Evidence for the role of mast cells in colon-bladder cross organ sensitization

Abstract

This study examined the contribution of mast cells to colon-bladder cross organ sensitization induced by colon irritation with trinitrobenzene sulfonic acid (TNBS-CI). In urethane anesthetized rats 12 days after TNBS-CI, the voiding interval was reduced from 357 s to 201 s and urothelial permeability, measured indirectly by absorption of sodium fluorescein from the bladder lumen, increased six-fold. These effects were blocked by oral administration of ketotifen (10 mg/kg, for 5 days), a mast cell stabilizing agent. TNBS-CI in wild type mice produced a similar decrease in voiding interval (from 319 s to 209 s) and a 10-fold increase in urothelial permeability; however this did not occur in KitªWª/KitªW-vª mast cell deficient mice. Contractile responses of bladder strips elicited by Compound 48/80 (50 μg/ml), a mast cell activating agent, were significantly larger in strips from rats with TNBS-CI (145% increase in baseline tension) than in control rats (55% increase). The contractions of strips from rats with TNBS-CI were reduced 80-90% by pretreatment of strips with ketotifen (20 μM), whereas contractions of strips from control animals were not significantly changed. Bladder strips were pretreated with SLIGRL-NH2 (100 μM) to desensitize PAR-2, the receptor for mast cell tryptase. SLIGRL-NH2 pretreatment reduced by 60-80% the 48/80 induced contractions in strips from rats with TNBS-CI but did not alter the contractions in strips from control rats. These data indicate that bladder mast cells contribute to the bladder dysfunction following colon-bladder cross-sensitization.

Fig. 1

The number of mast cells (per 20× field) in the bladder is significantly increased 12 days after initiation of TNBS colitis. Ketotifen (10 mg/kg/day) administered for 5 days in control animals (n=4) did not alter mast cell numbers (one way ANOVA, p>0.05). Ketotifen administered for 5 days between days 7 and 12 after TNBS-CI (n=5) did not significantly (p>0.05) reduce the increased numbers of bladder mast cells after TNBS treatment (n=4). Both TNBS treatment groups had significantly increased numbers of mast cells compared to the control (vehicle/no treatment) group (p<0.05).

Fig. 2

Effect of TNBS-CI and ketotifen 10 mg/kg/day on voiding interval during continuous infusion cystometry in urethane anesthetized rats. Four groups of animals (n=3 per group) consisting of control (vehicle/no treatment), ketotifen treated controls, TNBS treated and TNBS treated with ketotifen from day 7 to day 12 of TNBS-CI were used in this study. Ketotifen administered for 5 days did not alter voiding in controls but normalized hyperactive voiding measured on day 12 in rats with TNBS-CI (one way ANOVA, p≤0.05). Voiding interval in TNBS animals was significantly different from control animals (*) and from TNBS-CI animals treated with ketotifen (#) which were not different from controls (*p<0.05 vs. control, #p<0.05 vs. TNBS-CI+Ketotifen).

Fig. 3

Treatment with ketotifen 10 mg/kg/day administered for 5 days starting on day 7 or for 7 days starting on day 5 after TNBS-CI normalizes bladder urothelial permeability measured on day 12 in rats with TNBS-CI. Urothelial permeability was evaluated by measuring the plasma concentration of sodium fluorescein after intravesical administration. Experiments were conducted on five groups of animals (n=4 animals per group): controls (vehicle/no treatment), controls treated with ketotifen for 5 days, TNBS-CI for 10–12 days, TNBS-CI treated with ketotifen for 5 or 7 days. Plasma concentration of sodium fluorescein was significantly increased 12 days after TNBS-CI (one way ANOVA, *p<0.05 vs. control) but not after TNBS+ketotifen (#p>0.05 vs. control).

Fig. 4

Compound 48/80 (50 μg/ml) produces a small increase in the contractile activity of bladder smooth muscle strips from control rats (a), but this effect is enhanced in strips from rats with TNBS-CI. (b). The effect of Compound 48/80 in strips from rats with TNBS-CI is suppressed by pretreatment with ketotifen (20 μM) (c) or by desensitization of PAR-2 by 30 min pretreatment with SLIGRL-NH₂, (100 μM) a PAR-2 agonist (d). These recordings are from four separate experiments.

Fig. 5

The effect of Compound 48/80 (50 μg/ml) to enhance phasic contractions of bladder smooth muscle strips was larger in preparations from rats with TNBS-CI (n=9 strips) than in preparations from control rats (n=9 strips, vehicle/no treatment, two way ANOVA, *p<0.05). In all preparations the effect of Compound 48/80 (50 μg/ml) is greater at 15 min compared to 3 min after administration. Pretreatment with the mast cell stabilizer ketotifen (20 μM) (n=8 strips) reduced the responses to Compound 48/80 at 3 and 15 min after drug application in strips from rats with TNBS-CI (#p<0.05) but did not alter the response to Compound 48/80 in strips from control animals (n=9 strips). PAR-2 desensitization via SLIGRL-NH₂ (100 μM) also reduced the effect of Compound 48/80 at 15 min after drug application in strips from TNBS-CI rats (@p < 0.05) but did not alter the response in strips from control rats. Enhancement is expressed as percent change in amplitude of phasic contractions of bladder smooth muscle strips after administration of Compound 48/80 (50 μg/ml).

Fig. 6

The effect of TNBS-CI on voiding interval in urethane anesthetized mast cell deficient (Kit^W/Kit^W-v) and wild type (Kit^+/+) adult mice. Voiding interval was measured during continuous infusion saline cystometry (25 μl/min). Kit^+/+ (n=5) and Kit^W/Kit^W-v (n=3) animals in which TNBS vehicle was instilled intrarectally 7 days prior to the experiment exhibited similar voiding intervals. TNBS-CI 7 days prior to the experiment significantly reduced the voiding interval (n=5 per group) in Kit^+/+ mice but not in Kit^W/Kit^W-v mice, which have normal voiding intervals equivalent to those in wild type control littermates (one way ANOVA, p<0.05). Voiding interval in Kit^+/+ TNBS-CI is significantly different (*p<0.05) from that in wild type Kit^+/+ and significantly different (#p<0.05) than that in Kit^W/Kit^W-v TNBS-CI mice.

Fig. 7

The effect of TNBS-CI on urothelial permeability in urethane anesthetized mast cell deficient (Kit^W/Kit^W-v) and wild type (Kit⁺/⁺) adult mice. To estimate urothelial permeability plasma concentration of sodium fluorescein was measured 15 min after infusion of the tracer solution (0.1 ml, 10 mg/ml) into the bladder. Wild type Kit^+/+ (n=5) and untreated Kit^W/Kit^W-v (n=3) animals in which TNBS vehicle was instilled intrarectally 7 days prior to the experiment exhibited similar urothelial permeabilities. TNBS-CI 7 days prior to the experiment significantly increased urothelial permeability in wild type Kit^+/+ mice (n=4) but not in mast cell deficient mice (Kit^W/Kit^W-v) which have normal urothelial permeability equivalent to that in wild type littermates (one way ANOVA, p<0.05). Voiding interval in Kit^+/+ TNBS-CI is significantly less (* p<0.05) from that in Kit^+/+ and significantly different (#p<0.05) from that in Kit^W/Kit^W-v TNBS-CI mice.

Fig. 8

Putative mechanisms involved in mast cell contribution to bladder dysfunction in the TNBS-CI model. Dorsal root ganglion (DRG) neurons co-innervating the colon and bladder are activated by TNBS colitis. Action potentials (AP) propagating from the colon to the bladder release neuropeptides (NP) that can activate receptors (neurokinin receptor, NKR) in smooth muscle (SM), urothelium (UT), mast cells and afferent nerve terminals. The stimulated mast cell release tryptase (tryp) that activates protease activated receptor-2 (PAR-2) in SM, afferent nerves and UT to induce a contraction (“?” in the figure implies that the UT induced SM contraction mechanism is unknown). Compound 48/80 releases Tryp from mast cells and SLIGRL-NH₂ activates and desensitizes PAR-2. See text for further description of chemical interactions between the various cell types.