| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS
Department of 1Clinical Physiopathology, 2Physiological Sciences, 3Pharmacology, 4Urology, University of Florence, Florence; 5Bioxell, Milan; and 6National Institute of Biostructures and Biosystems, Rome, Italy
 |
ABSTRACT |
|---|
 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
1C-subunit. In the bladder from Sprague-Dawley rats, BAY K 8644 induced a dose-dependent increase in tension, which was significantly enhanced by elocalcitol treatment (30 µg·kg–1·day–1, 2 wk). In conclusion, elocalcitol upregulated Ca2+ entry through L-type Ca2+ channels in hBCs, thus balancing its inhibitory effect on RhoA/Rho kinase signaling and suggesting its possible efficacy for the modulation of bladder contractile mechanisms. vitamin D analogue; human bladder smooth muscle cells; voltage-gated calcium channel; overactive bladder
Elocalcitol (also known as BXL-628) is a vitamin D receptor (VDR) agonist able to inhibit RhoA/ROCK signaling in both SHR bladder strips and human bladder cells (21), thus, suggesting the use of this molecule in controlling bladder contractile activity. Interestingly, in the normal bladder from Sprague-Dawley (SD) rats, elocalcitol treatment delays the contractile effect of carbachol without changing maximal responsiveness (21). This observation suggests that elocalcitol-induced inhibition of RhoA/ROCK signaling could be balanced by an upregulation of other intracellular contractile pathways to maintain the overall contractile function of the bladder. The key role played by Ca2+ influx via L-type Ca2+ channel for bladder contraction (1) has been clearly demonstrated in mice deficient for the L-type Ca2+ channel pore-forming subunit Cav1.2 (32). In these mice, K+- and carbachol-induced bladder contractions were significantly reduced compared with wild-type mice and partially compensated by ROCK hyperactivity, as demonstrated by an increased sensitivity to Y27632. The reverse could occur under elocalcitol dosing, which reduces ROCK signaling. Since bladder strips from elocalcitol-treated rats exhibited a higher sensitivity to the selective L-type Ca2+ channel antagonist isradipine (21), we hypothesized an upregulation of L-type Ca2+ channel activity. To confirm this hypothesis, we investigated the effects of elocalcitol on bladder smooth muscle L-type calcium channels. Here we report results from electrophysiological, molecular, and intracellular calcium transient studies performed on primary cell cultures of human bladder smooth muscle cells (hBCs), which has been previously characterized (11, 21). Functional studies of in vitro contractility in urinary bladder strips from rats chronically treated with elocalcitol are also described.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
-fluoro-25-hydroxy-16,23E-diene-26,27-bishomo-20-epi-cholecalciferol (referred to as elocalcitol) at a fixed concentration (10 nM) for 6, 24, and 48 h (gene expression experiments) or for 48 h (protein expression experiments). Elocalcitol was provided by Dr. Milan Uskokovic (BioXell, Nutley, NJ). For cytosolic free calcium concentration measurements ([Ca2+]i) and electrophysiological studies, hBCs were seeded in their growth medium onto sterile glass coverslips and cultured until 70–80% confluence. Experiments were performed as described below. [Ca2+]i measurements in hBCs. [Ca2+]i was measured in hBCs using fura-2 applied to microfluorescent digital video imaging, as previously described (14). Subconfluent cells, grown on round glass coverslips, were starved for 24 h in serum-free medium and then were loaded with 4 µmol/l of fura 2-AM and 15% Pluronic F-127 (Molecular Probes, Eugene, OR) for 40 min at 22°C. [Ca2+]i was measured in fura 2-loaded cells in HEPES-NaHCO3 buffer containing (in mmol/l) 140 NaCl, 4.8 KCl, 0.5 NaH2PO4, 12 NaHCO3, 1.2 MgCl2, 1.5 CaCl2, 10 HEPES, and 10 glucose (pH 7.4). Verapamil (Sigma-Aldrich, St. Louis, MO) treatment (10–6 M) was performed for 48 h before [Ca2+]i measurements. Experiments performed in the absence of extracellular calcium were performed in a buffer of a similar composition as above, where in nominally calcium-free buffer EDTA 0.5 mM was added. Thapsigargin (3 x 10–8 M, Sigma-Aldrich) and different concentrations of elocalcitol (10–12-10–7 M) were directly added into the perfusion chamber (time 0 in the time-course graphics) after recording of the [Ca2+]i basal value. Ratio images (340–380 nm excitation, 510 nm emission) were collected every 3 s; calibration curves were obtained as previously described (14). At least eight cells found in the same optical field (using x40 magnification objective) were analyzed for each different treatment, and the value obtained from each single cell was pooled within the same experiment. Each treatment was repeated using three different cell preparations; the number of different experiments is indicated as n.
Electrophysiological recordings.
The electrophysiological records were obtained by the whole cell patch-clamp technique in voltage-clamp conditions. Coverslips with adherent cells (hBCs) were superfused at a rate of 1.8 ml/min with an external TEA-Ca2+ bath solution that blocked any ionic current but Ca2+ current and with the following composition (in mM): 10 CaCl2, 145 TEA-Br, and 10 HEPES. The patch pipettes were filled with an internal solution (in mM): 150 CsBr, 5 MgCl2, 10 EGTA, and 10 HEPES. pH was 7.4 and 7.2 for the bath and pipette solution, respectively. When filled, the resistance of the pipettes measured 3–7 M
. The details of the technique, set up, and electronics are described in Benvenuti et al. (3). Briefly, the patch pipette was connected to a micromanipulator and an Axopatch 200B amplifier (Axon Instruments, Union City, CA). Voltage-clamp protocol generation and data acquisition were controlled by using an output and an input of the A/D-D/A interfaces (Digidata 1200; Axon Instruments) and Pclamp 9 software (Axon Instruments). Currents were low-pass filtered with a Bessel filter at 2 kHz. For the activation pulse protocol the cell was held at –90 mV, and 4-s long step pulses from –80 to 50 mV were applied in 10-mV increments; the protocol used an interval of 20 s between stimulating episodes for recovery. The steady-state inactivation for Ca2+ current (ICa) was studied by two-pulse protocol with a 1-s prepulse to different voltages, 1-s test pulse fixed to 10 mV, and interpulse of 200 ms. We used prepulses and test pulse of 1 s duration since L-type ICa did not completely inactivate: its decay reached a steady-state value after about 0.8 s in control and 0.6 or less after BAY K 8644 or elocalcitol treatments. The interpulse interval to the holding potential of 200 ms was chosen to both prevent substantial recovery from inactivation between activating pulses and allow the activation kinetics of Ca2+ permeability to return to its resting state. Again, in the two-pulse protocol, we used an interval of 20 s between stimulating episodes for recovery. Every activation and inactivation protocols were repeated twice. The steady-state ionic current of activation was evaluated by Ia(V) = Gmax(V – Vrev)/{1 + exp[(Va – V)/ka]} and steady-state inactivation by Ih(V) = I/{1 + exp[–(Vh – V)/kh]}, where Gmax is the maximal conductance for the Ia; Vrev is the apparent reversal potential; Va and Vh are the potentials that elicit, respectively, the half-maximal activation and inactivation values; and ka and kh are the steepness factors. The amount of Ca2+ entry was evaluated by the time integral of ICa time course elicited by voltage step at 0 mV. To compare the contribution of the early rising phase respect to that of the falling phase, we calculated the time integral of the first 20 ms and of the following 4 s of the current trace. The membrane capacitance (Cm) was used as an index of cell-surface area assuming that membrane-specific capacitance is constant at 1 µF/cm2. To allow comparison of test current recorded from different cells, the current amplitude and membrane conductance (Gm) were normalized to Cm. Experiments were performed at 22°C.
Real-time quantitative RT-PCR.
Isolation of RNA from hBCs was performed using RNAeasy kit (Quiagen, Valencia, CA). cDNA synthesis were performed as previously described (11). The mRNA quantitative analysis (qRT-PCR) was performed according to the fluorescent TaqMan methodology (19). PCR primers and probes specific for mRNA sequence of target genes (CACNA1C/1C, CACNA1D/1D, and CACNA1S/1S) were purchased from Applied Biosystems (Foster City, CA). GAPDH was chosen as reference gene and selected among the endogenous control provided by Applied Biosystems. Amplification and detection were performed with the ABI Prism 7700 Sequence Detection System, as previously reported (21). Each measurement was carried out in duplicate. Data analysis was based on the comparative Ct method according to the manufacturer's instructions (Applied Biosystems).
SDS-PAGE/Western blot analysis. After treatment, cells were washed in phosphate-buffered saline and scraped in 1x MLB lysis buffer (5x lysis buffer: 125 mM HEPES, pH 7.5, 750 mM NaCl, 5% Igepal CA-630, 50 mM MgCl2, 5 mM EDTA, and 10% glycerol) Aliquots containing 80 µg of proteins measured by Bradford's method using Coomassie reagent (Bio-Rad Labs, Hercules, CA) were diluted in reducing sample buffer (62.5 mM Tris, pH 6.8, 10% glycerol, 20% SDS, 2.5% pyronin, and 100 mM dithiothreitol) and loaded onto 7% SDS-PAGE. After separation by SDS-PAGE, proteins were transferred to polyvinyldifluoride membrane (Immobilon-P, Millipore, Bedford, MA). Membranes were blocked overnight at 4°C in 3% BSA in 1x PBS (Sigma-Aldrich)-0.01% Tween and incubated for 5 h with primary antibodies anti-human Cav1.2 (1:500 dilution, Alomone Labs, Jerusalem, Israel) and anti-actin (1:1,000 dilution, Santa Cruz Biotechnology) in 0.2% BSA in 1x PBST followed by peroxidase-conjugated secondary IgG (1:10,000, Santa Cruz Biotechnology). Finally, bands were visualized by home-made chemiluminescence reagent (10) and autoradiographed using hyperfilm (GE Healthcare, Buckinghamshire, UK). Densitometric analysis of band intensity was performed using Photoshop 5.5 software (Adobe Systems, Agrate Brianza, Milan, Italy).
Contractility studies. Male SD rats (275–300 g; Harlan Italy, San Pietro al Natisone, Udine, Italy) were divided into two groups: 1) untreated and 2) treated with elocalcitol, 30 µg/kg by daily oral gavage, for a total of 9 administrations in 2 wk, as previously described (10). Blood for serum calcium measurements was obtained at the end of experimental protocol. Animals were euthanized by cervical dislocation, and the urinary bladders were removed for in vitro contractility studies. Bladder strips, longitudinally dissected, were mounted in 10-ml vertical tissue baths as previously reported (21). High potassium salt solution (KCl), made by equimolar substitution of sodium by potassium, increased the tonic tension of bladder strips, with the maximum effect obtained at 80 mM. The increase in basal tension elicited by a fixed dose of carbachol (10 µM) was referred to that evoked by 80 mM KCl, taken as 100%. The maximal tonic tension elicited by 10 µM carbachol (Sigma-Aldrich) was then taken as 100%, and the contractile effect induced by different concentrations of S-(–)-1,4-dihydro-2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl)phenyl]-3-pyridinecarboxylic acid methyl ester (BAY K 8644, Sigma-Aldrich) was referred to this value. Stock solutions of BAY K 8644 were made in ethanol; carbachol was freshly dissolved in double-distilled water and further dilutions of all substances were made in Krebs solution. Control experiments with BAY K 8644 showed that the final concentrations of ethanol did not modify the vasoconstrictor response to BAY K 8644.
Experiments were performed in accordance to the Italian Ministerial Law no. 116/92 and approved by the Institutional Animal Care and Use Committee of the University of Florence, Florence, Italy.
Serum calcium measurements. Serum calcium levels were measured using a commercially available colorimetric assay (Sigma-Aldrich), according to the manufacturer's instructions, as previously reported (10).
Statistical analysis. Results are expressed as means ± SE. Statistical analysis was performed with one-way ANOVA test followed by Tukey-Kramer post hoc analysis, or, for electrophysiological data, by two-way ANOVA with Bonferroni's correction for multiple comparisons. P < 0.05 was taken as significant. Half-maximal response effective concentration (EC50) was calculated using the computer program ALLFIT (12).
|
RESULTS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
Voltage-gated Ca2+ currents in hBCs.
hBCs exhibited voltage-activated ICa. ICa traces representative of three different cell preparations are shown in Fig. 2A (holding potential, HP = –90 mV). The inward transient current recorded from –50 mV indicates the occurrence of T-type ICa (Fig. 2, A–F). This current at –40 mV showed a time to peak (tp) at 55 ms and decayed with a time constant (
i) of about 50 ms. The slower decay observed starting from –30 mV, with i = 130 ms at 0 mV, indicates the activation of L-type Ca2+ channels. The inactivating current was evaluated by fitting the ICa decay by an exponential function plus a constant such as ICa(t) = ICa,decay exp(–t/i) + ICa,ss, where ICa,ss is a constant. The value of ICa,ss was equal to zero for T-type but different to zero for L-type ICa. This inactivating current value is indicated in Table 1. The peak current of ICa versus V plot (ICa-V) agrees with the occurrence of T- and L-type ICa due to a change in steepness between –30 and –20 mV (Fig. 2G). Accordingly, the selective L-type Ca2+ channel antagonists isradipine (10–7 M, Fig. 2, B and G) or verapamil (10–7 M, data not shown), only blocked the slow L-type ICa. Hence, the decaying phase was faster (i about 50 ms). The residual ICa, corresponding to T-type ICa, could be reduced in size, using the two-pulse protocol, by prepulsing the test voltage step to 10 mV and was completely blocked by prepulses above –30 mV (Fig. 2B).
|
|
i from 130 to 104 ms; see Table 1, P < 0.05 vs. control). Moreover, it increased the steady-state Ca2+ current (ICa,ss) value. Notably, with respect to control, the Boltzmann parameters of activation and inactivation curves were shifted of 5 (Va, P < 0.01) and 10 mV (Vh, P < 0.001) toward more negative potentials, respectively (Table 1). Because the ka and Vrev values are not substantially different with respect to control, the stronger increase of ICa/Cm with respect to Gm/Cm agrees with the shift of Va (Fig. 3C).
|
i of the exponential decay at 0 mV was 130, 104, and 60 ms in control, BAY K 8644, and elocalcitol, respectively (Table 1). Elocalcitol increased L-type ICa size and the specific conductance (Gm/Cm), but less than BAY K 8644 (Fig. 3C and Table 1). Finally, elocalcitol determined a significant negative shift of the activation and inactivation curves (see Va and Vh value, respectively, in Table 1), as observed with BAY K 8644. Interestingly, elocalcitol differed from BAY K 8644 also in a time-dependent Ca2+ entry, as evaluated by the time integral of ICa: Ca2+ entry induced by elocalcitol, which was higher in the first 10 ms but lower at later time points (4 s, see Fig. 3D and ICa,ss/Cm value in Table 1). T- and L-type ICa did not change significantly during the different protocols denoting that the effect of elocalcitol persisted at least more than 40 min (that is the experiment duration in a single cell) .
Long-term effects of elocalcitol on Ca2+ currents.
The long-term effect of elocalcitol on Ca2+ currents was evaluated in hBCs treated for 48 h with the VDR agonist (10–8 M). Again, as shown in Figs. 2, E and G, and 3A, elocalcitol did not affect T-type but only L-type ICa. In L-type ICa, a further significant shift of Va and Vh toward more negative potentials was observed in long-term stimulated cells compared with acute treatments (Table 1). Moreover, both Gm/Cm and ICa/Cm were significantly increased (P < 0.05) in long-term versus acute treatments (Figs. 2, E and G and 3C; elocalcitol 48 h). Interestingly, treating hBCs with 10–8 M elocalcitol for 48 h did not prevent further activation of the Ca2+ channels by 10–5 M BAY K 8644. In fact, this agonist did not modify Va, Vh, and i values (Table 1) but caused a significant (P < 0.05) further increase of Gm/Cm, ICa/Cm, and time integral at the early 20 ms (Figs. 2, F and G, and 3, C and D; Table 1). These results suggest that BAY K 8644 and elocalcitol operate through different mechanisms on L-type Ca2+ channels. When experiments were performed in hBCs pretreated with verapamil (10–7 M, 48h), elocalcitol did not induce any effect (Fig. 3C).
L-type calcium channel expression in hBCs.
To test whether elocalcitol effects on Ca2+ currents in hBCs were associated to changes in the expression of L-type Ca2+ channel, we analyzed gene and protein expression of the corresponding pore-forming 1 subunit. By qRT-PCR we analyzed in hBCs the mRNA levels of different isoforms of the L-type 1 subunits, 1C, 1D, and 1S. In good agreement with the known tissue distribution of the L-type 1 isoforms in smooth muscle cells (13), we found that the mRNA level for 1C (also referred to as Cav1.2) was the most abundant in hBCs, being 1,000-fold more expressed than the 1D isoform, whereas mRNA for the 1S (skeletal muscle isoform) was not detectable, as expected (Fig. 4A). Time-course experiments (6, 24, and 48 h) in hBCs showed that treatment with 10–8 M elocalcitol significantly induced mRNA expression for 1C only at 48 h (Fig. 4B; P < 0.01, n = 5) and did not affect 1D expression levels (data not shown). Protein expression results obtained by Western blot analysis were consistent with those from qRT-PCR, confirming the elocalcitol-induced increase in Cav1.2 expression. As shown in Fig. 4C, a band corresponding to the expected molecular mass for Cav1.2 protein, just over 200 kDa, was detected in hBCs. This immunopositivity was significantly increased (P < 0.01, n = 4) in protein extracts from elocalcitol-treated (10–8 M, 48 h) compared those with untreated hBCs (Fig. 4D).
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
L-type Ca2+ channels are heteromeric macromolecules composed of a pore-forming 1 (or CaV1) subunit that can generate Ca2+ currents, plus ancillary 2/
, β, and, in skeletal muscle, 
subunits (see Ref. 9 for review). According to the proposed nomenclature of voltage-gated Ca2+ channels, the CaV1 family (CaV1.1 through CaV1.4) includes channels containing 1S, 1C, 1D, and 1F, which specifically mediate L-type Ca2+ currents, with the 1C (also referred to as CaV1.2) being the most expressed in smooth muscle cells (13). Accordingly, hBCs showed a predominant 1C mRNA expression.
L-type Ca2+ channels belong to the "high-threshold" activation type of calcium channels, because they open under large depolarization of the plasma membrane. In hBCs, we found that both BAY K 8644 and elocalcitol activate calcium currents, a novel observation compatible with L-type Ca2+ channel activation. Moreover, the steady-state activation and inactivation curves relative to the L-type Ca2+ channel opening were clearly shifted more toward negative potential by acute exposure to elocalcitol and, to a greater extent, after long-term treatment. These findings suggest an improved and earlier excitability of hBCs in the presence of elocalcitol. Thus elocalcitol may induce influx of Ca2+ into hBCs at potentials close to the resting value. In addition, the faster inactivation kinetics observed in elocalcitol-treated versus untreated hBCs may be responsible for a time-limited faster change of intracellular calcium that could promote phasic contractions and conversely could inhibit a sustained contraction. About twofold increase of specific channel conductance and about 2.5-fold faster time course of L-type Ca2+ currents were observed under elocalcitol stimulus. In good agreement with our findings, 1,25-dihydroxyvitamin D3, 1,25(OH)2D3, the natural vitamin D3 hormone, facilitates the opening of L-type Ca2+ channels in osteoblastic-like osteosarcoma ROS 17/2.8 cells at membrane potentials close to the resting value (8). Indeed, effects of 1,25(OH)2D3 on cell excitability via modulation of calcium influx through L-type Ca2+ channels have been described in a variety of cell systems, including neuronal (6, 7) and bone (8, 18, 29, 34) cells, as well as diverse muscle cell types, such as skeletal (31), cardiac (27), and vascular smooth muscle (4). In osteoblasts, along with genomic effects on gene transcription, 1,25(OH)2D3 also promotes nongenomic rapid responses by acting on L-type Ca2+ channel activities (8, 18, 29, 34). In hippocampal neurons, 1,25(OH)2D3 reduces age-related changes associated with Ca2+ dysregulation (6) and, in particular, confers neuroprotection by downregulating L-type Ca2+ channel expression (7). Finally, in isolated cardiac muscle cells the L-type Ca2+ channel blockers nifedipine and verapamil abolished the increase in Ca2+ uptake produced by 1,25(OH)2D3 (27).
In the present studies, in addition to rapid, nongenomic actions on Ca2+ fluxes, we found that both gene and protein expression levels of pore-forming 1C subunit were significantly upregulated in hBCs after 48 h treatment with elocalcitol, suggesting also long-term genomic effects of the VDR agonist on L-type Ca2+ channel expression. In contrast to our results, studies performed in osteoblastic (20) and neuronal (7) cells showed that chronic treatment with 1,25(OH)2D3 downregulates 1C mRNA expression. This apparent discrepancy may reflect the cell-type selectivity of elocalcitol-mediated actions, which depend on coactivators and corepressors selectively expressed in different cell types (15). Alternatively, this discrepancy may reflect a different functional role played by calcium entry through L-type channels in different cell types.
In any case, the molecular evidence for increased Cav1.2/1C expression by elocalcitol treatment of hBCs is further strengthened by our electrophysiological data, obtained in hBCs chronically treated with the VDR agonist. Indeed, a significant shift of the activation and inactivation membrane potentials toward more negative values was observed in long-term stimulated cells compared with acute treatments, along with an increase of L-type ICa amplitude and specific channel conductance (Gm/Cm). In addition, the functional enhancement in sensitivity to L-type calcium agonist (BAY K 8644, present study) and antagonist (isradipine; Ref. 21) in the bladder of SD rats chronically treated for 2 wk with elocalcitol, further support a positive effect of elocalcitol on L-type Ca2+ channel.
This novel property of elocalcitol regulating opening and functional activity of L-type Ca2+ channel in hBCs could open new opportunities for VDR ligation in bladder pharmacology. Precisely coordinated contractions of different smooth muscle components are required to ensure a normal bladder function, which involves not only the ability to fill and store urine at low pressure, but also to generate sufficient force for complete bladder emptying. The contractile force of bladder smooth muscle cells is dependent on several mechanisms that regulate the phosphorylated status of MLC, through a balance between the activity of MLC kinase (calcium dependent) and MLC phosphatase. The activity of the latter is negatively regulated by calcium-independent mechanisms, mainly RhoA/ROCK-mediated. Elocalcitol inhibits the RhoA/ROCK signaling, thus reducing calcium sensitizing-mediated contractions (21), but also activates L-type calcium channels, as shown in the present study, thus promoting calcium-dependent contractile mechanisms. These apparently antithetic activities of elocalcitol could explain its overall lack of effect on carbachol-stimulated bladder contractility, as observed after chronic dosing in different rat models (21, 26, and present study). Therefore, elocalcitol does not simply suppress bladder contractions, but modulates bladder contractility, by decreasing calcium sensitization and increasing L-type-mediated calcium entry.
In conclusion, the present studies identify a novel action of the VDR agonist elocalcitol, by showing its capacity to regulate calcium entry through L-type Ca2+ channels in the hBCs. This suggests a possible efficacy of elocalcitol for the modulation of bladder contractile mechanisms. Promising preclinical studies performed in a rat model of bladder outlet obstruction (BOO) have demonstrated the efficacy of elocalcitol in reducing the negative functional changes of the bladder smooth muscle associated with BOO, thus preserving the emptying ability of the bladder (26). Moreover, a proof of concept clinical study has recently demonstrated the ability of elocalcitol to ameliorate overactive bladder symptoms compared with placebo (30).
|
FOOTNOTES |
|---|
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
|
REFERENCES |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
2. Andersson KE, Wein AJ. Pharmacology of the lower urinary tract: basis for current and future treatments of urinary incontinence. Pharmacol Rev 56: 581–631, 2004.
3. Benvenuti S, Saccardi R, Luciani P, Urbani S, Deledda C, Cellai I, Francini F, Squecco R, Rosati F, Danza G, Gelmini S, Greeve I, Rossi M, Maggi R, Serio M, Peri A. Neuronal differentiation of human mesenchymal stem cells: changes in the expression of the Alzheimer's disease-related gene seladin-1. Exp Cell Res 312: 2592–2604, 2006.[Web of Science][Medline]
4. Bian K, Ishibashi K, Bukoski RB. 1,25(OH)2D3 modulates intracellular Ca2+ and force generation in resistance arteries. Am J Physiol Heart Circ Physiol 270: H230–H237, 1996.
5. Bing W, Chang S, Hypolite JA, DiSanto ME, Zderic SA, Rolf L, Wein AJ, Chacko S. Obstruction-induced changes in urinary bladder smooth muscle contractility: a role for Rho kinase. Am J Physiol Renal Physiol 285: F990–F997, 2003.
6. Brewer LD, Porter NM, Kerr DS, Landfield PW, Thibault O. Chronic 1alpha,25-(OH)2 vitamin D3 treatment reduces Ca2+-mediated hippocampal biomarkers of aging. Cell Calcium 40: 277–286, 2006.[CrossRef][Web of Science][Medline]
7. Brewer LD, Thibault V, Chen KC, Langub MC, Landfield PW, Porter NM. Vitamin D hormone confers neuroprotection in parallel with downregulation of L-type calcium channel expression in hippocampal neurons. J Neurosci 21: 98–108, 2001.
8. Caffrey JM, Farach-Carson MC. Vitamin D3 metabolites modulate dihydropyridine-sensitive calcium currents in clonal rat osteosarcoma cells. J Biol Chem 264: 20265–20274, 1989.
9. Catterall W. Structure and regulation of voltage-gated Ca2+ channels. Annu Rev Cell Dev Biol 16: 521–555, 2000.[CrossRef][Web of Science][Medline]
10. Crescioli C, Ferruzzi P, Caporali A, Mancina R, Comerci A, Muratori M, Scaltriti M, Vannelli GB, Smiroldo S, Mariani R, Villari D, Bettuzzi S, Serio M, Adorini L, Maggi M. Inhibition of spontaneous and androgen-induced prostate growth by a nonhypercalcemic calcitriol analog. Endocrinology 144: 3046–3057, 2003.
11. Crescioli C, Morelli A, Adorini L, Ferruzzi P, Luconi M, Vannelli GB, Marini M, Gelmini S, Fibbi B, Donati S, Villari D, Forti G, Colli E, Andersson KE, Maggi M. Human bladder as a novel target for vitamin D receptor ligands. J Clin Endocrinol Metab 90: 962–972, 2005.
12. De Lean A, Munson PJ, Rodbard D. Simultaneous analysis of families of sigmoidal curves: application to biossay, radioligand assay, and physiological dose-response curves. Am J Physiol Endocrinol Metab Gastrointest Physiol 235: E97–E102, 1978.
13. Ertel EA, Campbell KP, Harpold MM, Hofmann F, Mori Y, Perez-Reyes E, Schwartz A, Snutch TP, Tanabe T, Birnbaumer L, Tsien RW, Catterall WA. Nomenclature of voltage-gated calcium channels. Neuron 25: 533–535, 2000.[CrossRef][Web of Science][Medline]
14. Failli P, Rocco C, De Franco R, Caligiuri A, Gentilizi A, Giotti A, Gentilizi P, Pinzani M. The mitogenic effect of platelet-derived growth factor in human hepatic stellate cells requires calcium influx. Am J Physiol Cell Physiol 269: C1133–C1139, 1995.
15. Fibbi B, Morelli A, Marini M, Zhang XH, Mancina R, Vignozzi L, Filippi S, Chavalmane A, Silvestrini E, Colli E, Adorini L, Vannelli GB, Maggi M. Atorvastatin but not elocalcitol increases sildenafil responsiveness in Spontaneously Hypertensive Rats by regulating the RhoA/ROCK pathway. J Androl 29: 70–84, 2007.[CrossRef][Web of Science][Medline]
16. Fleichman M, Schneider T, Fetscher C, Michel MC. Signal transduction underlying carbachol-induced contraction of rat urinary bladder. II. Protein kinases. J Pharmacol Exp Ther 308: 54–58, 2004.
17. Hegde SS. Muscarinic receptors in the bladder: from basic research to therapeutics. Br J Pharmacol 147: S80–S87, 2006.[CrossRef][Web of Science][Medline]
18. Li W, Farach-Carson MC. Parathyroid hormone-stimulated resorption in calvaria cultured in serum-free medium is enhanced by the calcium-mobilizing activity of 1,25-dihydroxyvitamin D(3). Bone 29: 231–235, 2001.[Medline]
19. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)). Methods 25: 402–408, 2001.[CrossRef][Web of Science][Medline]
20. Meszaros JG, Karin NJ, Akanbi K, Farach-Carson MC. Down-regulation of L-type Ca2+ channel transcript levels by 1,25-dihyroxyvitamin D3. Osteoblastic cells express L-type alpha1C Ca2+ channel isoforms. J Biol Chem 271: 32981–32985, 1996.
21. Morelli A, Vignozzi L, Filippi S, Vannelli GB, Ambrosini S, Mancina R, Crescioli C, Donati S, Fibbi B, Colli E, Adorini L, Maggi M. BXL-628, a vitamin D receptor agonist effective in benign prostatic hyperplasia treatment, prevents RhoA activation and inhibits RhoA/Rho kinase signaling in rat and human bladder. Prostate 67: 234–247, 2007.[CrossRef][Web of Science][Medline]
22. Rajasekaran M, White S, Baquir A, Wilkes N. Rho-kinase inhibition suppresses bladder hyperactivity in spontaneously hypertensive rats. J Androl 26: 182–188, 2005.
23. Ratz PH, Miner AS. Length-dependent regulation of basal myosin phosphorylation and force in detrusor smooth muscle. Am J Physiol Regul Integr Comp Physiol 284: R1063–R1070, 2003.
24. Rivera L, Brading AF. The role of Ca2+ influx and intracellular Ca2+ release in the muscarinic-mediated contraction of mammalian urinary bladder smooth muscle. BJU Int 98: 868–875, 2006.[CrossRef][Web of Science][Medline]
25. Schneider T, Fetscher C, Krege S, Michel MC. Signal transduction underlying carbachol-induced contraction of human urinary bladder. J Pharmacol Exp Ther 309: 1148–1153, 2004.
26. Schröder A, Colli E, Maggi M, Andersson KE. Effects of a vitamin D(3) analogue in a rat model of bladder outlet obstruction. BJU Int 98: 637–642, 2006.[CrossRef][Web of Science][Medline]
27. Selles J, Boland R. Rapid stimulation of calcium uptake and protein phosphorylation in isolated cardiac muscle by 1,25-dihydroxyvitamin D3. Mol Cell Endocrinol 77: 67–73, 1991.[CrossRef][Web of Science][Medline]
28. Takahashi R, Nishimura J, Hirano K, Seki N, Naito S, Kanaide H. Ca2+ sensitization in contraction of human bladder smooth muscle. J Urol 172: 748–752, 2004.[CrossRef][Web of Science][Medline]
29. Takeuchi K, Guggino SE. 24R,25-(OH)2 vitamin D3 inhibits 1alpha,25-(OH)2 vitamin D3 and testosterone potentiation of calcium channels in osteosarcoma cells. J Biol Chem 271: 33335–33343, 1996.
30. Tiwari A, Naruganahalli KS. Current and emerging investigational medical therapies for the treatment of overactive bladder. Expert Opin Investig Drugs 15: 1017–1037, 2006.[CrossRef][Web of Science][Medline]
31. Vazquez G, de Boland AR, Boland RL. 1alpha,25-dihydroxy-vitamin-D3-induced store-operated Ca2+ influx in skeletal muscle cells. Modulation by phospholipase C, protein kinase C, and tyrosine kinases. J Biol Chem 273: 33954–33960, 1998.
32. Wegener JW, Schulla V, Lee TS, Koller A, Feil S, Feil R, Kleppisch T, Klugbauer N, Moosmang S, Welling A, Hofmann F. An essential role of Cav1.2 L-type calcium channel for urinary bladder function. FASEB J 18: 1159–1161, 2004.
33. Wibberley A, Chen Z, Hu E, Hieble JP, Westfall TD. Expression and functional role of Rho-kinase in rat urinary bladder smooth muscle. Br J Pharmacol 138: 757–766, 2003.[CrossRef][Web of Science][Medline]
34. Zanello LP, Norman A. 1alpha,25(OH)2 Vitamin D3 actions on ion channels in osteoblasts. Steroids 71: 291–297, 2006.[CrossRef][Web of Science][Medline]
This article has been cited by other articles:
|
|
 |
|
  M. T. Mizwicki and A. W. Norman The Vitamin D Sterol-Vitamin D Receptor Ensemble Model Offers Unique Insights into Both Genomic and Rapid-Response Signaling Sci. Signal., June 16, 2009; 2(75): re4 - re4. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Ordonez-Moran, M. J. Larriba, H. G. Palmer, R. A. Valero, A. Barbachano, M. Dunach, A. G. de Herreros, C. Villalobos, M. T. Berciano, M. Lafarga, et al. RhoA-ROCK and p38MAPK-MSK1 mediate vitamin D effects on gene expression, phenotype, and Wnt pathway in colon cancer cells J. Cell Biol., November 17, 2008; 183(4): 697 - 710. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
Ratio (
). B: effect of Elo in hBCs pretreated (dashed line) or untreated (solid line) with verapamil (10–6 M, 48 h). C: effect of Elo (solid line) or buffer alone (dotted line), administered at the first black diamond (
P < 0.05 between the indicated bars.
200 kDa; top) and actin (molecular mass 44 kDa, bottom) protein expression in hBCs treated or not with Elo (10–8 M for 48 h). Images are representative 7% SDS-PAGE run for protein extracts of two distinct cell preparations (untreated- and Elo-1; untreated- and Elo-2). D: densitometric computer-assisted analysis for Cav1.2 band intensity normalized over actin in hBCs treated or not with Elo (10–8 M for 48 h). Results are expressed as percentage with respect to untreated cells and are reported as means ± SE of 4 experiments.