Intraluminal pressure elevates intracellular calcium and contracts CNS pericytes: Role of voltage-dependent calcium channels.

Arteriolar smooth muscle cells (SMCs) and capillary pericytes dynamically regulate blood flow in the central nervous system in the face of fluctuating perfusion pressures. Pressure-induced depolarization and Ca2+ elevation provide a mechanism for regulation of SMC contraction, but whether pericytes participate in pressure-induced changes in blood flow remains unknown. Here, utilizing a pressurized whole-retina preparation, we found that increases in intraluminal pressure in the physiological range induce contraction of both dynamically contractile pericytes in the arteriole-proximate transition zone and distal pericytes of the capillary bed. We found that the contractile response to pressure elevation was slower in distal pericytes than in transition zone pericytes and arteriolar SMCs. Pressure-evoked elevation of cytosolic Ca2+ and contractile responses in SMCs were dependent on voltage-dependent Ca2+ channel (VDCC) activity. In contrast, Ca2+ elevation and contractile responses were partially dependent on VDCC activity in transition zone pericytes and independent of VDCC activity in distal pericytes. In both transition zone and distal pericytes, membrane potential at low inlet pressure (20 mmHg) was approximately -40 mV and was depolarized to approximately -30 mV by an increase in pressure to 80 mmHg. The magnitude of whole-cell VDCC currents in freshly isolated pericytes was approximately half that measured in isolated SMCs. Collectively, these results indicate a loss of VDCC involvement in pressure-induced constriction along the arteriole-capillary continuum. They further suggest that alternative mechanisms and kinetics of Ca2+ elevation, contractility, and blood flow regulation exist in central nervous system capillary networks, distinguishing them from neighboring arterioles.

Urothelium-derived prostanoids enhance contractility of urinary bladder smooth muscle and stimulate bladder afferent nerve activity in the mouse.

The transitional epithelial cells (urothelium) that line the lumen of the urinary bladder form a barrier between potentially harmful pathogens, toxins, and other bladder contents and the inner layers of the bladder wall. The urothelium, however, is not simply a passive barrier, as it can produce signaling factors, such as ATP, nitric oxide, prostaglandins, and other prostanoids, that can modulate bladder function. We investigated whether substances produced by the urothelium could directly modulate the contractility of the underlying urinary bladder smooth muscle. Force was measured in isolated strips of mouse urinary bladder with the urothelium intact or denuded. Bladder strips developed spontaneous tone and phasic contractions. In urothelium-intact strips, basal tone, as well as the frequency and amplitude of phasic contractions, were 25%, 32%, and 338% higher than in urothelium-denuded strips, respectively. Basal tone and phasic contractility in urothelium-intact bladder strips were abolished by the cyclooxygenase (COX) inhibitor indomethacin (10 µM) or the voltage-dependent Ca2+ channel blocker diltiazem (50 µM), whereas blocking neuronal sodium channels with tetrodotoxin (1 µM) had no effect. These results suggest that prostanoids produced in the urothelium enhance smooth muscle tone and phasic contractions by activating voltage-dependent Ca2+ channels in the underlying bladder smooth muscle. We went on to demonstrate that blocking COX inhibits the generation of transient pressure events in isolated pressurized bladders and greatly attenuates the afferent nerve activity during bladder filling, suggesting that urothelial prostanoids may also play a role in sensory nerve signaling.NEW & NOTEWORTHY This paper provides evidence for the role of urothelial-derived prostanoids in maintaining tone in the urinary bladder during bladder filling, not only underscoring the role of the urothelium as more than a barrier but also contributing to active regulation of the urinary bladder. Furthermore, cyclooxygenase products greatly augment sensory nerve activity generated by bladder afferents during bladder filling and thus may play a role in perception of bladder fullness.

Afferent nerve activity in a mouse model increases with faster bladder filling rates in vitro, but voiding behavior remains unaltered in vivo.

Storage and voiding functions in urinary bladder are well-known, yet fundamental physiological events coordinating these behaviors remain elusive. We sought to understand how voiding function is influenced by the rate at which the bladder fills. We hypothesized that faster filling rates would increase afferent sensory activity and increase micturition rate. In vivo, this would mean animals experiencing faster bladder filling would void more frequently with smaller void volumes. To test this hypothesis, we measured afferent nerve activity during different filling rates using an ex vivo mouse bladder preparation and assessed voiding frequency in normally behaving mice noninvasively (UroVoid). Bladder afferent nerve activity depended on the filling rate, with faster filling increasing afferent nerve activity at a given volume. Voiding behavior in vivo was measured in UroVoid cages. Male and female mice were given access to tap water or, to induce faster bladder filling rates, water containing 5% sucrose. Fluid intake increased dramatically in mice consuming 5% sucrose. As expected, micturition frequency was elevated in the sucrose group. However, even with the greatly increased rate of urine production, void volumes were unchanged in both genders. Although faster filling rates generated higher afferent nerve rates ex vivo, this did not translate into more frequent, smaller-volume voids in vivo. This suggests afferent nerve activity is only one factor contributing to the switch from bladder filling to micturition. Together with afferent nerve activity, higher centers in the central nervous system and the state of arousal are likely critical to coordinating the micturition reflex.

The KV 7 channel activator retigabine suppresses mouse urinary bladder afferent nerve activity without affecting detrusor smooth muscle K+ channel currents.

KEY POINTS: KV 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 KV 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 KV 7 channel currents. However, the KV 7 channel agonist retigabine nearly abolished sensory nerve outflow from the urinary bladder during bladder filling. We conclude that KV 7 channel activation likely affects urinary bladder function by blocking afferent nerve outflow to the brain, which is key to sensing bladder fullness. ABSTRACT: KV 7 channels are voltage-dependent K+ channels that open in response to membrane depolarization to regulate cell excitability. KV 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 KV 7 channels as regulators of murine urinary bladder function. The KV 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 KV 7 channels. The KV 7 inhibitor XE-991 (10 µM) inhibited UBSM K+ currents; the properties of these currents, however, were typical of KV 2 channels and not KV 7 channels. Retigabine inhibited voltage-dependent Ca2+ 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 KV 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 KV 2 channels.

TRPV4 blockade reduces voiding frequency, ATP release, and pelvic sensitivity in mice with chronic urothelial overexpression of NGF.

Transient receptor potential vanilloid family member 4 (TRPV4) transcript and protein expression increased in the urinary bladder and lumbosacral dorsal root ganglia of transgenic mice with chronic urothelial overexpression of nerve growth factor (NGF-OE). We evaluated the functional role of TRPV4 in bladder function with open-outlet cystometry, void spot assays, and natural voiding (Urovoid) assays with the TRPV4 antagonist HC-067047 (1 µM) or vehicle in NGF-OE and littermate wild-type (WT) mice. Blockade of TRPV4 at the level of the urinary bladder significantly (P ≤ 0.01) increased the intercontraction interval (2.2-fold) and void volume (2.6-fold) and decreased nonvoiding contractions (3.0-fold) in NGF-OE mice, with lesser effects (1.3-fold increase in the intercontraction interval and 1.3-fold increase in the void volume) in WT mice. Similar effects of TRPV4 blockade on bladder function in NGF-OE mice were demonstrated with natural voiding assays. Intravesical administration of HC-067047 (1 µM) significantly (P ≤ 0.01) reduced pelvic sensitivity in NGF-OE mice but was without effect in littermate WT mice. Blockade of urinary bladder TRPV4 or intravesical infusion of brefeldin A significantly (P ≤ 0.01) reduced (2-fold) luminal ATP release from the urinary bladder in NGF-OE and littermate WT mice. The results of the present study suggest that TRPV4 contributes to luminal ATP release from the urinary bladder and increased voiding frequency and pelvic sensitivity in NGF-OE mice.

Development of stress-induced bladder insufficiency requires functional TRPV1 channels.

Social stress causes profound urinary bladder dysfunction in children that often continues into adulthood. We previously discovered that the intensity and duration of social stress influences whether bladder dysfunction presents as overactivity or underactivity. The transient receptor potential vanilloid type 1 (TRPV1) channel is integral in causing stress-induced bladder overactivity by increasing bladder sensory outflow, but little is known about the development of stress-induced bladder underactivity. We sought to determine if TRPV1 channels are involved in bladder underactivity caused by stress. Voiding function, sensory nerve activity, and bladder wall remodeling were assessed in C57BL/6 and TRPV1 knockout mice exposed to intensified social stress using conscious cystometry, ex vivo afferent nerve recordings, and histology. Intensified social stress increased void volume, intermicturition interval, bladder volume, and bladder wall collagen content in C57BL/6 mice, indicative of bladder wall remodeling and underactive bladder. However, afferent nerve activity was unchanged and unaffected by the TRPV1 antagonist capsazepine. Interestingly, all indices of bladder function were unchanged in TRPV1 knockout mice in response to social stress, even though corticotrophin-releasing hormone expression in Barrington's Nucleus still increased. These results suggest that TRPV1 channels in the periphery are a linchpin in the development of stress-induced bladder dysfunction, both with regard to increased sensory outflow that leads to overactive bladder and bladder wall decompensation that leads to underactive bladder. TRPV1 channels represent an intriguing target to prevent the development of stress-induced bladder dysfunction in children.

Inhibition of vascular smooth muscle inward-rectifier K+ channels restores myogenic tone in mouse urinary bladder arterioles.

Prolonged decreases in urinary bladder blood flow are linked to overactive and underactive bladder pathologies. However, the mechanisms regulating bladder vascular reactivity are largely unknown. To investigate these mechanisms, we examined myogenic and vasoactive properties of mouse bladder feed arterioles (BFAs). Unlike similar-sized arterioles from other vascular beds, BFAs failed to constrict in response to increases in intraluminal pressure (5-80 mmHg). Consistent with this lack of myogenic tone, arteriolar smooth muscle cell membrane potential was hyperpolarized (-72.8 ± 1.4 mV) at 20 mmHg and unaffected by increasing pressure to 80 mmHg (-74.3 ± 2.2 mV). In contrast, BFAs constricted to the thromboxane analog U-46619 (100 nM), the adrenergic agonist phenylephrine (10 µM), and KCl (60 mM). Inhibition of nitric oxide synthase or intermediate- and small-conductance Ca2+-activated K+ channels did not alter arteriolar diameter, indicating that the dilated state of BFAs is not attributable to overactive endothelium-dependent dilatory influences. Myocytes isolated from BFAs exhibited BaCl2 (100 µM)-sensitive K+ currents consistent with strong inward-rectifier K+ (KIR) channels. Notably, block of these KIR channels "restored" pressure-induced constriction and membrane depolarization. This suggests that these channels, in part, account for hyperpolarization and associated absence of tone in BFAs. Furthermore, smooth muscle-specific knockout of KIR2.1 caused significant myogenic tone to develop at physiological pressures. This suggests that 1) the regulation of vascular tone in the bladder is independent of pressure, insofar as pressure-induced depolarizing conductances cannot overcome KIR2.1-mediated hyperpolarization; and 2) maintenance of bladder blood flow during bladder filling is likely controlled by neurohumoral influences.

Purinergic signalling underlies transforming growth factor-ß-mediated bladder afferent nerve hyperexcitability.

KEY POINTS: The sensory components of the urinary bladder are responsible for the transduction of bladder filling and are often impaired with neurological injury or disease. Elevated extracellular ATP contributes, in part, to bladder afferent nerve hyperexcitability during urinary bladder inflammation or irritation. Transforming growth factor-ß1 (TGF-ß1) may stimulate ATP release from the urothelium through vesicular exocytosis mechanisms with minimal contribution from pannexin-1 channels to increase bladder afferent nerve discharge. Bladder afferent nerve hyperexcitability and urothelial ATP release with CYP-induced cystitis is decreased with TGF-ß inhibition. These results establish a causal link between an inflammatory mediator, TGF-ß, and intrinsic signalling mechanisms of the urothelium that may contribute to the altered sensory processing of bladder filling. ABSTRACT: The afferent limb of the micturition reflex is often compromised following bladder injury, disease and inflammatory conditions. We have previously demonstrated that transforming growth factor-ß (TGF-ß) signalling contributes to increased voiding frequency and decreased bladder capacity with cystitis. Despite the functional presence of TGF-ß in bladder inflammation, the precise mechanisms of TGF-ß mediating bladder dysfunction are not yet known. Thus, the present studies investigated the sensory components of the urinary bladder that may underlie the pathophysiology of aberrant TGF-ß activation. We utilized bladder-pelvic nerve preparations to characterize bladder afferent nerve discharge and the mechanisms of urothelial ATP release with distention. Our findings indicate that bladder afferent nerve discharge is sensitive to elevated extracellular ATP during pathological conditions of urinary bladder inflammation or irritation. We determined that TGF-ß1 may increase bladder afferent nerve excitability by stimulating ATP release from the urothelium via vesicular exocytosis mechanisms with minimal contribution from pannexin-1 channels. Furthermore, blocking aberrant TGF-ß signalling in cyclophosphamide-induced cystitis with TßR-1 inhibition decreased afferent nerve hyperexcitability with a concomitant decrease in urothelial ATP release. Taken together, these results establish a role for purinergic signalling mechanisms in TGF-ß-mediated bladder afferent nerve activation that may ultimately facilitate increased voiding frequency. The synergy between intrinsic urinary bladder signalling mechanisms and an inflammatory mediator provides novel insight into bladder dysfunction and supports new avenues for therapeutic intervention.

Social stress in mice induces urinary bladder overactivity and increases TRPV1 channel-dependent afferent nerve activity.

Social stress has been implicated as a cause of urinary bladder hypertrophy and dysfunction in humans. Using a murine model of social stress, we and others have shown that social stress leads to bladder overactivity. Here, we show that social stress leads to bladder overactivity, increased bladder compliance, and increased afferent nerve activity. In the social stress paradigm, 6-wk-old male C57BL/6 mice were exposed for a total of 2 wk, via barrier cage, to a C57BL/6 retired breeder aggressor mouse. We performed conscious cystometry with and without intravesical infusion of the TRPV1 inhibitor capsazepine, and measured pressure-volume relationships and afferent nerve activity during bladder filling using an ex vivo bladder model. Stress leads to a decrease in intermicturition interval and void volume in vivo, which was restored by capsazepine. Ex vivo studies demonstrated that at low pressures, bladder compliance and afferent activity were elevated in stressed bladders compared with unstressed bladders. Capsazepine did not significantly change afferent activity in unstressed mice, but significantly decreased afferent activity at all pressures in stressed bladders. Immunohistochemistry revealed that TRPV1 colocalizes with CGRP to stain nerve fibers in unstressed bladders. Colocalization significantly increased along the same nerve fibers in the stressed bladders. Our results support the concept that social stress induces TRPV1-dependent afferent nerve activity, ultimately leading to the development of overactive bladder symptoms.

Opposing roles of smooth muscle BK channels and ryanodine receptors in the regulation of nerve-evoked constriction of mesenteric resistance arteries.

In depolarized smooth muscle cells of pressurized cerebral arteries, ryanodine receptors (RyRs) generate "Ca2+ sparks" that activate large-conductance, Ca2+ -, and voltage-sensitive potassium (BK) channels to oppose pressure-induced (myogenic) constriction. Here, we show that BK channels and RyRs have opposing roles in the regulation of arterial tone in response to sympathetic nerve activation by electrical field stimulation. Inhibition of BK channels with paxilline increased both myogenic and nerve-induced constrictions of pressurized, resistance-sized mesenteric arteries from mice. Inhibition of RyRs with ryanodine increased myogenic constriction, but it decreased nerve-evoked constriction along with a reduction in the amplitude of nerve-evoked increases in global intracellular Ca2+. In the presence of L-type voltage-dependent Ca2+ channel (VDCC) antagonists, nerve stimulation failed to evoke a change in arterial diameter, and BK channel and RyR inhibitors were without effect, suggesting that nerve- induced constriction is dependent on activation of VDCCs. Collectively, these results indicate that BK channels and RyRs have different roles in the regulation of myogenic versus neurogenic tone: whereas BK channels and RyRs act in concert to oppose myogenic vasoconstriction, BK channels oppose neurogenic vasoconstriction and RyRs augment it. A scheme for neurogenic vasoregulation is proposed in which RyRs act in conjunction with VDCCs to regulate nerve-evoked constriction in mesenteric resistance arteries.

Sympathetic nerve stimulation induces local endothelial Ca2+ signals to oppose vasoconstriction of mouse mesenteric arteries.

It is generally accepted that the endothelium regulates vascular tone independent of the activity of the sympathetic nervous system. Here, we tested the hypothesis that the activation of sympathetic nerves engages the endothelium to oppose vasoconstriction. Local inositol 1,4,5-trisphosphate (IP(3))-mediated Ca(2+) signals ("pulsars") in or near endothelial projections to vascular smooth muscle (VSM) were measured in an en face mouse mesenteric artery preparation. Electrical field stimulation of sympathetic nerves induced an increase in endothelial cell (EC) Ca(2+) pulsars, recruiting new pulsar sites without affecting activity at existing sites. This increase in Ca(2+) pulsars was blocked by bath application of the α-adrenergic receptor antagonist prazosin or by TTX but was unaffected by directly picospritzing the α-adrenergic receptor agonist phenylephrine onto the vascular endothelium, indicating that nerve-derived norepinephrine acted through α-adrenergic receptors on smooth muscle cells. Moreover, EC Ca(2+) signaling was not blocked by inhibitors of purinergic receptors, ryanodine receptors, or voltage-dependent Ca(2+) channels, suggesting a role for IP(3), rather than Ca(2+), in VSM-to-endothelium communication. Block of intermediate-conductance Ca(2+)-sensitive K(+) channels, which have been shown to colocalize with IP(3) receptors in endothelial projections to VSM, enhanced nerve-evoked constriction. Collectively, our results support the concept of a transcellular negative feedback module whereby sympathetic nerve stimulation elevates EC Ca(2+) signals to oppose vasoconstriction.

Adenosine signaling activates ATP-sensitive K+ channels in endothelial cells and pericytes in CNS capillaries.

The dense network of capillaries composed of capillary endothelial cells (cECs) and pericytes lies in close proximity to all neurons, ideally positioning it to sense neuron- and glial-derived compounds that enhance regional and global cerebral perfusion. The membrane potential (VM) of vascular cells serves as the physiological bridge that translates brain activity into vascular function. In other beds, the ATP-sensitive K+ (KATP) channel regulates VM in vascular smooth muscle, which is absent in the capillary network. Here, with transgenic mice that expressed a dominant-negative mutant of the pore-forming Kir6.1 subunit specifically in brain cECs or pericytes, we demonstrated that KATP channels were present in both cell types and robustly controlled VM. We further showed that the signaling nucleotide adenosine acted through A2A receptors and the Gαs/cAMP/PKA pathway to activate capillary KATP channels. Moreover, KATP channel stimulation in vivo increased cerebral blood flow (CBF), an effect that was blunted by expression of the dominant-negative Kir6.1 mutant in either capillary cell type. These findings establish an important role for KATP channels in cECs and pericytes in the regulation of CBF.

Unique properties of muscularis mucosae smooth muscle in guinea pig urinary bladder.

The muscularis mucosae, a type of smooth muscle located between the urothelium and the urinary bladder detrusor, has been described, although its properties and role in bladder function have not been characterized. Here, using mucosal tissue strips isolated from guinea pig urinary bladders, we identified spontaneous phasic contractions (SPCs) that appear to originate in the muscularis mucosae. This smooth muscle layer exhibited Ca(2+) waves and flashes, but localized Ca(2+) events (Ca(2+) sparks, purinergic receptor-mediated transients) were not detected. Ca(2+) flashes, often in bursts, occurred with a frequency (∼5.7/min) similar to that of SPCs (∼4/min), suggesting that SPCs are triggered by bursts of Ca(2+) flashes. The force generated by a single mucosal SPC represented the maximal force of the strip, whereas a single detrusor SPC was ∼3% of maximal force of the detrusor strip. Electrical field stimulation (0.5-50 Hz) evoked force transients in isolated detrusor and mucosal strips. Inhibition of cholinergic receptors significantly decreased force in detrusor and mucosal strips (at higher frequencies). Concurrent inhibition of purinergic and cholinergic receptors nearly abolished evoked responses in detrusor and mucosae. Mucosal SPCs were unaffected by blocking small-conductance Ca(2+)-activated K(+) (SK) channels with apamin and were unchanged by blocking large-conductance Ca(2+)-activated K(+) (BK) channels with iberiotoxin (IbTX), indicating that SK and BK channels play a much smaller role in regulating muscularis mucosae SPCs than they do in regulating detrusor SPCs. Consistent with this, BK channel current density in myocytes from muscularis mucosae was ∼20% of that in detrusor myocytes. These findings indicate that the muscularis mucosae in guinea pig represents a second smooth muscle compartment that is physiologically and pharmacologically distinct from the detrusor and may contribute to the overall contractile properties of the urinary bladder.

The Role of PIEZO1 in Urinary Bladder Function and Dysfunction in a Rodent Model of Cyclophosphamide-Induced Cystitis.

In the urinary bladder, mechanosensitive ion channels (MSCs) underlie the transduction of bladder stretch into sensory signals that are relayed to the PNS and CNS. PIEZO1 is a recently identified MSC that is Ca2+ permeable and is widely expressed throughout the lower urinary tract. Recent research indicates that PIEZO1 is activated by mechanical stretch or by pharmacological agonism via Yoda1. Aberrant activation of PIEZO1 has been suggested to play a role in clinical bladder pathologies like partial bladder outlet obstruction and interstitial cystitis/bladder pain syndrome (IC/BPS). In the present study, we show that intravesical instillation of Yoda1 in female Wistar rats leads to increased voiding frequency for up to 16 hours after administration compared to vehicle treatment. In a cyclophosphamide (CYP) model of cystitis, we found that the gene expression of several candidate MSCs (Trpv1, Trpv4, Piezo1, and Piezo2) were all upregulated in the urothelium and detrusor following chronic CYP-induced cystitis, but not acute CYP-induced cystitis. Functionally with this model, we show that Ca2+ activity is increased in urothelial cells following PIEZO1 activation via Yoda1 in acute and intermediate CYP treatment, but not in naïve (no CYP) nor chronic CYP treatment. Lastly, we show that activation of PIEZO1 may contribute to pathological bladder dysfunction through the downregulation of several tight junction genes in the urothelium including claudin-1, claudin-8, and zona occludens-1. Together, these data suggest that PIEZO1 activation plays a role in dysfunctional voiding behavior and may be a future, clinical target for the treatment of pathologies like IC/BPS.

Nerve-released acetylcholine contracts urinary bladder smooth muscle by inducing action potentials independently of IP3-mediated calcium release.

Nerve-released ACh is the main stimulus for contraction of urinary bladder smooth muscle (UBSM). Here, the mechanisms by which ACh contracts UBSM are explored by determining Ca(2+) and electrical signals induced by nerve-released ACh. Photolysis of caged inositol 1,4,5-trisphosphate (IP(3)) evoked Ca(2+) release from the sarcoplasmic reticulum. Electrical field stimulation (20 Hz) induced Ca(2+) waves within the smooth muscle that were present only during stimulus application. Ca(2+) waves were blocked by inhibition of muscarinic ACh receptors (mAChRs) with atropine and depletion of sarcoplasmic reticulum Ca(2+) stores with cyclopiazonic acid (CPA), and therefore likely reflect activation of IP(3) receptors (IP(3)Rs). Electrical field stimulation also increased excitability to induce action potentials (APs) that were accompanied by Ca(2+) flashes, reflecting Ca(2+) entry through voltage-dependent Ca(2+) channels (VDCCs) during the action potential. The evoked Ca(2+) flashes and APs occurred as a burst with a lag time of approximately 1.5 s after onset of stimulation. They were not inhibited by blocking IP(3)-mediated Ca(2+) waves, but by blockers of mAChRs (atropine) and VDCCs (diltiazem). Nerve-evoked contractions of UBSM strips were greatly reduced by blocking VDCCs, but not by preventing IP(3)-mediated Ca(2+) signaling with cyclopiazonic acid or inhibition of PLC with U73122. These results indicate that ACh released from nerve varicosities induces IP(3)-mediated Ca(2+) waves during stimulation; but contrary to expectations, these signals do not appear to participate in contraction. In addition, our data provide compelling evidence that UBSM contractions evoked by nerve-released ACh depend on increased excitability and the resultant Ca(2+) entry through VDCCs during APs.

Nerve-evoked purinergic signalling suppresses action potentials, Ca2+ flashes and contractility evoked by muscarinic receptor activation in mouse urinary bladder smooth muscle.

Contraction of urinary bladder smooth muscle (UBSM) is caused by the release of ATP and ACh from parasympathetic nerves. Although both purinergic and muscarinic pathways are important to contraction, their relative contributions and signalling mechanisms are not well understood. Here, the contributions of each pathway to urinary bladder contraction and the underlying electrical and Ca(2+) signalling events were examined in UBSM strips from wild type mice and mice deficient in P2X1 receptors (P2X1(-/-)) before and after pharmacological inhibition of purinergic and muscarinic receptors. Electrical field stimulation was used to excite parasympathetic nerves to increase action potentials, Ca(2+) flash frequency, and force. Loss of P2X1 function not only eliminated action potentials and Ca(2+) flashes during stimulation, but it also led to a significant increase in Ca(2+) flashes following stimulation and a corresponding increase in the force transient. Block of muscarinic receptors did not affect action potentials or Ca(2+) flashes during stimulation, but prevented them following stimulation. These findings indicate that nerve excitation leads to rapid engagement of smooth muscle P2X1 receptors to increase action potentials (Ca(2+) flashes) during stimulation, and a delayed increase in excitability in response to muscarinic receptor activation. Together, purinergic and muscarinic stimulation shape the time course of force transients. Furthermore, this study reveals a novel inhibitory effect of P2X1 receptor activation on subsequent increases in muscarinic-driven excitability and force generation.

PACAP38-Mediated Bladder Afferent Nerve Activity Hyperexcitability and Ca2+ Activity in Urothelial Cells from Mice.

Pituitary adenylate cyclase-activating polypeptide (PACAP; Adcyap1) and its cognate PAC1 receptor (Adcyap1r1) have tissue-specific distributions in the lower urinary tract (LUT). The afferent limb of the micturition reflex is often compromised following bladder injury, disease, and inflammatory conditions. We have previously demonstrated that PACAP signaling contributes to increased voiding frequency and decreased bladder capacity with cystitis. Thus, the present studies investigated the sensory components (e.g., urothelial cells, bladder afferent nerves) of the urinary bladder that may underlie the pathophysiology of aberrant PACAP activation. We utilized bladder-pelvic nerve preparations and urothelial sheet preparations to characterize PACAP-induced bladder afferent nerve discharge with distention and PACAP-induced Ca2+ activity, respectively. We determined that PACAP38 (100 nM) significantly (p ≤ 0.01) increased bladder afferent nerve activity with distention that was blocked with a PAC1/VPAC2 receptor antagonist PACAP6-38 (300 nM). PACAP38 (100 nM) also increased Ca2+ activity in urothelial cells over that observed in control preparations. Taken together, these results establish a role for PACAP signaling in bladder sensory components (e.g., urothelial cells, bladder afferent nerves) that may ultimately facilitate increased voiding frequency.

TRPV4 forms a novel Ca2+ signaling complex with ryanodine receptors and BKCa channels.

Vasodilatory factors produced by the endothelium are critical for the maintenance of normal blood pressure and flow. We hypothesized that endothelial signals are transduced to underlying vascular smooth muscle by vanilloid transient receptor potential (TRPV) channels. TRPV4 message was detected in RNA from cerebral artery smooth muscle cells. In patch-clamp experiments using freshly isolated cerebral myocytes, outwardly rectifying whole-cell currents with properties consistent with those of expressed TRPV4 channels were evoked by the TRPV4 agonist 4alpha-phorbol 12,13-didecanoate (4alpha-PDD) (5 micromol/L) and the endothelium-derived arachidonic acid metabolite 11,12 epoxyeicosatrienoic acid (11,12 EET) (300 nmol/L). Using high-speed laser-scanning confocal microscopy, we found that 11,12 EET increased the frequency of unitary Ca2+ release events (Ca2+ sparks) via ryanodine receptors located on the sarcoplasmic reticulum of cerebral artery smooth muscle cells. EET-induced Ca2+ sparks activated nearby sarcolemmal large-conductance Ca2+-activated K+ (BKCa) channels, measured as an increase in the frequency of transient K+ currents (referred to as "spontaneous transient outward currents" [STOCs]). 11,12 EET-induced increases in Ca2+ spark and STOC frequency were inhibited by lowering external Ca2+ from 2 mmol/L to 10 micromol/L but not by voltage-dependent Ca2+ channel inhibitors, suggesting that these responses require extracellular Ca2+ influx via channels other than voltage-dependent Ca2+ channels. Antisense-mediated suppression of TRPV4 expression in intact cerebral arteries prevented 11,12 EET-induced smooth muscle hyperpolarization and vasodilation. Thus, we conclude that TRPV4 forms a novel Ca2+ signaling complex with ryanodine receptors and BKCa channels that elicits smooth muscle hyperpolarization and arterial dilation via Ca2+-induced Ca2+ release in response to an endothelial-derived factor.

Rhythmic Calcium Events in the Lamina Propria Network of the Urinary Bladder of Rat Pups.

The lamina propria contains a dense network of cells, including interstitial cells (ICs), that may play a role in bladder function by modulating communication between urothelium, nerve fibers and smooth muscle or acting as pacemakers. Transient receptor potential vanilloid 4 (TRPV4) channels allow cation influx and may be involved in sensing stretch or chemical irritation in urinary bladder. Urothelium was removed from rats (P0-Adult), cut into strips, and loaded with a Ca2+ fluorescent dye (Fluo-2 AM leak resistant or Cal 520) for 90 min (35-37°C) to measure Ca2+ events. Ca2+ events were recorded for a period of 60 seconds (s) in control and after drug treatment. A heterogeneous network of cells was identified at the interface of the urothelium and lamina propria of postnatal rat pups, aged ≤ postnatal (P) day 21, with diverse morphology (round, fusiform, stellate with numerous projections) and expressing platelet-derived growth factor receptor alpha (PDGFRα)- and TRPV4-immunoreactivity (IR). Ca2+ transients occurred at a slow frequency with an average interval of 30 ± 8.6 s. Waveform analyses of Ca2+ transients in cells in the lamina propria network revealed long duration Ca2+ events with slow upstrokes. We observed slow propagating waves of activity in the lamina propria network that displayed varying degrees of coupling. Application of the TRPV4 agonist, GSK1016790 (100 nM), increased the duration of Ca2+ events, the number of cells with Ca2+ events and the integrated Ca2+ activity corresponding to propagation of activity among cells in the lamina propria network. However, GSK2193874 (1 µM), a potent antagonist of TRPV4 channels, was without effect. ATP (1 µM) perfusion increased the number of cells in the lamina propria exhibiting Ca2+ events and produced tightly coupled network activity. These findings indicate that ATP and TRPV4 can activate cells in the laminar propria network, leading to the appearance of organized propagating wavefronts.

Lack of direct effect of adiponectin on vascular smooth muscle cell BKCa channels or Ca2+ signaling in the regulation of small artery pressure-induced constriction.

The aim of this study was to investigate mechanisms by which adiponectin influences vascular Ca2+ signaling, K+ channel activity and thus contractile tone of small arteries. Vasodilation to adiponectin was studied in mesenteric resistance arteries constricted with intraluminal pressure. Ca2+ signals were characterized using high speed confocal microscopy of intact arteries. Patch clamp investigated the effect of adiponectin on individual VSMC potassium (K+) channel currents. Adiponectin dilated arteries constricted with pressure-induced tone by approximately 5% and the induced vasodilation was only transient. The dilation to adiponectin was reduced by pharmacological interruption of the Ca2+ spark/large conductance activated K+ (BK) channel pathway but from a physiological perspective, interpretation of the data was limited by the small effect. Neither Adiponectin nor the presence of intact perivascular adipose tissue (PVAT) influenced Ca2+ spark or Ca2+ wave frequency or characteristics. Studied using a perforated patch approach, Adiponectin marginally increased current through the VSMC BK channel but this effect was lost using the whole cell technique with dialysis of the cytoplasm. Adiponectin did not change the frequency or amplitude of Ca2+ spark-induced transient outward currents (STOC). Overall, our study shows that Adiponectin induces only a small and transient dilation of pressure constricted mesenteric arteries. This vasodilatory effect is likely to be independent of Ca2+ sparks or direct BK channel activation.