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Published as doi: 10.1096/fj.06-7326com.
 (The FASEB Journal. 2007;21:2818-2828.)
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Sphingosine-1-phosphate and the immunosuppressant, FTY720-phosphate, regulate detrusor muscle tone

Kenneth R. Watterson, Krystina M. Berg, Dmitri Kapitonov, Shawn G. Payne, Amy S. Miner, Robert Bittman{ddagger}, Sheldon Milstien{dagger}, Paul H. Ratz1 and Sarah Spiegel1,2

Department of Biochemistry and Molecular Biology, Virginia Commonwealth University School of Medicine, Richmond, Virginia, USA;
{dagger} National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland, USA; and

{ddagger} Department of Chemistry and Biochemistry, Queens College of The City University of New York, Flushing, New York, USA

2Correspondence: Department of Biochemistry and Molecular Biology, VCU School of Medicine, 1101 E. Marshall St., 2011 Sanger Hall, Richmond, VA 23298, USA. E-mail: sspiegel{at}vcu.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Overactive bladder syndrome (OBS) results from disturbances of bladder function. Bladder smooth muscle (detrusor) exhibits spontaneous rhythmic activity (tone) independent of neurogenic control, which is enhanced in patients with OBS. We have now uncovered a prominent role for the bioactive sphingolipid metabolite, sphingosine-1-phosphate (S1P), in regulating rabbit detrusor smooth muscle tone and contraction. S1P-induced contraction of detrusor muscle was dependent on stretch and intracellular calcium. Although detrusor expresses the S1P receptors S1P1 and S1P2, only S1P2 appeared to be involved in S1P-induced contraction, since SEW2871 (S1P1 agonist) and dihydro-S1P (potent agonist for all S1P receptors except S1P2) were poor contractile agents. In agreement, the S1P2 antagonist JTE013 inhibited S1P-induced contraction. The fast, transient muscle contraction (phasic) mediated by S1P was dependent on phospholipase C (PLC) whereas the slower, sustained contraction (tonic) was not. Surprisingly, the immunosuppressant FTY720-phosphate, an agonist for all S1P receptors except S1P2, had distinct contractile properties and also induced slow, sustained contraction. Thus, FTY720-phosphate and/or S1P may regulate calcium channels in an S1P receptor-independent manner. Collectively, our results demonstrate that S1P may regulate detrusor smooth muscle tone and suggest that dysregulation of complex S1P signaling might contribute to OBS.—Watterson, K. R., Berg, K. M., Kapitonov, D., Payne, S. G., Miner, A. S., Bittman, R., Milstien, S., Ratz, P. H., Spiegel, S. Sphingosine-1-phosphate and the immunosuppressant, fty720-phosphate, regulate detrusor muscle tone.


Key Words: sphingolipid metabolite • calcium channels


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THE URINARY BLADDER IS RESPONSIBLE FOR the storage and emptying of urine. Integral to this function is the ability of the bladder to contract and relax. During storage of urine, the bladder smooth muscle (detrusor) relaxes, whereas emptying of the bladder involves coordinated contraction of detrusor smooth muscle and relaxation of the urethra. These processes are tightly regulated by central and peripheral nerves. Stimulation of postganglionic neurons in the pelvic nerve releases acetylcholine (ACh), which acts on muscarinic receptors on detrusor smooth muscle and induces contraction, whereas parasympathetic fibers from the pelvic nerve to the urethra mediate relaxation. Disturbances of bladder function lead to lower urinary tract symptoms, including urgency and frequency, resulting in overactive bladder syndrome (OBS). OBS is also a key feature of interstitial cystitis, a chronic disease characterized by suprapubic pain related to bladder filling.

Several recent studies suggest that the bioactive sphingolipid metabolite, sphingosine-1-phosphate (S1P), plays an important role in contraction of various types of smooth muscle, including airway, visceral, vascular, and gastrointestinal (1 2 3 4 5 6 7 8 9) . S1P is present at high levels in plasma and serum, and most of its actions are mediated by binding to a family of G-protein-coupled receptors, denoted S1P1–5 (10) . These receptors are abundantly expressed on smooth muscle cells. However, the importance of each receptor subtype appears to be muscle type specific. For example, S1P-induced contractions of human airway smooth muscle (2) and esophageal smooth muscle (4) are mediated via S1P2. Both S1P1 and S1P2 have been implicated in S1P-mediated contraction of gastrointestinal muscle (7 , 11) . Recently, vascular dysfunction was observed in S1P2 knockout mice, and intact S1P2-deficient aortas displayed blunted contractile responses to KCl and phenylephrine compared with wild-type aortas (12) . Vascular disturbance within the stria vascularis appears to lead to deafness in the S1P2 receptor null mice (13) . In contrast, S1P3 but not S1P2 receptors mediate the potent constriction of coronary arteries by S1P (14) . Moreover, activation of either S1P1 or S1P3 expressed on endothelial cells can also induce NO-dependent vasorelaxation (15 16 17) . Similar to other potent lipid mediators, S1P can also act in an autocrine or paracrine manner, and many external stimuli have been shown to stimulate sphingosine kinase, the enzyme that forms S1P, resulting in its increased production and secretion (18) . In agreement, muscarinic receptor-dependent airway smooth muscle contraction is linked to activation of sphingosine kinase and formation of S1P (5) . Similarly, reduced vascular tone observed after inhibition of sphingosine kinase, the enzyme that produces S1P, in vascular smooth muscle cells suggests that S1P is generated by these cells themselves (19) .

Detrusor smooth muscle is known to exhibit spontaneous rhythmic activity (tone) independent of neurogenic control, which is enhanced in patients with OBS and interstitial cystitis. Because calcium influx plays a central role in smooth muscle contractions and several studies suggest that S1P is important for calcium homeostasis of smooth muscle cells (reviewed in refs. 10 , 20 ), it was of interest to examine its role in the contraction of rabbit detrusor muscle in which there is a marked increase in rhythmic tone, a characteristic of human bladder overactivity. In this study, we found that S1P regulates detrusor smooth muscle tone and also uncovered a novel biological action of the immunosuppressant drug, FTY720-phosphate, a S1P mimetic. Moreover, our results indicate that S1P- and FTY720-phosphate may directly regulate calcium channels independent of S1P receptors. Because contractile tone is enhanced in patients with OBS, the regulation of detrusor tone by S1P has important clinical implications.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Tissue preparation
Whole bladders from adult female New Zealand white rabbits were washed, cleaned of adhering tissue, including fat and serosa, and placed in a cold physiological salt solution (PSS) containing 140 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 1.6 mM CaCl2, 1.2 mM Na2HPO4, 2.0 mM MOPS, pH 7.4, 0.02 mM Na2-EGTA, and 5.6 mM dextrose. PSS will be referred to as a "Ca2+-containing solution" while PSS with no CaCl2 and the addition of 1 mM EDTA to chelate Ca2+ as a "Ca2+-free solution."

Longitudinal detrusor strips free of underlying urothelium were cut from the wall of the bladder above the trigone. Each muscle strip was incubated in aerated PSS at 37°C and secured by small muscle clips to a micrometer for manual length adjustments and a force transducer (Harvard Bioscience, Holliston, MA, USA; and Radnotti Glass Technology, Monrovia, CA, USA) for measurement of isometric contraction as described (21) .

Determination of optimal length (Lo) for contraction of isolated detrusor strips
Isometric contraction was measured exactly as described previously (21) . Briefly, voltage signals were digitized (PCI-6024E National Instruments) and data were analyzed with multichannel data integration software (DMC from Aurora Scientific, Aurora, Ontario, Canada; and DASYLab from National Instruments, Austin, TX, USA). Muscle tissues were secured to clips and the initial (cold) zero preload length was ~ 5 mm. The chamber was then equilibrated for 1 h at 37°C to permit development of spontaneous rhythmic contraction (tone) (22) . The media was replaced with Ca2+-free PSS to eliminate spontaneous contractile activity; muscles were stretched in 0.5 mm steps and allowed to stress-relax with each increase until a stable preload between 0.025 g and 0.05 g above zero was established (slack length, or Ls). Basal tone was regained by incubating tissues in Ca2+-containing PSS and the maximal force produced by 110 mM KCl at Ls (Fs) was determined. Tissues were then stretched to various lengths above Ls or relaxed below Ls in Ca2+-free PSS, and the new tissue length (L) was expressed as a ratio to Ls (L/Ls). On regaining tone in PSS, detrusor contractions at various lengths (F) were induced by either 110 mM KCl, 1 µM S1P, or 1 µM carbachol. Detrusor contractions were then expressed as a proportion of the KCl-induced contraction at Ls (F/Fs). S1P-induced detrusor contraction was highly dependent on stretch. S1P does not produce significant muscle contraction at lengths less than or equal to an L/Ls value of 1. As the muscle was stretched, the tone of the muscle, defined as the average force produced by the muscle at rest, increased in a manner related to the typical length-tension relationship of various smooth muscle types, including detrusor. S1P produced ~50% of a KCl-induced contraction at slack length when L/Ls = 2, as opposed to 10% at L/Ls = 1. Muscle tone at L/Ls = 2 was ~20% of Fs and the tone at L/Ls = 1 was close to 0. All experiments were carried out using the L/Ls value of 1.6 since S1P-induced contraction was significant at this length and maximal contraction produced by 110 mM KCl remained at ~100% of Fs. This was therefore defined as the optimal length (Lo) for detrusor strip agonist-induced contraction. Data were expressed as the active force (total force minus the unstimulated basal force).

Measurement of intracellular calcium
Detrusor muscle strips were stretched to Lo, then loaded for 2 h with 7.5 µM Fura-2-PE3-AM and 0.01% (w/v) pluronic F-127 (Teflabs, Austin, TX, USA) to enhance solubility. Tissues were then washed for 30 min in three changes of PSS. The fluorescence emission at a wavelength of 510 nm was collected for alternating excitations at 340 and 380 nm and expressed as a ratio of emission fluorescence (F340/F380) using a fluorometer. Agonist-induced changes in intracellular free calcium were quantified as a fraction of the maximal signal produced upon stimulation with 110 mM KCl, and the minimum signal produced in a Ca2+-free solution containing 5 mM EGTA plus 10 µM ionomycin was corrected for background fluorescence by incubating tissues in 4 mM MnCl2 quench solution containing 10 µM ionomycin.

RT-PCR
Rabbit detrusor smooth muscle strips with removed urothelium were frozen in liquid nitrogen and crushed to a fine powder using a prechilled mortar and pestle. Total RNA was isolated using TRIzol Reagent (Life Technologies, Gaithersburg, MD, USA), treated with RNase-free DNase I (Qiagen, Valencia, CA, USA), and purified with RNeasy kit (Qiagen, Valencia, CA, USA). RNA was reverse transcribed with gene-specific primers using M-MuLV reverse transcriptase (New England Biolabs, Ipswich, MA, USA) at 45°C for 1 h. The resulting cDNA was amplified by PCR with the following cycle parameters: 94°C, 20 s; 56°C, 30 s; 68°C, 30 s for 35 cycles. DMSO was added to a final concentration of 10% in both reverse transcription and PCR reactions. S1PR-specific primers were designed based on homologous sequences of human, rat, mouse, pig, dog, and sheep (where available). The sequences of the primers used are provided in Supplemental Table 1. The PCR products were separated by horizontal polyacrylamide gel electrophoresis, stained with ethidium bromide, and visualized by ultraviolet fluorescence using an Innotech imaging system (Alphatech, Washington, DC, USA). The PCR products were confirmed by sequencing.

Immunoblotting
Powdered rabbit detrusor smooth muscle prepared as described above was resuspended in lysis buffer (50 mM sodium HEPES, pH 7.5, 150 mM sodium chloride, 5 mM EDTA, 10 mM sodium fluoride, 10 mM sodium phosphate, 1% (v/v) Triton X-100, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, 5 µg/ml aprotinin), then Dounce homogenized. After centrifugation, equal amounts of soluble protein (20 µg) were separated by 12% (w/v) SDS-PAGE and proteins were transferred to nitrocellulose membranes. Membranes were blocked for 1 h in 5% (w/v) nonfat milk in Tris-buffered saline containing 0.5% Tween-20, then incubated overnight at 4°C with anti-S1P2 antibody (1:500, Exalpha, Watertown, MA, USA). Protein expression was detected by enhanced chemiluminescence (Pierce, Rockford, IL, USA).

Reagents
S1P, dihydro-S1P and U73433 were from Biomol Research Laboratory (Plymouth Meeting, PA, USA). The active enantiomer (S)-FTY720-P was prepared as described previously (23) . Carbachol was from Sigma Aldrich (St. Louis, MO, USA). Nifedipine, 2-aminoethoxydiphenyl borate (2-APB), and cyclopiazonic acid (CPA) were from Calbiochem (La Jolla, CA, USA). U73122 and JTE013 were from Tocris (Ellisville, MO, USA). LOE908 was from Boeheringer Ingelheim Pharmaceuticals; SEW9720 was from Avanti Polar Lipids (Alabaster, AL, USA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
S1P-induced detrusor contraction is dose dependent and increases rhythm and basal tone
Similar to other agonists, S1P produced an initial fast phasic contraction in rabbit detrusor strips, which then decreased with time, yielding an early peak response. The rapid phasic phase was followed by a slower increase in force, producing a sustained, tonic response (Fig. 1 A, B). Unstimulated (i.e., basal) detrusor strips display spontaneous rhythmic contractions and muscle tone (22) . Spontaneous rhythmic contraction and muscle tone can be seen in Fig. 1A in the force tracing just before the addition of S1P (the rhythmic contraction tracing and the force response produced when tissues are exposed to a calcium-free solution are expanded in the inset of Fig. 1A for better visualization of the periodic nature of the spontaneous rhythmic contraction). Rhythmic contraction and muscle tone are abolished by treatment of tissues with a calcium-free solution (Fig. 1A and ref. 22 ).


Figure 1
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  		Figure 1. S1P induces detrusor contraction. A, B) Detrusor contraction was induced by 1 µM S1P and expressed as the ratio of the KCl-induced contraction at Lo (F/Fo). Data are means ± SE, n = 6. Representative tracing of contractile response produced by 1 µM S1P is shown in panel A. Inset: The rhythmic contraction tracing produced by tissues exposed to a calcium-containing solution and the force response produced when tissues are exposed to a Ca2+-free solution ("line" at zero F/Fo), are expanded. The very low level of mechanical "noise" is shown in the Ca2+-free force tracing. C) Detrusor strips were treated with increasing concentrations of S1P. Fitting of S1P concentration-contraction data to a sigmoidal curve revealed that the EC50 of S1P in negative log M units was 6.6 ± 0.3 (249 nM), the slope of the curve was 1.4 ± 0.5 and the maximum active force was 0.2 ± 0.03 F/Fo, n = 3. D) Detrusor strips were either untreated or exposed to vehicle (DMSO), 1 µM S1P, or 1 µM carbachol (CCh) for 2 min. Strips were then washed three times and the active force (F/F0) of each was measured after 5 and 30 min after washout. Data are means ± SE, n = 5. *P < 0.05 vs. basal tone.

In addition, 1 µM S1P produced an ~ 50% increase in the frequency of basal rhythm (51.7%±14.7%, n=4, P<0.05). Although S1P-induced contraction was only ~40% of that induced by 1 µM carbachol, it was significantly greater than the basal rhythmic tone (Fig. 1D ). S1P-induced detrusor contraction was concentration dependent, with an EC50 of 249 nM (Fig. 1C ). The effective concentration range for S1P was also within the physiological range required to stimulate the S1P-specific cell surface receptors, suggesting that either one or more of these receptors were responsible for mediating this response. In agreement with earlier studies (24 , 25) , contraction induced by carbachol was virtually abolished after a 5 min washout. In contrast, S1P-induced contraction was still significantly higher than the initial resting tone even after 30 min of washout (Fig. 1D ). This persistent effect of S1P could be due to difficulties arising from washing out the lipid compared with carbachol, which is water soluble. Alternatively, S1P may produce persistent effects that continue even when S1P has been removed in a manner similar to that of endothelin-1 (ET-1) -induced detrusor contraction, which is resistant to washout (26) .

Role of calcium in S1P-induced detrusor contraction
Increased intracellular calcium concentration is an important mediator of muscle contraction, and the basal tone of detrusor muscle is dependent on the presence of extracellular calcium (27 , 28) . We therefore examined the role of calcium in S1P-induced detrusor contraction. Similar to other types of smooth muscle, including gastrointestinal muscle (7) and vascular smooth muscle (29) , S1P induced a significant increase in intracellular calcium concentration (Fig. 2 A). The increase in calcium was smaller and had different kinetics than that produced by carbachol (Fig. 2A ). Peak contractile responses induced by S1P, in contrast to carbachol, were absent in Fura-2-treated detrusor strips (Fig. 2B ). Thus, this phasic contraction may be caused by mobilization of a labile pool of calcium, which is buffered by Fura-2. The tonic contractile response induced by S1P was retained and the degree of force was proportional to the degree of increase in calcium (Fig. 2C ). The greater force:calcium ratio produced by carbachol suggests that S1P did not cause calcium sensitization (30) .


Figure 2
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  		Figure 2. S1P-induced increases in intracellular calcium. Fura-2-loaded detrusor strips were stimulated with carbachol and S1P as indicated. A) A representative Ca2+ trace of intracellular calcium concentrations; B) a representative force trace. C) S1P- and carbachol-induced changes in force and intracellular calcium concentration. Data are means ± SE, n = 3.

The role of S1P receptors in detrusor contraction
Most of the biological effects of S1P are mediated through its receptors. To elucidate the importance of specific S1P receptor subtypes in S1P-mediated contraction, we utilized specific S1P receptor agonists. In contrast to S1P, the S1P1 receptor-specific agonist, SEW2871, did not significantly increase the basal tone of detrusor strips (Fig. 3 A). Similarly, dihydro-S1P, an analog of S1P with similar affinities to S1P for all the S1P receptors except S1P2, with which it has ~1 log-fold less affinity, also failed to significantly contract the muscle (Fig. 3B ). Moreover, pretreatment with the S1P2 antagonist JTE013 (31) abolished the ability of S1P to cause detrusor contraction (Fig. 3C ).


Figure 3
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  		Figure 3. Involvement of S1P receptors in detrusor contraction. Detrusor strips were treated with 1 µM SEW2871 followed by 1 µM S1P (A), 1 µM dihydro-S1P followed by 1 µM S1P (B), 1 µM JTE013 followed by 1 µM S1P (C), 1 µM FTY720-phosphate followed by 1 µM S1P (D), and 1 µM S1P followed by a second addition of 1 µM S1P (E). Representative active force traces are shown. F, G) Maximum contractions induced by the indicated agonists. Data are means ± SE, n = 3. *P < 0.05. H) S1P receptor expression in detrusor muscle was determined by RT-PCR using receptor-specific primers in the presence (+) or absence (–) of reverse transcriptase (RT). In the absence of RT, there were no significant PCR products detected. I) S1P2 protein expression was examined by Western blot with anti-S1P2 antibody.

To confirm that the lack of responses to SEW2871 and dihydro-S1P were due to their agonist properties and not to differences in contractile efficiency of the detrusor strips, 1 µM S1P was added without a washout after a steady state was reached. Indeed, subsequent addition of S1P to strips that were treated with SEW2871 or dihydro-S1P contracted the muscle strips to an extent similar to that of S1P alone (Fig. 3A, B, G ). In sharp contrast, a second response to S1P was blunted when S1P was added during the tonic phase of a first S1P contraction (Fig. 3E, G ). Collectively, these results point to the involvement of S1P2 in S1P-mediated detrusor contraction. RT-PCR revealed the expression of mRNA for S1P1 and S1P2 whereas S1P3, S1P4, and S1P5 were not detected (Fig. 3H ). The predicted amino acid sequences from the PCR products of S1P1 and S1P2 were identical to the corresponding amino acid sequences of rabbit S1PRs in the database. Moreover, Western blot analysis confirmed the expression of the S1P2 receptor in detrusor muscle (Fig. 3I ).

Surprisingly, however, FTY720-phosphate, which can bind and activate all S1P receptors except S1P2, significantly contracted detrusor muscle. Of note, FTY720-phosphate caused a monotonic contraction lacking the rapid early peak phase exhibited by S1P (Fig. 3D ). It is also important to note that the contractile profile of FTY720-phosphate differed markedly from dihydro-S1P despite their similar affinities for the S1P receptors. Moreover, when S1P was added at the steady state of an FTY720-phosphate contraction, although the phasic phase of S1P-induced contraction was preserved (Fig. 3D, G ), the tonic phase of contraction was absent (Fig. 3D ). Since the pharmacological difference between S1P- and FTY720-phosphate similar is that S1P can also activate S1P2, this result suggests that the phasic contractile response to S1P may be mediated through the S1P2 receptor.

The phasic phase of S1P-induced detrusor contraction is mediated by PLC-dependent calcium mobilization
Given that S1P2 is coupled to activation of PLC, we next determined its involvement in S1P-induced detrusor contraction. The phasic contraction in response to S1P was eliminated by the PLC inhibitor U73122 (Fig. 4 B, E), whereas the strong tonic contraction was unaffected. As expected, the inactive analog U73343 had no significant effects on contractile responses to S1P (Fig. 4A, E ). Moreover, inhibition of PLC had no significant effect on FTY720-phosphate-induced contraction (Fig. 4D, F ).


Figure 4
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  		Figure 4. Effect of PLC inhibitors on S1P- and FTY720-phosphate-induced detrusor contraction. Detrusor strips were pretreated with 10 µM PLC inhibitor U73122 or 10 µM inactive analog U73443, then treated with 1 µM S1P or FTY720-phosphate followed by addition of S1P, as indicated. A–D) Representative active force traces). E, F) Maximal contractions for panels A, B and C, D, respectively. Data are means ± SE of 6 independent experiments. *P < 0.05.

The transient S1P-induced contraction observed when S1P was added during the steady state of a FTY720-phosphate-induced contraction (see Fig. 3D ) was abolished in the presence of U73122 (Fig. 4D, F ), but not in the presence of U73343 (Fig. 4C, F ). These data support the notion that the early peak phase of an S1P-induced contraction was due to PLC activation, inositol-1,4,5-trisphosphate (InsP3) generation, and calcium mobilization from sarcoplasmic reticulum (SR) pools and that the tonic phase was due to a PLC-independent mechanism. FTY720-phosphate-induced contractions were entirely independent of PLC.

Distinct roles of calcium channels in S1P- and FTY720-phosphate-induced contraction
Having established that S1P increases intracellular calcium in detrusor smooth muscle, it was important to examine the role of calcium channels and intracellular calcium stores. Nifedipine, a specific antagonist of L-type voltage-operated calcium channels (VOCC), abolished S1P-induced contraction (Fig. 5 A), suggesting that L-type calcium channels play an important role. Moreover, S1P-induced muscle contraction was also inhibited by a low concentration (1 µM) of LOE-908, a nonselective cation channel (NSCC) blocker, albeit to a lesser extent than nifedipine (Fig. 5A ). In addition, 2-APB, at a relatively low concentration (10 µM) that mainly inhibits transient receptor potential (TRP) channels (32 33 34) , significantly reduced smooth muscle contraction induced by S1P- and FTY720-phosphate (Fig. 5A ). FTY720-phosphate-induced contraction was also virtually abolished in the presence of nifedipine but was not significantly affected by 1 µM LOE-908 (Fig. 5A ). These data suggest that S1P- and FTY720-phosphate may activate 2-APB-sensitive TRPs in addition to VOCCs to permit strong force maintenance in tonic detrusor smooth muscle, with S1P also affecting NSCCs. The inhibitory effects of 2-APB and nifedipine on the phasic phase of S1P-induced contraction were significantly altered in the presence of FTY720-phosphate (Fig. 5B ). S1P-induced contraction in the presence of either 2-APB or nifedipine was significantly greater when S1P was preceded by FTY720-phosphate treatment compared with S1P-induced contraction in the presence of the inhibitors alone, with the most striking difference observed with 2-APB. These data support the notion that FTY720-phosphate activates calcium entry via calcium channels and that this calcium entry supports the refilling of intracellular calcium pools utilized by S1P to produce the early peak contractile response. Since the protective effect of FTY720-phosphate on S1P-induced phasic contraction was more pronounced with 2-APB than with nifedipine, this would suggest that the regulation of VOCCs by FTY720-phosphate in the refilling of calcium stores is more prominent than FTY720-phosphate-dependent regulation of TRP channels.


Figure 5
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  		Figure 5. The effect of calcium channel blockers on S1P and FTY720-phosphate-induced detrusor contraction. Detrusor strips were either pretreated for 20 min with vehicle, 10 µM 2-APB, 1 µM LOE-908, or with 0.1 µM nifedipine for 10 min. The tissues were then exposed to 1 µM S1P (open bars) or 1 µM FTY720-phosphate (hatched bars) (A). Significant inhibition in the presence of the channel inhibitors compared with agonist treatment alone is denoted by an asterisk (P<0.05). B) On reaching a steady tonic phase of contraction after the addition of FTY720-phosphate, detrusor strips were subsequently treated with 1 µM S1P and the peak response was recorded (gray bars). Data are means ± SE (n=3) of agonist-induced maximal contractions in the presence or absence of the calcium channel antagonists. B) Inset: a representative active force trace. *P < 0.05.

The role of SR calcium stores in S1P- and FTY720-phosphate-induced detrusor contraction
CPA is a potent inhibitor of calcium-dependent ATPases and depletes the intracellular membrane stores of calcium in smooth muscle and other cell types. Hence, CPA was used to further examine the contribution of the SR calcium pool to S1P-induced detrusor muscle contraction. Emptying of calcium stores with CPA resulted in sustained muscle contraction (Fig. 6 A), which was inhibited by nifedipine (Fig. 6B, D ) but not by 2-APB (Fig. 6C, D ). This indicates that SR pool replenishment requires VOCCs but not necessarily TRP channels in rabbit detrusor smooth muscle. Moreover, pretreatment with CPA blocked S1P- and FTY720-phosphate-induced contraction (Fig. 7 A, B), reinforcing an important role for the SR pool in both S1P- and FTY720-phosphate-induced contraction. Although prior treatment with S1P reduced CPA-induced contraction (Fig. 7D, E ), prior treatment with FTY720-phosphate did not have a significant effect on CPA-induced contraction (Fig. 7C, E ), suggesting that S1P activated the SR calcium pool and that FTY720-phosphate refilled it.


Figure 6
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  		Figure 6. Nifedipine but not 2-APB inhibits CPA-induced contraction. Detrusor strips were treated with 10 µM CPA, and a representative active force trace is shown in panel A. Detrusor strips were preincubated with 0.1 µM nifedipine (B, D) or 10 µM 2-APB (C, D) for 20 min, then treated with 10 µM CPA, as indicated. D) Maximal contractions for panels A–C. Data are means ± SE (n=3). *P < 0.05.


Figure 7
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  		Figure 7. Effect of calcium store depletion on S1P- and FTY720-phosphate-induced detrusor contraction. A, B) Detrusor strips were treated with 10 µM CPA. On reaching a steady tonic phase of contraction, 1 µM S1P or FTY720-phosphate was added. A representative active force trace is shown in panel A. B) Mean values for maximal contractions. Data are means ± SE (n=3). *P < 0.05. C–E) Detrusor strips were treated with FTY720-phosphate, 10 µM CPA, and 1 µM S1P in the order indicated. Representative active force traces are shown in panels C, D and maximal contractions are expressed as means ± SE (n=3) are shown in panel E. *P < 0.05.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In normal human bladder, detrusor contraction is regulated predominantly by acetylcholine, whereas the contributions of nonadrenergic, noncholinergic (NANC) mechanisms are relatively small. In contrast, when bladder function is abnormal such as occurs in OBS, the contribution of NANC components becomes more significant. For example, an NANC response in bladder strips was detected only in patients with detrusor overactivity (35 , 36) . Here we report that S1P produces both phasic and tonic contractions of detrusor muscle, adding this bioactive sphingolipid metabolite to the list of known physiological NANC mediators that contract detrusor muscle, which includes ATP, endothelin-1 (ET-1), and prostanoids (37) .

Because detrusor muscles express S1P1 and S1P2, it was of interest to determine which of these S1P receptors was involved in S1P-induced contraction. Several lines of evidence point to a critical role for S1P2. First, in contrast to S1P, dihydro-S1P, which is identical to S1P except that it lacks one double bond and acts as an agonist for all S1P receptors but with a 20-fold lower affinity for S1P2, was a much weaker contractile agonist than S1P. Second, SEW2871, a specific agonist for S1P1, did not significantly contract detrusor strips. Moreover, JTE013, a S1P2-specific antagonist, blocked the contractile effects of S1P.

S1P2 has been linked to activation of Gq and it was previously shown that JTE013 specifically inhibits S1P2-, but not S1P1- or S1P3-mediated increases in intracellular free calcium induced by S1P (38) . Calcium is a key component of smooth muscle contraction and, in agreement with other studies in numerous cell types (2 , 7 , 39 , 40) , we found that S1P increased [Ca2+]i in detrusor muscle strips. This increase correlated with increased contraction. In gastrointestinal smooth muscle, S1P liberates calcium from internal stores via PLC- and InsP3-dependent calcium release, which is responsible for initiating phasic muscle contraction (7 , 11) . In agreement, the PLC inhibitor U73122 blocked the fast, phasic phase of detrusor contraction induced by S1P whereas the slow, tonic phase of contraction was unaltered, suggesting that the phasic phase is dependent on calcium release from intracellular stores.

Although these results point to a critical role of S1P2 in S1P-mediated detrusor contraction, FTY720-phosphate, a potent S1P receptor agonist with comparable affinity to S1P for all the S1P receptors except S1P2, induced slow, sustained contraction that was similar in magnitude to the tonic contraction induced by S1P. Moreover, treatment with S1P after FTY720-phosphate-induced detrusor contraction produced only a transient phase of contraction. In contrast to its effect on S1P-induced contraction, U73122 did not block FTY720-phosphate-induced contraction, suggesting that it could be independent of calcium store release (Fig. 8 ).


Figure 8
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  		Figure 8. Relationship between calcium regulation and S1P- and FTY720-phosphate-mediated detrusor contraction. S1P- and FTY720-phosphate-mediated detrusor contraction is dependent on control of the concentration of intracellular calcium. Contraction by FTY720-phosphate is mediated predominantly by both TRP and VOCC channels independent of PLC-dependent liberation of calcium stores, suggesting that FTY720-phosphate may directly activate these channels. In the case of S1P, the tonic phase of contraction is entirely dependent on the action of TRP and VOCC channels. However, S1P also produces a transient, phasic contraction via PLC-dependent liberation of calcium from internal stores. This calcium, in turn, may also regulate calcium entry to mediate the tonic phase of S1P-induced detrusor contraction. FTY720-phosphate is a potent agonist for all S1P receptors except S1P2. In addition, dihydro-S1P, a relatively weak agonist for S1P2, is also a poor contractile agent. Thus, it is likely that S1P-dependent liberation of calcium from internal stores is mediated via activation of S1P2.

In detrusor muscle, voltage-dependent L-type calcium channels (VOCCs) are required for both S1P- and FTY720-phosphate-mediated detrusor contraction since contraction in each case was blocked by nifedipine. VOCCs are critical for myogenic reactivity and tone during arterial blood pressure regulation, and previous studies have established their integral role in development of detrusor tone as well as in agonist-mediated contraction (27 , 28 , 41 , 42) . Detrusor muscle from mice deficient in the smooth muscle Cav1.2 calcium channel lacked spontaneous contractile activity, and K+- and carbachol-induced contraction were both reduced ~10-fold (28) . These mice also had severely reduced micturition, indicating an essential role of Cav1.2 L-type calcium channel for normal urinary bladder function.

While nifedipine completely blocked both S1P- and FTY720-phosphate-induced contraction, 2-APB and LOE908 also inhibited contraction, but to a lesser degree, suggesting that other calcium channel subtypes, such as TRPs and NSCCs, may also be involved in S1P- and FTY720-phosphate-induced contraction. Although 2-APB was originally reported to be a selective InsP3 receptor inhibitor (43) , subsequent studies revealed that this effect is variable (reviewed in ref. 44 ). Recent studies provide compelling evidence that at concentrations of < 30 µM, 2-APB inhibits calcium influx by direct inhibition of TRP channels independently of the function of InsP3 receptors (33 , 34 , 44 45 46 47 48 49 50) . However, 2-APB was recently shown to block specific gap junction channels formed by different connexins (51) . FTY720-phosphate has a S1P receptor affinity profile similar to that of dihydro-S1P, and so it was surprising to find such a striking difference in their contractile potencies. This may be an indication that FTY720-phosphate can regulate calcium channels independent of S1P receptors and suggests a novel target for FTY720-phosphate (Fig. 8) . The only reported effect of FTY720-phosphate on channel activity involves atrial myocytes, where it activates the cardiac G-protein-gated potassium channel I (KACh), resulting in bradycardia (52) . It is also possible that both S1P- and FTY720-phosphate can regulate calcium channels independently of S1P receptors, perhaps by direct modulation of calcium channel activity or an indirect action on K+ channel regulation. A recent study reported that S1P activates Ca2+-activated K+ (BKCa) channels in a G-protein-coupled, receptor-independent manner in endothelial cells (53) . Moreover, S1P has been identified as an activator of TRPC5 in vascular smooth muscle, and it has been suggested that TRPC5 is an ionotropic receptor for intracellular S1P (29) .

An important concept in the regulation of smooth muscle tone and contraction is the superficial buffer barrier (54) . This is where the peripheral SR acts as a calcium buffer, accumulating a fraction of Ca2+ entering cells during high K+ stimulation before it reaches the myofilaments. This study has demonstrated that S1P- and FTY720-phosphate-induced contraction appears to involve a complex modulation of SR release and replenishment. For instance, FTY720-phosphate appears to "protect" the phasic phase of S1P-induced contraction from the inhibitory effect of either nifedipine or 2-APB. This suggests that FTY720-phosphate promotes calcium entry, resulting in the filling of the SR pool that is utilized by S1P to induce the phasic phase of contraction. In contrast, the phasic contractile response induced by S1P was absent in the presence of Fura-2, further suggesting that this peak contraction may be caused by mobilization of a labile pool of calcium that is buffered by Fura-2. Preincubation with CPA to inhibit calcium-dependent ATPases and cause emptying and blockade of the replenishment of the SR pool resulted in complete inhibition of both S1P- and FTY720-phosphate-induced detrusor contraction. However, a large part of the CPA-induced contraction was mediated primarily via VOCCs. In agreement, it has been reported that ~90% of CPA-induced detrusor contraction can be blocked by verapamil (24) . Collectively, our results indicate that S1P-induced contraction is attributed to fast release of calcium from internal stores and subsequent sequestration of calcium into the stores, resulting in decreased muscle tone.

Since contractile tone is enhanced in patients with OBS, the regulation of detrusor tone has important clinical implications, and hence S1P signaling represents a novel, potentially important clinical target in the treatment of OBS.


   ACKNOWLEDGMENTS
 
This work was supported by National Institutes of Health Grants R37 GM043880 (S.S.) and R01DK059620 (P.H.R.), and by the NIMH Intramural Research Program (S.M.).


   FOOTNOTES
 
1 These authors contributed equally to this work.

Received for publication September 12, 2006. Accepted for publication March 26, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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