Endothelin activates the mitogen-activated protein kinase pathway through endothelin receptor B in prostate stromal cells

P13

BPH:BASICRESEARCH Sunday, February 24,15.30-17.00

ENDOTHELIN ACTIVATES THE MITOGEN-ACTIVATED PROTEIN KINASE PATHWAY THROUGH ENDOTHELIN RECEPTOR B IN PROSTATE STROMAL CELLS Klocker

Helmut, Bartsch Georg

Urology, University

of Innsbruck,

Innsbruck,

226

225

hrs, Room F

ATP POTENTIATES HUMAN PROSTATIC CONTRACTIONS VIA P2XRECEPTORS Calvert Robert’, Banks Frederick*. Robert’, Burnstock Geoffrey’

Thompson

Cecil’,

SMOOTH Mikhailidis

MUSCLE

Dimitri’,

Morgan

‘Autonomic Neuroscience Institute, Royal Free Hap&xl, London, United Kingdom, ‘Urology, Royal Free Hospital, London, United Kmgdom, ‘Clinical Biochemistry. Royal Free Hospital, London, United Kingdom

Austria

INTRODUCTION & OBJECTIVES: Endothelin receptors are expressed in the human prostate and the vasoactive endothelin peptides have been implicated in the pathophysiology of benign prostate hyperplasia (BPH) and prostate cancer (PCs). In cultured prostatic smooth muscle cells endothelin-I induces cell contraction and an endothelin receptor A (ETA) antagonists blocks this effect. In this study we investigated intracellular signalling of endothelin in cultured prostate stromal cells. MATERIAL & METHODS: Prostate stromal cells exhibiting a smooth muscle cell phenotype were established from tissue derived from the transition zone of radical prostatectomy specimens. Expression of endothelin receptors was analysed by immunoblotting. Cells were treatment with entothelin-1 or BQ320 an endothelin receptor B (ETB) specific agonist. or a combination of agonists with ETA or ETB specific inhibitors, respectively, and activation of the mitogenactivated protein (MAP) kinases erkl and erk2 and protein kinase B (PKBI Akt- I). a kinase of a major survival pathway, were analysed by immunoblotting employing phosphoprotein-specific antibodies. Cell proliferation was assessed by MTT assay. RESULTS: Cultured prostate stromal cells express both endothelin receptors, ETA and ETB. Treatment with endothelin-I or the ETB agonist BQ320 resulted in a profound activation of erk-I ,2. Activation was blocked by an ETB antagonist but not by an ETA-specific antagonist, revealing that MAP kinase activation is mediated through receptor ETB. Despite MAP kinase activation, treatment with endothelin or BQ320 for 72 hours did not have a proliferative effect on prostate stromal cells. Activation of the PKBIAkt-I survival pathway by endothelin or BQ320 was marginal only. Only in some stromal cell strains a weak increase of PKB/Akt- I phosphorylation was detected.

INTRODUCTION & OBJECTIVES: In addition to its intracellular role in energy metabolism, adenosine 5’.triphosphate (ATP) acts as a neurotransmitter which mediates smooth muscle contraction or relaxation in a number of organs. including the bladder, vas deferens, uretera and gut [I]. Sympathetic nerves that utilise noradrenaline and ATP as cotrnnsmmers supply the prostate and pharmacological blockade of adrenoceptors on prostatic smooth muscle has been successfully used to relax smooth muscle tone and improve the symptoms and flow rates of men suffering from benign prostatic obstruction. The purpose of this study was to immunohistochemically characterise the expression of receptors for ATP (purinoceptor subtypes) on prostatic smooth muscle and to examine their pharmacological functions. MATERIAL & METHODS: Following ethical approval and consent, prostatic chips were taken from men undergoing transureteral resection for benign prostatic hyperplasia. n=6. lmmunohistochemistry was performed on frozen sections using primary antibodies against P2XI, 2,3,4,5,6,7 and P2Y I, 2 and 4-purinoceptors. An avldin-biotin complex technique was used and negative control5 performed. Strips of prostate containing smooth muscle were dissected from fresh prostatic chips and mounted in organ baths in oxygenated Krebs solution at 37 centigrade. Functional studies were performed using ATP, 2methylthioADP. PPADS and phenylephrine, see results section. RESULTS: Prostatic smooth muscle cells showed strong immune-staining for antibodles to P2X I and P2Y I receotors and weaker staining for the anti-P2X2 antibodv. In control experiments preabsorp&on wth the nascent pepti& reduced staining. ATP did not affect the tone of the prostate strips under basal conditions. However in pre-contracted prostatic strips (by 0.1 mM phenylephrine) further contraction was Induced by addition of ATP. This was dose dependent and amounted to 38% potentiation (mean) of contraction at ImM ATP. The response was competitively inhibited by the P2-purinoceptor antagonist PPADS (0.03 mM) and the P2Y I-agonist. Zmethyl-thioADP had no effect.

CONCLUSIONS: The vasoactive peptides stimulate not only cell contraction in prostate smooth muscle cells but also activate the MAP kinase signalling pathway. This warrants further examination of the role of endothelins in the dysfunction of the stroma in BPH.

CONCLUSIONS: P2X1, P2X2 and P2Y 1 receptors are expressed on prostatic smooth muscle. ATP has a synergistic effect on adrenergic tone. This is likely to be mediated by either P2XI or P2X2 receptor!, as a P2YI-selective agonist had no effect and a P2X antagonist reduced the action. Antagonists to P2X qignaliing might be targeted to nnprove the efficacv, of aloha-blockade in a combination theraov for benign obstruction. We - orostatic & . speculate that the P2Y I receptor (which is a well-recognised mediator of mitogenesis in other tiswes) may be involved in the trophic changes that occur in benign prostatic hyperplasla. References [I] Bumstock G. The past, present and future of purine nucleotides as FignaIling molecules. (Neuropharmacology 1997; 36: 1127-1139)

227

228

&,

P2Xl RECEPTOR ACTIVATION RESULTS IN DECREASED INTRACELLULAR CALCIUM INFLUX IN OBSTRUCTED RAT URINARY BLADDER SMOOTH MUSCLE

DOXAZOSIN INDUCED CHANGES IN THE PROSTATE OF SPONTANEOUS HYPERTENSIVE RATS: A STEREOLOGICAL STUDY

Siuve R.2, Uvelius B.2,AmerA.’

‘Urology. Hospital universitarto de Getafe. Madrid, Spain, *Basic Research Unit, Hospital universitario de Getafe, Madrid, Spain

IDepartment 2Department

of Physiological Sciences, Lund University, of Urology, Lund University, Lund, Sweden

Lund,

Sweden,

INTRODUCTION & OBJECTIVES: Previous studies have shown altered contractile response to purinergic activation in the urinary bladder in various conditions, e.g. cystitis or outflow obstruction. The present study was undertaken to determine if the lowered contractile response to ATP in obstructed rat urinary bladder was due to a decreased influx of calcium, and whether the P2X purinoceptor density was changed. MATERIAL & METHODS: Urinary outflow obstruction in the rat was created by a partial ligature of the uretera (0), with non-obstructed rats serving as controls (C). Activation with 80 mM K+ served as an internal control for subsequent ATP contractions. Intact bladder strips were activated with I mM of ATP. Force was registered and intracellular calcium was measured with Fura-2. Bladder smooth muscle was homogenised and subjected to Western blotting and immunostaining using a polyclonal P2XI antibody. Bladder tissue material was sectioned for light and electron microscopy. The outlines of at least 300 cell contours were measured for each bladder. RESULTS: The bladder weight increased about 3-fold from 66+3 mg (C) to 206+17 (0) (n=5). Activation with ATP gave a transient contractile response where the maximum force relative the K+ contraction was decreased in the obstructed strips (C: 152+21%; 0: 62516%). Peak intracellular calcium concentration in the hypertrophic preparations was about 50% compared to the control preparations(C: 63 12126; 0: 335&59 nM). Basal calcium was unchanged in the two preparations. T’/r for calcium influx was decreased in the obstructed bladders strips (C: I ,0*0,3: 0: 2,1&0,5 s). In the western blot analysis the amount of immunoreactivity was similar in the control and obstructed groups. Cell membrane area per unit wet weight was decreased in the obstructed bladders (66% of the control value). CONCLUSIONS: The cell membrane area per unit weight was decreased in the obstructed bladders as indicated by the morphometry. This would suggest that an upregulation of the P2X I receptors has occurred despite the decreased response to ATP. The decreased and slower influx of calcium after activation with ATP. could be explained by an increased diffusion distance in the hypertrophic muscle cells from cell membrane to contractile units.

Lunn Marcos’, Moreno Antonio’, Paez Alvaro’, Ferruelo Antonio?. Gamer Jose Miguel’, Berenguer Antonio’

INTRODUCTION & OBJECTIVES: The spontaneous hypertensive rat (SHR) model haa been previously proposed to study benign prostate hyperplasia (BPH) in the murine setting. These animals develop prostate changes described as hyperplasia. The objectives of the present study are to validate the SHR murine model for BPH basic research, a? well as to address doxazosin-induced prostatic structural changes. MATERIAL & METHODS: From an initial number of 60 rats (3 months of age) equally distributed in four cohorts, a final number of 59 rats entered the study. and allocated in the following arms: I) SHR rats with oral doxazosin given during 3 months (n=15); 2) SHR rats with unilateral pelvic ganglion excision (n=l4): 3) SHR rats with no manipulation (n=l4); 4) standard Wistar-Kyoto (WKY) non-manipulated rats were used as controls (n=l6). The age of all animals when sacrtficed was 6 months. Doxarosin mesylate (Cardurana, kindly supplied by Pfizer Inc., Madrid) was uniformly compacted in standard rat food (Panlab Export S.L.). Final daily dosage was 0.03 mg (I .83 g of drug/kg of food), calculations were based in maximal therapeutical dosage in humans,, rat body weight, and oral doxazosin bioavailability in rats. Unilateral major pelvic ganghon excision was performed with the aid of a surgical microscope under total anaesthesia. All animals were weighed at the beginning of the experiments and before sacrifice. All food administered in the doxarosm arm was also weighed. Right prostatic ventral lobe was excised after sacrifice and weighed. Stereologlcal analysis was performed from light microscopy hematoxilin-eosin qlides, and volume density of tissue compartments (epithelial. luminal, glandular and stromal) were calculated according to D&se’s principle. RESULTS: In all rats in the doxnosin arm daily drug intake was between 0.039 mg and 0.045 mg (mean 0.041 mg), therefore drug intake was in all cases above the amount expected. No statistical differences were found with regard to ventral prostatic lobe weight. A greater development of glandular prostatic epithelium was found in all cohorts of SHR rats. An atrophy of prostatic epithelium could be observed after microsurgical denervation. Finally, a reduction of the volume density of epithelial or glandular compartments after doxazosine exposure could not be demonstrated (p=O.429), but a significant reduction of the volume of luminal compartment was observed after doxazosin therapy.

I 641

Volume densities (x1000) Epithelial Glandular

894

Luminal Stromal

253 (p = 0,002) 107

II s25

III 586

421

IV

841

911

744 (p
316 159

325 89

323 256 (p>o,OOI)

(p
CONCLUSIONS: The validity of the SHR model for benign prostate hyperplasia basic research has been confirmed in this study. An atrophy of prostate epithelium can be seen in denervated rata, but could not be demonstrated in the same strain after doxazosin admmiwation. A reduction in the lummal volume was found after drug exposure, probably with relation to an additional effect on prostate secretion. European

Urology Supplements

1 (2002) No. 1, pp. 59