The rat urinaryprostacyclin as well as other

Prostaglandins Leukotrienes and Medicine 16: 235-248, 1984

J.Y. Jeremy, D.P. Mikhailidis and P. Dandona, Department of Chemical Pathology and Human Metabolism, Royal Free Hospital & School of Medicine, London. lW3 2QG U.K. (reprint requests to PD)

It has hitherto been assumed that urinary prostanoid excretion reflects renal and/or systemic prostanoid synthesis. Since the bladder forms an integral part of the urinary tract, we investigated whether this organ was capable of synthesising prostanoids. The rat urinary bladder was found to generate large amounts of 6-oxo-prostaglandin FE (the stable, spontaneous metabolite of prostacyclin) in vitro; it also produced smaller amounts of prostaqlandin E2 and thromboxane B2 (the stable, spontaneous metabolite of thromboxane AZ). Distension of the bladder and changes in pS and osmolarity of the incubate were found to markedly alter the production of these prostanoids. Urinary prostanoids may, therefore, reflect not merely renal and/or systemic prostanoid synthesis but also local synthesis and release by the bladder. The presence of these prostanoids in the bladder suggests that they may play a local role in cytoprotection and the regulation of bladder tone.

The complex methodology involved in the measurement of plasma concentrations of 6-oxo-prostaglandin Fla (6-oxo-PGFla: the stable, spontaneous metabolite of prostacyclin, pGI2), the inconsistency of the initial results obtained and the recent agreement that only very low plasma concentrations can be detected has led several workers to measure the urinary concentrations of this prostaqlandin and interpret them as indicators of its production by renal tissue and/or systemic vasculature1'27. However, none have considered the possibility that the urinary bladder may also be a source of PGI2. Local production by

235

the bladder, especially if influenced by urine dependent variables (osmolarity; pH; distention), may alter the interpretation of urinary prostaglandin levels by making a significant contribution to the total amount of prostanoids in the urine. The present study therefore investigates whether the rat urinary bladder produces significant amounts of PC12 and other prostanoids.

Male Sprague-Uawley rats (300 g) were decapitated, their urinary bladders excised and placed in Krebs-Ringer bicarbonate buffer (KHB; pregassed to pH 7.4 with 5/95; CO2/02), on melting ice. The KEB consisted of (mmol/l): NaCl, 118.6; KCl, 4.75; CaC12, 2.54: KH2PO4, 1.19; MgS04, 1.19; NaHCO3, 2.46: glucose 5.56. In some experiments whole bladders were used, while in others bladder minces were used.

16-gauge Abbocath-T teflon catheters (Abbott Ireland Ltd., Sligo, Eire) were inserted into the bladder cavity through the bladder neck and secured with silk sutures. A syringe (2 ml) was then attached to the luer end of the catheter, and the bladder washed with KBB. The following experiments were then carried out: (a)

Intnatftion

of the effect

of bla@!et diMon

The bladders were filled with between 200 to 1600 ul KEB. The bladders were'then immersed in KBB at 37°C and incubated for 60 min. Eight bladders were tested at each distention volume (see table 1). (b)

Invemtigatlon of the

effect of oralarity

(u&ngswxv~)

The bladders were filled with 750 ul KRB containing 0 - 1600 mmol/l sucrose. The bladders were then incubated in KHB at 37'C for 60 min. Eight bladders were tested at each sucrose concentration (see table 2). (cl

Invmtiq8tion

of

the

effwt

of

omxarity

(\uies

urea)

Urea was dissolved in KBB over a range of O-1600 mmol/l. Incubation was carried out as described above. Eight bladders were tested at each urea concentration (see table 3). For (a), (b) and (c) above, at the end of the incubations the KBB in the bladders was removed with a syringe, care being taken not to touch the urothelium with the tip of the catheter. These KRB samples were stored at -70 'C until measurements of prostaglandin concentrations by specific radioimmunoassay as described below.

236

Bladders were opened and cut into approximately 1 mm squares with a scalpel blade on a teflon block and then rinsed with ice-cold EBB. Tissue (20 mg) was placed in Eppendorf centrifuge tubes containing 1 ml KRB. These tubes were kept in melting ice until the start of the following experiments: (a)

Investigation of

the

eff00t

Of

pB

The pH of the EEB was adjusted with dilute hydrochloric acid or sodium hydroxide over a range of 4 - 10 units. Incubation was carried out at 37 "C for 30 min. Eight experiments were carried out at each pH (see table 4). (b) Investigation

of the effeat

of mlarity

(\uias urea)

Urea was dissolved in KRB over a range of 0 - 1600 mmol/l. Incubations were carried out as described in (a) above. Eight experiments were carried out at each urea concentration (see table 5). ( cl

Inwmtigation of the effect

of mluity

(using

mmro8d

Sucrose was dissolved in ERB over a concentration range of 0 1600 mmol/l. Incubation was carried out as described above. Eight experiments were carried out at each sucrose concentration (see table 6). At the end of the incubation for (a), (b) and (c) above, the tubes were centrifuged in an Eppendorf microcentrifuge. The supernatants were then stored at -70 'C until measurement of prostaglandin concentrations by specific radioimmunoassay as described below.

In addition, rat bladder minces were incubated as described in (a) above (pE 7.01, but a 10 min pre-incubation phase with indomethacin (final concentration: 100 mg/l) was included.

Promtsglandln

X2

(PGE2)

Antisera for PGE2 assays were purchased from the Louis Pasteur Institute, Paris, France; [3H]-PGB2 (160 Ci/mmol) from New England Nuclear. Bceton, USA; unlabelled PGE2 from Sigma Chemicals, Poole, Dorset, UK. Assays were carried out according to protocols obtained from the Louis Pasteur Institute, Paris, France. Thro&oxaneB:,

(TEB7) amay

Thromboxane A2 (TEA21 production by the rat bladder was assesaed by measurement of TEB2 levels, since TEB2 is the stable, spontaneous metabolite of TEA2. TEB2 levels were measured as previously described26r using a specific radioimmunoassay. 237

6-oxo-prostaglandin FIq:(B-oxo-PGFl~) FGI2 production by the rat bladder was assessed by measuring the concentrations of 6-0x0-PGFla, the stable, spontaneous metabolite of FGI2, using a specific radioimmunoassay as previously described26.

To confirm our radioimmunoassay data, we also assessed the ability of the rat bladder to convert [14Cl-arachidonicacid ( [14Cl-AA) into prostanoids, using a thin layer chromatography (TLC) technique, as previously describeda7g2*. Briefly, 50 mg minces of bladder were incubated with 50 nCi [14C)-AA (New England Nuclear; 56 mCi/nnnol)in 200 ul TRIS-HCl buffer, pH 8.0 (containing 0.9% NaCl and 1 mmol/l EDTA). Following incubation for 90 min at 37'C, the tissue was extracted with ethanol and the supernatant evaporated in vacua. The residue was redissolved in 50 ul ethanol/water (50:50, v/v), and applied on to TLC plates (Silica gel G precoated plastic plates, Merck Darmstadt, Germany), and develqed in the organic phase of ethyl acetate acetic acid/2,2,4 trimethylpentane /water: 110:20:50:100 v/v/ mixture. Standards of PGE2, 6-oxo-PGFla and TXB2 were also applied to the plate and detected with iodine vapour. The plates were divided laterally into 5 mm sections and counted for radioactivity. Statixtical hnalpcrirr Mann-Whitney tests (two-tailed) were used to obtain P values. Results are expressed as median and range.

(a)

Invut4ation

of tin efhat

of

blamer

v

(Table 1)

Sampling the KRB contained in the bladder revealed that increasing distention caused a significant increase in 6-oxo-PGFla production. Distention was also associated with an increase in PGE2 and TXB2 production but quantitatively the main product was 6-0x0-PGFld.

238

Table

The effect of distention

1.

whole

rat bladders.

Results

on prostanoid

are expressed

production

as median

by

(range)

Prostanoid raleued

into

Volume of Km

the bladder

distending the rat bladders (pl)

lumen (ng/bO mid

200

400

800

6-oxo-PGF1=

11

20'

34*+*

(6

PGC2

-

231

(10

33)

-

(16

0.6'

0.4 (0.2 - 0.8)

-

(0.2 - 0.8)

1200

59'1.e 56)

(33 - 94)

(0.Q - 2.0)

(0.Q - 2.4)

0.8'"*

o.b***

( 0.1 - 0.2)

(0.1 - 0.3)

(0.2 - 0.41

(0.4 - 0.7)

Mann-Whitney

* p

??

test comparing

- 134)

(0.3 - 1.2)

0.3.'

p
(33

1.4*++

0.2**

??

6a*+'

0.8"

0.1

TXR2

1600

0.02;

** p

??

prostanoid

1.1)**+

(0.6 - 1.3)

0.002

production

obtained

400, 800 and 1600 ul KRB with those obtained

at

at

200 ~1 PSS for the same prostanoid.

Sucrose over the aoncentration range 0 - 1600 nmcl/l prugreeeively increamed the eecretion of 6-oxo-FGF~, IGl!f2 and TXl.9into the bladder lumen.

239

Table

2.

The effect of osmolarity production

(using sucrose)

on prostanoi?

by whole rat hlarldt~rs.

Results are expressed

as median

(range).

Prostanoid released

in Concentration

bladder

of eucro*e

(mmol/l)

lUIS6.n

(ng160 dn)

6-oxo-PGF1a

PGE2

0

200

400

800

1600

27

31

39

45

52"'

(12.0-58.0)

(13.0-56.0)

(24.0-58.0)

0.8

0.7

1.3

(0.4-1.3)

TXR2

(0.5-1.0)

0.3 (0.1-0.6)

* p(O.03;

Mann-Whitney

prostanoid

200, 400, 800 and 1600 mmol/l

(c) xn-*h

Of t&

of sucrose

tit-t

0.02;

1.6** (0.8-2.5)

0.6"

(0.3-0.6)

** p

test comparing

in the absence

(0.9-2.4)

0.5

(0.2-0.6)

(38.0-76.0)

1.4'

(0.4-2.1)

0.3

(28.0-72.0)

(0.3-0.7)

*** p

0.6** (0.3-1.2)

0.01

production

obtained

at

sucrose with those obtained for the same prostanoid.

Of Omwity

(Wing urea) (Table 3)

Urea over the concentration range 0 - 1600 nnnol/lprogressively increased the secretion of 6-0x0-Pops and KZIE2into thQ bladder lumen, although this increase only achieved statistical significance for FGE2. Urea over the same concentration range had no significant effect on the secretion of TX%2 into the bladder lumen.

240

The effect of osmolarity (using urea) on prostanoid production

Table 3.

as median (range). wholerat bladders.Resultsare expressed

by Prostanoid

released into bladder lumen ng/60 min 6-oxo-PGFlo

0

200

400

800

1600

27

24

26

32

44

(12.0-46.0) (12.0-40.0) (15.0-45.0) (18.0-44.0) (21.0-72.0)

PGE2

0.7

0.6

0.9

0.9

1.4'

(0.4-1.5)

(0.3-1.3)

(0.6-1.1)

(0.6-1.0)

(0.7-2.1)

TXB2

0.2

0.3

(0.1-0.3)

(0.1-0.6)

??

0.2

p

(0.1-0.7)

0.3

0.3

(0.2-0.4)

(0.3-1.1)

(0.04

Mann-Whitney test comparing prostanoid production at 200, 400, 800 and 1600 mnol/l urea with that obtained in the absence of urea for the same prostanoid.

4) PGE2 am3 TXB2 secretion was significant diminution of production of all three prostanoids at pH values below and above this range. Table 4.

The effect of pH on prostanoid production by Results are expressed as median (range).

rat bladder minces. Prostanoid produced

ng/

pH range

20 mg tissue/

6-oxo-PGFla

18'*

26

(14-22)

PGE2

32 (22-41)

(18-32)

0.4.' (0.2-0.6)

TXR2

7.0

6.0

4.0

30 lin

0.2** (0.1-0.2)

??

8.0

32 (21-38)

0.6'

0.9

0.8

(0.3-0.8)

(0.7-1.2)

(0.5-1.1)

0.3

0.3

0.3

(0.2-0.4)

(0.3-0.4)

(0.2-0.4)

p (0.01;

10.0

18'* (14-25)

0.4** (0.2-0.6)

0.1** (0.1-0.2)

* p = 0.002.

??

Mann-Whitney test comparing peak prostanoid production (pN 7) with production at the other pH values for the same prostanoid.

241

(b)

Inveatiqation of the effect of oemolarity (wing urea) (Table 5) Urea over the concentration range studied CO-1600 mnol/l) had no consistent effect on the production of 6-oxo-PGFlcC. PGEZ orTxB2 production.

Table

5.

The effect of osmolarity

by rat bladder

minces.

Results

(using urea) on prostanoid are expressed

as median

production (range).

Prostanoid Urea concantratim (al/l)

produced n9/20 mg rimaue/30

Jn

6-0x0-PGPlq

PGE2

TXB2

0

,200

400

33

31

36

(20-43)

(18-41)

(20-52)

800

1600

40

37

(20-64)

(24-49)

1.1

0.9

1.2

1.2

1.3

(0.5-1.4)

(0.6-1.2)

(0.7-1.4)

(0.7-1.7)

(0.8-1.8)

0.1

0.1

( 0.1-0.3)

( 0.1-0.3)

All p

values = not

0.2

0.2

0.2

(0.1-0.4)

(0.1-0.3)

(0.1-0.5)

significant

Mann-Whitneytest comparingprostanoid 400, 800 and 1600 mmol/l absence

Production

urea with that obtained

at 200, in the

of urea for the same prostanoid.

(Table 6) progressively increarred the sscretfon of 6-oam-WFla , P6E2 and TXB~ by bladder minces, although this increaee only achieved

statistical significance for 6-~o-#Jlrla and TXB2.

242

Table 6.

Effect of osmolarity (using sucrose) on prostanoid production by rat bladder minces Results are

expressed as median (range).

Proetanoid produced (w/20

mg Conoentration of Socrome (ml/l)

tiaaue/30 min) 0

6-oxo-PGFla

26 (17.0-44.0)

FGE2

0.9

(0.6-1.4)

TXR2

0.2 (0.1-0.4)

200

400

27

36'

(13.0-40.0)

(24.0-48.0)

0.8

0.9

(0.6-1.3)

(0.6-1.4)

0.2

0.2

(0.1-0.3)

'p

(0.1-0.4)

800 37. (20.0-53.0)

1.0 (0.6-1.4)

0.4' (0.1-0.5)

1600 39* (22.0-58.0)

1.2 (0.7-1.5)

0.4* (0.1-0.6)

(0.02

Harm-Whitney test comparing prostanoid production at 200, 400, 800 and 1600 mmol/l urea with that in the absence of urea for the same prostanoid.

Pre-incubation with indomethacin significantly reduced the amount of prostanoids measured in the incubates. The maximal inhibition observed was never complete (approximately 80%) and was similar to that previously published by us for indomethacin using vascular tissue models2g.

Using the TLC technique, the major prostaglandinprodumd was 6-0x0-PGIpla. Radioactivity peaks corresponding to #;Ez and TXQ were also apparent, but they were quantitavely lower than 6-ox0-pGLr~~,thus confirming our radioimmunoassay data.

243

DISCUSSION

We have demonstrated that the rat urinary bladder produces significant amounts of PGI2, and that the secretion of this prostaglandin, like that of PGE2 and TXB2, is altered markedly by the degree of stretch to which the bladder wall is subjected. The other two fluctuating variables, osmolarity and pH, to which the urinary bladder is continuously subjected, were also shown to significantly alter the secretion of the prostanoids. The production of PG12 (measured as 6-0x0-PGFla) by rat bladder minces (32 ng/20 mg tissue/min) is of a similar order to production by rat aortic rings (56 ng/20 mg tissue/min) obtained from the same animals (unpublished observations). However, comparisons are not strictly valid because of differences in tissue structure and preparation. Nevertheless, these findings clearly demonstrate that the bladder has the potential to produce considerable amounts of PGI2. Since PG12 and TXB2 have opposing effects on smooth muscle activity30t31, it is possible that the relative concentrations of these two prostanoids are involved in the maintenance of bladder and sphincter tone. Indeed, urinary TXB2 was reported to be elevated during episodes of ureteric c01ic~~. However, the latter study did not state whether any concomitant changes in 6-0x0-PGFlg production were also observed. Another important implication of our observations is that PGI2 may play a role in the cytoprotection of the vesical mucosa, which is exposed to large variations of osmolarity, pH, stretch, toxins, drugs and chemicals. The role of PGE2 and PGI2 in cytoprotection of another mucosal surface, the gastric mucosa, has already been demonstrated32-35 and analogues of PGE2 have been used successfully in the treatment of peptic ulcers35. It is also of interest that PGE2 has been used successfully to improve symptoms caused by schistosomal ulcers of the urinary bladder36. In addition, two patients have recently been successfully treated for cyclophosphamiae-inducedhaemorrhagic cystitis by using intravesical infusions of PGE237. It is also tempting to speculate that the association of heavy smoking with bladder cancer3* may be mediated through altered prostanoid secretion. Nicotine is known to inhibit vascular PG12 production39; and may therefore also inhibit prostanoid production by the urinary bladder, thus resulting in imparied local cytcprotection and increased susceptibility to carcinogens. In addition, certain prostanoids have been shown to have anti-mitotic effects46r41. Ws have also confirmed that urinary bladders from another species, the cat, proauce XXII2(2 specialenstested; urpnbliehed observations). Ws can, therefore, no longer assume that urinary levels of prostanoids merely reflect vascular and/or renal PG12 A significant contribution to 6-oxo-PGFla and other production.

244

ptostaglandins contained in the urine of humans and experimental animals may therefore be derived from the urinary bladder. Furthermore, this local production may be significantly altered by several urine aepenasnt variables (e.g. pLI,distention). Bffects of distention and potassium and calcium concentration on vascular tissue PGI2 synthesis have already been reported by ~~~7~~2144.

We are currently investigating the effect of several other variables, including drugs and their metabolites, on bladder prostaglandin synthesis.

Part of the results reported in the present paper have been published in the form of an abstract (MeedicalResearch Society Meeting, London, January 1904)4s.

Acknowledgements: DPM is a Wellcam Trust Research Fellow. JYJ is funded by the Peter Samuels Fund, Royal Free Hospital. We thank Pamela Dale for secretarial assistance.

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