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L-type Ca2+ channel sparklets revealed by TIRF microscopy in mouse urinary bladder smooth muscle.

ABSTRACT: Calcium is a ubiquitous second messenger in urinary bladder smooth muscle (UBSM). In this study, small discrete elevations of intracellular Ca(2+), referred to as Ca(2+) sparklets have been detected in an intact detrusor smooth muscle electrical syncytium using a TIRF microscopy Ca(2+) imaging approach. Sparklets were virtually abolished by the removal of extracellular Ca(2+) (0.035 ± 0.01 vs. 0.23 ± 0.07 Hz/mm(2); P<0.05). Co-loading of smooth muscle strips with the slow Ca(2+) chelator EGTA-AM (10 mM) confirmed that Ca(2+) sparklets are restricted to the cell membrane. Ca(2+) sparklets were inhibited by the calcium channel inhibitors R-(+)-Bay K 8644 (1 ?M) (0.034 ± 0.02 vs. 0.21 ± 0.08 Hz/mm(2); P<0.05), and diltiazem (10 ?M) (0.097 ± 0.04 vs. 0.16 ± 0.06 Hz/mm(2); P<0.05). Ca(2+) sparklets were unaffected by inhibition of P2X1 receptors ?,?-meATP (10 ?M) whilst sparklet frequencies were significantly reduced by atropine (1 ?M). Ca(2+) sparklet frequency was significantly reduced by PKC inhibition with Gö6976 (100 nM) (0.030 ± 0.01 vs. 0.30 ± 0.1 Hz/mm(2); P<0.05), demonstrating that Ca(2+) sparklets are PKC dependant. In the presence of CPA (10 ?M), there was no apparent change in the overall frequency of Ca(2+) sparklets, although the sparklet frequencies of each UBSM became statistically independent of each other (Spearman's rank correlation 0.2, P>0.05), implying that Ca(2+) store mediated signals regulate Ca(2+) sparklets. Under control conditions, inhibition of store operated Ca(2+) entry using ML-9 (100 ?M) had no significant effect. Amplitudes of Ca(2+) sparklets were unaffected by any agonists or antagonists, suggesting that these signals are quantal events arising from activation of a single channel, or complex of channels. The effects of CPA and ML-9 suggest that Ca(2+) sparklets regulate events in the cell membrane, and contribute to cytosolic and sarcoplasmic Ca(2+) concentrations.

SUBMITTER: Sidaway P

PROVIDER: S-EPMC3974850 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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L-type Ca2+ channel sparklets revealed by TIRF microscopy in mouse urinary bladder smooth muscle.

Sidaway Peter P Teramoto Noriyoshi N

PloS one 20140403 4

Calcium is a ubiquitous second messenger in urinary bladder smooth muscle (UBSM). In this study, small discrete elevations of intracellular Ca(2+), referred to as Ca(2+) sparklets have been detected in an intact detrusor smooth muscle electrical syncytium using a TIRF microscopy Ca(2+) imaging approach. Sparklets were virtually abolished by the removal of extracellular Ca(2+) (0.035 ± 0.01 vs. 0.23 ± 0.07 Hz/mm(2); P<0.05). Co-loading of smooth muscle strips with the slow Ca(2+) chelator EGTA-AM ...[more]

PMID: 24699670

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Project description:Urinary incontinence is associated with enhanced spontaneous phasic contractions of the detrusor smooth muscle (DSM). Although a complete understanding of the etiology of these spontaneous contractions is not yet established, it is suggested that the spontaneously evoked action potentials (sAPs) in DSM cells initiate and modulate the contractions. In order to further our understanding of the ionic mechanisms underlying sAP generation, we present here a biophysically detailed computational model of a single DSM cell. First, we constructed mathematical models for nine ion channels found in DSM cells based on published experimental data: two voltage gated Ca2+ ion channels, an hyperpolarization-activated ion channel, two voltage-gated K+ ion channels, three Ca2+-activated K+ ion channels and a non-specific background leak ion channel. The ion channels' kinetics were characterized in terms of maximal conductances and differential equations based on voltage or calcium-dependent activation and inactivation. All ion channel models were validated by comparing the simulated currents and current-voltage relations with those reported in experimental work. Incorporating these channels, our DSM model is capable of reproducing experimentally recorded spike-type sAPs of varying configurations, ranging from sAPs displaying after-hyperpolarizations to sAPs displaying after-depolarizations. The contributions of the principal ion channels to spike generation and configuration were also investigated as a means of mimicking the effects of selected pharmacological agents on DSM cell excitability. Additionally, the features of propagation of an AP along a length of electrically continuous smooth muscle tissue were investigated. To date, a biophysically detailed computational model does not exist for DSM cells. Our model, constrained heavily by physiological data, provides a powerful tool to investigate the ionic mechanisms underlying the genesis of DSM electrical activity, which can further shed light on certain aspects of urinary bladder function and dysfunction.

Project description: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.

Project description:Mice with smooth muscle (SM)-specific knockout of Na(+)/Ca(2+) exchanger type-1 (NCX1(SM-/-)) and the NCX inhibitor, SEA0400, were used to study the physiological role of NCX1 in mouse mesenteric arteries. NCX1 protein expression was greatly reduced in arteries from NCX1(SM-/-) mice generated with Cre recombinase. Mean blood pressure (BP) was 6-10 mmHg lower in NCX1(SM-/-) mice than in wild-type (WT) controls. Vasoconstriction was studied in isolated, pressurized mesenteric small arteries from WT and NCX1(SM-/-) mice and in heterozygotes with a global null mutation (NCX1(Fx/-)). Reduced NCX1 activity was manifested by a marked attenuation of responses to low extracellular Na(+) concentration, nanomolar ouabain, and SEA0400. Myogenic tone (MT, 70 mmHg) was reduced by approximately 15% in NCX1(SM-/-) arteries and, to a similar extent, by SEA0400 in WT arteries. MT was normal in arteries from NCX1(Fx/-) mice, which had normal BP. Vasoconstrictions to phenylephrine and elevated extracellular K(+) concentration were significantly reduced in NCX1(SM-/-) arteries. Because a high extracellular K(+) concentration-induced vasoconstriction involves the activation of L-type voltage-gated Ca(2+) channels (LVGCs), we measured LVGC-mediated currents and Ca(2+) sparklets in isolated mesenteric artery myocytes. Both the currents and the sparklets were significantly reduced in NCX1(SM-/-) (vs. WT or NCX1(Fx/-)) myocytes, but the voltage-dependent inactivation of LVGCs was not augmented. An acute application of SEA0400 in WT myocytes had no effect on LVGC current. The LVGC agonist, Bay K 8644, eliminated the differences in LVGC currents and Ca(2+) sparklets between NCX1(SM-/-) and control myocytes, suggesting that LVGC expression was normal in NCX1(SM-/-) myocytes. Bay K 8644 did not, however, eliminate the difference in myogenic constriction between WT and NCX1(SM-/-) arteries. We conclude that, under physiological conditions, NCX1-mediated Ca(2+) entry contributes significantly to the maintenance of MT. In NCX1(SM-/-) mouse artery myocytes, the reduced Ca(2+) entry via NCX1 may lower cytosolic Ca(2+) concentration and thereby reduce MT and BP. The reduced LVGC activity may be the consequence of a low cytosolic Ca(2+) concentration.

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L-type Ca2+ channel sparklets revealed by TIRF microscopy in mouse urinary bladder smooth muscle.

Publications

L-type Ca2+ channel sparklets revealed by TIRF microscopy in mouse urinary bladder smooth muscle.

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