17β-Hydroxysteroid oxidoreductase in epithelium and stroma of human prostate

Steroi~Btockem. Molec. Biol. Vol.46, No. Printedin GreatBritain.All rightsreNrved J.

I,

pp. 91-101, 1993

0960-0760/93$6.00 + 0.00 Copyright~ 1993PerpmonPromLtd

17/~-HYDROXYSTEROID EPITHELIUM

AND

OXIDOREDUCTASE

STROMA

OF

HUMAN

IN

PROSTATE

S. TUI~,'* H. SCHULZ~and M, KRIEGj Ilmtitute of Clinical Chemistryand Laboratory Medka'ne,UniversityClinic Bergmanmheil, 4630 Bochum 1 and 2Ikpartment of Urology, UniversityClinic Marienhmpital, 4690 Herne 1, Germany (Receit~d 12 October 1992; accepted 1 March 1993)

S i m a r y - - l t is conceivable that androstenedione contributes indirectly to 5a-dihydrotutosterone formation in human prostate by its intraprostatic conversion to testosterone. This revers/ble conversion is catalyzed by the enzyme 17p-hydroxysteroid oxidoreductase (17#-HSOR). At present, rather limited information on kinetic parameters like sp~fic concentration (Vat), affinity to steroid substrates ( K s ) and to pyridine nueleotides (K,,,,) of 17p-HSOR is available. Thus, we determined those aforementioned kinet~ ~ in epithelium and stroma of normal human prostate (NPR) and benign prmtatic hypegplama (BPH). The main results were: (1) the mean K,~ of 17~-HSOI~/NADPH was significantly (P < 0.0001) lower than those of all other 17p-HSORs. (2) In almost all cases the mean g... was higher in BPH than NPR. (3) In all cases, the mean Vm~K,s rafim of 17~-HSORm were higher than those of 17~-HSOI~. The highest ratio was found regarding 17~-HSOI~/ NADPH in BPH stroma. (4) In stroma, a significantly positive correlation of V~JK,~ of 17~-HSOI~/NADPH with age was found. (5) The lowest K,, was found regarding NADP +, followed by NADPH. It is concluded that in human prostate the balance of the revertible conversion of testosterone to androstenedione is shifted potentially towards testosterone, particularly in BPH stroma.

However, due to rather fimited information on kinetic parameters like specific concentration It is believed that 5~-dihydrotestosterone (Vmx), affinity to steroid substrates (Kms) and to (DHT) is the most potent androgen at the pyridine nucleotides (K s) of ! 7p-HSOR, it is cellular level of the normal (NPR) and hyper- as yet unclear whether the reduction of androplastic human prostate (BPH), whereas testos- stenedione to testosterone or the oxidation of terone from blood seems to be its most testosterone to androstenedione is the preferred important precursor [1]. Moreover, it is conceiv- reaction in epithelium and stroma of N P R and able that the circulating androstenedione con- BPH. Furthermore, in light of strong associtributes indirectly to D H T formation because it ation between B P H development and aging['/] has been shown that this androgen can be the question arises as to whether an association tmmformed to testosterone in BPH tissue [2-6]. exists between aging and prostatic 17p-HSOR The enzyme that catalyzes this reversible reac- activity. tion is known as 17p-hydroxysteroid oxidoTo address those open questions, in reductase (17/~-HSOR). study we determined steroid and pyridiue nucleotide specificity of 17p-HSOR in epi*To whom correspondenceshould be addressed. thelium and stroma of NPR and BPH. In Abbrev/at/ons: 17#-HSOIt/NADPOl), 17#-hydmxystemid oxidoreductue, 17#-hydroxysteroid:NADP+ 17-oxido- addition, we correlated the data with the reductase, EC 1.I.1.64; 17#-HSOR/NAD(H), 17#- donor's age. hydroxysteroid oxidoreducta~, 17#-hydroxysteroid: NAD + 17-oxidoreducta~,EC 1.1.1.63; 30¢~)-HSOR, 3~- and 3p-hydroxysternid:NAD(P)+ oxidoreductaae, EXPERIMENTAL EC 1.1.1.50 and 1.1.1.51; 5¢t-reduota~ 3-oxo-Scgsteroid: NADP+-4-erie oxidoreducta~, EC 1.3.1.22; Chemica/s testmterone, 17~8-hydroxy4-andrmten.3-one; DHT, 17~-hydroxy-Se¢-andrmtu-3-onz; amkmtmaglion, an[la,2~-3H]tmtosterone(sp.act. 1,73-7,00 TBq/ drmt-4-ene-3,17-dione;K~, mmnigyto ~goid mbttrate; mmol) and [ 1,2,6,7-3H]andrmt-4-ene-3,17-dione K s, affinityto pyridine nucleofide; the reductive and oxidativedirectionof the enzymaticreactionsare shown ( ~ . act. 2,69-3,40 TBq/mmol) were purchased by the indices "red" and "ox', ~ v e l y . from Amersham Buchler (Braunschweig, INTRODUCTION

91

92

S. TUNN et aL

Germany). The unlabeled steroids were purchased from Sigma (St Louis, MO, U.S.A.), the eluent for high pressure liquid chromatography (HPLC) from Baker (Grog Gerau, Germany), the scintillation solution Ready Solv HP from Beckman (Miinchen, Germany), and all other chemicals from Merck AG (Darmstadt, Germany), Serva (Heidelberg, Germany), and Boehringer (Mannheim, Germany).

Tissue preparation NPR tissue was obtained from four braindead kidney donors, aged 35-56 years. BPH tissue was obtained from 10 men, aged 55-87 years, by suprapubic prostatectomy. Each donor gave written consent to this study. After surgical extirpation the tissue was chilled immediately in ice-cold 0.9% NaCI. All tissue specimens were divided into small pieces and stored at - 196°C. For each tissue specimen the respective histology was proven by an experienced pathologist. Prostatic epithelium and stroma were separated mechanically and homogenized according to Cowan et al. [8] with modifications as described previously [9]. The purity of the epithelial and stromal fractions was tested by measuring acid phosphatase as a marker for epithelial cells and: hydroxyproline as a marker for collagenous connective tissue, i.e. stromal elements [I0]. The mean acid phosphatase content (mU/mg protein + SEM) in epithelium and stroma was 1146-[-195 ( n = 12) and 2 4 8 + 5 9 (n -- 12), respectively. The mean hydroxyproline content (/zg/mg wet wt + SEM) in epithelium and stroma was 0.5 + 0.06 (n = 6) and 8.7 + 0.8 (n = 6), respectively. Thus, the relative purity of epithelial and stromal fractions was on average 94 and 79%, respectively. For enzyme measurement, aliquots of the frozen homogenates were pulverized in a porcelain mortar chilled with liquid nitrogen. The powder was allowed to thaw in small tubes which were kept in an ice-bath overnight.

Measurement of 17[3-HSOR activity After optimization of assay conditions using BPH tissue (see Results), all enzyme activities of this study were determined in an incubation mixture (final vol 202#1) composed of buffer diluted epithelial or stromal homogenate (0.3 to 0.7mg protein/incubation mixture), and varying concentrations of the respective steroid substrate (either as tritium labeled steroid alone or tritium labeled plus unlabeled steroid). For

the determination of 17fl-HSOR~d and 17flHSORo~ androstenedione and testosterone were used as substrates, respectively. For determination of the affinity of 17//H S O R ~ and 17//-HSORo~ to their steroid substrates (Kms), androstenedione was used in a concentration range of 35-5250 nM, testosterone in a concentration range of 80-5050 nM. The reaction was started by addition of the respective pyridine nucleotide [NADPH and NADH: 500#M, using a regenerating system (5 mM glucose-6-phosphate, 0.6 U glucose-6phosphate dehydrogenase), or NADP + and NAD + : 500 #M]. For determination of the affinity of 17//HSOR,od and 17fl-HSORo~ to the pyridine nucl¢otides (KmN), NADPH, NADH, NADP +, and NAD + were used in a concentration range of 0.5-10, 20-1000, 0.1-5 and 10-1000#M, respectively. In these experiments, a nonsaturating steroid substrate concentration of about 70 nM was used throughout. (Therefore, only K~,,, values are presented in the Results.) The reaction was started by addition of the respective nucleotide. All mixtures were incubated at 37°C either for 30 (17B-HSOR~) or 60 min (17B-HSORo~). The reactions were stopped by addition of 3 ml ether. The metabolites were extracted twice with ether (2 x 60 s). The ether phases were evaporated to dryness (Vortex Evaporator, Haake Buchler, Saddle Brook, N J, U.S.A.), redissolved in 500 #1 ether, and again evaporated to dryness. The evaporated metabolites were redissolved in 50 ~1 acetonitrile containing 100 #g of the following steroids as tracer: testosterone, androstenedione, DHT, and androstane-3,17dione. Next, the steroids were separated by HPLC (refractive index detector, fraction collector with peak slope detector, PharmaciaLKB, Freiburg, Germany), using a reversed phase column (Lichrosorb RP 18, PharmaciaLKB) and filtered and helium degassed eluents of either acetonitrile-H20 (50: 50, v/v) for determination of 17//-HSORr~ or methanol-H20 (40:60, v/v) for determination of 17B-HSORo~. The recovery was on average 70% of the starting material. Regarding chromatography, further details have been previously reported [9]. The enzyme activities were calculated from the percentage of radioactively labeled metabolites, taking into account recovery, blank values, the specific activity of tritium labeled steroids, and the ratio of added tritium labeled to unlabeled steroid. Furthermore, due to the presence of

17p-Hydroxysteroidoxidoreductasein human prostate relatively high 5=-reductase activity that metabolizes to a considerable extent androstenedione to androstane-3,17-dione, the substrate concentration being used for calculation of the activity of 17/~-HSOI~d was obtained by subtracting the mean concentration of formed androstane3,17-dione from the initially added androstenedione concentration. Each measurement was performed in duplicate. K , and Vm~ values were derived from Lineweaver-Burk plots [11], computing the regression lines by the method of least squares.

Other methodz Protein was determined according to the method of Lowry et aL[12], using bovine serum albumin as standard. The activity of acid phosphatase (EC 3.1.3.2) was measured by the method of Walter and Schtitt[13]. Hydroxyproline was measured as described by Stegemann [14] with minor modifications as described previously [9]. As has been described already for other prostatic enzymes [15, 16], the potential steroid converting capacity (Vm~/K=s of the respective enzymes) of the 17jf-HSORs

.~

,

4

Optimization of assay conditions for enzyme determination The activities of 17fl-HSOR~d and 17flHSORo, were measured in epithelium and

4 0

.001 .01

o.

.1

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17 8 - H S O R ox

1.0"]

101 8-1

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RESULTS

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was estimated by the equation v = V ~ . S / ( K , s +S), where S is the endogenous steroid substrate concentration (androstenedione for 17fi-HSOR=d and testosterone for 17fi-HSORo,). Since under /n vivo conditions the Kms values of the 17~-HSORs are much greater than S[17, 18], it follows that K, s + S ,~ Kms and v = Vm/K,s . S. Under the assumption of identical steroid substrate concentrations, the potential enzymatic capacity is given by the V=JK=s ratio [15, 16]. The statistical significance of the differences between the means was determined by Student's t-test. Such calculation was performed only in the case of n >I 4. The significance of age-dependent changes was determined by the Spearman-rank correlation coefficient (R). P < 0.05 was considered significant.

17 8 - HSOR r ~

i '°1, 1~

93

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.001 .01

.1

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Fig. 1. Activities(percentage of formed metabolites) of 17~-HSORmd(upper panels) and 17~-HSOI~ (lower panels) in epithelium ( I ) and stroma (17) of one BPH. as a function of pyridine nucleotide concentration.The proteinconcentrationrangedfrom 0.30 to 0.38ms/incubationmixture,the incubation time was 30 and 60rain for 17p-HSOR~ anti 17p-HSOI~, ~ v e l y . The substrate concentration ranged from 50 to 150nM. All experimentswere performedin duplicate and were corrected for blank values. For further details, see Experimental.

S. TUNNet al.

94

stroma of BPH under variations of protein concentration (range: 0.2-l.lmg/incubation mixture), incubation time (range: 10-120min), and pyridine nucleotide concentration (range: 0.1-10raM). Concerning protein, linearity of enzyme activity was found over the whole range. Routinely, protein concentrations used were between 0.3 and 0.7rag/incubation mixture. Concerning incubation time, linearity was proven up to 120 rain. Routinely, for determination of 17~-HSOR~d an incubation time of 30 min was used, whereas for determination of

l Tp-HSORox due to the relatively low amount of formed metabolites an incubation time of 60 rain was selected. As shown in Fig. l, the optimal pyridine nucleotide concentrations were found to be equal or higher than 0.5 raM. Routinely, a concentration of 0.5 mM was used.

Kinetic parameters (K,s, V,~) of 1713-HSOR~ and 17~-HSOR~ in epithelium and stroma of NPR and BPH The activity, i.e. the velocity of formation of metabolites, of 1713-HSOR~=dand 1713-HSORox

Epithelium

178-HSORred NADPH

Stroma

1.0.

1.0'

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0.4

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[ 11nM]

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corrected for blank values.

17#-Hydroxysteroid oxidoreductase in h u m a n prostate

was measured under optimized incubation conditions in epithelium and stroma of NPR and BPH as a function of the respective steroid substrate concentration, whereby either a phosphorylated or non-phosphorylated pyridine nucleotide has been added. Figure 2 shows typical Lineweaver-Burk plots according to the Michaelis-Menten model exemplarily for one NPR. All K.s and Vmax values were determined by such plots. The mean ( + SEM) K.s and Vm~ values are summarized in Table 1. The significances between the means are given in Table 2. The main results are: (1) In no case were significant differences in K=s between epithelium and stroma found. (2) The mean K.s value of 17/~H S O R ~ / N A D P H was significantly lower than those of all other 17/~-HSORs. (3) Comparing NPR and BPH, higher mean K.s values were always found in BPH for 1 7 ~ - H S O R ~ N A D H and 17/~-HSORox/NADP +. On the other hand, the mean K,s of 17/~-HSOR~/NADPH was lower in BPH stroma than in stroma of NPR. (4) In almost all cases the mean Vm~ values were higher in BPH than NPR. (5) In BPH, the mean Vm~ of 17fl-HSOR,,JNADPH was significantly higher in stroma than epithelium, while regarding 17fl-HSOR=dNADH and 17flHSORox/NAD + their mean Vm~ values were higher in epithelium. (6) In both NPR and BPH the mean Vm,~ values of the epithelial 17fl-HSOR~/NADH were distinctly higher than those of 17fl-HSOR~/NADPH. The same holds true if the mean V=~,of the epithelial 17flHSORoJNAD + and 17fl-HSORoJNADP + are compared.

95

4444+144

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| +14-I+I+I

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V~..JK.s of 17~-HSOR,,d and 17~-HSORox in epithelium and stroma of NPR and BPH The potential capacity of an enzyme to convert its substrate can be estimated by the ratio Vm~/K.s [15, 16]. Such ratios were calculated for 17/~-HSOR~ and 17/~-HSORox (Table 3). The significances between the means are given in Table 4. The main results are: (1) In all cases the mean Vm,JK.s ratios of 17/~-HSOR=d were higher than those of 17/~-HSORo~. (2) The highest mean V~/K.s was found for the 17/~-HSOR~d/NADPH of BPH stroma. This ratio was significantly higher than the V~/K.s ratios of both the 17/~-HSOR~/NADPH in BPH epithelium and 1 7 ~ - H S O ~ A D H in BPH stroma. (3) The mean Vm~/K.s ratios of 17~-HSORo,fNAD + were significantly SBMB 46/I--H

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.

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i iiii

96

S. TUNN et al.

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1 7 / 1 - H y d r o x y s t e r o i d o x i d o r e d u c t a s e in h u m a n p r o s t a t e Table 3. Mean ( + S E M )

Vm,JK,~values of

17~-HSOR in epithelium and stroma of N P R and BPH

BPH

NPR

Enzyme 17p-HSOR~ 17p-HSOR~ 17p-HSORo~ 17~-HSOI~

V=dX,,s

V.,I K=s

Steroid substrate

Nucleotide

n"

Epithelium

Stroma

n

Androstenedione Androstenedione Testosterone Testosterone

NADPH NADH NADP + NAD +

2/4 2 4 3

0.044 0.032 0.002 + 0.001 0.006 + 0.001

0.025 + 0.009 0.006 0.002 + 0.001 0.005 + 0.003

10 4 10 4

Epithelium 0.049 + 0.075 + 0.003 + 0.020 +

0.011 0.016 0.001 0.010

Stroma 0.128 + 0.011 + 0.002 + 0.006 +

0.006 0.001 0.0003 0.001

"Statistical significance of diffcmncas between means was calculated only in case of n I> 4.

how the 17/]-HSOR is catalyzing, in detail, this reversible reaction. Looking at the specific concentration (Vm~) of 17p-HSOR, as well as at the affinities of 17~-HSOR to its steroid substrates (Kms) and to its pyridine nucleotides (K,~N), the present study clearly indicates that this enzymatic reaction in the human prostate is rather Relationship between V~x/K, s and age If all Vm~/K=s ratios of 17/~-HSOR~ and complex. Concerning the affinities of 17p-HSOR to the 17/1-HSORo~ were plotted against the age steroid substrates (K=s), significant differences of the donors (Fig. 3), a significantly posibetween epithelium and stroma of NPR and tive correlation was found only regarding BPH were not found. Thus, in epithelium and 17/]-HSOR~/NADPH in stroma. The positive stroma the conversion of testosterone to ancorrelation of that Vm~/K=s ratio is due to a drostenedione and vice versa seems to be under statistically significant negative correlation with the control of an identical complex of enzymes. age of the corresponding Kms and a trend to An identical Kms in epithelium and stroma was higher values with age of the corresponding Vm~ also found by Bartsch et al. [4] regarding the (Fig. 4). All other K,s and Vm~ values of this 17~-HSOR~/NADH. However, other Kms data study showed no significant correlation with in our study indicates that in the human age, either in epithelium or in stroma (data not prostate the reversible interconversion of anshown). drostenedione to testosterone appears to be catalyzed by a heterogenous enzyme group. Determination of the affinity of 17fl-HSOR to To this direction points the fact that 17ppyridine nucleotides (KmM) HSOR~d/NADPH has a significantly higher For estimation of the affinity of 17fl-HSOR affinity to androstenedione than 17/~-HSORod to pyridine nucleotides (K~#) like NADPH, NADP + to testosterone. Furthermore, when NADH, NADP + and NAD +, the 17fl-HSOR compared to NPR stroma, in stroma of BPH activity, i.e. the velocity of formation of metab- the substrate affinity of 17p-HSOR~/NADPH olites, was measured exemplarily in epithelium is higher, whereas it is lower regarding 17/~and stroma of two BPH as a function of the HSORox/NADP +. Finally, the affinity of concentration of these pyridine nucleotides. The 17/~-HSOR~/NADPH to androstenedione is K=N values (Table 5) were derived from 5- to 15-fold higher than the affinities of 17pLineweaver-Burk plots. The lowest mean K, N HSOR~d/NADH, 17/~-HSORoJNADP +, and value, i.e. the highest affinity, was found regard- 17p-HSORox/NAD + to their steroid substrates. ing NADP +. The affinity of 17fl-HSOR to Such striking differences in K , s values might be NADPH was at least 3-fold lower, and the due either to the existence of different substrate affinity to NAD + and N A D H at least 10-fold binding sites at the same enzyme molecule or to lower. the existence of structurally different enzymes. In this context, it is interesting to note that the 17~-HSOR from human testis seems to possess DISCUSSION different binding sites [19], whereas in rat testis It has been known for a long time that the two different enzymes are probably responsible human prostate is potentially capable of con- for the reversibility of this pathway [20]. Moreverting androstenedione to testosterone and vice over, with respect to the human placental estraversa [2, 3]. In this context, the question arises of diol-linked 17~-HSOR, by means of enzyme higher than those of 17fl-HSORoJNADP +. (4) In epithelium the mean Vm~JK.,s ratios of 1 7 ~ - H S O R J N A D + and 17~-HSORr~/NADH were significantly higher than those of the stroma.

e. e,e,~.e,e.e, e.e,e.e.e. e,e.e.e. ~'

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17#-HSOR,,s/NADPH, NPR, E

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17O-HSOR,,~NADPH, BPH, E

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17p-H$OR,,jNADPH, NPR, S

~~~.

17p-HSOR,~ADPH, DPH, S

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~~-~.

1 7 p - H S O ~ A D H , BPH, E

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I?p-HSOR~dNADH, NPR, S

ig I

m.

IVp-HSORm~qADH, BPH, S

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O

17~-HSORo,fNADP +, NPR, E

~~~-

17B-HSORo~iADP +, NPR, S

~-

17fl-HSORo,]NADP+, BPH, S

17~-HSORo~NADP +, BPH, E

L

17p-HSORaJNAD+, NPR, E 17p-HSOR~/NAD+, BPH, E 17p-HSOR~/NAD+, NPR, S

17,8-HSOI~,/NAD+, BPH, S

•/v la NN~I. "S

86

17/~-Hydroxysteroid oxidoreductase in human prostate

Epithelium

99

Stroma

O.20 M

17B-HSOR red

S

0.15

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0.10

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0 20 40 60 80 100

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0.03 0.01

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.......... 0 20 40 6O e0100

Age

0.01

0.00 20400050100

[years]

Fig. 3. Correlation between V,=.JK.s o f 1 7 g - H S O ~ A D P H , 17g-HSORm]NADH, 17g-HSOR.=J N A D P + , and 1 7 p - H S O R = / N A D + in epithelium and stroma o f N P R ( I ) and BPH (1"7) and the age of the donor. The significance of aBe-related ¢Jumges w u determined by Spearman-tank correlation coetBcient (R).

purification it has been found that three different monomers are forming six dimers [21]. Thus, in the case of the prostatic 17p-HSOR, its purification from cellular as well as subcellular compartments is needed to describe in more detail the reason for such complex enzymatic reaction. As well as the affinity of 17~-HSOR to its steroid substrate (/~,s), its affinity to phosphorylated and non-phosphorylated pyridine nucleotides (K.N) like NAD(H) and NADP(H) also strongly controls catalysis. In human prostate, this study reveals significant differences in K.,~ of 17~-HSOR between epithelium and stroma as well as between the phosphorylated and non-phosphorylated pyridine nucleo-

tides. That is, NADP(H) is bound with higher affinity to 17~-HSOR than NAD(H). If in human prostate a similar concentration of pyridine nucleotides is present as in rat prostate [22], the K=Mvalues of our study suggest that the 17~-HSOR is more saturated by the phosphoryated as compared to the nonphosphorylated pyridine nucleotides. Thus, as regards 17p-HSOR~, NADPH instead of NADH is the preferred nucleotide, underlining the particular role of 17p-HSOR~/NADPH in converting androstenedione to testosterone. However, more quantitative data on the respective nucleotides in the various cellular and subcellular compartments of the human prostate

S. TUNN et al.

100

Epithelium

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1000

1ooo

800

800

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[ years

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uu 20 40 60 80100 |

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]

Fig. 4. Correlation between K ~ (upper panels) and Vm~ (lower panels) of IT~8-HSORmd/NADPH in epithelium and stroma o f N P R (m) and BPH (17) and the age of the donor. The significanee of age-related changes was determined by Spearman-rank correlation coefficient (R).

are needed to better understand the potential role of those nucleotides in the normal and diseased human prostate. Turning to the Vm~ values, i.e. to the amount of 17p-HSORs in human prostate, there is considerable evidence that 17~-HSOR~d/ NADPH is located predominantly in stroma, whereas 17p-HSOR=d/NADH is found mainly in epithelium. Thus, the earlier data of Harper et al. [3] and Bartsch et al. [4] are confirmed. As compared to 17p-HSOR.~, it appears that the corresponding 17p-HSORo~ is almost always present in a significantly lower amount in human prostate. Hence, in view of the Vm~ values the balance of the reversible conversion of testosterone to androstenedione in human prostate is shifted potentially towards testosterone. Such

a shift is also discernible as far as the Vm~/K. s ratios of this study are concerned. That is, the highest ratios were found regarding 17flHSOR~, As pointed out in studies on 5~,-reductase and 3=~)-HSOR, the quotient V , ~ / K = s is a useful index to estimate the potential enzymatic capacity in converting a substrate to its product [15, 16]. Moreover, the aformaamtioned shift of the enzymatic reaction towanJs testosterone in human prostate seems to apply particularly to BPH stroma because in mammalian tissues the ratio of NADPH to NADP+ is high in favor of the reduced counterpart, whereas in the case of NAD(H) the opposite holds true [22]. Finally, the age,dependent ~ t i v e correlation of K. s of 17~,HS6R,~NAD~PH in prostatic

Table 5. Mean affinity (KMs) of 17p-HSOR in epithelium and stroma of BPH to pyridine nucleotides ~m N

(vM) Enzyme 17p-HSORn~ 1?p-HSORn,d 17p-HSORo~ 17,8-HSORo,

Steroid substrate Nucleotide Andro~ione NADPH At~dro#tenedione NADH Testmtm'cme NADP ÷ Testosterone NAD +

n 2 2 2 2

Epithelium , 1.70 199 0.49 117

Stroma , ,p 23.6 215 0.1 I 238

17p-Hydroxysteroid oxidoreductase in human prostate s t r o m a indicate t h a t with a g i n g the affinity o f 17p-HSOR,JNADPH to a n d r o s t e n e d i o n e increases, I n o u r o w n p r e v i o u s studies o n 5 a r e d u c t a s e a n d 3 u ( ~ ) - H S O R we were able to d e m o n s t r a t e t h a t in the h u m a n p r o s t a t e aging h a s a significant i m p a c t o n kinetic p a r a m e t e r s like V ~ a n d K , s. Hence, in p a r t also the V ~ / l ~ s values were a g e - d e p e n d e n t [15, 16]. I n the p r e s e n t s t u d y a positive c o r r e l a t i o n o f V ~ / I ~ s with age was f o u n d with respect to 17~-HSOR,JNADPH. However, looking at B P H s t r o m a alone, the V n ~ / K , s values a r e n o t significantly c o r r e l a t e d with age. Thus, further studies o n N P R a n d B P H f r o m p a t i e n t s in the age r a n g e o f 4 0 - 6 0 years are n e e d e d to clarify w h e t h e r the significant a g e - r e l a t e d increase o f V~/K,s of stromal 17~-HSOR~/NADPH is related to aging p e r se o r to specific B P H processes. Be t h a t as it m a y , since the o t h e r V m u / K , s r a t i o s o f this s t u d y a r e n o t f o u n d to be correlated w i t h age, in B P H s t r o m a the t e s t o s t e r o n e f o r m a t i o n f r o m a n d r o s t e n e d i o n e increasingly exceeds all o t h e r e n z y m a t i c a l l y r e g u l a t e d interc o n v e r s i o n s between t e s t o s t e r o n e a n d and r o s t e n e d i o n e . H o w e v e r , the p o t e n t i a l steroid c o n v e r t i n g c a p a c i t y o f p r o s t a t i c enzymes like 5 ~ - r e d u c t a s e a n d 3 u ( ~ ) - H S O R are m a n i f o l d higher[15, 16]. Hence, in c o n c e r t with those p o w e r f u l enzymes, a t p r e s e n t it r e m a i n s u n c l e a r w h e t h e r the t e s t o s t e r o n e f o r m a t i o n f r o m and r o s t e n e d i o n e /n vivo c o n t r i b u t e s significantly to the i n t r a p r o s t a t i c t e s t o s t e r o n e level. O n the o t h e r h a n d , it is conceivable t h a t 17/1-HSOR,~/ NADPH c o m p e n s a t e s to a certain extent the well k n o w n a g e - d e p e n d e n t decrease o f bioa v a i l a b l e p e r i p h e r a l t e s t o s t e r o n e [23]. REFERENCES

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