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Keywords:

  • bladder;
  • interstitial cystitis;
  • adenosine triphosphate;
  • epithelium;
  • dimethyl sulphoxide;
  • heparin

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Authors

Objective  To determine whether dimethyl sulphoxide (DMSO) and heparin reduce the greater stretch-activated ATP release in interstitial cystitis (IC), as ATP serves as a nocio-neurotransmitter in the bladder, and thus explain their beneficial effects in patients with IC (a disease characterized by hypersensory bladder symptoms).

Materials and methods  Bladder epithelia in IC release more ATP in response to stretch than do control samples. Both DMSO and heparin are used intravesically to treat IC; such agents can modulate urothelial function because they directly contact bladder urothelium. Biopsies taken from patients with IC and from control subjects were grown in primary cultures using established cell-culture techniques. Cultured urothelial cells were stretched with the Flexcell device (Flexcell International Corp., McKeesport, PA, USA) and supernatant ATP was measured, using a luciferin-luciferase assay. DMSO (0.1%, 0.5% and 1%) or heparin (50, 200, 800 and 1600 U) was added to the cells at the start of the stretch experiments and the ATP released into the supernatant measured. Cell viability was also determined using Trypan Blue staining.

Results  IC cells released significantly more ATP in response to stretch than did control cells. This increased release of ATP by stretched IC cells was significantly blocked by adding DMSO or heparin at all concentrations used. Heparin appeared to have a greater dose-dependent effect on ATP release than did DMSO.

Conclusions  These findings are consistent with the hypothesis that the urothelium provides sensory input via ATP release and that this process is increased in IC. Furthermore, stretch-activated ATP release was blocked by adding DMSO and heparin, both intravesical agents commonly used to treat the symptoms of IC. This study supports the notion that purinergic-targeted therapy is warranted in treating IC. Further studies are needed to determine the mechanisms of increased ATP release by IC urothelial cells.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Authors

IC is a syndrome of pelvic and/or perineal pain, urinary urgency and frequency; it may be a multifactorial syndrome and not caused by one condition [1,2]. Previous studies have already highlighted the central role of bladder urothelium in the pathophysiology of IC [3,4] but the aetiology of IC remains elusive.

There is growing evidence to suggest that extracellular ATP serves as a sensory neurotransmitter mediating nociception (pain). ATP can activate pain-sensing nerves through specific receptors (nociceptors), informing the brain about the status of visceral organs and protecting them from injury. Burnstock [5] proposed a novel hypothesis for purine-mediated mechanosensory transduction. Extracellular ATP, released by epithelial cells lining hollow viscus organs (bladders, gut, etc.) acts on P2X3 receptors on subepithelial nerve endings. This initiates impulses that are relayed via the spinal cord to pain centres in the brain. Findings from animal models have shown that ATP released by bladder urothelia during bladder distension or stretch may be involved in activating pelvic nerve afferents [6–10]. These studies support the hypothesis that ATP mediates the sensory pathway in the micturition reflex.

Because IC is a hypersensory bladder condition, it can be hypothesized that the bladder purinergic system is augmented. Urine specimens from patients with IC contained significantly higher ATP concentrations than those from control patients. Also, when urothelial cells were grown in culture and stretched, IC cells released significantly more ATP than control cells in a stretch-intensity dependent fashion [11]. By using established urothelial cell-culture techniques and an in vitro urothelial cell-stretch model, we determined the effect of DMSO and heparin, two commonly used intravesical agents to treat IC symptoms, on stretch-activated ATP release. Future novel treatments for IC could be agents designed specifically to decrease the augmented ATP release by IC urothelial cells.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Authors

Biopsies from four patients with IC, as defined by criteria for IC [12], were obtained during cystoscopy and hydrodistension. Four controls (patients with no voiding symptoms but undergoing other surgery) also had cystoscopic bladder biopsies taken. Patients were under regional or general anaesthesia for cystoscopy, hydrodistension of bladder and biopsies. The Institutional Research Board approved this study and subjects were consented using the Board protocols.

The cold-cup biopsy technique was used to obtain bladder biopsies, the tissues then placed in physiological saline and delivered to the laboratory within 10 min for processing. The technique of establishing bladder urothelial cell cultures was described previously [13]. Staining for pancytokeratin (Clone AE1/AE3 cocktail, Signet Laboratories Inc., Dedham, MA, USA) showed that the cells stained positively for cytokeratin, consistent with the epithelial origin of the cell cultures. Cells used for the experiments underwent fewer than six passages.

For the cell-stretch experiment, a Flexcell FX-2000 (Flexcell International Inc., McKeesport, PA, USA) unit was used to stretch a confluent culture of bladder urothelial cells. As described previously, there are several variables of stretch that can be varied in this model [11,14,15]. These include percentage elongation (or intensity of stretch), duration of stretch and duration of rest between stretching. In these experiments, IC and control urothelial cells were stretched using no stretch and 20% elongation. In each stretch-relaxation cycle, stretch occurred for 2 s followed by 1 s of relaxation; cells were stretched for a total of 96 h.

DMSO or heparin was added at the beginning of the stretch experiment. The concentrations of DMSO used were 0.1%, 0.5% and 1% (v/v per cell well), and for heparin, 50, 200, 800 or 1600 U (per cell well; each well contained 2 mL of growth buffer solution). These concentrations were used because higher concentrations of DMSO or heparin caused either cell death (based on Trypan Blue staining, 0.4%, Gibco BRL, Grand Island, NY, USA) or cell detachment from the growth surface of the stretch plates, preventing the stretch stimulus from being imparted on a monolayer of cells. More importantly, the concentrations of these agents used in these experiments did not interfere with measurement of ATP.

Each experiment was repeated twice and independently, and supernatant ATP was measured independently twice. For each experiment, ATP data were normalized to that experiment's time 0 value. Cell viability was measured using Trypan Blue staining and a haemocytometer was used to count the cells.

ATP was measured using the Bioluminescence Somatic Cell Assay Kit (Sigma Chemicals, St. Louis, MO, USA). A standard curve with known concentrations of ATP was constructed for each experiment; the r2 linear correlation coefficient obtained from the standard curves ranged from 0.992 to 0.999 over a concentration range of 105.

The mean values were compared using the two-sided Student's t-test, with P < 0.05 considered to indicate statistical significance. The values are presented as the mean (sem).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Authors

ATP was measured in the supernatant sampled at different times during the stretch; the amount released by IC cells was significantly greater (P < 0.05) within 24 h of stretch than in unstretched IC cells (Fig. 1a and Table 1). Stretched normal cells did not show this dramatic increase in ATP release and furthermore, unstretched IC cells and unstretched normal cells showed no significant change in ATP release (Fig. 1a).

image

Figure 1. The effect of DMSO or heparin on stretch-activated mean ATP release by urothelial cells. Stretched IC cells not exposed to DMSO or heparin (a ; open green circles in all plots) released significantly more ATP than unstretched IC cells (red open circles) after 24 h, stretched normal cells (light green closed circles) and unstretched normal cells (light red closed squares) after 48 h. DMSO at all concentrations (b ; 0.1%, light green closed circles; 0.5%, red open squares, 1%, light red closed squares) eliminated this augmented ATP release. Heparin attenuated this augmented release in a dose-dependent fashion (c , light green closed circles 50 U; red open squares, 200 U; light red closed squares, 800 U; black open triangles, 1600 U). There were no significant changes in ATP release when DMSO or heparin were added in other cell conditions (unstretched IC cells, stretched and unstretched normal cells); the data from these experiments are not shown, for clarity. Error bars are the sem , but for clarity are not plotted where they were smaller than the symbol size.

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Table 1.  The significance of stretch on ATP released by IC and normal cells is shown in the first set of values with no DMSO or heparin. The significance of DMSO and heparin on ATP released by stretched IC cells is given in the second and third set of values
ComparisonsSample time
24487296
  1. N, normal (control) urothelial cells; IC, urothelial cells from bladders with IC; S, stretched; US, unstretched. Bold values represent significant P values (< 0.05).

No DMSO or heparin
S-IC vs S-N0.132   0.0118   0.0038< 0.001
S-IC vs US-IC0.0013   0.0118   0.0015< 0.001
S-IC vs US-N0.363   0.038   0.006< 0.001
S-N vs US-N0.298   0.252   0.074   0.008
S-N vs US-IC0.376   0.804   0.005   0.005
DMSO, %
S-IC vs S-IC+0.10.85   0.38< 0.001   0.002
S-IC vs S-IC+0.50.41   0.084< 0.001< 0.001
S-IC vs S-IC+10.039   0.61   0.001< 0.001
Heparin, U
S-IC vs S-IC+500.60< 0.0010.019   0.0013
S-IC vs S-IC+2000.017< 0.0010.006< 0.001
S-IC vs S-IC+8000.11   0.0050.008< 0.001
S-IC vs S-IC+16000.097   0.0020.002< 0.001

Table 1 compares the different experimental conditions, using Student's t -test to obtain the respective P values. Stretch is an effective stimulus causing significant ATP release by both IC and control cells, but within 24 h for IC cells and 96 h for control cells. Stretched IC cells released significantly more ATP than stretched control cells after 48 h.

DMSO at all concentrations effectively abolished the increase in ATP release by stretched IC cells (Fig. 1b), with statistically significant differences (P < 0.05) at 24 h (1% DMSO) and 72–96 h (0.1%, 0.5% and 1%) of stretch (Table 1). In control experiments (unstretched IC cells, stretched and unstretched normal cells), DMSO at all concentrations did not affect ATP release.

Heparin at all concentrations effectively attenuated ATP release (Fig. 1c) with statistically significant (P < 0.05) differences at 48–96 h of stretch (Table 1). There appeared to be a dose effect, with the lowest concentration of heparin (50 U) diminishing ATP release the least (Fig. 1c). Heparin did not alter ATP release in the control experiments (unstretched IC cells, stretched and unstretched normal cells).

To determine whether changes in ATP release by cells were a result of changes in cell number during stretch, cells were counted in all experiments. The number of cells was not statistically different among the different conditions throughout the duration of stretch (data not shown) and therefore changes in ATP do not reflect changes in cell number. Furthermore, cell viability assays with Trypan Blue showed that the cell viability was similar across all experimental conditions.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Authors

Although many causes have been proposed the pathophysiology of IC remains unknown. While there are many different theories there are data that highlight the potential role of the urothelium in the pathophysiology of IC [3,4]. The traditional view of the function of bladder urothelium is the protection of the bladder stroma from urinary noxious substances. However, the urothelium may also act as a stretch-sensitive sensory organ initiating the afferent pathway in the micturition reflex. To date, potential bladder sensory neurotransmitters include substance P, calcitonin gene-related peptide, neurokinins, nitric oxide, PGs and ATP [16–18].

Extracellular ATP could be the primary bladder nociotransmitter. ATP can activate pain-sensing nerves through specific receptors (e.g. P2X3) located on sensory nerve terminals which terminate near the bladder urothelium [19]. P2X3‘knock-out’ mice have a greater bladder capacity and lower voiding frequency than the wild-type controls, suggesting that these receptors are important in maintaining bladder sensory thresholds [9]. Furthermore, in these animals, both P2X3 knock-out and wild type, ATP release was stretch-dependent [10]. This ATP-P2X3 sensory mechanism might be part of both the sensory [11] and motor pathophysiology of IC [20].

The treatment of IC is empirical; intravesically applied agents have the benefit of establishing high concentrations at the intended target tissue (e.g. urothelium), with a reduced risk of systemic side-effects. DMSO has been approved for intravesical use in IC, but the mechanism(s) is unknown and might be a result of its anti-inflammatory actions. Few prospective studies of the use of DMSO in IC have been published [21,22]. Although the effects of DMSO on nitric oxide mechanisms in the urothelium have been described [23], its effects on urothelial purinergic mechanisms have not been studied. The present study suggests that DMSO interfered with the increased release of ATP by stretched IC cells.

Heparin is also used intravesically for IC; the theory supporting its use is that there is a decreased glycosaminoglycan (GAG) layer in the apical surface of the bladder urothelium in IC [24]. This purported deficiency in GAG allows urinary potassium to permeate the suburothelium to depolarize sensory nerves [25]. Theoretically, heparin replenishes this deficient GAG layer [26,27] but it also has anti-inflammatory effects and inhibits the growth of fibroblasts, smooth muscle and blood vessels. Results from the present study suggest that heparin also affected purinergic mechanisms in urothelial cells from patients with IC.

In the in vitro experiments, the DMSO concentrations used were 0.1–1% and the heparin concentrations 50–1600 U; these doses are lower than those used clinically. Typically, clinical intravesical doses of DMSO and heparin are 50% and 10 000 U, respectively. However, concentrations of DMSO of > 1% and heparin of > 1600 U in the in vitro experiments resulted in the detachment of the entire monolayer of cells from the growth surface. This was confirmed by changes in cell morphology from spindle-shaped to rounded, which did not happen at lower concentrations of DMSO and heparin. Once the cells become physically detached from the growth surface, the stretch force generated by the Flexcell device cannot be transmitted to the cells.

The present results showed that DMSO and heparin, both commonly used to treat the symptoms of IC, attenuated stretch-activated ATP release in cultured IC bladder urothelial cells. These observations, with previous results [11], suggest that bladder urothelial purinergic mechanisms are important in bladder sensory function and are altered in IC. New treatments for IC could focus on agents that modulate bladder sensory nerve via inhibition of urothelial purinergic signalling, and further detailed mechanistic studies into bladder urothelial purinergic mechanisms are warranted which may lead to novel and effective treatments for hypersensory bladder disorders like IC.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Authors
  • 1
    Nickel JC. Interstitial cystitis. Etiology, diagnosis, and treatment. Can Fam Physician 2000; 46: 24304
  • 2
    Erickson DR. Interstitial cystitis: update on etiologies and therapeutic options. J Womens Health Gender Based Med 1999; 8: 74558
  • 3
    Keay S, Warren JW. A hypothesis for the etiology of interstitial cystitis based upon inhibition of bladder epithelial repair. Med Hypotheses 1998; 51: 7983
  • 4
    Leppilahti M, Kallioinen M, Tammela TL. Duration of increased mucosal permeability of the urinary bladder after acute overdistension: an experimental study in rats. Urol Res 1999; 27: 2726
  • 5
    Burnstock G. Release of vasoactive substances from endothelial cells by shear stress and purinergic mechanosensory transduction. J Anat 1999; 194: 33542
  • 6
    Usune S, Katsuragi T, Furukawa T. Effects of PPADS and suramin on contractions and cytoplasmic Ca2+ changes evoked by AP4A, ATP and alpha, beta-methylene ATP in guinea-pig urinary bladder. Br J Pharmacol 1996; 117: 698702
  • 7
    Ferguson DR, Kennedy I, Burton TJ. ATP is released from rabbit urinary bladder epithelial cells by hydrostatic pressure changes – a possible sensory mechanism? J Physiol 1997; 505: 50311
  • 8
    Namasivayam S, Eardley I, Morrison JF. Purinergic sensory neurotransmission in the urinary bladder: an in vitro study in the rat. BJU Int 1999; 84: 85460DOI: 10.1046/j.1464-410x.1999.00310.x
  • 9
    Cockayne DA, Hamilton SG, Zhu QM et al. Urinary bladder hyporeflexia and reduced pain-related behaviour in P2X3-deficient mice. Nature 2000; 407: 10115
  • 10
    Vlaskovska M, Kasakov L, Rong W et al. P2X3 knock-out mice reveal a major sensory role for urothelially released ATP. J Neurosci 2001; 21: 56707
  • 11
    Sun Y, Keay S, DeDeyne P, Chai TC. Augmented stretch activated adenosine triphosphate release from bladder urothelial cells in patients with interstitial cystitis. J Urol 2001; 166: 19516
  • 12
    Gillenwater JY, Wein AJ. Summary of the National Institute of Arthritis, Diabetes, Digestive and Kidney Diseases Workshop on Interstitial Cystitis, National Institutes of Health, Bethesda, Maryland, August 28–29, 1987. J Urol 1988; 140: 2036
  • 13
    Trifillis AL, Cui X, Jacobs S, Warren JW. Culture of bladder epithelium from cystoscopic biopsies of patients with interstitial cystitis. J Urol 1995; 153: 2438
  • 14
    Steers WD, Broder SR, Persson K et al. Mechanical stretch increases secretion of parathyroid hormone-related protein by cultured bladder smooth muscle cells. J Urol 1998; 160: 90812
  • 15
    Park JM, Borer JG, Freeman MR, Peters CA. Stretch activates heparin-binding EGF-like growth factor expression in bladder smooth muscle cells. Am J Physiol 1998; 275: C124754
  • 16
    Lecci A, Carini F, Tramontana M et al. Urodynamic effects induced by intravesical capsaicin in rats and hamsters. Auton Neurosci 2001; 91: 3746
  • 17
    Birder LA, Apodaca G, De Groat WC, Kanai AJ. Adrenergic- and capsaicin-evoked nitric oxide release from urothelium and afferent nerves in urinary bladder. Am J Physiol 1998; 275: F2269
  • 18
    Campbell DJ, Tenis N, Rosamilia A, Clements JA, Dwyer PL. Urinary levels of substance P and its metabolites are not increased in interstitial cystitis. BJU Int 2001; 87: 358DOI: 10.1046/j.1464-410x.2001.00990.x
  • 19
    Birder LA, Kanai AJ, De Groat WC et al. Vanilloid receptor expression suggests a sensory role for urinary bladder epithelial cells. PNAS 2001; 98: 13396401
  • 20
    Palea S, Artibani W, Ostardo E, Trist DG, Pietra C. Evidence for purinergic neurotransmission in human urinary bladder affected by interstitial cystitis. J Urol 1993; 150: 200712
  • 21
    Perez-Marrero R, Emerson LE, Feltis JT. A controlled study of dimethyl sulfoxide in interstitial cystitis. J Urol 1988; 140: 369
  • 22
    Fowler JE. Prospective study of intravesical dimethyl sulfoxide in treatment of suspected early interstitial cystitis. Urology 1981; 18: 216
  • 23
    Birder LA, Kanai AJ, De Groat WC. DMSO effect on bladder afferent neurons and nitric oxide release. J Urol 1997; 158: 198995
  • 24
    Parsons CL, Boychuk D, Jones S, Hurst R, Callahan H. Bladder surface glycosaminoglycans: an epithelial permeability barrier. J Urol 1990; 143: 13942
  • 25
    Parsons CL, Greenberger M, Gabal L, Bidair M, Barme G. The role of urinary potassium in the pathogenesis and diagnosis of interstitial cystitis. J Urol 1998; 159: 18626
  • 26
    Parsons CL, Shrom SH, Fritz R, Mulholland SG. The protective effect of heparin in experimental bladder infection. J Surg Res 1978; 25: 3249
  • 27
    Parsons CL. The therapeutic role of sulfated polysaccharides in the urinary bladder. Urol Clin North Am 1994; 21: 93100

Authors

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Authors

Y. Sun, PhD, Post-doctoral Fellow.

T.C. Chai, MD, FACS, Assistant Professor.

T.C. Chai, Division of Urology, University of Maryland, 22 South Greene Street, S8D18, Baltimore, MD 21201, USA. e-mail: tchai@smail.umaryland.edu