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ABSTRACT: 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.
ChemMedChem 08/2012; 7(10):1763-9. · 3.15 Impact Factor
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ABSTRACT: 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. Doyle C, Sergeant GP, Hollywood MA, McHale NG, and Thornbury KD. Effects of phenylephrine on spontaneous activity and L-type Ca(2+) current in isolated corpus cavernosum myocytes. J Sex Med **;**:**-**.
Journal of Sexual Medicine 07/2012; · 3.55 Impact Factor
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ABSTRACT: 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.
Journal of Cellular and Molecular Medicine 12/2011; 16(4):691-700. · 4.13 Impact Factor
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ABSTRACT: We have characterized the native voltage-dependent K(+) (K(v)) current in rabbit urethral smooth muscle cells (RUSMC) and compared its pharmacological and biophysical properties with K(v)2.1 and K(v)2.2 channels cloned from the rabbit urethra and stably expressed in human embryonic kidney (HEK)-293 cells (HEK(Kv2.1) and HEK(Kv2.2)). RUSMC were perfused with Hanks' solution at 37°C and studied using the patch-clamp technique with K(+)-rich pipette solutions. Cells were bathed in 100 nM Penitrem A (Pen A) to block large-conductance Ca(2+)-activated K(+) (BK) currents and depolarized to +40 mV for 500 ms to evoke K(v) currents. These were unaffected by margatoxin, κ-dendrotoxin, or α-dendrotoxin (100 nM, n = 3-5) but were blocked by stromatoxin-1 (ScTx, IC(50) ∼130 nM), consistent with the idea that the currents were carried through K(v)2 channels. RNA was detected for K(v)2.1, K(v)2.2, and the silent subunit K(v)9.3 in urethral smooth muscle. Immunocytochemistry showed membrane staining for both K(v)2 subtypes and K(v)9.3 in isolated RUSMC. HEK(Kv2.1) and HEK(Kv2.2) currents were blocked in a concentration-dependent manner by ScTx, with estimated IC(50) values of ∼150 nM (K(v)2.1, n = 5) and 70 nM (K(v)2.2, n = 6). The mean half-maximal voltage (V(1/2)) of inactivation of the USMC K(v) current was -56 ± 3 mV (n = 9). This was similar to the HEK(Kv2.1) current (-55 ± 3 mV, n = 13) but significantly different from the HEK(Kv2.2) currents (-30 ± 3 mV, n = 11). Action potentials (AP) evoked from RUSMC studied under current-clamp mode were unaffected by ScTx. However, when ScTx was applied in the presence of Pen A, the AP duration was significantly prolonged. Similarly, ScTx increased the amplitude of spontaneous contractions threefold, but only after Pen A application. These data suggest that K(v)2.1 channels contribute significantly to the K(v) current in RUSMC.
AJP Cell Physiology 08/2011; 301(5):C1186-200. · 3.54 Impact Factor
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ABSTRACT: 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.
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.
Stimulating intramural nerves in urethral smooth muscle yielded contractions that were inhibited by the broad spectrum P2 receptor inhibitor pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate and the P2X receptor agonist α,β-methylene adenosine triphosphate but not by the P2Y receptor antagonist MRS2500. When studied under voltage clamp at a holding potential of -60 mV, interstitial cells of Cajal showed spontaneous transient inward currents that were increased in frequency by adenosine triphosphate but not by α,β-methylene adenosine triphosphate. In contrast, smooth muscle cells were quiescent but responded to adenosine triphosphate and α,β-methylene adenosine triphosphate by producing a single transient inward current. Currents evoked by adenosine triphosphate in smooth muscle cells were inhibited by α,β-methylene adenosine triphosphate, pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate and suramin, and by a decrease in extracellular Na+ from 130 to 13 mM.
Stimulating purinergic nerves in rabbit urethral smooth muscle induces contractions via the activation of P2X receptors on smooth muscle cells.
The Journal of urology 06/2011; 186(2):745-52. · 4.02 Impact Factor
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ABSTRACT: 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.
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.
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.
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.
The Journal of urology 12/2009; 183(2):801-11. · 4.02 Impact Factor
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ABSTRACT: 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.
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.
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+).
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.
These results provide a new basis for understanding detumescent tone in the corpus cavernosum and its inhibition by NO.
Journal of Sexual Medicine 01/2009; 6(4):958-66. · 3.55 Impact Factor
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ABSTRACT: The aim of the present study was to investigate the properties and role of capacitative Ca(2+) entry (CCE) in interstitial cells (IC) isolated from the rabbit urethra. Ca(2+) entry in IC was larger in cells with depleted intracellular Ca(2+) stores compared with controls, consistent with influx via a CCE pathway. The nonselective Ca(2+) entry blockers Gd(3+) (10 microM), La(3+) (10 microM), and Ni(2+) (100 microM) reduced CCE by 67% (n = 14), 65% (n = 11), and 55% (n = 9), respectively. These agents did not inhibit Ca(2+) entry when stores were not depleted. Conversely, CCE in IC was resistant to SKF-96365 (10 microM), wortmannin (10 microM), and nifedipine (1 microM). Spontaneous transient inward currents were recorded from IC voltage-clamped at -60 mV. These events were not significantly affected by Gd(3+) (10 microM) or La(3+) (10 microM) and were only slightly decreased in amplitude by 100 microM Ni(2+). The results from this study demonstrate that freshly dispersed IC from the rabbit urethra possess a CCE pathway. However, influx via this pathway does not appear to contribute to spontaneous activity in these cells.
AJP Cell Physiology 10/2005; 289(3):C625-32. · 3.54 Impact Factor
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ABSTRACT: The back-illuminated electron multiplying charge-coupled device (EMCCD) camera is having a profound influence on the field of low-light dynamic cellular microscopy, combining highest possible photon collection efficiency with the ability to virtually eliminate the readout noise detection limit. We report here the use of this camera, in 512 x 512 frame-transfer chip format at 10-MHz pixel readout speed, in optimizing a demanding ultra-low-light intracellular calcium flux microscopy setup. The arrangement employed includes a spinning confocal Nipkow disk, which, while facilitating the need to both generate images at very rapid frame rates and minimize background photons, yields very weak signals. The challenge for the camera lies not just in detecting as many of these scarce photons as possible, but also in operating at a frame rate that meets the temporal resolution requirements of many low-light microscopy approaches, a particular demand of smooth muscle calcium flux microscopy. Results presented illustrate both the significant sensitivity improvement offered by this technology over the previous standard in ultra-low-light CCD detection, the GenIII+intensified charge-coupled device (ICCD), and also portray the advanced temporal and spatial resolution capabilities of the EMCCD.
Journal of Biomedical Optics 9(6):1244-52. · 3.16 Impact Factor
