ß3-adrenoceptor agonists inhibit carbachol-evoked Ca2+ oscillations in murine detrusor myocytes.

OBJECTIVE: To test if carbachol (CCh)-evoked Ca2+ oscillations in freshly isolated murine detrusor myocytes are affected by ß3-adrenoceptor (ß-AR) modulators. MATERIALS AND METHODS: Isometric tension recordings were made from strips of murine detrusor, and intracellular Ca2+ measurements were made from isolated detrusor myocytes using confocal microscopy. Transcriptional expression of ß-AR sub-types in detrusor strips and isolated detrusor myocytes was assessed using reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time quantitative PCR (qPCR). Immunocytochemistry experiments, using a ß3-AR selective antibody, were performed to confirm that ß3-ARs were present on detrusor myocytes. RESULTS: The RT-PCR and qPCR experiments showed that ß1-, ß2- and ß3-AR were expressed in murine detrusor, but that ß3-ARs were the most abundant sub-type. The selective ß3-AR agonist BRL37344 reduced the amplitude of CCh-induced contractions of detrusor smooth muscle. These responses were unaffected by addition of the BK channel blocker iberiotoxin. BRL37344 also reduced the amplitude of CCh-induced Ca2+ oscillations in freshly isolated murine detrusor myocytes. This effect was mimicked by CL316,243, another ß3-AR agonist, and inhibited by the ß3-AR antagonist L748,337, but not by propranolol, an antagonist of ß1- and ß2-ARs. BRL37344 did not affect caffeine-evoked Ca2+ transients or L-type Ca2+ current in isolated detrusor myocytes. CONCLUSION: Inhibition of cholinergic-mediated contractions of the detrusor by ß3-AR agonists was associated with a reduction in Ca2+ oscillations in detrusor myocytes.

The role of Ca2+-activated Cl- current in tone generation in the rabbit corpus cavernosum.

Rabbit corpus cavernosum smooth muscle (RCCSM) cells express ion channels that produce Ca2+-activated Cl- (IClCa) current, but low sensitivity to conventional antagonists has made its role in tone generation difficult to evaluate. We have reexamined this question using two new generation IClCa blockers, T16Ainh-A01 and CaCCinh-A01. Isolated RCCSM cells were studied using the perforated patch method. Current-voltage protocols revealed that both L-type Ca2+ current and IClCa T16Ainh-A01 and CaCCinh-A01 (10 µM) reduced IClCa by ~85%, while 30 µM abolished it. L-type Ca2+ current was unaffected by 10 µM CaCCinh-A01 but was reduced by 50% at 30 µM CaCCinh-A01, 46% at 10 µM T16Ainh-A01, and 78% at 30 µM T16Ainh-A01. Both drugs reduced spontaneous isometric tension in RCCSM strips, by 60-70% at 10 µM and >90% at 30 µM. Phenylephrine (PE)-enhanced tension was also reduced (ED50 = 3 µM, CaCCinh-A01; 14 µM, T16Ainh-A01). CaCCinh-A01 at 10 µM had little effect on 60 mM KCl contractures, though they were reduced by 30 µM CaCCinh-A01 and T16Ainh-A01 (10 µM and 30 µM) consistent with their effects on L-type Ca2+ current. Both drugs also reversed the stimulatory effect of PE on intracellular Ca2+ waves, studied with laser scanning confocal microscopy in isolated RCCSM cells. In conclusion, although both drugs were effective blockers of IClCa, the effect of T16Ainh-A01 on L-type Ca2+ current precludes its use for evaluating the role of IClCa in tone generation. However, 10 µM CaCCinh-A01 selectively blocked IClCa versus L-type Ca2+ current and reduced spontaneous and PE-induced tone, suggesting that IClCa is important in maintaining penile detumescence.

Effects of new-generation TMEM16A inhibitors on calcium-activated chloride currents in rabbit urethral interstitial cells of Cajal.

Interstitial cells of Cajal (ICC) isolated from the rabbit urethra exhibit Ca2+-activated Cl- currents (I ClCa) that are important for the development of urethral tone. Here, we examined if TMEM16A (ANO1) contributed to this activity by examining the effect of "new-generation" TMEM16A inhibitors, CACCinh-A01 and T16Ainh-A01, on I ClCa recorded from freshly isolated rabbit urethral ICC (RUICC) and on contractions of intact strips of rabbit urethra smooth muscle. Real-time quantitative PCR experiments demonstrated that TMEM16A was highly expressed in rabbit urethra smooth muscle, in comparison to TMEM16B and TMEM16F. Single-cell RT-PCR experiments revealed that only TMEM16A was expressed in freshly isolated RUICC. Depolarization-evoked I ClCa in isolated RUICC, recorded using voltage clamp, were inhibited by CACCinh-A01 and T16Ainh-A01 with IC50 values of 1.2 and 3.4 µM, respectively. Similarly, spontaneous transient inward currents (STICs) recorded from RUICC voltage clamped at -60 mV and spontaneous transient depolarizations (STDs), recorded in current clamp, were also inhibited by CACCinh-A01 and T16Ainh-A01. In contrast, spontaneous Ca2+ waves in isolated RUICC were only partially reduced by CACCinh-A01 and T16Ainh-A01. Finally, neurogenic contractions of strips of rabbit urethra smooth muscle (RUSM), evoked by electric field stimulation (EFS), were also significantly reduced by CACCinh-A01 and T16Ainh-A01. These data are consistent with the idea that TMEM16A is involved with CACCs in RUICC and in contraction of rabbit urethral smooth muscle.

Differential efficacy of GoSlo-SR compounds on BKα and BKαγ1-4 channels.

Large conductance, voltage and Ca2+ activated K+ channels (BK channels) are abundantly expressed throughout the body and are important regulators of smooth muscle tone and neuronal excitability. Their dysfunction is implicated in various diseases including overactive bladder, hypertension and erectile dysfunction. Therefore, BK channel openers bear significant therapeutic potential to treat the above diseases. GoSlo-SR compounds were designed to be potent and efficacious BK channel openers. Although their structural activity relationships, activation in both BKα and BKαß channels and the hypothetical mode of action of these compounds has been studied in detail in recent years, their effectiveness to open the BKαγ channels still remains unexplored. In this study, we have examined the efficacy of 3 closely related GoSlo-SR openers, GoSlo-SR-5-6 (SR-5-6), GoSlo-SR-5-44 (SR-5-44) and GoSlo-SR-5-130 (SR-5-130) using inside out patches on BKα channels coexpressed with 4 different LRRC (γ1-4) subunits in HEK293 cells. Our data suggests that the activation effects due to SR-5-6 were not significantly affected in the presence of γ1-4 subunits. Interestingly, the effects of more efficacious BK channel opener SR-5-44 were altered by different γ subunits. In cells expressing BKα channels, the shift in V1/2 (ΔV1/2) induced by SR-5-44 (3 µM) was -76 ± 3 mV, whereas it was significantly reduced by ∼70 % in BKαγ1 channels (ΔV1/2= -23 ± 3, p < 0.001, ANOVA). In BKαγ2 channels the ΔV1/2 was -36 ± 1 mV, which was less than that observed in BKαγ3 and BKαγ4 channels where the ΔV1/2 was -47 ± 5 mV, and -82 ± 5 mV, respectively. Additionally, the excitatory effects of a 'ß specific' BK channel opener, SR-5-130 were only partially restored in the patches containing BKαγ1-4 channels. Together this data highlights that subtle modifications in GoSlo-SR structures alter their effectiveness on BK channels with accessory γ subunits and this study might provide a scaffold for the development of more tissue specific BK channel openers.

Muscarinic Receptor Induced Contractions of the Detrusor are Mediated by Activation of TRPC4 Channels.

PURPOSE: Muscarinic receptor mediated contractions of the detrusor rely on Ca2+ influx through voltage-gated Ca2+ channels but to our knowledge the mechanism linking stimulation of M3Rs to the activation of voltage dependent Ca2+ channels has not been established. TRPC4 channels are receptor operated cation channels that couple muscarinic receptor activation to depolarization of intestinal smooth muscle cells, voltage-activated Ca2+ influx and contraction. We investigated whether TRPC4 channels are involved in cholinergic mediated contractions of the detrusor. MATERIALS AND METHODS: Isometric tension recordings were made on strips of murine detrusor and intracellular Ca2+ measurements were made on isolated detrusor myocytes using confocal microscopy. Transcriptional expression of TRPC and IP3R subtypes in intact detrusor strips and isolated detrusor myocytes was assessed using reverse transcriptase-polymerase chain reaction. RESULTS: Cholinergic stimulation of the detrusor induced by electrical field stimulation or exogenous application of carbachol or neostigmine evoked contractions consisting of a transient plus a tonic response, which was blocked by ML204, an inhibitor of TRPC4 channels. A phasic oscillatory component was blocked by the IP3R inhibitor 2-APB. Carbachol evoked reproducible Ca2+ responses in isolated detrusor myocytes, consisting of an initial Ca2+ transient followed by Ca2+ oscillations. ML204 inhibited the initial Ca2+ transient whereas 2-APB inhibited the Ca2+ oscillations. Reverse transcriptase-polymerase chain reaction experiments showed that TRPC4ß, TRPC6 and IP3R1 were selectively expressed in isolated detrusor myocytes. Control experiments demonstrated that ML204 did not affect L-type Ca2+ or BK current amplitude, caffeine induced Ca2+ transients or KCl induced contractions of the detrusor. CONCLUSIONS: Muscarinic receptor mediated contractions of the detrusor involve the activation of TRPC4ß channels.

Regulation of nerve-evoked contractions of rabbit vas deferens by acetylcholine.

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

The role of Ca(2+) influx in spontaneous Ca(2+) wave propagation in interstitial cells of Cajal from the rabbit urethra.

KEY POINTS: Tonic contractions of rabbit urethra are associated with spontaneous electrical slow waves that are thought to originate in pacemaker cells termed interstitial cells of Cajal (ICC). ICC pacemaker activity results from their ability to generate propagating Ca(2+) waves, although the exact mechanisms of propagation are not understood. In this study, we have identified spontaneous localised Ca(2+) events for the first time in urethral ICC; these were due to Ca(2+) release from the endoplasmic reticulum (ER) via ryanodine receptors (RyRs) and, while they often remained localised, they sometimes initiated propagating Ca(2+) waves. We show that propagation of Ca(2+) waves in urethral ICC is critically dependent upon Ca(2+) influx via reverse mode NCX. Our data provide a clearer understanding of the intracellular mechanisms involved in the generation of ICC pacemaker activity. Interstitial cells of Cajal (ICC) are putative pacemaker cells in the rabbit urethra. Pacemaker activity in ICC results from spontaneous propagating Ca(2+) waves that are modulated by [Ca(2+)]o and whose propagation is inhibited by inositol tri-phosphate receptor (IP3 R) blockers. The purpose of this study was to further examine the role of Ca(2+) influx and Ca(2+) release in the propagation of Ca(2+) waves. Intracellular Ca(2+) was measured in Fluo-4-loaded ICC using a Nipkow spinning disc confocal microscope at fast acquisition rates (50 fps). We identified previously undetected localised Ca(2+) events originating from ryanodine receptors (RyRs). Inhibiting Ca(2+) influx by removing [Ca(2+)]o or blocking reverse mode sodium-calcium exchange (NCX) with KB-R 7943 or SEA-0400 abolished Ca(2+) waves, while localised Ca(2+) events persisted. Stimulating RyRs with 1 mm caffeine restored propagation. Propagation was also inhibited when Ca(2+) release sites were uncoupled by buffering intracellular Ca(2+) with EGTA-AM. This was reversed when Ca(2+) influx via NCX was increased by reducing [Na(+)]o to 13 mm. Low [Na(+)]o also increased the frequency of Ca(2+) waves and this effect was blocked by tetracaine and ryanodine but not 2-aminoethoxydiphenyl borate (2-APB). RT-PCR revealed that isolated ICC expressed both RyR2 and RyR3 subtypes. We conclude: (i) RyRs are required for the initiation of Ca(2+) waves, but wave propagation normally depends on activation of IP3 Rs; (ii) under resting conditions, propagation by IP3 Rs requires sensitisation by influx of Ca(2+) via reverse mode NCX; (iii) propagation can be maintained by RyRs if they have been sensitised to Ca(2+).

Molecular mechanisms underlying the effect of the novel BK channel opener GoSlo: involvement of the S4/S5 linker and the S6 segment.

GoSlo-SR-5-6 is a novel large-conductance Ca(2+)-activated K(+) (BK) channel agonist that shifts the activation V1/2 of these channels in excess of -100 mV when applied at a concentration of 10 µM. Although the structure-activity relationship of this family of molecules has been established, little is known about how they open BK channels. To help address this, we used a combination of electrophysiology, mutagenesis, and mathematical modeling to investigate the molecular mechanisms underlying the effect of GoSlo-SR-5-6. Our data demonstrate that the effects of this agonist are practically abolished when three point mutations are made: L227A in the S4/S5 linker in combination with S317R and I326A in the S6C region. Our data suggest that GoSlo-SR-5-6 interacts with the transmembrane domain of the channel to enhance pore opening. The Horrigan-Aldrich model suggests that GoSlo-SR-5-6 works by stabilizing the open conformation of the channel and the activated state of the voltage sensors, yet decouples the voltage sensors from the pore gate.

The role of cAMP dependent protein kinase in modulating spontaneous intracellular Ca²âº waves in interstitial cells of Cajal from the rabbit urethra.

Interstitial cells of Cajal (ICC) serve as electrical pacemakers in the rabbit urethra. Pacemaking activity in ICC results from spontaneous intracellular Ca(2+) waves that rely on Ca(2+) release from endoplasmic reticulum (ER) stores. The purpose of this study was to investigate if the action of protein kinase A (PKA) affected the generation of Ca(2+) waves in ICC. Intracellular [Ca(2+)] was measured in fluo-4 loaded ICC, freshly isolated from the rabbit urethra using a Nipkow spinning disc confocal microscope. Application of the PKA inhibitor H-89 (10 µM) significantly inhibited the generation of spontaneous Ca(2+) waves in ICC and this was associated with a significant decrease in the ER Ca(2+) load, measured with 10mM caffeine responses. Ca(2+) waves could be rescued in the presence of H-89 by stimulating ryanodine receptors (RyRs) with 1mM caffeine but not by activation of inositol 1,4,5 tri-phosphate receptors (IP3Rs) with 10 µM phenylephrine. Increasing intracellular PKA with the cAMP agonists forskolin and 8-bromo-cAMP failed to yield an increase in Ca(2+) wave activity. We conclude that PKA may be maximally active under basal conditions in ICC and that inhibition of PKA with H-89 leads to a decreased ER Ca(2+) load sufficient to inactivate IP3Rs but not RyRs.

The effect of high [K(+)]o on spontaneous Ca(2+) waves in freshly isolated interstitial cells of Cajal from the rabbit urethra.

Interstitial cells of Cajal (ICC) act as putative pacemaker cells in the rabbit urethra. Pacemaker activity in ICC results from spontaneous global Ca(2+) waves that can be increased in frequency by raising external [K(+)]. The purpose of this study was to elucidate the mechanism of this response. Intracellular [Ca(2+)] was measured in fluo-4-loaded smooth muscle cells (SMCs) and ICC using a Nipkow spinning disk confocal microscope. Increasing [K(+)]o to 60 mmol/L caused an increase in [Ca(2+)]i accompanied by contraction in SMCs. Raising [K(+)]o did not cause contraction in ICC, but the frequency of firing of spontaneous calcium waves increased. Reducing [Ca(2+)]o to 0 mmol/L abolished the response in both cell types. Nifedipine of 1 µmol/L blocked the response of SMC to high [K(+)]o, but did not affect the increase in firing in ICC. This latter effect was blocked by 30 µmol/L NiCl2 but not by the T-type Ca(2+) channel blocker mibefradil (300 nmol/L). However, inhibition of Ca(2+) influx via reverse-mode sodium/calcium exchange (NCX) using either 1 µmol/L SEA0400 or 5 µmol/L KB-R7943 did block the effect of high [K(+)]o on ICC. These data suggest that high K(+) solution increases the frequency of calcium waves in ICC by increasing Ca(2+) influx through reverse-mode NCX.

Pharmacological characterization of TMEM16A currents.

Recent studies have shown that transmembrane protein 16 A (TMEM16A) is a subunit of calcium-activated chloride channels (CACCs). Pharmacological agents have been used to probe the functional role of CACCs, however their effect on TMEM16A currents has not been systematically investigated. In the present study, we characterized the voltage and concentration-dependent effects of 2 traditional CACC inhibitors (niflumic acid and anthracene-9-carboxcylic acid) and 2 novel CACC / TMEM16A inhibitors (CACC(inh)A01 and T16A(inh)A01) on TMEM16A currents. The whole cell patch clamp technique was used to record TMEM16A currents from HE K 293 cells that stably expressed human TMEM16A. Niflumic acid, A-9-C, CACC(inh)A01 and T16A(inh)A01 inhibited TMEM16A currents with IC50 values of 12, 58, 1.7 and 1.5 µM, respectively, however, A-9-C and niflumic acid were less efficacious at negative membrane potentials. A-9-C and niflumic acid reduced the rate of TMEM16A tail current deactivation at negative membrane potentials and A-9-C (1 mM) enhanced peak TMEM16A tail current amplitude. In contrast, the inhibitory effects of CACC(inh)A01 and T16A(inh)A01 were independent of voltage and they did not prolong the rate of TMEM16A tail current deactivation. The effects of niflumic acid and A-9-C on TMEM16A currents were similar to previous observations on CACCs in vascular smooth muscle, strengthening the hypothesis that they are encoded by TMEM16A. However, CACC(inh)A01 and T16A(inh)A01 were more potent inhibitors of TMEM16A channels and their effects were not diminished at negative membrane potentials making them attractive candidates to interrogate the functional role of TMEM16A channels in future studies.

Development of GoSlo-SR-5-69, a potent activator of large conductance Ca2+-activated K+ (BK) channels.

We have designed, synthesised and characterised the effects of a number of novel anthraquinone derivatives and assessed their effects on large conductance, Ca(2+) activated K(+) (BK) channels recorded from rabbit bladder smooth muscle cells using the excised, inside/out configuration of the patch clamp technique. These compounds are members of the GoSlo-SR family of compounds, which potently open BK channels and shift the voltage required for half maximal activation (V1/2) negatively. The efficacy of the anilinoanthraquinone derivatives was enhanced when the size of ring D was increased, since the cyclopentane and cyclohexane derivatives shifted the V1/2, by -24 ± 6 mV and -54 ± 8 mV, respectively, whereas the cycloheptane and cyclooctane derivatives shifted the V1/2 by -61 ± 6 mV and -106 ± 6 mV. To examine if a combination of hydrophobicity and steric bulking of this region further enhanced their ability to open BK channels, we synthesised a number of naphthalene and tetrahydro-naphthalene derivatives. The tetrahydro-2-naphthalene derivative GoSlo-SR-5-69 was the most potent and efficacious of the series since it was able to shift the activation V1/2 by greater than -100 mV when applied at a concentration of 1 µM and had an EC50 of 251 nM, making it one of the most potent and efficacious BK channel openers synthesised to date.

ATP evokes inward currents in corpus cavernosum myocytes.

INTRODUCTION: Although adenosine triphosphate (ATP) has often been reported to relax the corpus cavernosum, this may be mediated by indirect effects, such as release of nitric oxide from the endothelium. Recent data suggest that P2X(1) receptors may be up-regulated in diabetes, and these exert an anti-erectile effect by causing the corpus cavernosum smooth muscle cells (CCSMCs) to contract. However, to date, there is no functional evidence that ATP can directly stimulate CCSMC. AIMS: This study aims to (i) to directly examine the effect of ATP on membrane currents in freshly isolated CCSMC, where influences of endothelium and other cells are absent; and (ii) to determine the receptor subtypes, ionic currents, and Ca(2+) signals stimulated by ATP. METHODS: CCSMCs were enzymatically dispersed from male New Zealand White rabbits for patch clamp recording and measurement of intracellular Ca(2+) in fluo-4-loaded cells using spinning disk confocal microscopy. MAIN OUTCOME MEASURES: Patch clamp recordings were made of ATP-evoked membrane currents and spontaneous membrane currents. Spinning disk confocal imaging of intracellular Ca(2+) was performed, and the response to ATP was recorded. RESULTS: ATP evoked repeatable inward currents in CCSMC (1st application: -675 ± 101 pA; 2nd application: -694 ± 120 pA, N = 9, P = 0.77). ATP-induced currents were reduced by suramin from -380 ± 121 to -124 ± 37 pA (N = 8, P < 0.05), by α,ß-methylene ATP from -755 ± 235 to 139 ± 49 pA (N = 5, P < 0.05), and by NF449 from -419 ± to -51 ± 13 pA (N = 6, P < 0.05). In contrast, MRS2500, a P2Y1(1,12,13) antagonist, had no effect on ATP responses (control: -838 ± 139 pA; in MRS2500: -822 ± 184 pA, N = 13, P = 0.84) but blocked inward currents evoked by 2-MeSATP, a P2Y1,12,13 agonist (control: -623 ± 166 pA; in MRS2500: -56 ± 25 pA, N = 6, P < 0.05). The ATP-evoked inward current was unaffected by changing the transmembrane Cl(-) gradient but reversed in direction when extracellular Na(+) was reduced, indicating that it was a cation current. CONCLUSIONS: ATP directly stimulates CCSMC by evoking a P2X-mediated cation current.

Mechanisms underlying activation of transient BK current in rabbit urethral smooth muscle cells and its modulation by IP3-generating agonists.

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

Structure-activity relationships of a novel group of large-conductance Ca(2+)-activated K(+) (BK) channel modulators: the GoSlo-SR family.

Opening up ion channels: We synthesised a series of anthraquinone analogues, called the GoSlo-SR family. Their effects on bladder smooth muscle BK channels were examined and, as shown, shifted voltage dependent activation >-100 mV (at 10 µM). They were more efficacious than NS11021 and could provide a new scaffold for the design of efficacious BK openers.

Effects of phenylephrine on spontaneous activity and L-type Ca2+ current in isolated corpus cavernosum myocytes.

INTRODUCTION: Norepinephrine is important in maintaining detumescent tone in the corpus cavernosum, although the mechanism is incompletely understood. As α-adrenoceptor-induced tone is antagonized by L-type Ca(2+) channel blockers, it is usually assumed that direct modulation of this current is involved. However, the effects of α-adrenoceptor agonists have never been directly examined on L-type current in corpus cavernosum myocytes (CCSMC), leaving open other possibilities. In particular, CCSMC are now known to develop spontaneous tone via a pacemaker mechanism involving spontaneous Ca(2+) waves that activate Cl(-) currents, causing depolarization and voltage-dependent activation of L-type channels. We hypothesized that phenylephrine modulates tone via this system, rather than by directly activating L-type channels. AIMS: Examine in freshly isolated CCSMC the effect of phenylephrine on: (i) spontaneous Cl(-) currents and depolarizations; (ii) cytosolic Ca(2+) waves; and (iii) L-type current. METHODS: CCSMC were enzymatically dispersed from male New Zealand White rabbits for patch clamp recording and real time Ca(2+) imaging. MAIN OUTCOME MEASURES: Spontaneous Cl(-) currents, spontaneous depolarizations, cytosolic Ca(2+) and L-type current. RESULTS: Phenylephrine enhanced the amplitude and frequency of spontaneous Cl(-) currents, increased the duration and frequency of spontaneous depolarizations and increased the frequency of spontaneous Ca(2+) waves. These effects were blocked by 2-aminoethoxy diphenylborate (2-APB), suggesting that they were mediated by IP(3) -induced Ca(2+) release from intracellular stores. In contrast, 2-APB had no effect on Ca(2+) transients evoked by releasing stored Ca(2+) with caffeine, suggesting that it had little effect on store Ca(2+) content. Phenylephrine depressed L-type current by around 30%. This effect was removed by blocking with 2-APB. Notably, phenylephrine failed to enhance the current, even in the presence of 2-APB. Furthermore, the phorbol ester, phorbol 12-myristate 13-acetate, had no effect on L-type current. CONCLUSION: Phenylephrine effects on the corpus cavernosum are mediated by modulation of the spontaneous pacemaker mechanism, rather than by direct stimulation of L-type channels.

Platelet-derived growth factor receptor-α cells in mouse urinary bladder: a new class of interstitial cells.

Specific classes of interstitial cells exist in visceral organs and have been implicated in several physiological functions including pacemaking and mediators in neurotransmission. In the bladder, Kit(+) interstitial cells have been reported to exist and have been suggested to be neuromodulators. More recently a second interstitial cell, which is identified using antibodies against platelet-derived growth factor receptor-α (PDGFR-α) has been described in the gastrointestinal (GI) tract and has been implicated in enteric motor neurotransmission. In this study, we examined the distribution of PDGFR-α(+) cells in the murine urinary bladder and the relation that these cells may have with nerve fibres and smooth muscle cells. Platelet-derived growth factor receptor-α(+) cells had a spindle shape or stellate morphology and often possessed multiple processes that contacted one another forming a loose network. These cells were distributed throughout the bladder wall, being present in the lamina propria as well as throughout the muscularis of the detrusor. These cells surrounded and were located between smooth muscle bundles and often came into close morphological association with intramural nerve fibres. These data describe a new class of interstitial cells that express a specific receptor within the bladder wall and provide morphological evidence for a possible neuromodulatory role in bladder function.

P2X receptor currents in smooth muscle cells contribute to nerve mediated contractions of rabbit urethral smooth muscle.

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

Novel excitatory effects of adenosine triphosphate on contractile and pacemaker activity in rabbit urethral smooth muscle.

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

Spontaneous Ca2+ waves in rabbit corpus cavernosum: modulation by nitric oxide and cGMP.

INTRODUCTION: Detumescent tone and subsequent relaxation by nitric oxide (NO) are essential processes that determine the erectile state of the penis. Despite this, the mechanisms involved are incompletely understood. It is often assumed that the tone is associated with a sustained high cytosolic Ca(2+) level in the corpus cavernosum smooth muscle cells, however, an alternative possibility is that oscillatory Ca(2+) signals regulate tone, and erection occurs as a result of inhibition of Ca(2+) oscillations by NO. AIMS: The aim of this study is to determine if smooth muscle cells displayed spontaneous Ca(2+) oscillations and, if so, whether these were regulated by NO. METHODS: Male New Zealand white rabbits were euthanized and smooth muscle cells were isolated by enzymatic dispersal for confocal imaging of intracellular Ca(2+) (using fluo-4AM) and patch clamp recording of spontaneous membrane currents. Thin tissue slices were also loaded with fluo-4AM for live imaging of Ca(2+). MAIN OUTCOME MEASURE: Cytosolic Ca(2+) was measured in isolated smooth muscle cells and tissue slices. Results. Isolated rabbit corpus cavernosum smooth muscle cells developed spontaneous Ca(2+) waves that spread at a mean velocity of 65 microm/s. Dual voltage clamp/confocal recordings revealed that each of the Ca(2+) waves was associated with an inward current typical of the Ca(2+)-activated Cl(-) currents developed by these cells. The waves depended on an intact sarcoplasmic reticulum Ca(2+) store, as they were blocked by cyclopiazonic acid (Calbiochem, San Diego, CA, USA) and agents that interfere with ryanodine receptors and IP(3)-mediated Ca(2+) release. The waves were also inhibited by an NO donor (diethylamine NO; Tocris Bioscience, Bristol, Avon, UK), 3-(5-hydroxymethyl-2-furyl)-1-benzyl indazole (YC-1) (Alexis Biochemicals, Bingham, Notts, UK), 8-bromo-cyclic guanosine mono-phosphate (Tocris), and sildenafil (Viagra, Pfizer, Sandwich, Kent, UK). Regular Ca(2+) oscillations were also observed in whole tissue slices where they were clearly seen to precede contraction. This activity was also markedly inhibited by sildenafil, suggesting that it was under NO regulation. CONCLUSIONS: These results provide a new basis for understanding detumescent tone in the corpus cavernosum and its inhibition by NO.