LIPOPOLYSACCHARIDE UPREGULATES BRADYKININ 1 RECEPTORS IN THE ISOLATED MOUSE BLADDER

0022-5347/98/1606-2267$03.00/0 T H E Jf)I'RN.U. OF UROLOGY

Vol. 160, 2267-2273, December 1998 Printed in U S A .

Copyright 0 1998 by AMERICAN UROLOCICAL ASSOCIATION, INC.

LIPOPOLYSACCHARIDE UPREGULATES B W Y K I N I N 1RECEPTORS IN THE ISOLATED MOUSE BLADDER B. W. BUSSER, T. G . HAMMOND, D. E. BJORLING

AND

R. SABAN*

From the Smooth Muscle Laboratory, School of Veterinary Medicine, University of Wisconsin, Madison, Wisconsin and the Nephrology Section, Tulane University School of Medicine and Tulane Environmental Astrobiology Center, New Orleans, Louisiana

ABSTRACT

Purpose: Bradykinin 1 (B1) receptors have been shown to be upregulated a t sites of inflammation. The purpose of this study was to determine the effect of lipopolysaccharide (LPS) on B1 receptor modulation in the isolated mouse bladder. Materials and Methods: The contractile responses of isolated mouse bladder to B1 and B2 agonists were determined in vitro following prolonged incubation with LPS or saline. Results: Bradykinin (BK), a B2 agonist, but not des-Argg-bradykinin (DABK), a B1 agonist, was found to be a potent contractile agonist of the mouse urinary bladder under basal conditions. However, both sensitivity and maximal response to DABK increased during a second exposure to the agonist in a time-dependent manner. In vivo or in vitro treatment with LPS increased both sensitivity and maximal response of isolated bladders to DABK, whereas bladder contraction to BK and other peptides remained the same. Treatment of tissues with a B1 receptor antagonist 45 minutes prior to second exposure to DABK, or the prostaglandin synthesis inhibitor, indomethacin, 30 minutes prior to LPS or saline incubation, significantly inhibited the increase of both maximal response and sensitivity. Conclusions: These results indicate that bladder B1 receptors can be upregulated by LPS, and that prostaglandins seem to mediate the effects of the B1 receptor activation in the isolated mouse bladder. KEYWORDS:peptidase inhibitors, interstitial cystitis, mouse bladder

Kinins are known to be involved in many physiological and pathological processes, including blood pressure regulation, nociception, the contraction or relaxation of smooth muscle, and the regulation of inflammation. The action of kinins is mediated by bradykinin receptors. Regoli and Barabe' proposed the existence of two bradykinin receptors, B1 and B2, over 15 years ago. Receptor cloning and pharmacological studies with highly specific B1 or B2 antagonists have validated their conclusions. These studies have revealed that bradykinin (BK) acts preferentially on B2 receptors, whereas the active carboxypeptidase metabolite of BK, des-Argg-BK (DABK), acts preferentially on B1 B2 receptors are known to be constitutively expressed in numerous animal tissues; whereas B 1receptors are inducible. B1 receptors can be induced following tissue trauma, in vitro incubation, systemic lipopolysaccharide (LPS) treatment, chemical colitis or cystitis, and immune complex art h r i t i ~ . * *Systemic ~-~ LPS treatment has been shown to increase B 1 receptor populations in the rabbit cardiovascular system,2 rat peripheral vasculature,' and rat stomach fund ~In addition, . ~ Bouthillier et a1.l' have shown that LPS administration in vitro increased B1 receptor levels in the rabbit isolated aorta. A role for the kallikrein-kinin system in bladder inflammation has been suggested based on increased kallikrein activity in the urine of patients with interstitial cystitis."-'*

More recently, a preliminary report of histochemical analysis of B1 receptors suggested an upregulation of this receptor in the urinary bladder during interstitial cystitis. l3 Animal studies to confirm functional B1 receptor activity in the isolated urinary bladder have produced conflicting results. Interestingly, a study by Allogho et al.I4 suggested that the mouse urinary bladder is a bradykinin monoreceptor system for the B2 receptor. However, other animal models have demonstrated functional B1 receptor activity in the urinary bladder. For example, B1 receptor activity was demonstrated following chemical cystitis in rats,7 and in rabbit bladder strips following prolonged in vitro incubation6 Due to the lack of demonstrating functional B1 receptor activity in the mouse bladder, the purpose of this research was to determine if prolonged in vitro incubation with E. coli LPS alters B1 receptor activity in the isolated mouse urinary bladder. MATERIALS AND METHODS

Tissue preparation. Female Balb/c mice (8 weeks old) were anaesthetized with ketamine HC1 (1.8 mg./mouse) and xylazine (0.24 mg./mouse), and the urinary bladder was removed. Whole bladders were suspended as ring segments between two stainless steel stirrups in 10 ml. organ baths (37C) containing aerated (95% 0, and 5% CO,), physiological salt solution (PSS, pH 7.4) of the following composition (mM): NaC1, 119; NaH,PO,, 1; KCl, 4.7; CaCl,, 2.5; MgCl,, 0.5; Accepted for publication June 19, 1998. NaHCO,; and glucose, 25. The tissues were attached to force * Requests for reprints: Enteric Neuromuscular Disease Labora- displacement transducers (Grass FT-03; Grass Instruments, tory, Division of Gastroenterology, Department of Internal Medicine, University of Texas, Medical Branch, 1108 Strand, Room 219, Quincy, MA, USA), and changes in tension were displayed on polygraphs (Grass Model 79). Initial tension was set at 1gm. Galveston, TX 77555-0632. Supported by National Institutes of Health grants NIDDK which was found t o be the optimal tension for this preparaDK49471-04, DK53216-01 and DK 51392-01, and the Interstitial tion in preliminary experiments. Tissues were allowed t o Cystitis Association. Presented in part at the Experimental Biology Meeting, San Fran- equilibrate for 1 hour, during which PSS was changed and replaced with fresh buffer and tension readjusted every 15 cisco, California, April 18-22, 1998. 2261

2268

UPREGULATION OF BLADDER B1 RECEPTORS

minutes. At the completion of each experiment, maximal peptide analogues were stored as a stock in sodium metbladder contraction was induced with carbachol (10 pM). abisulfite and frozen at -4C; these drugs were diluted in Concentration-response Curves were analyzed as a percent Of saline for use on the day of the experiment. Phosphoramidon, captopril and lipopolysaccharide were stored as stock solumaximal contraction in response to carbachol. Effect of endogenous peptidase activity on BK- and DABK- tion in saline at -4C; these drugs were diluted in saline for induced bladder contraction. Tissues were incubated in the use on the day of the experiment. Indomethacin was prepresence or absence of phosphoramidon (1 pM), a neutral en- pared fresh on the day of the experiment by dissolution at pH dopeptidase inhibitor, and captopril (1 pM), an angiotensin- 10.0 followed by pH titration to physiologic pH and dilution converting enzyme inhibitor, for 1hour to investigate the role to desired concentrations. Statistical analysis. The concentration of BK that produced of endogenous peptidases on BK- or DABK-induced mouse bladder contraction. The preparations were then exposed to 50% of the maximal effect (EC50) was determined from inincreasing concentrations of BK (lo-'' M) or DABK dividual concentration-response curves to BK. Because the (10- loM) to generate cumulative concentration- CRC to DABK did not reach a stable plateau at the concentrations used, the potency of this peptide was evaluated a t response curves (CRC). In uitro effect of E. coli LPS. To determine the effect of in 10% (EC10) of the maximal response obtained with carbavitro exposure to LPS on kinin-induced contraction of mouse chol, as described before for other peptides.lS-l6 The EClO or bladder, CRC were determined for DABK o r BK in the ab- EC50 values were determined by interpolation according to sence of peptidase inhibitors. The tissues were then washed Fleming et al.17 EC values were converted to the negative with fresh PSS (37C) for 30 minutes to remove DABK or BK, logarithm, and the geometric means of EC were determined and incubated with LPS (100 pg./ml.) or saline for 1, 3 or 5 and expressed as -log molar EC.17 The mean and SEM were hours. CRC were determined again for the same agonist. The calculated for all data. Means were compared by Student's t specificity of LPS action was determined by comparing its test. A value of p <0.05was considered indicative of signifieffects on bladder contractile responses t o kinins, substance cant difference. lS P, neurokinin A, and carbachol. Only one agonist was tested in each preparation. RESULTS Effect of inhibition of prostaglandin synthesis. To investigate the influence of prostanoids on DABK-induced contracEffect ofpeptidase inhibitors on BKor DABK-induced bladtion of mouse bladder, CRC were obtained for DABK, and the der contraction. Peptidase inhibitors increased the sensitivtissues were washed with fresh PSS for 30 minutes to remove ity of isolated bladders to BK, but not DABK (fig. 1). This was DABK and incubated with a cyclooxygenase inhibitor (indo- indicated by a significant 7-fold leftward shift of the CRC for methacin; 45 pM) for 30 minutes. LPS (100 pg./ml.) or an BK (measured a t EC501, but no significant shift was observed equivalent volume of saline was then added to the tissue bath for DABK (measured at EC10). The maximal response to BK for 1hour, subsequent to which CRC to DABK were obtained. was not significantly changed when comparing the peptidase Effect of B1 receptor antagonist. To confirm that the effect inhibitor-treated preparations t o saline-treated, whereas the of LPS or saline on the increased contractile response of the bladder to DABK is mediated through the B1 receptor, a first CRC to DABK was obtained, tissues were washed with fresh PSS for 30 minutes, incubated with LPS (100 pgJm1.1 or BK-SALINE saline for 15 minutes, and then exposed to a B1 receptor antagonist (des-Argg-[Leu8-]-BK 10 pM) or an equivalent BK-PEPTIDASE I NHIBlTORS volume of saline for a n additional 45 minutes. CRC for DABK were determined again. Yo Cch + DABK-SALINE Effect of in vivo intravesical instillation of LPS. To deter- maximum mine the effect of in vivo intravesical instillation of LPS on & DABK-PEPTIDASE INHIBlTORS DABK-induced contraction of the isolated mouse bladder, 6o mice were anaesthetized with ketamine HCl(1.8 mg./mouse) and xylazine (0.24 mg./mouse). A 24 gauge, 3/4 inch polypro50 T pylene catheter (Tom Cat Catheter, Shenvood Medical, St. Louis, MO) was introduced transurethrally into the bladder until the first drop of urine appeared. Light pressure on the abdomen was applied to remove urine, and 0.05 ml. of either 100 pg./ml. LPS or pyrogen-free saline was instilled into the bladder. Thirty minutes later, the LPS or saline was removed by light pressure on the abdomen, and 0.05 ml. of the same substance was added for another 30 minutes to ensure con* stant contact of the substances with the bladder. Mice were allowed to wake up. Animals were sacrificed 24 hours later, and the urinary bladders were studied in vitro as described above. Tissues were allowed to equilibrate for 1 hour and incubated with phosphoramidon (1pM) and captopril(1 pM) for 1 hour. Bladders were exposed to increasing concentra-12 -11 -10 -9 -8 -7 -6 -5 tions of DABK (lo-'' - lo-" M) and CRC were obtained. Chemicals. The following compounds were purchased from log [peptide] M Sigma Chemical Co. (St. Louis, Missouri): E. coli LPS strain FIG. 1. Effect of peptidase inhibitors on bladder responses to BK 055:B5; bradykinin; des-Argg-bradykinin(DABK); indomethand DABK. Tissues were incubated in PSS with peptidase inhibitors acin ~l[p-chlorobenzoyll-5-methoxy-2-methylindole-3-acetic (phosphoramidon 1 WMand captopril 1 wM) or vehicle (saline) for 1 acid); phosphoramidon (N-(a-rhamnopyranosyloxy-hydroxy- hour prior to exposure to BK or DABK. Results are means 2 S.E.M. of 4 to 6 experiments for each group, and shown a s percentage of phosphiny1)leu-trp; captopril ( [2Sl-l-[3-mercapto-2-methylpropionyll-L-proline; and carbamylcholine chloride (carba- maximum response to carbachol, which corresponded to 4.54 t 0.7 gm.contraction. Asterisks indicate a statistical significant difference chol). B1 antagonist (des-Argg-[Leus-]-BK)was purchased when comparing peptidase inhibitor versus saline-treated preparafrom Peninsula Laboratories (Belmont, CA). PeDtide and tions (n < O Of;)

1

2269

UPREGULATION OF BLADDER B 1 RECEPTORS

maximal response to DABK was significantly greater. These results are summarized in table 1. Potency of B l and B2 agonists on isolated mouse bladder. Our results indicate that BK is a potent agonist of the mouse bladder. BK induced bladder contraction in concentrations as low as 10-l' M. Maximal response to BK was obtained a t M and corresponded to 45 t 5% of the maximal response to carbachol (fig. 1). Initial bladder responses to DABK were observed at lo-' M, and the maximal response, within the range of concentrations studied, corresponded only t o 20 -t 2% of the maximum obtained with carbachol (fig. 1). Time-dependent increase of mouse bladder reponsiveness to DABK. To study the role of time on the increased responsiveness of isolated mouse bladder to DABK, CRC were generated to DABK, preparations were washed for 30 minutes to remove the agonist and incubated in PSS for 1,3, or 5 hours. CRC were determined again for DABK. As shown in fig. 2, A, incubation of the preparations with PSS for 1, 3, or 5 hours potentiated DABK activity and maximal responses as compared to the initial CRC (0 hours of incubation). The second CRC to DABK presented a leftward shift of 32-fold at 1hour, 375-fold a t 3 hours, and 801-fold after 5 hours (measured a t EClO level). This data is summarized in table 2. The specificity of the time-dependent increase in responsiveness of the bladder to DABK was confirmed through the use of other contractile peptides. In vitro incubation with PSS for 1 hour did not alter the maximal response to BK. However, it caused a slight, but significant, desensitization in a second CRC to BK. This was represented by a 4-fold shift of the BK CRC to the right (measured a t EC50; fig. 3, table 2). In contrary, 1hour incubation with PSS did not alter bladder maximal responses (data not shown) and sensitivity to other contractile peptides such as substance P and neurokinin A. The EClO values for substance P were found to be 3.0 t 1 X M and 2.8 t 1 x M before and after in vitro saline, respectively (n = 4). The EC50 values for neurokinin A were determined to be 4.1 ? 1 x lo-' M and 2.0 2 1 X lo-' M before and after in vitro saline, respectively (n = 4). Effect of in vitro administration of LPS on isolated mouse bladder. In vitro incubation of isolated bladders with LPS increased the sensitivity to DABK in a time-dependent manner. This was represented by a 282-fold leftward shift of the CRC after one hour of incubation with LPS, a 439-fold shift after 3 hours, and a 2868-fold shift after 5 hours (measured at EC10; fig. 2, B). In addition, in vitro treatment with LPS significantly increased maximal bladder responses to DABK (table 2). In vitro treatment with LPS did not alter the sensitivity of isolated bladders t o BK during the 1-hour period investi-

TABLE1. Effect ofpeptidase inhibitors on bladder responses to BK and DABK DABK Pretreatment

EClO IwM)"

saline oentidnw inhihitod

0.015 i 0.010 0.005 t 0.003

Maximum (%

Cch)

9.5 ? 1.4 19.8 ? 2.4'

BK Pretreatment

EC50 (pMF

Maximum (lo Cch)

saline 0.067 2 0.010 44.0 2 5.0 peptidase inhibitorsb 0.009 2 0.001' 45.0 ? 5.0 EClO (effective concentration 10%) Peptidase inhibitors M phosphoramidon and M captopril; 1 hour incubation) ' EC50 (effective concentration 50%) Asterisks indicate a significant difference ( p <0.05) between the peptidase inhibitors and saline groups.

gated, although LPS significantly increased the maximal contractile response to BK (table 2). In addition, in vitro LPS failed to alter bladder maximal responses (data not shown) and sensitivity to other peptides such as substance P and neurokinin A. The EClO values for substance P were deterand 5.4 ? 1.5 x 10 M before mined to be 6.0 t 1.5 x and after in vitro incubation with LPS, respectively (n = 4). The EC50 values for neurokinin A were found to be 3.0 z 1.0 x lo-' and 3.5 -t 1.0 X lo-' M before and after in vitro incubation with LPS, respectively (n = 4). Effect of a B l antagonist on increased sensitivity of isolated mouse bladder to DABK. The B1 antagonist, des-Arg9-[LeuHlbradykinin (DALBK), inhibited LPS- and time-induced upregulation of bladder responses to DABK (measured a t EClO level; fig. 4, table 3). In addition, the maximal contractile responses to DABK were not significantly different from the first CRC to the second CRC for both LPS and time- treatment (fig. 4, table 3). Influence ofprostanoids on the increased activity of isolated mouse bladder to DABK. A prostaglandin synthesis inhibitor, indomethacin, inhibited the increased sensitivity to DABK following LPS and saline treatment (measured a t EC10; fig. 5, table 3). In addition, the maximal contractile response elicited by DABK during the first CRC was not significantly different from the second following LPS or saline treatment (table 3). Effect of in vivo intravesical bladder instillation with LPS or saline on bladder responses to DABK. Bladders studied 24 hours after in vivo intravesical instillation with LPS demonstrated an increased contractile response to DABK when compared to bladders isolated from saline-treated animals (fig. 6 , table 4). LPS treatment produced a significant 17-fold leftward shift (measured at EC10) of the CRC as compared to mice treated with saline. In addition, in vivo administration of LPS significantly increased the maximal response to DABK when compared with saline (table 4). Effect of i n vitro and in vivo treatments on maximum carbachol tissue contraction. To normalize among tissue and animal differences in responsiveness to peptides, data were analyzed as a percent of the maximal contraction obtained with carbachol (Cch; 10 pM.). None of the treatments, either in vitro or in vivo, had a significant effect on the Cch maximum (measured in grams) elicited at the end of the experiment (data not shown). DISCUSSION

Because pain and urinary urgency are such prominent components of cystitis, a role for BK in the pathogenesis of interstitial cystitis (IC) has been suggested." In addition, it has been shown that kallikrein activity is increased in the urine of patients with IC.'l-l' In support of this hypothesis, we previously presented evidence that BK is spontaneously released from the urinary bladder and that peptidases play an important role in its degradation.'" The present results indicate that peptidase inhibitors increased the contractile responses to BK, but not DABK. Others have found that peptidase inhibitors did not alter the response of the isolated rabbit bladder to DABK.' It seems that removal of one arginine molecule from BK decreases susceptibility of the new peptide to enzymatic degradation. Our results also indicate that although the sensitivity of the bladder to DABK was not altered by peptidase inhibitors, there was a trend for an increase in maximal responses to DABK. This may indicate that at higher concentrations, DABK may release other endogenous substances which are substrate for these enzymes. Therefore, during peptidase inhibition, this indirect effect might be accentuated. Other studies suggest that BK initiates neurogenic inflammation in vivo,20and inhibition of B2 receptors significantly reduced plasma extravasation and detrusor hyperreflexia

UPREGULATION OF BLADDER B 1 RECEPTORS

2270 % Cch maximum

*

25 20 -

%Cch maximum

SALINE

+ 1HOUR

40 -

+ 3HOURS 15-

50 -

OHOURS

--O-

--m-

OHOURS

-E--

1HOUR

*p

+ BHOURS

/ 30 -

5HOURS

LPS

-0- 5HOURS

10-

20 -

5-

10-

0-

0-11

-10

-9

A

-8

-7

-6

-10

-11

-5

-9

-8

-7

-6

-5

log [DABK] M

log [DABK] M

FIG.2. Time-course of alterations on CRC for DABK. CRC were obtained to DABK, tissues were washed for 30 minutes, and then incubated in PSS with LPS (100 fig./ml.)or vehicle (saline) for 1,3 or 5 hours. CRC were obtained for DABK again. Results presented are first (0 hours) and second CRC to DABK following 1hour, 3 hours, or 5 hours incubation with saline (A) or LPS ( B ) .Results are presented as means 2 S.E.M. of 4 experiments for each group, and shown as percentage of maximum response to carbachol, whlch corresponded to 4.342 0.5 gm. contraction.Asterisks indicate a statistical significant difference when comparing first CRC to second CRC to DABK ( p <0.05). Note that different scales were used in A and B to allow for comparison within same figure.

TABLE2. Effect of in vitro administration

of

LPS on mouse bladder responses to DABK

DABK First CRC

Second CRC

EClO (@MI"

Maximum (% Cch)

1.2 t 1 1.2 t 1 1.2 2 1 1.3 t 1

2.7 t 0.5 3.5 t 0.5 2.1 t 0.3 3.6 t 0.7

Treatment

SAL (1 hour) SAL ( 3 hour) LPS (1 hour) LPS ( 3 hour) BK

First CRC EC50 (@MY

EClO (pM) 0.038 2 0.040* 0.003 2 0.001" 0.004 i 0.001* 0.002 t 0.001'

Dose Ratiob

Maximum ( % Cch)

31 375 282 439

6.7 t 1.3' 12.2 c 1.0" 11.7 t 2.1' 19.7 t 1.5".

Dose Ratio"

Maximum (7 Cch)

Second CRC Maximum (c/o Cch)

Treatment

EC50 (pM)

0.03 t .01 49.1 -t 5 SAL ( 1hour) 0.15 t 0.12 0.23 0.09 t .01 38.1 i 4 LPS (1 hour) 0.73 2 0.12 1.34 A EClO (effective concentration 10%) Ratio is defined as the EC value for the second CRC divided by the EC value for the first CRC. EC50 (effective concentration 50%) Asterisk indicate a significant difference (p <0.05) between the first CRC and the second CRC maximum to the same agonist.

55.6 I 3 64.1 L 6'

associated with cyclophosphamide-induced cystitis in tion during long periods of incubation in vitro or after tissue In addition, antagonists of NK1 receptors for sub- injury or infection, a n effect that seems t o be associated with stance P (CP 96,345) and bradykinin B2 receptors (HOE 140) their de novo synthesis.' Recent evidence indicates that upreduced the inflammatory response of the rat urinary blad- regulation of B1 receptors may represent a component of the response to LPS. Upregulation of B1 receptors in response to der to catheterization and xylene i r r i t a t i ~ n . ' ~ The existence of two subtypes of bradykinin receptor, B1 LPS has been described in both vascularz7 and non-vascular and B2, has been confirmed through the use of high affinity smooth muscle,' and during fever," edema,' and pain.29 Our peptide and nonpeptide receptor antagonists, radioligand results indicate that this phenomenon also occurs in the binding studies and, recently, receptor cloning and expres- isolated mouse bladder following prolonged incubation with sion ~ t u d i e s . ' Evidence ~ suggests a role for B2 receptors in LPS or 24 hours after intravesical instillation with LPS. more classical acute inflammatory events, such as edema and These observations may be specifically important in chronic inflammatory pain, whereas B1 receptors appear to be in- inflammatory conditions, because the BK metabolite, desvolved in chronic inflammatory responses, including certain Argg-BK, acquires a n active and relevant role with the proforms of persistent hyperalge~ia.~.BK causes alteration in gressive expression of B1 receptors that occurs in such states. vascular tone via B1 and B2 receptors'" and modulates inIn this study, we present evidence suggesting upregulation creased vascular permeability in response to LPS via B2 of mouse bladder B1 receptors. First, we have demonstrated receptors.'" that the contractile responses of isolated mouse bladder to A particular characteristic of B1 receptors is their induc- DABK increased in a time-dependent manner. Second, the

2271

UPREGULATION OF BLADDER B1 RECEPTORS

80 -

-m-

FIRSTCRC

-+--

2NDCRC/LPS

DALBK

-a-

2ND CRC/SALINE + DALBK

-o-

2ND CRC/LPS + DALBK

0

60 -

+ 2ND CRC/LPS + SALINE 0

40 -

20 -

0-

FIG.3. Effect of in vitro treatment with LPS on CRC to BK. CRC were generated for BK, tissues were washed for 30 minutes to remove agonist and incubated in PSS with LPS (100 pg./ml.) or vehicle (saline)for 1hour. CRCs were generated a second time to the same agonist. Results are means 2 S.E.M. of 4 experiments for each group, and shown as percentage of maximum response to carbachol, which corresponded to 4.82 2 0.8 contraction. Asterisks indicate a statistical significant difference when comparing first CRC to second CRC to BK (p <0.05).

results obtained in the presence of a B1 receptor antagonist (DALBK) further support the involvement of this receptor. The specificity of this alteration was also indicated by the findings that bladder responses to other contractile peptides, such as substance P and neurokinin A, or carbachol, were not concomitantly altered. Lastly, our conclusion is supported through other studies documenting that B1 receptor populations increase in a time-dependent manner following in vitro incubation,6. 10,30,31.32 Interestingly, in rabbit bladder strips, the maximal responses to DABK were seen after 3 hours of incubation in vitro.6 In this study, we demonstrated that in the mouse bladder, contraction continued to increase past 3 hours up to the 5 hour time point investigated. This may be due to the different method of tissue preparation and suspension for pharmacological analysis. In this study, we used fresh, whole mucosa-intact bladders suspended as ring segments. A study with rabbit bladders6 used mucosa-free, longitudinal strips from bladders that were removed previously and stored at 4C overnight. It could also be argued that the mouse is simply a better model than the rabbit for studying bladder B1 receptor upregulation because the response to DABK continues to increase in the mouse bladder when the response plateaus in the rabbit bladder. In this study, we also demonstrated that the mouse bladder is not a bradykinin monoreceptor system, as suggested by Allogho et al.14 Rather, following in vitro incubation, isolated mouse bladder is a direceptor system and the functional responses seem to be modulated by both B1 and B2 receptors. The difference between our results and those of Allogho et al. may be due to the fact that we used the whole mouse bladder suspended as a ring segment, whereas Allogho et al.14 used the mouse bladder cut in longitudinal strips. Another explanation for the different finding is that

FIG. 4. Inhibition of increased responses to DABK by B1 receptor antagonist. Followin generation of first CRC, tissues were washed for 30 minutes, and tien incubated in PSS with LPS (100Fg./ml.) or vehicle (saline) for 15 minutes, followed by addition of DALBK (10 pM) or vehicle control (saline) for additional 45 minutes. CRC were generated for a second time to the same agonist. Results are means 2 S.E.M of 4 experiments for each group, and shown as percentage of maximum response to carbachol, which corresponded to 4.62 2 0.5 gm. contraction. Asterisks indicate statistical significant difference when comparing first CRC to second CRC to DABK (p <0.05).

Allogho et al. did not allow tissues to incubate for a sufficient time in vitro to investigate the responses to DABK. It is interesting to note that the responses to DABK in longitudinal bladder strips6,l4are not as evident as the ones observed with ring segments. It is possible that the whole bladder, as a ring segment, is a better method for the pharmacological analysis of urinary bladder contraction, though this assertion would require further study. We also present evidence suggesting that LPS potentiates the increase in responsiveness and sensitivity of isolated mouse bladder to DABK. This effect was seen after in vitro or in vivo administration of LPS. Again, this effect seems to be mediated at the B1 receptor level, because incubation of the preparations with DALBK significantly abolished the effect of DABK. In addition, LPS failed to upregulate bladder responses to BK, substance P, neurokinin A, or carbachol, thus further supporting the notion that this effect is mediated at the B1 receptor level. The effect of LPS on B1 receptors in the mouse bladder is in agreement with reports that in vitro or in vivo administration of LPS t o rabbits increases B1 receptor populations." In addition, intravenous administration of LPS has been shown to increase B1 receptor levels in the rat stomach fundus ex vivog and rat peripheral vasculature in viv0.s We also explored the possible involvement of prostaglandins in mediating the contractile responses to DABK. Our

UPREGULATION O F BLADDER B1 RECEPTORS

2272

TABLE 3. Inhibition of bladder responses to DABK following incubation with a B l antagonist or an inhibitor of cyclooxygenase DABK ~

~~

Second CRC

First CRC EClO IFMI"

Treatment

Maximum ( 5 Cch)

EClO (pM)

Maximum (% Cchl

Dose Ratio" 1.2 2.0 0.7 1.0

0.7 2 0.1 SAIJDALBK' 4.8 2 1.1 ? 1.0 0.6 2 0.1 4.8 t 1.1 LPSDALBK 1.2 t 1.0 1.2 t 1.0 0.8 2 0.1 6.1 2 0.5 SALIINDOd LPSnNDO 0.5 2 0.1 0.i I 0.1 6.9 2 1.9 EClO (effective concentration 10%). Ratio is defined as the EC value for the second CRC divided by the EC value for the first CRC. DALBK tdes-Arg--[Leu81-BK.B1 receptor antagonist; 10." M, 60 minutes incubation). INDO tindomethacin, cyclooxygenase inhibitor; 4.5 x 10.' M. 90 minutes incubation). 1.2

9.1 2 7.8 2 5.0 2 4.7 5

2.0 0.8

0.6 1.4

'

INDOMETHACIN

.-.- LPS

% Cch maximum

FIRST CRC

-u-

SALINE

2ND CRC/SALINE +INDOMETHACIN 2ND CRCRPS + INDOMETHACIN

*

2ND CRCRPS + SALINE

7

10-

T 5-

0-10

3

O J -10

-9

-8

-7

-6

-5

log [DABK] M FIG. 5. Inhibition of increased responses to DABK by indomethacin. Following generation of first CRC, tissues were washed for 30 minutes and then incubated with indomethacin (45 pM)or vehicle control (saline) for 30 minutes. Following that period, tissues were exposed to LPS (100pg./ml.) or vehicle (saline) for 60 minutes. CRC were generated for a second time to the same agonist. Results are means % S.E.M. of 4 experiments for each group, and shown as percentage of maximum response to carbachol, which corresponded to 4.85 -t 0.8 gm.contraction. Asterisks indicate statistical significant difference when comparing first CRC to second CRC to DABK (p <0.05).

results indicate that indomethacin reduced mouse bladder responses to DABK. It seems that prostaglandins are important in mediating the contractile responses to DABK in select tissues. In the archetypal B1 receptor preparation, the rabbit isolated aorta, indomethacin had no effect on B1 responses.3o Further, indomethacin was shown to have no effect on B1 responses in the rat stomach fundusg and human umbilical vein.:" On the other hand, Cabrini et al. provided evidence that indomethacin abolishes contractile responses to DABK in isolated guinea pig gall bladder."' However, the researchers proposed that this effect is mediated by B2 receptors. In other isolated tissue preparations, indomethacin has been

-9

-a

-7

-6

-5

log [DABK] M FIG. 6. CRC to DABK in isolated mouse bladder 24 hours after in vivo intravesical instillation with LPS (100 pg./ml.) or saline. All responses were obtained in presence of peptidase inhibitors. Results are means t S.E.M. of 8 experiments for LPS instillation and 4 experiments for saline instillation, and shown as percentage of maximum response to carbachol, which corresponded to 3.33 % 0.5 gm. contraction. Asterisks indicate statistical significant difference when comparing LPS to saline instilled bladders (p c0.05).

TABLE4. Effect of in uivo intrauesical instillation of LPS or saline on bladder resnonses to DABK Pretreatment

EClO ( u M ) ~

Maximum (% Cch)

Intravesical saline" 1.30 5 1.00 4.1 t 0.7 Intravesical LPS" 0.07 ir 0.02* 9.7 2 2.1% Anaesthesized mice were intravesically instilled with 100 pg./ml. LPS or saline 24 hours prior to in vitro experiment. EClO (effective concentration 10%). Asterisks indicate a significant difference (p <0.05) between the saline and LPS treated groups.

shown to significantly inhibit vascular smooth muscle contraction in response to DABK in the isolated pig coronary artery,33 rat portal vein,34 and dog renal artery without end~thelium.~" In this report, we present evidence supporting a role of prostaglandins in mediating contractile responses to DABK in a non-vascular smooth muscle tissue preparation, the isolated mouse urinary bladder. Our results are supported by the study by Marceau et al.7 documenting

UPREGULATION OF BLADDER B1 RECEPTORS

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