrated responses to PAR activation in whole muscles. Cells were isolated and ICC and PDGFRα(+) cells were identified by constitutive expression of fluorescent reporters. Thrombin (PAR1 agonist) and trypsin (PAR2 agonist) caused biphasic responses in colonic muscles: transient hyperpolarization and relaxation followed by repolarization and excitation. The inhibitory phase was blocked by apamin, revealing a distinct excitatory component. Patch clamp studies showed that the inhibitory response was mediated by activation of small conductance calcium-activated K(+) channels in PDGFRα(+) cells, and the excitatory response was mediated by activation of a Cl(-) conductance in ICC. SMCs contributed little to PAR responses in colonic muscles. In summary, PARs regulate the excitability of colonic muscles; different conductances are activated in each cell type of the SMC-ICC-PDGFRα(+) cell (SIP) syncytium. Motor responses to PAR agonists are integrated responses of the SIP syncytium., (© 2014 The Authors. The Journal of Physiology © 2014 The Physiological Society.)

Key Points: Force production and maintenance in smooth muscle is largely controlled by myosin regulatory light chain (RLC) phosphorylation, which relies on a balance between Ca(2+)/calmodulin-dependent myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) activities. MYPT1 is the regulatory subunit of MLCP that biochemically inhibits MLCP activity via T694 or T852 phosphorylation in vitro. Here we separately investigated the contribution of these two phosphorylation sites in bladder smooth muscles by establishing two single point mutation mouse lines, T694A and T852A, and found that phosphorylation of MYPT1 T694, but not T852, mediates force maintenance via inhibition of MLCP activity and enhancement of RLC phosphorylation in vivo. Our findings reveal the role of MYPT1 T694/T852 phosphorylation in vivo in regulation of smooth muscle contraction., Abstract: Force production and maintenance in smooth muscle is largely controlled by different signalling modules that fine tune myosin regulatory light chain (RLC) phosphorylation, which relies on a balance between Ca(2+)/calmodulin-dependent myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) activities. To investigate the regulation of MLCP activity in vivo, we analysed the role of two phosphorylation sites on MYPT1 (regulatory subunit of MLCP) that biochemically inhibit MLCP activity in vitro. MYPT1 is constitutively phosphorylated at T694 by unidentified kinases in vivo, whereas the T852 site is phosphorylated by RhoA-associated protein kinase (ROCK). We established two mouse lines with alanine substitution of T694 or T852. Isolated bladder smooth muscle from T852A mice displayed no significant changes in RLC phosphorylation or force responses, but force was inhibited with a ROCK inhibitor. In contrast, smooth muscles containing the T694A mutation showed a significant reduction of force along with reduced RLC phosphorylation. The contractile responses of T694A mutant smooth muscle were also independent of ROCK activation. Thus, phosphorylation of MYPT1 T694, but not T852, is a primary mechanism contributing to inhibition of MLCP activity and enhancement of RLC phosphorylation in vivo. The constitutive phosphorylation of MYPT1 T694 may provide a mechanism for regulating force maintenance of smooth muscle., (© 2014 The Authors. The Journal of Physiology © 2014 The Physiological Society.)

The mechanisms of mechanosensitivity underlying the response of the human bladder to stretch are poorly understood. Animal data suggest that stretch-activated two-pore-domain (K2P) K(+) channels play a critical role in bladder relaxation during the filling phase. The objective of this study was to characterize the expression and function of stretch-activated K2P channels in the human bladder and to clarify their physiological role in bladder mechanosensitivity. Gene and protein analysis of the K2P channels TREK-1, TREK-2 and TRAAK in the human bladder revealed that TREK-1 is the predominantly expressed member of the mechano-gated subfamily of K2P channels. Immunohistochemical labelling of bladder wall identified higher levels of expression of TREK-1 in detrusor smooth muscle cells in comparison to bladder mucosa. Functional characterization and biophysical properties of the predominantly expressed member of the K2P family, the TREK-1 channel, were evaluated by in vitro organ bath studies and the patch-clamp technique. Electrophysiological recordings from single smooth muscle cells confirmed direct activation of TREK-1 channels by mechanical stretch and negative pressure applied to the cell membrane. Inhibition of TREK-1 channels in the human detrusor significantly delayed relaxation of the stretched bladder smooth muscle strips and triggered small-amplitude spontaneous contractions. Application of negative pressure to cell-attached patches (-20 mmHg) caused a 19-fold increase in the open probability (NPo) of human TREK-1 channels. l-Methionine (1 mm), a specific TREK-1 inhibitor, dramatically decreased the NPo of TREK-1 channels from 0.045 ± 0.003 to 0.008 ± 0.001 (n = 8, P ≤ 0.01). Subsequent addition of arachidonic acid (10 μm), a channel opener, increased the open probability of methionine-inhibited unitary currents up to 0.43 ± 0.05 at 0 mV (n = 9, P ≤ 0.05). The results of our study provide direct evidence that the response of the human detrusor to mechanical stretch is regulated by activation of mechano-gated TREK-1 channels. Impaired mechanosensation and mechanotransduction associated with the changes in stretch-activated K2P channels may underlie myogenic bladder dysfunction in humans., (© 2014 The Authors. The Journal of Physiology © 2014 The Physiological Society.)

Ca(2+) sensitization of contraction has typically been investigated by bathing muscles in solutions containing agonists. However, it is unknown whether bath-applied agonists and enteric neurotransmission activate similar Ca(2+) sensitization mechanisms. We investigated protein kinase C (PKC)-potentiated phosphatase inhibitor protein of 17 kDa (CPI-17) and myosin phosphatase targeting subunit 1 (MYPT1) phosphorylation in murine gastric fundus muscles stimulated by bath-applied carbachol (CCh) or cholinergic motor neurotransmission. CCh increased MYPT1 phosphorylation at Thr696 (pT696) and Thr853 (pT853), CPI-17 at Thr38 (pT38), and myosin light chain at Ser19 (pS19). Electrical field stimulation (EFS) only increased pT38. In the presence of neostigmine, EFS increased pT38, pT853 and pS19. In fundus muscles of W/W(v) mice, EFS alone increased pT38 and pT853. Atropine blocked all contractions and all increases in pT696, pT853, pT38 and pS19. The Rho kinase (ROCK) inhibitor SAR1x blocked increases in pT853 and pT696. The PKC inhibitors Go6976 and Gf109203x or nicardipine blocked increases in pT38 and pT696. These findings suggest that cholinergic motor neurotransmission activates PKC-dependent CPI-17 phosphorylation. Bath-applied CCh recruits additional ROCK-dependent MYPT1 phosphorylation due to exposure of the agonist to a wider population of muscarinic receptors. Intramuscular interstitial cells of Cajal (ICC-IMs) and cholinesterases restrict ACh accessibility to a select population of muscarinic receptors, possibly only those expressed by ICC-IMs. These results provide the first biochemical evidence for focalized (or synaptic-like) neurotransmission, rather than diffuse 'volume' neurotransmission in a smooth muscle tissue. Furthermore, these findings demonstrate that bath application of contractile agonists to gastrointestinal smooth muscles does not mimic physiological responses to cholinergic neurotransmission.

Nitric oxide (NO) and cGMP have been shown to be important mediators of penile erection. Erectile dysfunction may result from reduced or non-functional signal transduction within this cascade. There is, however, some inconsistency in the available data as mice lacking NO synthases (endothelial and neuronal nitric oxide synthase, or both) appear to be fertile whereas mice deficient in cGMP-dependent protein kinase I (PKGI) suffer from erectile dysfunction. To clarify this discrepancy we performed studies on mice lacking the NO receptor NO-sensitive guanylyl cyclase (NO-GC). In addition, we generated cell-specific NO-GC knockout (KO) lines to investigate the function of NO in individual cell types. NO-GC was specifically deleted in smooth muscle or endothelial cells (SM-guanylyl cyclase knockout (SM-GCKO) and EC-GCKO, respectively) and these KO lines were compared with total knockouts (GCKO) and wild-type animals. We investigated expression of NO-GC, NO-induced relaxation of corpus cavernosum smooth muscle and their ability to generate offspring. NO-GC-positive immunostaining was detected in smooth muscle and endothelial cells of murine corpus cavernosum but not in interstitial cells of Cajal. NO released from NO donors as well as from nitrergic neurons failed to relax precontracted corpus cavernosum from GCKO mice in organ bath experiments. Similar results were obtained in corpus cavernosum from SM-GCKO mice whereas deletion of NO-GC in endothelial cells did not affect relaxation. The lack of NO-induced relaxation in GCKO animals was not compensated for by guanosine 3,5-cyclic monophosphate (cGMP) signalling. To our surprise, GCKO males were fertile although their ability to produce offspring was decreased. Our data show that deletion of NO-GC specifically in smooth muscle cells abolishes NO-induced corpus cavernosum relaxation but does not lead to infertility.

Adenosine 5′-triphosphate (ATP) has long been considered to be the purine inhibitory neurotransmitter in gastrointestinal (GI) muscles, but recent studies indicate that another purine nucleotide, β-nicotinamide adenine dinucleotide (β-NAD(+)), meets pre- and postsynaptic criteria for a neurotransmitter better than ATP in primate and murine colons. Using a small-volume superfusion assay and HPLC with fluorescence detection and intracellular microelectrode techniques we compared β-NAD(+) and ATP metabolism and postjunctional effects of the primary extracellular metabolites of β-NAD(+) and ATP, namely ADP-ribose (ADPR) and ADP in colonic muscles from cynomolgus monkeys and wild-type (CD38(+/+)) and CD38(−/−) mice. ADPR and ADP caused membrane hyperpolarization that, like nerve-evoked inhibitory junctional potentials (IJPs), were inhibited by apamin. IJPs and hyperpolarization responses to ADPR, but not ADP, were inhibited by the P2Y1 receptor antagonist (1R,2S,4S,5S)-4-[2-iodo-6-(methylamino)-9H-purin-9-yl]-2-(phosphonooxy)bicyclo[3.1.0]hexane-1-methanol dihydrogen phosphate ester tetraammonium salt (MRS2500). Degradation of β-NAD(+) and ADPR was greater per unit mass in muscles containing only nerve processes than in muscles also containing myenteric ganglia. Thus, mechanisms for generation of ADPR from β-NAD(+) and for termination of the action of ADPR are likely to be present near sites of neurotransmitter release. Degradation of β-NAD(+) to ADPR and other metabolites appears to be mediated by pathways besides CD38, the main NAD-glycohydrolase in mammals. Degradation of β-NAD(+) and ATP were equal in colon. ADPR like its precursor, β-NAD(+), mimicked the effects of the endogenous purine neurotransmitter in primate and murine colons. Taken together, our observations support a novel hypothesis in which multiple purines contribute to enteric inhibitory regulation of gastrointestinal motility.

Activation of enteric inhibitory motor neurons causes inhibitory junctional potentials (IJPs) and muscle relaxation in mammalian gastrointestinal (GI) muscles, including humans. IJPs in many GI muscles are bi-phasic with a fast initial hyperpolarization (fIJP) due to release of a purine neurotransmitter and a slower hyperpolarization component (sIJP) due to release of nitric oxide. We sought to characterize the nature of the post-junctional receptor(s) involved in transducing purinergic neural inputs in the murine colon using mice with genetically deactivated P2ry1. Wild-type mice had characteristic biphasic IJPs and pharmacological dissection confirmed that the fIJP was purinergic and the sIJP was nitrergic. The fIJP was completely absent in P2ry1(−/−) mice and the P2Y1 receptor antagonist MRS2500 had no effect on electrical activity or responses to electrical field stimulation of intrinsic nerves in these mice. Contractile experiments confirmed that purinergic responses were abolished in P2ry1(−/−) mice. Picospritzing of neurotransmitter candidates (ATP and its primary metabolite, ADP) and β-NAD (and its primary metabolite, ADP-ribose, ADPR) caused transient hyperpolarization responses in wild-type colons, but responses to β-NAD and ADPR were completely abolished in P2ry1(−/−) mice. Hyperpolarization and relaxation responses to ATP and ADP were retained in colons of P2ry1(−/−) mice. Video imaging revealed that transit of fecal pellets was significantly delayed in colons from P2ry1(−/−) mice. These data demonstrate the importance of purinergic neurotransmission in regulating colonic motility and confirm pharmacological experiments suggesting that purinergic neurotransmission is mediated via P2Y1 receptors.

Nitric oxide (NO) blunts the myogenic response (MR) in renal blood flow (RBF) autoregulation. We sought to clarify the roles of NO synthase (NOS) isoforms, i.e. neuronal NOS (nNOS) from macula densa, endothelial NOS (eNOS) from the endothelium, and inducible NOS (iNOS) from smooth muscle or mesangium. RBF autoregulation was studied in rats and knockout (ko) mice in response to a rapid rise in renal artery pressure (RAP). The autoregulatory rise in renal vascular resistance within the first 6 s was interpreted as MR, from ∼6 to ∼30 s as tubuloglomerular feedback (TGF), and ∼30 to ∼100 s as the third regulatory mechanism. In rats, the nNOS inhibitor SMTC did not significantly affect MR (67 ± 4 vs. 57 ± 4 units). Inhibition of all NOS isoforms by l-NAME in the same animals markedly augmented MR to 78 ± 4 units. The same was found when SMTC was combined with angiotensin II to reproduce the hypertension and vasoconstriction seen with l-NAME (58 ± 3 vs. 54 ± 7 units, l-NAME 81 ± 2 units), or when SMTC was replaced by the nNOS inhibitor NPA (57 ± 5 vs. 56 ± 7 units, l-NAME 79 ± 4 units) or by the iNOS inhibitor 1400W (50 ± 1 vs. 55 ± 4 units, l-NAME 81 ± 3 units). nNOS-ko mice showed the same autoregulation as wild-types (MR 36 ± 4 vs. 38 ± 3 units) and the same response to l-NAME (111 ± 9 vs. 114 ± 10 units). eNOS-ko had similar autoregulation as wild-types (44 ± 8 vs. 33 ± 4 units), but failed to respond to l-NAME (37 ± 7 vs. 78 ± 16 units). We conclude that the attenuating effect of NO on MR depends on eNOS, but not on nNOS or iNOS. In eNOS-ko mice MR is depressed by NO-independent means.

Smooth muscle of the uterus stays remarkably quiescent during normal pregnancy to allow sufficient time for development of the fetus. At present the mechanisms leading to uterine quiescence during pregnancy and how the suppression of activity is relieved at term are poorly understood. Myometrial excitability is governed by ion channels, and a major hypothesis regarding the regulation of contractility during pregnancy has been that expression of certain channels is regulated by hormonal influences. We have explored the expression and function of stretch-dependent K+ (SDK) channels, which are likely to be due to TREK channels, in murine myometrial tissues and myocytes using PCR, Western blots, patch clamp, intracellular microelectrode and isometric force measurements. TREK-1 is more highly expressed than TREK-2 in myometrium, and there was no detectable expression of TRAAK. Expression of TREK-1 transcripts and protein was regulated during pregnancy and delivery. SDK channels were activated in response to negative pressure applied to patches. SDK channels were insensitive to a broad-spectrum of K+ channel blockers, including tetraethylammonium and 4-aminopyridine, and insensitive to intracellular Ca2+. SDK channels were activated by stretch and arachidonic acid and inhibited by reagents that block TREK-1 channels, l-methionine and/or methioninol. Our data suggest that uterine excitability and contractility during pregnancy is regulated by the expression of SDK/TREK-1 channels. Up-regulation of these channels stabilizes membrane potential and controls contraction during pregnancy and down-regulation of these channels induces the onset of delivery.

Hydrophobic bile salts are thought to contribute to the disruption of gallbladder smooth muscle (GBSM) function that occurs in gallstone disease, but their mechanism of action is unknown. The current study was undertaken to determine how hydrophobic bile salts interact with GBSM, and how they reduce GBSM activity. The effect of hydrophobic bile salts on the activity of GBSM was measured by intracellular recording and calcium imaging using wholemount preparations from guinea pig and mouse gallbladder. RT-PCR and immunohistochemistry were used to evaluate expression of the G protein-coupled bile acid receptor, GPBAR1. Application of tauro-chenodeoxycholate (CDC, 50-100 microm) to in situ GBSM rapidly reduced spontaneous Ca(2+) flashes and action potentials, and caused a membrane hyperpolarization. Immunoreactivity and transcript for GPBAR1 were detected in gallbladder muscularis. The GPBAR1 agonist, tauro-lithocholic acid (LCA, 10 microm) mimicked the effect of CDC on GBSM. The actions of LCA were blocked by the protein kinase A (PKA) inhibitor, KT5720 (0.5-1.0 microm) and the K(ATP) channel blocker, glibenclamide (10 microm). Furthermore, LCA failed to disrupt GBSM activity in Gpbar1(/) mice. The findings of this study indicate that hydrophobic bile salts activate GPBAR1 on GBSM, and this leads to activation of the cyclic AMP-PKA pathway, and ultimately the opening of K(ATP) channels, thus hyperpolarizing the membrane and decreasing GBSM activity. This inhibitory effect of hydrophobic bile salt activation of GPBAR1 could be a contributing factor in the manifestation of gallstone disease.

Interstitial cells of Cajal (ICC) isolated from the rabbit urethra exhibit pacemaker activity that results from spontaneous Ca(2+) waves. The purpose of this study was to investigate if this activity was influenced by Ca(2+) uptake into mitochondria. Spontaneous Ca(2+) 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 microm) and antimycin A (5 microm) abolished Ca(2+) waves and increased basal Ca(2+) levels. Similar results were achieved when mitochondria membrane potential was collapsed using the protonophores FCCP (0.2 microm) and CCCP (1 microm). Spontaneous Ca(2+) waves were not inhibited by the ATP synthase inhibitor oligomycin (1 microm), 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 microm) also inhibited STICS. Stimulation of Ca(2+) uptake into mitochondria using the plant flavonoid kaempferol (10 microm) induced a series of propagating Ca(2+) waves. The kaempferol-induced activity was inhibited by application of caffeine (10 mm) or removal of extracellular Ca(2+), but was not significantly affected by the IP(3) receptor blocker 2-APB (100 microm). These data suggest that spontaneous Ca(2+) waves in urethral ICC are regulated by buffering of cytoplasmic Ca(2+) by mitochondria.

Emerging evidence indicates a nociceptive role of vagal afferents. A distinct oesophageal innervation in the rat, with muscular and mucosal afferents travelling predominantly in the recurrent (RLN) and superior laryngeal nerve (SLN), respectively, enabled characterization of mucosal afferents with nociceptive properties, using novel isolated oesophagus-nerve preparations. SLN and RLN single-fibre recordings identified 55 and 14 units, respectively, with none conducting faster than 8.7 m s(-1). Mucosal response characteristics in the SLN distinguished mechanosensors (n = 13), mechanosensors with heat sensitivity (18) from those with cold sensitivity (19) and a mechanoinsensitive group (5). The mechanosensitive fibres, all slowly adapting, showed a unimodal distribution of mechanical thresholds (1.4-128 mN, peak approximately 5.7 mN). No difference in response characteristics of C and Adelta fibres was encountered. Mucosal proton stimulation (pH 5.4 for 3 min), mimicking gastro-oesophageal reflux disease (GORD), revealed in 31% of units a desensitizing response that peaked around 20 s and faded within 60 s. Cold stimulation (15 degrees C) was proportionally encoded but the response showed slow adaptation. In contrast, the noxious heat (48 degrees C) response showed no obvious adaptation with discharge rates reflecting the temperature's time course. Polymodal (69%) mucosal units, > 30% proton sensitive, were found in each fibre category and were considered nociceptors; they are tentatively attributed to vagal nerve endings type I, IV and V, previously morphologically described. All receptive fields were mapped and the distribution indicates that the posterior upper oesophagus may serve as a 'cutbank', detecting noxious matters, ingested or regurgitated, and triggering nocifensive reflexes such as bronchoconstriction in GORD.

Using mutant mice genetically lacking certain subtypes of muscarinic receptor, we have studied muscarinic signal pathways mediating cationic channel activation in intestinal smooth muscle cells. In cells from M2 subtype-knockout (M2-KO) or M3-KO mice, carbachol (100 microM) evoked a muscarinic cationic current (mI(Cat)) as small as approximately 10% of mI(Cat) in wild-type (WT) cells. No appreciable current was evoked in M2/M3 double-KO cells. All mutant type cells preserved normal G-protein-cationic channel coupling. The M3-KO and WT mI(Cat) each showed a U-shaped current-voltage (I-V) relationship, whereas the M2-KO mI(Cat) displayed a linear I-V relationship. Channel analysis in outside-out patches recognized 70-pS and 120-pS channels as the major muscarinic cationic channels. Active patches of M2-KO cells exhibited both 70-pS and 120-pS channel activity usually together, either of which consisted of brief openings (the respective mean open times O(tau) = 0.55 and 0.23 ms). In contrast, active M3-KO patches showed only 70-pS channel activity, which had three open states (O(tau) = 0.55, 3.1 and 17.4 ms). In WT patches, besides the M2-KO and M3-KO types, another type of channel activity was also observed that consisted of 70-pS channel openings with four open states (O(tau) = 0.62, 2.7, 16.9 and 121.1 ms), and patch current of this channel activity showed a U-shaped I-V curve similar to the WT mI(Cat). The present results demonstrate that intestinal myocytes are endowed with three distinct muscarinic pathways mediating cationic channel activation and that the M2/M3 pathway targeting 70-pS channels, serves as the major contributor to mI(Cat) generation. The delineation of this pathway is consistent with the formation of a functional unit by the M2-Go protein and the M3-PLC systems predicted to control cationic channels.

The objectives of this study were to evaluate the physiological importance of the flow and shear generated by phasic contractions of lymphatic vessels and the mechanisms responsible for the influences of such shear on lymphatic pumping. Lymphatic segments of the rat thoracic duct were isolated, cannulated and pressurized. The diastolic diameters were measured in phasically non-active segments. The diastolic and systolic diameters, half-relaxation time (HRT), contraction frequency, ejection fraction and fractional pump flow were determined in phasically active segments. Since imposed flow was excluded, flow and shear occurred only as a result of the intrinsic contractions in phasically active segments whereas in phasically non-active segments contraction-generated flow and shear were absent. The influences of incrementally increased transmural pressure (from 1 to 5 cmH(2)O) were examined in control conditions and after NO synthase blockade (l-NAME 10(-4) m) or cyclooxygenase blockade (indomethacin 10(-5) m). The spontaneous phasic contractions produced a flow-dependent diastolic relaxation. This reduction of the lymphatic tone is a regulatory mechanism that maintains pumping in thoracic duct in an energy-saving/efficient mode: it improves diastolic filling (enhanced lusitropy - lowering HRT), makes lymphatic contractions stronger (enhanced inotropy - higher contraction amplitude) and propels more fluid forward during each contraction (elevated ejection fraction) while decreasing contraction frequency (reduced chronotropy). The findings also demonstrated that the NO pathway, not the cyclooxygenase pathway is responsible for this reduction of lymphatic tone and is the prevailing pathway responsible for the self-regulatory adjustment of thoracic duct pumping to changes in lymph flow pattern.

Gastrointestinal (GI) smooth muscles are influenced by many levels of regulation, including those provided by enteric motor neurones, hormones and paracrine substances. The integrated contractile responses to these regulatory mechanisms depend heavily on the state of excitability of smooth muscle cells. Resting ionic conductances and myogenic responses to agonists and physical parameters, such as stretch, are important in establishing basal excitability. This review discusses the role of 2-pore-domain K+ channels in contributing to background conductances and in mediating responses of GI muscles to enteric inhibitory nerve stimulation and stretch. Murine GI muscles express TREK-1 channels and display a stretch-dependent K+ (SDK) conductance that is also activated by nitric oxide via a cGMP-dependent mechanism. Cloning and expression of mTREK-1 produced an SDK conductance that was activated by cGMP-dependent phosphorylation at serine-351. GI muscle cells also express TASK-1 and TASK-2 channels that are inhibited by lidocaine and external acidification. These conductances appear to provide significant background K+ permeability that contributes to the negative resting potentials of GI muscles.

Smooth muscles from the urethra and bladder display characteristic patterns of spontaneous contractile activity in the filling phase of the micturition cycle. Tonic contractions are seen in the urethral smooth muscles, and phasic contractions occur in the detrusor. Overactivity in the detrusor is a common clinical problem. The ion channels in the smooth muscle membranes play an important role in determining the functional properties, and are obvious targets for treatment of the overactive bladder. Recent evidence suggests that interstitial cells may also play a role in determining the pattern of spontaneous activity, although their precise role is less well established in the urinary tract than in the gut. The ion channels involved in these cells are also of interest. This review discusses what is known of ion channels in these tissues, and their implications for function.

The basic functional unit of the large-conductance, voltage- and Ca2+-activated K+ (MaxiK, BK, BKCa) channel is a tetramer of the pore-forming alpha-subunit (MaxiKalpha) encoded by a single gene, Slo, holding multiple alternative exons. Depending on the tissue, MaxiKalpha can associate with modulatory beta-subunits (beta1-beta4) increasing its functional diversity. As MaxiK senses and regulates membrane voltage and intracellular Ca2+, it links cell excitability with cell signalling and metabolism. Thus, MaxiK is a key regulator of vital body functions, like blood flow, uresis, immunity and neurotransmission. Epilepsy with paroxysmal dyskinesia syndrome has been recognized as a MaxiKalpha-related disorder caused by a gain-of-function C-terminus mutation. This channel region is also emerging as a key recognition module containing sequences for MaxiKalpha interaction with its surrounding signalling partners, and its targeting to cell-specific microdomains. The growing list of interacting proteins highlights the possibility that associations with the C-terminus of MaxiKalpha are dynamic and depending on each cellular environment. We speculate that the molecular multiplicity of the C-terminus (and intracellular loops) dictated by alternative exons may modulate or create additional interacting sites in a tissue-specific manner. A challenge is the dissection of MaxiK macromolecular signalling complexes in different tissues and their temporal association/dissociation according to the stimulus.

Calcium (Ca2+) signals affect virtually every biological process, including both contraction and gene transcription in smooth muscle. Ca2+-regulated gene transcription is known to be important for both physiological and pathological responses in smooth muscle. The aim of this review is to discuss the current understanding of gene transcription regulated by excitation through Ca2+ signalling using a comparison of the two most characterized Ca2+-regulated transcription factors in smooth muscle, Ca2+-cyclic AMP response element binding protein (CREB) and nuclear factor of activated T-cells (NFAT). Recent studies have shown commonalities and differences in the regulation of CREB and NFAT through both voltage- and non-voltage-gated Ca2+ channels that lead to expression of smooth muscle cell specific differentiation markers as well as markers of proliferation. New insights into the regulation of specific genes through companion elements on the promoters of Ca2+-regulated genes have led to new models for transcriptional regulation by Ca2+ that are defined both by the source and duration of the Ca2+ signal and the composition of enhancer elements found within the regulatory regions of specific genes. Thus the combination of signalling pathways elicited by particular Ca2+ signals affect selective promoter elements that are key to the ultimate pattern of gene transcription.

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.

The observation of spontaneous sporadic releases of packets of stored calcium made 20 years ago has opened up a number of new concepts in smooth muscle physiology: (1) the calcium release sites are ryanodine and inositol 1,4,5-trisphosphate (IP3) receptor channels which contribute to cell-wide increases in [Ca2+]i in response to cell depolarization, activation of IP3-generating receptors, or other stimuli; (2) changes in [Ca2+]i act back on the cell membrane to activate or modulate K+, Cl- and cation channel activity so affecting contraction, in arterial smooth muscle for example affecting blood pressure; (3) IP3 production is voltage dependent and is believed to contribute to pacemaker potentials and to refractory periods which control the rhythmical motility of many hollow organs. Most smooth muscle tissues contain interstitial cells (ICs) in addition to contractile smooth muscle cells (SMCs). The interactions of these internal mechanisms, and in turn the interactions of SMCs and ICs in various smooth muscle tissues, are major factors in determining the unique physiological profiles of individual smooth muscles.

The adapter protein paxillin has been implicated in the regulation of cytoskeletal organization and cell motility. Paxillin undergoes tyrosine phosphorylation in response to the contractile stimulation of smooth muscle, and the depletion of paxillin by antisense inhibits smooth muscle contraction. In the present study, acetylcholine (ACh)-stimulation of tracheal smooth muscle tissues increased paxillin phosphorylation at tyr-31 and tyr-118 by three- to fourfold. The role of tyr-31 and tyr-118 phosphorylation of paxillin in smooth muscle was evaluated by introducing plasmids encoding wild type paxillin or paxillin mutants F31, F118 or F31/118 (phenylalanine substitution at tyrosine sites 31, 118) into tracheal smooth muscle strips by reversible permeabilization, and incubating the tissues for 2 days. The expression of recombinant proteins was confirmed by immunoblot and immunofluorescence analysis. Expression of the paxillin mutants F31, F118 or F31/118 inhibited the contractile response to ACh stimulation but did not inhibit the increase in myosin light chain phosphorylation. The expression of wild type paxillin had no significant affect on force or myosin light chain phosphorylation. ACh stimulation reduced G-actin/F-actin ratio in tissues expressing wild type paxillin; whereas the agonist-induced decrease in G-actin/F-actin was inhibited in strips expressing paxillin mutant F31/118. The paxillin mutant F31/118 showed a marked decrease in their interaction with the SH2/SH3 adaptor protein CrkII but not with vinculin or focal adhesion kinase. We conclude that paxillin phosphorylation at tyr-31 and tyr-118 regulates active tension development during contractile stimulation. Paxillin phosphorylation at these two sites may be important in regulating actin filament dynamics and organization during smooth muscle contraction.

It has been suggested that magnesium deficiency is correlated with many diseases. 31P NMR experiments were carried out in order to investigate the effects of Na+ substitution on Mg2+ depletion in smooth muscle under divalent cation-free conditions. In the taenia of guinea-pig caeci, the intracellular free Mg2+ concentration ([Mg2+]i) was estimated from the chemical shifts of (1) the beta-ATP peak alone and (2) beta- and gamma-ATP peaks. Both estimations indicated that [Mg2+]i decreased only very slowly in Mg(2+)-free, Ca(2+)-free solutions in which Na+ was substituted with large cations such as NMDG (N-methyl-D-glucamine) and choline. Furthermore, the measurements of tension development supported the suggestion of preservation of intracellular Mg2+ with NMDG substitution. Substituting extracellular Na+ with the small cation, Li+, also shifted the beta-ATP peak towards a lower frequency, but the frequency shift was significantly less than that seen upon Na+ substitution with K+. The estimated [Mg2+]i depletion was, however, comparable with that seen after Na+ substitution with K+ using the titration curves of metal-free and Mg(2+)-bound ATP obtained in Li(+)-based model solutions. It was concluded that Mg2+ rapidly decreases only when small cations were the major electrolyte of the extracellular medium. Na+ substitutions with NMDG, choline or Li+ had little effect on intracellular ATP concentration after 100 min treatment.

The p21-activated protein kinases (PAKs) have been implicated in cytoskeletal rearrangements and modulation of non-muscle contractility. Little, however, is known about the role of the PAK family members in smooth muscle contraction. Therefore, we investigated the effect of the predominant isoform in vascular smooth muscle cells, PAK1, on contraction and phosphorylation of the regulatory light chains of myosin (r-MLC) in Triton-skinned guinea-pig smooth muscle. We also investigated which of the three putative substrates at the contractile apparatus - MLCK, caldesmon or r-MLC - is phosphorylated by PAK1 in smooth muscle tissue. Incubation of Triton-skinned carotid artery and taenia coli from guinea-pig with an active mutant of PAK1 in relaxing solution for 30-60 min resulted in inhibition of submaximal force by about 50 %. The mechanism of inhibition of force was studied in the Triton-skinned taenia coli. In this preparation, inhibition of force was associated with a respective inhibition of r-MLC phosphorylation. In the presence of the myosin phosphatase inhibitor, microcystin-LR (10 microM), the rate of contraction and r-MLC phosphorylation elicited at pCa 6.79 were both decreased. Because under these conditions the rate of r-MLC phosphorylation is solely dependent on MLCK activity, this result suggests that the inhibitory effect of PAK1 on steady-state force and r-MLC phosphorylation is due to inhibition of MLCK. In line with this, we found that MLCK was significantly phosphorylated by PAK1 while there was very little 32P incorporation into caldesmon. PAK1 phosphorylated isolated r-MLC but not those in the skinned fibres or in purified smooth muscle myosin II. In conclusion, these results suggest that PAK1 attenuates contraction of skinned smooth muscle by phosphorylating and inhibiting MLCK.

Modulation of spontaneous electrical activities (slow waves, pacemaker potentials and follower potentials) in response to hyperpolarization produced by the ATP-sensitive K+ channel openers (KCOs) pinacidil or nicorandil was investigated in smooth muscle tissues of the guinea-pig stomach antrum. With hyperpolarization, the amplitude of slow waves and follower potentials was reduced and that of pacemaker potentials was increased, with a minor modulation of their frequency. The attenuation of slow waves was associated with an inhibition of the 1st component and abolition of the 2nd component. All these actions of KCOs were antagonized by glibenclamide. An increase in the extracellular K+ concentration prevented the KCO-induced hyperpolarization with partial restoration of slow waves, suggesting that the inhibition was produced mainly by a decrease in membrane resistance. Exposure of tissues to KCOs for a long period of time (> 20 min) resulted in the reappearance of slow waves displaying both 1st and 2nd components. The 2nd component of the slow wave, which displayed a slower recovery, was inhibited again by 5-hydroxydecanoic acid, an inhibitor of mitochondrial ATP-sensitive K+ channels. Noradrenaline hyperpolarized the membrane by activating apamin-sensitive K+ channels and increased the amplitude and frequency of slow waves through activation of alpha 1-adrenoceptors, actions different from those of KCOs. Thus, inhibition of slow waves by KCOs may be primarily related to the decrease in amplitude of a passive electrotonic component, possibly due to a reduction of the input resistance. The hyperpolarization shifted the threshold potential for generation of the 2nd component of slow waves to negative levels, presumably due to modulation of mitochondrial functions.

The seven amino acid insert in the smooth muscle myosin heavy chain is thought to regulate the kinetics of contraction, contributing to the differences between fast and slow smooth muscle. The effects of this insert on force and stiffness were determined in bladder tissue of a transgenic mouse line expressing the insert SMB at one of three levels: an SMB wild type (+/+), an SMA homozygous type (-/-) and a heterozygous type (+/-). For skinned muscle, an increase in MgADP or inorganic phosphate (Pi) should shift the distribution of crossbridges in the actomyosin ATPase (AMATPase) to increase the relative population of the crossbridge state prior to ADP release and Pi release, respectively. Exogenous ADP increased force and stiffness in a manner consistent with increasing the Ca2+ concentration in both the +/+ and +/- mouse types. However, the -/- type showed a significantly greater increase in force than in stiffness suggesting that immediately prior to ADP release, the AMATPase either has an additional force producing isomerization state or a slower ADP dissociation rate for the -/- type compared to the +/+ or +/- types. Exogenous Pi led to a significantly greater decrease in stiffness than in force for all three mouse types suggesting that there is a force producing state prior to Pi release. In addition, the increase in Pi showed similar changes in the +/+ and -/- types whereas in the +/- type the decreases in both force and stiffness were greater than the other two mouse types indicating that the insert can affect the cooperativity between myosin heads. In conclusion, the seven amino acid insert modulates the kinetics and/or states of the AMATPase, which could lead to differences in the kinetics of contraction between fast and slow smooth muscle.

Myosin phosphatase (MLCP) plays a critical regulatory role in the Ca(2+) sensitivity of myosin phosphorylation and smooth muscle contraction. It has been suggested that phosphorylation at Thr(695) of the MLCP regulatory subunit (MYPT1) and at Thr(38) of the MLCP inhibitor protein CPI-17 results in inhibition of MLCP activity. We have previously demonstrated that CPI-17 Thr(38) phosphorylation plays an important role in G-protein-mediated inhibition of MLCP in tonic arterial smooth muscle. Here, we attempted to evaluate the function of MYPT1 in phasic rabbit portal vein (PV) and vas deferens (VD) smooth muscles. Using site- and phospho-specific antibodies, phosphorylation of MYPT1 Thr(695) and CPI-17 Thr(38) was examined along with MYPT1 Thr(850), which is a non-inhibitory Rho-kinase site. We found that both CPI-17 Thr(38) and MYPT1 Thr(850) were phosphorylated in response to agonists or GTPgammaS concurrently with contraction and myosin phosphorylation in alpha-toxin-permeabilized PV tissues. In contrast, phosphorylation of MYPT1 Thr(695) did not increase. Comparable results were also obtained in both permeabilized and intact VD. The Rho-kinase inhibitor Y-27632 and the protein kinase C (PKC) inhibitor GF109203X suppressed phosphorylation of MYPT1 Thr(850) and CPI-17 Thr(38), respectively, in intact VD while MYPT1 Thr(695) phosphorylation was insensitive to both inhibitors. These results indicate that phosphorylation of MYPT1 Thr(695) is independent of stimulation of G-proteins, Rho-kinase or PKC. In the phasic PV, phosphorylation of CPI-17 Thr(38) may contribute towards inhibition of MLCP while the phasic visceral VD, which has a low CPI-17 concentration, probably utilizes other Ca(2+) sensitizing mechanisms for inhibiting MLCP besides phosphorylation of MYPT1 and CPI-17.

Ca2+ wave activity was monitored in the longitudinal (LM) layer of isolated murine caecum and proximal colon at 35 degrees C with fluo-4 AM and an iCCD camera. Both intracellular (within LM cells) and intercellular (also spreading from cell to cell) Ca2+ waves were observed. Intracellular Ca2+ waves were associated with a lack of muscle movement whereas intercellular Ca2+ waves, which were five times more intense than intracellular waves, were often associated with localized contractions. Several intracellular Ca2+ waves were present at the same time in individual LM cells. Waves in adjacent LM cells were not coordinated and were unaffected by TTX (1 microM) but were blocked by IP3 receptor antagonists xestospongin-C (Xe-C; 2 microM) or 2-aminoethyl diphenylborate (2-APB; 25 microM), and by ryanodine (10 microM). Caffeine (5 mM) restored wave activity following blockade with Xe-C. NiCl2 (1 mM) blocked intracellular Ca2+ waves, and nicardipine (2 microM) reduced their frequency and intensity, but did not affect their velocity, suggesting the sarcoplasmic reticulum may be fuelled by extracellular Ca2+ entry. Intercellular Ca2+ waves often occurred in bursts and propagated rapidly across sizeable regions of the LM layer and were blocked by heptanol (0.5 mM). Intercellular Ca2+ waves were dependent upon neural activity, external Ca2+ entry through L-type Ca2+ channels, and amplification via calcium-induced calcium release (CICR). In conclusion, intracellular Ca2+ waves, which may reduce muscle excitability, are confined to individual LM cells. They depend upon Ca2+ release from internal Ca2+ stores and are likely to be fuelled by extracellular Ca2+ entry. Intercellular Ca2+ waves, which are likely to underlie smooth muscle tone, mixing and propulsion, depend upon neural activity, muscle action potential propagation and amplification by CICR.

Possible interactions between different intracellular Ca(2+) release channels were studied in isolated rat gastric myocytes using agonist-evoked Ca(2+) signals. Spontaneous, local Ca(2+) transients were observed in fluo-4-loaded cells with linescan confocal imaging. These were blocked by ryanodine (100 microM) but not by the inositol 1,4,5-trisphosphate receptor (IP(3)R) blocker, 2-aminoethoxydiphenyl borate (100 microM), identifying them as Ca(2+) sparks. Caffeine (10 mM) and carbachol (10 microM) initiated Ca(2+) release at sites which co-localized with each other and with any Ca(2+) spark sites. In fura-2-loaded cells extracellular 2-aminoethoxydiphenyl borate and intracellular heparin (5 mg ml(-1)) both inhibited the global cytoplasmic [Ca(2+)] transient evoked by carbachol, confirming that it was IP(3)R-dependent. 2-Aminoethoxydiphenyl borate and heparin also increased the response to caffeine. This probably reflected an increased Ca(2+) store content since 2-aminoethoxydiphenyl borate more than doubled the amplitude of transients evoked by ionomycin. Ryanodine completely abolished carbachol and caffeine responses but only reduced ionomycin transients by 30 %, suggesting that blockade of carbachol transients by ryanodine was not simply due to store depletion. Double labelling of IP(3)Rs and RyRs demonstrated extensive overlap in their distribution. These results suggest that carbachol stimulates Ca(2+) release through co-operation between IP(3)Rs and RyRs, and implicate IP(3)Rs in the regulation of Ca(2+) store content.

A confocal Ca2+ imaging technique has been used to detect ATP release from individual sympathetic varicosities on the same nerve terminal branch. Varicose nerve terminals and smooth muscle cells in mouse vas deferens were loaded with the Ca2+ indicator Oregon Green 488 BAPTA-1. Field (nerve) stimulation evoked discrete, focal increases in [Ca2+] in smooth muscle cells adjacent to identified varicosities. These focal increases in [Ca2+] have been termed 'neuroeffector Ca2+ transients' (NCTs). NCTs were abolished by alpha,beta-methylene ATP (1 microM), but not by nifedipine (1 microM) or prazosin (100 nM), suggesting that NCTs are generated by Ca2+ influx through P2X receptors without a detectable contribution from L-type Ca2+ channels or alpha(1)-adrenoceptor-mediated pathways. Action potential-evoked ATP release was highly intermittent (mean probability 0.019 +/- 0.002; range 0.001-0.10) at 1 Hz stimulation, even though there was no failure of action potential propagation in the nerve terminals. Twenty-eight per cent of varicosities failed to release transmitter following more than 500 stimuli. Spontaneous ATP release was very infrequent (0.0014 Hz). No Ca2+ transient attributable to noradrenaline release was detected even in response to 5 Hz stimulation. There was evidence of local noradrenaline release as the alpha(2)-adrenoceptor antagonist yohimbine increased the probability of occurrence of NCTs by 55 +/- 21 % during trains of stimuli at 1 Hz. Frequency-dependent facilitation preferentially occurred at low probability release sites. The monitoring of NCTs now allows transmitter release to be detected simultaneously from each functional varicosity on an identified nerve terminal branch on an impulse-to-impulse basis.

To study the contribution of the Na(+)-Ca(2+) exchanger to Ca(2+) regulation and its interaction with the sarcoplasmic reticulum (SR), changes in cytoplasmic Ca(2+) concentration ([Ca(2+)](c)) were measured in single, voltage clamped, smooth muscle cells. Increases in [Ca(2+)](c) were evoked by either depolarisation (-70 mV to 0 mV) or by release from the SR by caffeine (10 mM) or flash photolysis of caged InsP(3) (InsP(3)). Depletion of the SR of Ca(2+) (verified by the absence of a response to caffeine and InsP(3)) by either ryanodine (50 microM), to open the ryanodine receptors (RyRs), or thapsigargin (500 nM) or cyclopiazonic acid (CPA, 10 microM), to inhibit the SR Ca(2+) pumps, reduced neither the magnitude of the Ca(2+) transient nor the relationship between the influx of and the rise in [Ca(2+)](c) evoked by depolarisation. This suggested that Ca(2+)-induced Ca(2+) release (CICR) from the SR did not contribute significantly to the depolarisation-evoked rise in [Ca(2+)](c). However, although Ca(2+) was not released from it, the SR accumulated the ion following depolarisation since ryanodine and thapsigargin each slowed the rate of decline of the depolarisation-evoked Ca(2+) transient. Indeed, the SR Ca(2+) content increased following depolarisation as assessed by the increased magnitude of the [Ca(2+)](c) levels evoked each by InsP(3) and caffeine, relative to controls. The increased SR Ca(2+) content following depolarisation returned to control values in approximately 12 min via Na(+)-Ca(2+) exchanger activity. Thus inhibition of the Na(+)-Ca(2+) exchanger by removal of external Na(+) (by either lithium or choline substitution) prevented the increased SR Ca(2+) content from returning to control levels. On the other hand, the Na(+)-Ca(2+) exchanger did not appear to regulate bulk average Ca(2+) directly since the rates of decline in [Ca(2+)](c), following either depolarisation or the release of Ca(2+) from the SR (by either InsP(3) or caffeine), were neither voltage nor Na(+) dependent. Thus, no evidence for short term (seconds) control of [Ca(2+)](c) by the Na(+)-Ca(2+) exchanger was found. Together, the results suggest that despite the lack of CICR, the SR removes Ca(2+) from the cytosol after its elevation by depolarisation. This Ca(2+) is then removed from the SR to outside the cell by the Na(+)-Ca(2+) exchanger. However, the exchanger does not contribute significantly to the decline in bulk average [Ca(2+)](c) following transient elevations in the ion produced either by depolarisation or by release from the store.

The transient rise of intracellular Ca(2+) in detrusor smooth muscle cells is due to the release of Ca(2+) from intracellular stores. However, it is not known how store refilling is maintained at a constant level to ensure constancy of the contractile response. The aim of these experiments was to characterise the role of L-type Ca(2+) channels in refilling. Experiments used isolated guinea-pig detrusor myocytes and store Ca(2+) content was estimated by measuring the magnitude of change to the intracellular [Ca(2+)] ([Ca(2+)](i)) after application of caffeine or carbachol using epifluorescence microscopy. Membrane potential was controlled when necessary by voltage clamp. After Ca(2+) stores were emptied they refilled with an exponential time course, with a time constant of 88 s. The value of the time constant was similar to that of the undershoot of [Ca(2+)](i) following store Ca(2+) release. The degree of store filling was enhanced by maintained depolarisation, or by transient depolarising pulses, and attenuated by L-type Ca(2+) channel antagonists. Inhibition of the sarcoplasmic reticular Ca(2+)-ATPase prevented refilling. Reduction of the resting [Ca(2+)](i) was accompanied by membrane depolarisation; under voltage clamp reduction of [Ca(2+)](i) decreased the number and magnitude of spontaneous transient outward currents. Ca(2+) release from intracellular stores, elicited by caffeine or carbachol, is independent of membrane potential under physiological conditions. However, store refilling occurs via Ca(2+) influx through L-type Ca(2+) channels. Ca(2+) influx is regulated by a feedback mechanism whereby a fall of [Ca(2+)](i) reduces the activity of Ca(2+)-activated K(+) channels, causing cell depolarisation and an enhancement of L-type Ca(2+) channel conductance.

1. Extracellular application of ATP generates two whole-cell currents in toad gastric smooth muscle cells: an immediate inward non-selective cation current (due to the activation of a P2X or P2Z-like receptor) and a slowly developing outward K(+) current. The inward non-selective cation current depends on the continuous presence of ATP while the outward K(+) current can last for minutes after ATP application ceases. 2. In cell-attached patches, application of ATP to the extra-patch membrane can activate K(+) channels in the patch indicating that a diffusible cellular messenger may be involved. The characteristics of these K(+) channels are similar to those of a previously described fatty acid-activated K(+) channel that is also a stretch-activated channel. 3. This whole-cell K(+) current can be induced by ATP in the absence of extracellular Ca(2+) (with EGTA present to chelate trace amounts). However, the current generated in the presence of extracellular Ca(2+) is considerably larger. 4. The pharmacological profiles for the activation of the non-selective cation current and the K(+) current are similar, suggesting that the same P2Z-like receptor could be mediating both responses. This type of plasma membrane receptor/channel-channel coupling by a process that does not appear to involve Ca(2+) flow through the receptor/channel or a subsequent membrane potential change may be representative of a new class of signalling mechanisms.

1. The role of the sarcoplasmic reticulum (SR) was investigated in spontaneous and agonist-induced uterine Ca2+ transients, by combining low- (mag-fluo-4) and high-affinity (fura-2) indicators to measure intraluminal SR ([Ca2+]L) and cytosolic ([Ca2+]i) calcium concentration, simultaneously, in single smooth muscle cells from pregnant rat uterus. 2. Carbachol or ATP, in the absence of extracellular Ca2+, decreased [Ca2+]L and increased [Ca2+]i. Although some replenishment (around 50 %) occurred in its absence, extracellular Ca2+ was required for full replenishment of the SR Ca2+. 3. In 4/15 cells, ATP evoked oscillations of [Ca2+]i. These were accompanied by successive release and re-uptake of SR Ca2+. Inhibition of the SR Ca2+-ATPase with thapsigargin abolished the oscillations and luminal changes. 4. Spontaneous [Ca2+]i transients produced no detectable changes in [Ca2+]L. The larger [Ca2+]i transients evoked by high-K+ depolarisation increased [Ca2+]L. Spontaneous activity was inhibited when [Ca2+]L was increased. 5. These data show that it is possible to simultaneously measure SR and cytosolic [Ca2+], and to investigate their response to agonist application and spontaneous activity.

The intracellular calcium concentration ([Ca2+]i) was monitored in fura-2-loaded myocytes isolated from the rat gastric antrum and voltage clamped at -60 1r1rqmV1qusing the perforated patch clamp technique. The rate of quench of fura-2 fluorescence by Mn2+ was used as a measure of capacitative Ca2+ entry. Cyclopiazonic acid (5 microM) did not affect the holding current but produced a sustained elevation in steady-state [Ca2+]i that was dependent on the presence of external calcium. Cyclopiazonic acid increased Mn2+ influx with physiological external [Ca2+], but not in Ca2+-free conditions. Cyclopiazonic acid increased the rate of [Ca2+]i rise following a rapid switch from Ca2+-free to physiological [Ca2+] solution. Sustained application of carbachol (10 microM) produced an elevation in steady-state [Ca2+]i that was associated with an increased rate of Mn2+ influx. Application of cyclopiazonic acid in the presence of carbachol further elevated steady-state [Ca2+]i without changing Mn2+ influx. Ryanodine (10 microM) elevated steady-state [Ca2+]i either on its own or following a brief application of caffeine (10 9i1s1sqmMc1q). Cyclopiazonic acid had no further effect when added to cells pre-treated with ryanodine. Neither caffeine nor ryanodine increased the rate of Mn2+ influx. When brief applications of ionomycin (25 microM) in Ca2+-free solution were used to release stored Ca2+, ryanodine reduced the amplitude of the resulting [Ca2+]i transients by approximately 30 %, indicating that intracellular stores were partially depleted. These findings suggest that continual uptake of Ca2+ by the sarcoplasmic reticulum Ca2+-ATPase into a ryanodine-sensitive store limits the bulk cytoplasmic [Ca2+]i under resting conditions. This pathway can be short circuited by 10 microM ryanodine, presumably by opening Ca2+ channels in the sarcoplasmic reticulum. Depletion of stores with cyclopiazonic acid or carbachol also activates capacitative Ca2+ entry.