Smooth Muscle Research Centre, Dundalk Institute of Technology, Dundalk, Ireland
Submitted 3 March 2005 ; accepted in final form 27 April 2005
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
smooth muscle; patch clamp; spontaneous transient inward currents
IC of the urethra are spontaneously active. When voltage-clamped at 60 mV, they develop spontaneous transient inward currents (STICs) due to activation of Ca2+-activated Cl channels (25). It was shown recently that these events are mediated by regularly occurring global Ca2+ oscillations that arise through periodic release of Ca2+ from intracellular stores (12). A feature of these events is that they are acutely sensitive to changes in the external Ca2+ concentration and cease immediately on application of Ca2+-free medium, suggesting that they are also dependent on Ca2+ influx. However, it appears that influx via L-type Ca2+ channels is not involved, as spontaneous Ca2+ oscillations in isolated IC were unaffected by application of nifedipine (12). The nature of the influx pathway involved therefore remains elusive; however, it is possible that a store-operated Ca2+ influx pathway may contribute to this process, as occurs in ICC in the murine small intestine (32). The aim of the present study was to investigate whether CCE is important for sustaining pacemaker activity in isolated urethral IC.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell dispersal. Strips of urethral smooth muscle (5 mm in width) were dissected, cut into 1-mm3 pieces, and stored in Ca2+-free Hanks' solution (see Solutions) at 4°C for 30 min before cell dispersal. Tissue pieces were incubated in dispersal medium containing (per 5 ml of Ca2+-free Hanks' solution) 15 mg of collagenase (Sigma type 1A), 1 mg of protease (Sigma type XXIV), 10 mg of bovine serum albumin (Sigma), and 10 mg of trypsin inhibitor (Sigma) for 1015 min at 37°C. Tissue was then transferred to Ca2+-free Hanks' solution and stirred for a further 1530 min to release single smooth muscle cells and IC. These cells were plated in petri dishes containing 100 µM Ca2+ Hanks' solution and stored at 4°C for use within 8 h.
Perforated-patch recordings from single cells.
Currents were recorded with the perforated patch-configuration of the whole cell patch-clamp technique as described previously (2527). This circumvented the problem of current rundown encountered when using the conventional whole cell configuration. The cell membrane was perforated with the antibiotic amphotericin B (600 µg/ml). Patch pipettes were initially frontfilled by dipping into pipette solution and then backfilled with the amphotericin B-containing solution. Pipettes were pulled from borosilicate glass capillary tubing (1.5-mm outer diameter, 1.17-mm inner diameter; Clark Medical Instruments) to a tip with a diameter of 11.5 µm and resistance of 24 M
.
Voltage clamp commands were delivered via an Axopatch 1D patch-clamp amplifier (Axon Instruments), and membrane currents were recorded by a 12-bit analog digital/digital analog converter (Axodata 1200 or Labmaster, Scientific Solutions) interfaced to an Intel computer running pCLAMP software. During experiments, the dish containing the cells was continuously perfused with Hanks' solution at 36 ± 1°C. Additionally, the cell under study was continuously superfused by means of a custom-built close delivery system with a pipette of 200-µm tip diameter placed
300 µm from the cell. The Hanks' solution in the close delivery system could be switched to a drug-containing solution with a "dead space" time of <5 s. In all experiments n refers to the number of cells studied, and each experimental set usually contained samples from a minimum of four animals. Summary data are presented as means ± SE, and statistical comparisons were made on raw data with Student's paired t-test, taking P < 0.05 levels as significant.
Ca2+ measurements with fura-2 microfluorimetry. Ca2+ measurements were made from IC incubated in fura-2 AM (5 µM) for 15 min at 37°C in Ca2+-free Hanks' solution. Cells were placed in a glass-bottomed dish and then mounted on the stage of an inverted microscope. The Ca2+ microfluorimetry system consisted of a dual monochromator passing 340 nm/380 nm light (5-nm bandwidth), a light chopper (PTI DeltaScan), and an inverted microscope with an oil immersion objective (x40, numerical aperture 1.3). The emission side of the microscope comprised an adjustable rectangular window, a filter (510 nm), and a photon-counting photomultiplier tube in the light path. Fluorescence equipment was controlled by PTI Felix software, which also performed storage and analysis of the acquired data. Before experimentation, cells were superfused with normal Hanks' solution for 10 min. Changes in cytosolic Ca2+ concentration ([Ca2+]i) were measured as the change in ratio of fluorescence at the 340- and 380-nm wavelengths. CCE was plotted as the total amplitude of the Ca2+ transient produced on introduction of 1.8 mM Ca2+ solution after incubation in Ca2+-free medium containing cyclopiazonic acid (CPA; 20 µM).
Solutions. The compositions of the solutions used were as follows (in mM): Ca2+-free Hanks' solution (for cell dispersal): 125 NaCl, 5.36 KCl, 10 glucose, 2.9 sucrose, 15.5 NaHCO3, 0.44 KH2PO4, 0.33 Na2HPO4, and 10 HEPES, pH adjusted to 7.4 with NaOH; Hanks' solution: 125 NaCl, 5.36 KCl, 10 glucose, 2.9 sucrose, 4.17 NaHCO3, 0.44 KH2PO4, 0.33 Na2HPO4, 0.4 MgSO4, 0.5 MgCl2, 1.8 CaCl2, and 10 HEPES, pH adjusted to 7.4 with NaOH; Ca2+-free Hanks' solution (superfusate for CCE measurement): 125 NaCl, 5.36 KCl, 10 glucose, 2.9 sucrose, 4.17 NaHCO3, 0.44 KH2PO4, 0.33 Na2HPO4, 0.4 MgSO4, 2.3 MgCl2, 10 glucose, 2.9 sucrose, 5.0 EGTA, and 10 HEPES, pH adjusted to 7.4 with NaOH; Cs+ perforated-patch solution: 133 CsCl, 1.0 MgCl2, 0.5 EGTA, and 10 HEPES, pH adjusted to 7.2 with CsOH.
Drugs. The following drugs were used: amphotericin B (Sigma); lanthanum chloride (Hopkin and Williams); 2-aminoethoxydiphenyl borate (2-APB, ACROS); gadolinium chloride, nickel chloride, and wortmannin (WT; Sigma); SKF-96365 and CPA (Calbiochem); and nifedipine (Bayer). All drugs were made up in the appropriate stock solution before being diluted to their final concentrations in Hanks' solution.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
The membrane-permeant IP3 receptor inhibitor 2-APB (14) was recently shown to block store-operated Ca2+ entry in several cell types (3, 11, 22). Our recent studies (26), however, showed that caffeine responses were unaffected by 2-APB in urethral IC, suggesting that stores can refill normally. We therefore performed experiments to test the effect of 2-APB on CCE in urethral IC directly. The results shown in Fig. 3E demonstrate that 2-APB produced a modest reduction in the amplitude of CCE in urethral IC. In eight cells CCE was reduced by 21 ± 7% by 100 µM 2-APB (P < 0.05).
A summary bar graph of the inhibitory effect of all these agents on CCE is plotted in Fig. 4. This pharmacological profile demonstrates that CCE in urethral IC was inhibited by Gd3+ (10 µM), La3+ (10 µM), and Ni2+ (100 µM) and, to a lesser extent, by 2-APB (100 µM) and Ni2+ (30 µM). However, it was not inhibited by SKF-96365 (10 µM), nifedipine (1 µM), or WT (10 µM).
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
A recent study by Torihashi et al. (32) suggested that pacemaker activity in ICC cultured from the murine small intestine is dependent on store-operated Ca2+ entry. They showed that removal of extracellular Ca2+ leads to cessation of spontaneous Ca2+ oscillations, suggesting that they are dependent on Ca2+ influx. This effect was not due to inhibition of L- or T-type Ca2+ channels, as nifedipine (1 µM) and Ni2+ (50 µM) did not affect the activity. The possibility that CCE was involved came from the observations that Ca2+ oscillations were inhibited by the putative CCE inhibitor SKF-96365 (4 µM) and that ICC were immunopositive for TRPC4 proteins, which had previously been described as store-operated Ca2+ channels in adrenal and endothelial cells (6, 20). The idea that a similar pathway could be responsible for sustaining spontaneous activity in urethral IC was prompted by the observations of Johnston et al. (12) that spontaneous Ca2+ oscillations were dependent not only on release of Ca2+ from intracellular stores but also on the extracellular concentration of Ca2+. Oscillations immediately ceased on addition of Ca2+-free medium and doubled in frequency when the concentration of Ca2+ was increased from 1.8 to 3.6 mM. This sensitivity to external Ca2+ implied a role for Ca2+ influx in mediating the activity, possibly by refilling the depleted Ca2+ stores.
The results of the present study, however, suggest that CCE is not involved in this process. IC were found to possess a CCE pathway characterized by sensitivity to low concentrations of La3+ and Gd3+ (10 µM) as well as relatively high concentrations of Ni2+ (100 µM). In contrast, Ca2+ entry induced in the absence of store depletion was unaffected by these agents, suggesting that IC possess a specific population of channels activated by store depletion that are inactive under resting conditions. Importantly, however, inhibition of CCE with these agents did not abolish STICs, suggesting that spontaneous activity in these cells is not completely reliant on CCE.
Perhaps this should not be surprising. Although several studies suggest that Ca2+ oscillations are driven by CCE (8, 13, 16), others have questioned this view. A review by Shuttleworth (29) pointed out that "if Ca2+ entry affects oscillation frequency by determining the rate at which stores recharge during the inter-spike interval, then inhibition of the SERCA-pump activity would be expected to slow oscillation frequency by extending the time required to recharge the stores." However, previous studies by Peterson et al. (19) showed that application of the SERCA pump inhibitor thapsigargin actually decreased the interspike interval, suggesting that the rate of refilling was not aligned with the oscillation frequency. Indeed, in urethral IC, application of CPA decreased the amplitude of STICs but had little effect on their frequency (26), suggesting that spontaneous activity is not sustained by CCE. This observation, in addition to the findings of the present study, points to an alternative "non-capacitative entry" pathway as a means for sustaining Ca2+ oscillations. At present we have little information with regard to the nature of this pathway in urethral IC. We know that in addition to the CCE inhibitors La3+, Gd3+, and Ni2+, STICs recorded in IC voltage-clamped at 60 mV were not inhibited by nifedipine (10 µM), suggesting that influx via L-type Ca2+ channels is also not involved (26).
Given the apparent lack of involvement of CCE in the generation of STICs in IC, the question arises as to what the role of CCE is in these cells. Once again, we have no definitive answer to the question; however, one possibility is that the amount by which Ca2+ stores are depleted during normal Ca2+ cycling is not sufficient to activate CCE. Such a model would be consistent with the findings of Parekh et al. (17), who concluded that activation of CCE is a threshold-dependent, all-or-nothing phenomenon. These investigators showed that intraluminal Ca2+ within IP3-sensitive stores had to fall to a particular threshold to activate Ca2+ release-activated current (ICRAC). It is possible, therefore, that a similar situation may exist in IC, although the exact physiological conditions under which this would occur are unknown. It is conceivable that stores are more fully depleted after activation of postjunctional 1-receptors on IC, which is known to cause depletion of IP3-sensitive stores in these cells (28). An alternative explanation may be that CCE exists as a protective mechanism to promote uptake into stores when intraluminal Ca2+ levels fall (29).
In summary, the data presented in this study show that a CCE pathway is present in urethral IC. However, it appears that this pathway is not critical for pacemaking in these cells and that inhibition of CCE does not account for effects of Ca2+-free medium in abolishing Ca2+ oscillations in IC. Further studies are needed to elucidate the exact role and molecular identity of CCE in IC as well as to investigate the Ca2+ influx pathways that contribute to pacemaker activity in IC.
![]() |
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
2. Bird GS and Putney JW Jr. Capacitative calcium entry supports calcium oscillations in human embryonic kidney cells. J Physiol 562: 697706, 2005.
3. Bootman MD, Collins TJ, Mackenzie L, Roderick HL, Berridge MJ, and Peppiatt CM. 2-Aminoethoxydiphenyl borate (2-APB) is a reliable blocker of store-operated Ca2+ entry but an inconsistent inhibitor of InsP3-induced Ca2+ release. FASEB J 16: 11451150, 2002.
4. Curtis TM and Scholfield CN. Nifedipine blocks Ca2+ store refilling through a pathway not involving L-type Ca2+ channels in rabbit arteriolar smooth muscle. J Physiol 532: 609623, 2001.
5. Flemming R, Xu SZ, and Beech DJ. Pharmacological profile of store-operated channels in cerebral arteriolar smooth muscle cells. Br J Pharmacol 139: 955965, 2003.[CrossRef][ISI][Medline]
6. Freichel M, Suh SH, Pfeifer A, Schweig U, Trost C, Weissgerber P, Biel M, Philipp S, Freise D, Droogmans G, Hofmann F, Flockerzi V, and Nilius B. Lack of an endothelial store-operated Ca2+ current impairs agonist-dependent vasorelaxation in TRP4/ mice. Nat Cell Biol 3: 121127, 2001.[CrossRef][ISI][Medline]
7. Gibson A, McFadzean I, Wallace P, and Wayman CP. Capacitative Ca2+ entry and the regulation of smooth muscle tone. Trends Pharmacol Sci 19: 266269, 1998.[CrossRef][ISI][Medline]
8. Girard S and Clapham D. Acceleration of intracellular calcium waves in Xenopus oocytes by calcium influx. Science 260: 229232, 1993.[ISI][Medline]
9. Hallam TJ, Jacob R, and Merritt JE. Influx of bivalent cations can be independent of receptor stimulation in human endothelial cells. Biochem J 259: 125129, 1989.[ISI][Medline]
10. Holda JR, Klishin A, Sedova M, Huser J, and Blatter LA. Capacitative calcium entry. News Physiol Sci 13: 157163, 1998.[ISI][Medline]
11. Iwasaki H, Mori Y, Hara Y, Uchida K, Zhou H, and Mikoshiba K. 2-Aminoethoxydiphenyl borate (2-APB) inhibits capacitative calcium entry independently of the function of inositol 1,4,5-trisphosphate receptors. Receptors Channels 7: 429439, 2001.[ISI][Medline]
12. Johnston L, Sergeant GP, Hollywood MA, Thornbury KD, and McHale NG. Calcium oscillations in interstitial cells of the rabbit urethra. J Physiol 565: 449461, 2005.
13. Kawanishi T, Blank LM, Harootunian AT, Smith MT, and Tsien RY. Ca2+ oscillations induced by hormonal stimulation of individual fura-2-loaded hepatocytes. J Biol Chem 264: 1285912866, 1989.
14. Maruyama T, Kanaji T, Nakade S, Kanno T, and Mikoshiba K. 2-Aminoethoxydiphenyl borate, a membrane-penetrable modulator of Ins(1,4,5)P3-induced Ca2+ release. J Biochem (Tokyo) 122: 498505, 1997.[Abstract]
15. Ng LC and Gurney AM. Store-operated channels mediate Ca2+ influx and contraction in rat pulmonary artery. Circ Res 89: 923929, 2001.
16. Paltauf-Doburzynska J, Frieden M, Spitaler M, and Graier WF. Histamine-induced Ca2+ oscillations in a human endothelial cell line depend on transmembrane ion flux, ryanodine receptors and endoplasmic reticulum Ca2+-ATPase. J Physiol 524: 701713, 2000.
17. Parekh AB, Fleig A, and Penner R. The store-operated calcium current ICRAC: nonlinear activation by InsP3 and dissociation from calcium release. Cell 89: 973980, 1997.[CrossRef][ISI][Medline]
18. Parekh AB and Penner R. Store depletion and calcium influx. Physiol Rev 77: 901930, 1997.
19. Petersen CC, Petersen OH, and Berridge MJ. The role of endoplasmic reticulum calcium pumps during cytosolic calcium spiking in pancreatic acinar cells. J Biol Chem 268: 2226222264, 1993.
20. Philipp S, Trost C, Warnat J, Rautmann J, Himmerkus N, Schroth G, Kretz O, Nastainczyk W, Cavalie A, Hoth M, and Flockerzi V. TRP4 (CCE1) protein is part of native calcium release-activated Ca2+-like channels in adrenal cells. J Biol Chem 275: 2396523972, 2000.
21. Putney JW Jr. A model for receptor-regulated calcium entry. Cell Calcium 7: 112, 1986.[CrossRef][ISI][Medline]
22. Putney JW Jr. Pharmacology of capacitative calcium entry. Mol Interv 1: 8494, 2001.
23. Sanders KM. A case for interstitial cells of Cajal as pacemakers and mediators of neurotransmission in the gastrointestinal tract. Gastroenterology 111: 492515, 1996.[ISI][Medline]
24. Sedova M, Klishin A, Huser J, and Blatter LA. Capacitative Ca2+ entry is graded with degree of intracellular Ca2+ store depletion in bovine vascular endothelial cells. J Physiol 523: 549559, 2000.
25. Sergeant GP, Hollywood MA, McCloskey KD, Thornbury KD, and McHale NG. Specialised pacemaking cells in the rabbit urethra. J Physiol 526: 359366, 2000.
26. Sergeant GP, Hollywood MA, McCloskey KD, McHale NG, and Thornbury KD. Role of IP3 in modulation of spontaneous activity in pacemaker cells of rabbit urethra. Am J Physiol Cell Physiol 280: C1349C1356, 2001.
27. Sergeant GP, Hollywood MA, McHale NG, and Thornbury KD. Spontaneous Ca2+ activated Cl currents in isolated urethral smooth muscle cells. J Urol 166: 11611166, 2001.[CrossRef][ISI][Medline]
28. Sergeant GP, Thornbury KD, McHale NG, and Hollywood MA. Characterization of norepinephrine-evoked inward currents in interstitial cells isolated from the rabbit urethra. Am J Physiol Cell Physiol 283: C885C894, 2002.
29. Shuttleworth TJ. What drives calcium entry during [Ca2+]i oscillations?challenging the capacitative model. Cell Calcium 25: 237246, 1999.[CrossRef][ISI][Medline]
30. Suzuki H, Takano H, Yamamoto Y, Komuro T, Saito M, Kato K, and Mikoshiba K. Properties of gastric smooth muscles obtained from mice which lack inositol trisphosphate receptor. J Physiol 525: 105111, 2000.
31. Thomas AP, Bird GS, Hajnoczky G, Robb-Gaspers LD, and Putney JW Jr. Spatial and temporal aspects of cellular calcium signaling. FASEB J 10: 15051517, 1996.
32. Torihashi S, Fujimoto T, Trost C, and Nakayama S. Calcium oscillation linked to pacemaking of interstitial cells of Cajal: requirement of calcium influx and localization of TRP4 in caveolae. J Biol Chem 277: 1919119197, 2002.
33. Ward SM, Ordog T, Koh SD, Baker SA, Jun JY, Amberg G, Monaghan K, and Sanders KM. Pacemaking in interstitial cells of Cajal depends upon calcium handling by endoplasmic reticulum and mitochondria. J Physiol 525: 355361, 2000.
34. Wilson SM, Mason HS, Smith GD, Nicholson N, Johnston L, Janiak R, and Hume JR. Comparative capacitative calcium entry mechanisms in canine pulmonary and renal arterial smooth muscle cells. J Physiol 543: 917931, 2002.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Visit Other APS Journals Online |