Anterior pituitary glandular kallikrein: Trypsin activation and estrogen regulation

Molecular and Cellular Endocrinologv, 46 (1986) 163-114 Elsevier Scientific Publishers Ireland, Ltd.

163

MCE 01495

Anterior

pituitary

glandular kallikrein: trypsin activation and estrogen regulation C. Andrew

Powers

Depurrmeni of Pkarmacologv. New York Medical College, Vulhaiiu, NY 10595 {C!S.A.J (Received

K
anterior

pituitary;

kallikrein;

trypsin:

17 January

1986: accepted

7 April 1986)

estrogen.

Summary Discrepant reports exist regarding the presence of glandular kallikrein or other trypsin-like serine proteases in the pituitary. The existence of pituitary kallikreins in latent forms could explain these discrepancies. I report that trypsin treatment of rat anterior pituitary homogenates activates two serine proteases which generate kinins from kininogen and selectively cleave chromogenic substrates for kallikreins. One protease (enzymatically and immunologically resembling glandular kallikrein) and activated 5-fold by trypsin and was 20 times more abundant in female than in male lobes due to hormonal regulation by ovarian estrogens. The second kallikrein (activated 20-fold by trypsin) was unaffected by estrogens. The results demonstrate that rat anterior pituitary kallikreins predominantly exist in latent forms requiring activation for detection. Additionally, glandular kallikrein is a major estrogen-induced protein in the rat anterior pituitary. No other member of this large protease family is known to be regulated by estrogens.

Introduction Glandular kallikreins are trypsin-like serine proteases usually characterized by their ability to generate kinins (potent vasoactive peptides) from kininogen (an inactive plasma glycoprotein) with a high degree of specificity (Schachter, 1980). These proteases have classically been suggested to function in local blood flow regulation via kinin generation. However, recent findings have shown that glandular kallikrein is a member of a large enzyme family which may have a role in the posttranslational processing of diverse biologically active peptides (Bothwell et al., 1979; Mason et al., 1983: Ashley and MacDonald, 1985). Furthermore, glandular kallikreins may be capable of processing 0303-7207/X6/$03.50

Q 1986 Elsevier Scientific

Publishers

Ireland.

precursor molecules other than kininogen (OleMoiYoi et al., 1979: Prado et al., 1984). Recently, the rat and pig pituitary have been found to contain kallikrein-like proteases which can generate bradykinin from kininogen and cleave synthetic kallikrein substrates such as D-Pro-PheArg-p-nitroanilide. The pig pituitary contains a novel kininogenase with similarities to plasma kallikrein (Powers and Nasjletti. 1982). as well as a kininogenase resembling glandular kallikrein (Polivka et al., 1982). In the rat pituitary, a kininogenase closely related or identical to glandular kallikrein is concentrated in the intermediate lobe (Powers and Nasjletti, 1983). In addition, the anterior lobe of the female rate pituitary contains abundant glandular kallikrein-like activity whereas Ltd.

164

the male anterior lobe contains practically none (Powers and Nasjletti, 1984a: Powers et al., 1984). The ability of kallikreins to generate active peptides from precursors and their localization in the pituitary has suggested an involvement in prohormone processing. However, some groups seeking prohormone processing enzymes in the pituitary have failed to detect serine protease which can cleave the kallikrein substrate Bz-Pro-PheArg-[‘dC]anilide (Chang et al., 1982; Loh and Gainer, 1982) or other synthetic substrates for trypsin-like enzymes (Pelaprat et al., 1984). Also, groups reporting trypsin-like serine proteases in the rat pituitary using substrates with broad specificities have not noted the major sex difference in the anterior lobe (Kenessey et al., 1979; McPartland et al., 1981a). Glandular kallikrein predominantly exists in latent forms in some tissues (e.g. exocrine pancreas) whereas only active forms exist in other tissues (e.g. submandibular gland) (Orstavik and Glenner, 1978; Schachter, 1980). Endocrine tissues have not previously been recognized to contain significant amounts of latent serine proteases requiring activation for detection. However, the pig pituitary kallikrein has recently been demonstrated to predominantly exist in a latent form activated by trypsin and certain other procedures (Powers and Nasjletti. 1984b). Thus, discrepancies in the detection of pituitary kallikreins may arise from their existence in latent forms variably detected by different extraction or assay procedures. Additionally, if the rat anterior pituitary contains significant amounts of latent kallikrein, then the previously reported sex difference may be due to an increased proportion of the enzyme in an active form rather than an actual difference in enzyme levels. For example, adrenal steroids can modulate the proportion of glandular kallikrein in an active form in rat urine (Noda et al., 1983). The purpose of this study was to: (1) determine whether the rate anterior pituitary contains latent kallikreins activated by trypsin, (2) examine whether the previously reported sex difference in anterior lobe glandular kallikrein is due to a differing proportion of the enzyme in an active form, and (3) determine the hormonal basis for the sex difference in anterior pituitary glandular kallikrein.

Materials and methods Animals

Male and female Sprague-Dawley rats (CD strain, Charles River Breeding Laboratories, Wilmington, MA) 60-70 days old were used. Some animals were gonadectomized or sham-operated 4 weeks prior to sacrifice. Some ovariectomized rats received one of the following treatments for 12 days prior to sacrifice: 10 pg estradiol benzoate every 48 h; 1 mg progesterone every 48 h; or 10 I-18 estradiol benzoate plus 1 mg progesterone every 48 h. Steroids were dissolved in benzyl alcohol. diluted in sesame oil, and injected subcutaneously in 0.1 ml volumes; controls received vehicle alone. The steroids (dissolved in ethanol) had no effect on proteolytic activity when added directly to pituitary homogenates to a concentration of 1 PM. Prepuration

of pituitrrry extructs

Rats were anesthetized with sodium pentobarbital (100 mg/kg) and transcardially perfused with 200-250 ml of 0.15 M NaCl to remove blood proteases from tissues prior to dissection. The neurointermediate anterior lobes of the pituitary were separated in situ with a needle, removed and gently washed in 0.15 M NaCl. Pools of 2-5 anterior lobes were homogenized in 0.4-1.0 ml of 50 mM sodium phosphate (pH 7.5) containing 0.15 M NaCl and 0.1% Triton X-100 using a microultrasonic cell disruptor (Kontes, Vineland, NJ; 5-10 s at 4.3 Watts in an ice bath). Homogenates were stored at -20°C until activation and assay for proteolytic activity. The protein concentration of homogenates was determined with the method of Lowry et al. (1951) using bovine serum albumin as the standard. Ttypsin activation

Pituitary homogenates were thawed, resonicated, and aliquots incubated with bovine trypsin (Sigma Chemical Co., St. Louis, MO; Type XIII, 12300 BAEE units/mg protein) at a final concentration of 10 pg/ml for O-20 min at room temperature. Reactions were stopped by adding lima bean trypsin inhibitor (LBTI; Sigma, Type II-L) to give a final concentration of 125 pg/ml. In some controls (0 min trypsin treatment), LBTI was added to homogenates immediately before trypsin and then incubated for 20 min.

165

Kj~i~-generating assays Untreated and trypsin-activated anterior pituitary extracts were assayed for kallikrein activity by measuring their ability to generate kinins from kininogen (kininogenase activity) as previously described (Powers and Nasjletti, 1982, 1983). Partially purified dog plasma kininogen was used as the substrate and kinins generated were measured by radioimmunoassay using synthetic bradykinin as the standard. Results were expressed as pmol kinins generated/ min/ mg protein.

Proteo~tic assays using ehro~~oge~icsubstrates Anterior pituitary extracts were also examined for their ability to cleave the following chromogenic peptide substrates (Kabi Diagnostica, Stockholm, Sweden) exhibiting relative specificity for kallikreins (Friberger et al., 1982): D-Pro-PheArg-p-nitroanilide (S-2302, plasma and glandular kallikrein substrate) and D-Val-Leu-Arg-p-nitroanilide (S-2266, glandular kallikrein substrate). The hydrolysis of a chromogenic substrate exhibiting specificity for trypsin (S-2222, Bz-Ile-Glu-GlyArg-~-nitroanilide) (Lottenberg et al., 1981) was also measured to insure that proteolytic activity detected was not due to residual uninhibited trypsin. The specificity of the chromogenic substrates was confirmed prior to use with highly purified preparations of porcine glandular kallikrein, human plasma kallikrein, human thrombin, human factor Xa. porcine plasmin, human urokinase, and bovine trypsin using the assay procedure described below. Proteolytic assays were conducted using an end-point method run at 37°C in 0.1 M Tris-HCl (pH 8.0) containing 25 pg./ml LBTI. Reactions were started by adding sufficient substrate to give the following final concentrations in a reaction volume of 250 ~1: S-2302, 300 PM; S-2246, 300 PM; S-2222, 200 PM. Reactions were stopped by adding 100 ~1 of 20% perchloric acid after lo-15% of the substrate had been hydrolyzed, or after 1-2 h of incubation, whichever came first. Free pnitroaniline generated was measured at 546 nm after conversion to a purple azo dye as previously described (Powers and Nasjletti, 1983). Control assays consisted of substrate or pituitary extracts incubated alone, or trypsin incubated with substrate in the presence of LBTI. Absorbance at 546

nm in controls was subtracted from that ples. Substrate hydrolysis was linear with directly proportional to the amount of extract incubated. Results were expressed p-nitroaniline released/ min/mg protein.

in samtime and pituitary as nmol

Inhibitor sensitivity and pH optirnu In some assays aprotinin (Sigma, Bovine lung), soybean trypsin inhibitor (Sigma, Type I-S), hirudin (Sigma, Grade IV), or (p-amidinophenyl)-methanesulfonyi fluoride (APMSF; Calbiochem, La Jolla, CA) were examined for their ability to inhibit proteolytic activities. Inhibitors were dissolved in 0.1 M Tris-HCl (pH 8.0) except for APMSF which was dissolved in methanol. Inhibitors were preincubated with pituitary extracts for 10 min prior to addition of substrate. The pH optima of the various proteases were examined using the following buffers: sodium acetate (pH 5). sodium phosphate (pH 6, pH 7) Tris-HCl (pH 8, pH 9) ammonium acetate (pH 10, pH 11). Controls included substrate incubated alone with each of the buffers to correct for spontaneous hydrolysis occurring at each pH value.

Some pituitary extracts were fractionated on DEAE-Sephadex using a modification of a procedure which separates glandular kallikrein in rat urine from two other urinary proteases (McPartland et al., 1981a,b). Pituitary homogenates (0.5 ml) were frozen and thawed twice, resonicated, trypsinized 20 min, and diluted with 2.0 ml H,O to give an extract containing 10 mM sodium phosphate (pH 7.5) and 30 mM NaCl. Following ~entrifugation (105000 x g for 60 min), 2.0 ml of the supernatant was applied to a 0.8 x 5 cm column of DEAE-Sephadex-A-50 equilibrated in 10 mM sodium phosphate (pH 7.5) containing 30 mM NaCl. The column was washed with 7 ml equilibration buffer and proteolytic activity was eluted with a two-step gradient consisting of 15 ml of 10 mM sodium phosphate (pH 7.5) containing 230 mM NaCl, and 12 ml of 10 mM sodium phosphate (pH 7.5) containing 530 mM NaCl. The flow rate during sample application and elution was 25-30 ml/h, and 1.5 ml fractions were collected and assayed for proteolytic activity as described above. Using this procedure, glandular

166

kallikrein

in rat urine

elutes with 530 mM NaCI.

Immunoprecipitations Aliquots from the various chromatography peaks were incubated for 1 h at 25°C with various dilutions of highly specific sheep or rabbit antiserum against glandular kallikrein from rat urine or non-immune rabbit or sheep serum. Rabbit antiserum (Carretero et al., 1978) was provided by Drs. O.A. Carretero and G. Scicli (Henry Ford Hospital, Detroit, MI) and sheep antiserum (Shimamoto et al., 1979) was provided by Drs. J. Chao and H.S. Margolius (Medical College of South Carolina, Charleston, SC). Immune complexes were precipitated with a Staphylococcus aureus-Protein A suspension (Pansorbin. Calbiothem). Sera had been acid- and heat-treated to destroy serum proteases and kallikrein inhibitors. Results Trypsin activation of proteolytic activity Untreated homogenates of the male anterior pituitary contained little kinin-generating (kininogenase) activity or activity towards chromogenic substrates for kallikreins (S-2302, D-Pro-Phe-Argand S-2266, D-Val-Leu-Arg-pp-nitroanilide, nitroanilide) (Fig. 1). Only the chromogenic substrate for trypsin (S-2222, Bz-Ile-Glu-Gly-Arg-pnitroanilide) exhibited an appreciable rate of hydrolysis in untreated homogenates. However, trypsin treatment (10 pg/ml for 5520 min followed by 125 pg/ml of lima bean trypsin inhibitor) caused a greater than 20-fold increase in kallikrein activity (kininogenase activity and S2302 and S-2266 hydrolysis) which was nearly maximal within 5 min (Fig. 1). In contrast, the hydrolysis of the trypsin substrate (S-2222) by anterior pituitary homogenates was only slightly increased by trypsin treatment. Addition of lima bean trypsin inhibitor to the homogenate before adding trypsin completely blocked the activation of proteolytic activity (Fig. 1). In addition, the increase in S-2222 hydrolysis caused by 20 min of trypsin treatment was less than 10-30s of the increase in S-2302 or S-2266 hydrolysis. respectively. Trypsin cleaves S-2222 8 times faster than S-2302 and 3.3 times faster than S-2266 at the substrate concentrations employed. Thus, the in-

S-2302 .--* I

c 0

/* KININOGEN

5 IO TIME (mid

15

20

Fig. 1. Trypsin-activation of proteolytic activity in male anterior pituitary homogenates. Homogenates were incubated O-20 min with 10 pg/ml trypsin and then LBTI was added to give 125 pg/ml. C had no trypsin added and the 0 min homogenate had LBTI added before trypsin. Proteolytic activity was measured at 37°C. pH 8 in the presence of 25 pg/ml LBTI using substrates for kallikreins (kininogen. S-2302 and S-2266) and trypsin (S-2222).

crease in kallikrein activity cannot be attributed to residual uninhibited trypsin. Trypsin also caused a massive activation of female anterior pituitary kallikrein activity with a similar time course (data not shown). Table 1 compares the proteolytic activities of untreated and trypsin-activated male and female anterior pituitary homogenates in order to determine whether the sex difference in kallikrein activity could be due to a differing proportion of glandular kallikrein in an active form. Untreated female homogenates cleaved the kallikrein substrates (kininogen, S-2302 and S-2266) 14 to 18 times faster than male homogenates. After trypsin activation. kininogenase activity increased about IO-fold in male homogenates but only 5-fold in female homogenates; S-2302 and S-2266 hydrolytic activity increased over 20-fold in male homogenates but only 7- to &fold in female homogenates. Thus, kallikrein activity in trypsinactivated homogenates was still 4 to 9 times higher in female homogenates than in male homogenates. However, the sex difference ratio (female activity/ male activity) was 2- to 3-fold larger in untreated

167

TABLE

1

COMPARISGN OF THE PROTEOLYTIC ACTIVITY ANTERIOR PITUITARY HOMOGENATES Pituitary homogenates were incubated for 20 then added to give 125 pg/ml. Aliquots were S-2266, and kininogen) and trypsin (S-2222). Values in parentheses show the sex difference for each substrate. Sex and treatment

Chromogenic (nmol/min/mg

OF UNTREATED

AND TRYPSIN-ACTIVATED

mm in the presence or absence of 10 pg/ml trypsin; lima bean trypsin inhibitor was assayed for proteolytic activity at 37°C pH 8 using substrates for kallikrein (S-2302, Each value represents the mean f SE of determinations on 3 pools of 5 lobes per pool. ratios (female activity/male activity) for untreated and trypsin-activated homogenates

peptide hydrolysis protein)

S-2302

S-2266

Kininogenase activity (pmol/h/mg

s-2222

Male untreated

0.037 * 0.004

0.024 f 0.001

Female untreated

0.550 + 0.040 * (14.9 kO.3)

0.359+ 0.033 * (15.0 kO.1)

0.203 F 0.043 (0.95 i 0.03)

0.994 i 0.084

0.522 + 0.050

0.363 f 0.025

Male trypsinized Female trypsinized

4.02 (4.04

* P 4 0.01 vs. corresponding ** P i: 0.01 vs. sex difference

MALE AND FE&MALE

kO.25 * kO.21) ** male homogenate. ratio of untreated

2.79 (5.34

kOo.06 * kO.32) **

17

0.21410.066

0.399 + 0.010 (1.10 jfO.06)

protein)

*2

309 (18.2

+12* + 1.1)

162

+2

+37 * 1551 (9.57 * 0.05) **

homogenate.

homogenates than in trypsin-activated homogenates (Table 1). Untreated or trypsin-activated anterior pituitary homogenates did not exhibit a sex difference in the cleavage of the trypsin substrate (S-2222). Also. S-2222 hydrolytic activity was only increased by about 90% following trypsin treatment of homogenates from either sex.

Separation of two kalIikrein-cake proteases using ~EAE-~ep~adex The larger sex difference ratio in kal~ikrein-like activity in untreated versus trypsin-activated homogenates may reflect either (I) a larger proportion of glandular kallikrein in an active form in female homogenates, or (2) the activation by trypsin of a second kallikrein-like protease whose levels are not sex dependent and which is almost completely inactive in untreated homogenates. Therefore, anterior pituitary homogenates were fractionated on DEAE-Sephadex in an attempt to separate any differing kallikrein-like activities. A step-gradient elution technique was used which separates glandular kallikrein in rat urine from two other urinary kallikrein-like proteases (Mc-

4

8

12

16

FRACTION

4

6

12

16

NUMBER

Fig. 2. DEAE-Sephadex chromatography of trypsin-activated male and female anterior pituitary extracts Pituitary extracts were applied to DEAE-Sephadex columns equilibrated in 30 mM NaCI, pH 7.5. No proteolytic activity eluted in the void volume or wash with 30 mM NaCI. Proteolytic activity was eluted with a step gradient of 230 mM NaCI followed by 530 mM NaC1; 1.5 ml fractions were collected and assayed for kallikrein activity using S-2302. The hydrolysis of a trypsin substrate (S-2222) was also measured. Arrows indicate the start of ehttion with 230 or 530 mM NaCI. Inset: kininogenase activity of fractions.

6

8

6

10

8

19

PH

Fig. 3. Proteolytic activity of pituitary kallikreins separated using DEAE-Sephadex as a function of pH. After trypsin treatment for 20 min. homogenates were fractionated as shown in Fig. 2. Protease peaks were assayed at the indicated pH. Activity measured at pH 8 was arbitrarily designated as 100% to facilitate comparisons.

Partland et al., 1981b). Glandular kallikrein elutes with 530 mM NaCl with the procedure used. Virtually all of the proteolytic activity in soluble extracts prepared from trypsin-activated male or female anterior pituitaries was bound to DEAE-Sephadex columns equilibrated in 30 mM NaCl. Fig. 4 shows the elution of proteolytic activity with 230 mM NaCl and 530 mM NaCl. A distinct peak of kallikrein activity (kininogenase and S-2302 hydrolytic activity) eluted with both 230 mM NaCl and 530 mM NaCl. The amount of kallikrein activity which eluted with 530 mM NaCl was 20 items greater for female extracts than for male extracts; in contrast, male and female extracts did not differ in the amount of activity eluting with 230 mM NaCI. The total recovery of kininogenase and S-2302 hydrolytic activity was greater than 95% for both male and female extracts. Inhibitor sensitivity, relutive substrate specificity, und pH optimum of separated kailikreins The kininogenase and S-2302 hydrolytic activity eiuting with 530 mM NaCl was inhibited by aprotinin and APMSF (a general serine protease inhibitor), and was resistant to soybean trypsin

I’500

I/l00 SERUM

WN I/500 I/20 DILUTION x 10.’

l/l00

ICC

Fig. 4. Precipitation of anterior pituitary kallikreins by antiserum against rat glandular kallikrein. Aliquots of DEAESephadex chromatography peaks from female anterior pituitary fractionations were incubated for 1 h with various dilutions of nonimmune or anti-glandular kallikrein serum. Following precipitation of immune complexes. the supernatant fluid was assayed for kalfikr~in-like activity using S-2302 (left columns of panels) or kin~nogen (right columns of panels) as the substrates. Upper rows of panels show the results obtained using sheep antiserum. and lower rows of panels show the results obtained using rabbit antiserum.

inhibitor and hirudin (a specific thrombin inhibitor) regardless of the sex of the anterior pituitary extract (Table 2); this profile of inhibitor sensitivity is characteristic of glandular kallikreins. In contrast, the kallikrein activity eluting with 230 mM NaCl was strongly inhibited by soybean trypsin inhibitor as well as by aprotinin and APMSF. and was resistant to hirudin. The kallikrein activity in unfractionated, untreated male homogenates was too low for accurate analysis of inhibitor sensitivity. However, the activity in similar female homogenates was identical to the activity eluting with 530 mM NaCl in its inhibitor sensitivity (data not shown). Thus, the smaller sex difference ratio in trypsin-activated homogenates can be attributed to an activation of a second kallikrein-like protease whose levels are not sex dependent and which is almost completely inactive in untreated homogenates.

169

TABLE

2

EFFECT OF VARIOUS INHIBITORS ON CHROMOGENIC PEPTIDE PEAK FRACTIONS FROM DEAE-SEPHADEX CHROMATOGRAPHY

HYDROLYSIS

AND

KININ

GENERATION

BY

Aliquots of the chromatography peaks shown in Fig. 2 were assayed for proteolytic activity in the presence of the indicated concentrations of aprotinin, soybean trypsin inhibitor (SBTI), hirudin, and ( p-amidinophenyl)-methanesulfonyl fluoride (APMSF) following a 10 min preincubation period with the inhibitors. Values are the mean of duplicate determinations. Extract sex

Substrate

Elution peak (mM NaCI)

Inhibition

of substrate

hydrolysis

Aprotinin

SBTI

(50 pg/mI)

(50 pg/ml)

(%) Hirudin (2 units/ml)

APMSF (200 PM)

Male Female

S-2302 S-2302

230 230

56.2 57.0

82.5 86.1

8.2 12.4

91.5 98.8

Male Female

S-2302 S-2302

530 530

82.7 98.0

19.5 9.2

3.8 0

94.0 98.9

Male Female

Kininogen Kininogen

230 230

80.8 61.8

16.7 0

88.2 100

Male Female

Kininogen Kininogen

530 530

100 98.3

26.2 26.9

0 11.2

100 99.1

Male Female

s-2222 s-2222

530 530

28.1 35.0

8.3 13.0

0 4.9

16.7 26.8

The two peaks of kallikrein activity were further compared for their ability to cleave kininogen and S-2266 relative to S-2302. The activity eluting with 530 mM NaCl cleaved S-2266 at 107% the rate of S-2302, and the ratio of kininogenase activity (pmol/h) to S-2302 hydrolysis (pmol/min) was 0.38: this profile of substrate specificity is virtually identical to that of glandular kallikrein from rat urine. In contrast, the activity eluting with 230 mM NaCl cleaved S-2266 at 46% the rate of S-2302 and the ratio of kininogenase activity to S-2302 hydrolysis was only 0.04. These differences in relative substrate specificities explain why the magnitude of the sex difference in unfractionated homogenates varies depending upon the kallikrein substrate used (see sex difference ratios in Table 1). In addition to the above substrate profiles, either kallikrein activity cleaved chromogenic substrates for trypsin (S-2222), plasmin (D-Val-LeuLys-p-nitroanilide), thrombin (D-Phe-pipecolylArg-p-nitroanilide), and urokinase (pyroglutamylGly-Arg-p-nitroanilide) at less than 10% the rate of S-2302. The pH optimum for kinin generation by the activity eluting with 530 mM NaCl was pH 8 whereas S-2302 and S-2266 hydrolysis was opti-

100 100

mal at pH 11 or greater (Fig. 3); glandular kallikreins characteristically cleave a number of tripeptide substrates at much higher pH values than required for kininogenase activity (Hofmann and Geiger, 1983; Uchida et al., 1980). In contrast, the activity eluting with 230 mM NaCl exhibited optimal S-2302 and S-2266 hydrolysis as well as kininogenase activity at pH 8 (Fig. 3). Immunoprecipitation with glandular kallikrein antiserum The above results indicated that the enzymatic properties of the kallikrein activity eluting with 530 mM NaCl were similar to those of glandular kallikrein while the activity eluting with 230 mM NaCl is a distinct protease. The two activities were further compared for immunological similarity to glandular kallikrein. Rabbit or sheep antiserum against glandular kallikrein from rat urine produced a potent, concentration-dependent precipitation of the kallikrein activity eluting from DEAE-Sephadex with 530 mM NaCl (Fig. 4). The potency of the two antisera in precipitating this activity was identical to that observed using similar concentrations of glandular kallikrein from rat urine (data not shown). Thus, the kallikrein activ-

170

ity eluting with 530 mM NaCl is immunologically identical or very closely related to glandular kallikrein from rat urine. In contrast, virtually none of the kallikrein activity eluting with 230 mM NaCI was precipitated by either rabbit or sheep anti-glandular kallikrein antiserum (Fig. 4). S-J?.?_3 hydrolytic activity About 70% of the activity cleaving the trypsin substrate (S-2222) eluted from DEAE-Sephadex with 530 mM NaCI; similar amounts of activity eluted from male and female lobes with a recovery of 55% (Fig. 2). The S-2222 hydrolysis was weakly inhibited by aprotinin and APMSF and was resistant to soybean trypsin inhibitor or hirudin (Table 2). This profile of inhibitor sensitivity is markedly different from that of trypsin (which is potently inhibited by aprotinin, soybean trypsin inhibitor and APMSF). S-2222 hydrolysis by unfractionated homogenates (either untreated or trypsin-activated) exhibited a similar profile of inhibitor sensitivity. Thus, not even S-2222 hydrolysis by trypsin-activated homogenates can be attributed to residual uninhibited trypsin. The S2222 hydrolytic activity eluting with 530 mM NaCl exhibited optimal activity at pH 8, and was not precipitated by glandular kallikrein antiserum (data not shown). IneSfctive activation procedures A number of treatments known to activate latent proteases in other tissues or plasma were tested for their ability to activate rat anterior pituitary proteases. Detergent treatment (0.1-l% sodium deoxycholate or Triton X-100) or repeated freeze-thaw cycles failed to activate proteolytic activity. Furthermore, trypsin massively activated prateolytic activity in soluble extracts (105 000 x g supernate) from detergent-treated homogenates (data not shown). Thus, the observed trypsin activation cannot be attributed to a solubilization of proteases from particulate fractions. Also ineffective were the following treatments capable of dissociating protein-protein complexes: (I) ion-exchange chromatography on DEAE-Sephadex, (2) dialysis against high salt concentrations (1 M NaCl or KCI), chelating agents (1 mM EDTA or 1, lo-phenanthroline). pH extremes (pH 3 or pH 11). or 10 mM Tris or phosphate buffers (pH 7.5), (3)

exposure to reducing agents (1 mM dithiothreitol) or nucleophilic reagents (500 mM hydroxyIamine or methylamine, pH 7.5). Assqs for pituitury inhibitors of kallikrein Pituitary homogenates were also tested for the presence of kallikrein inhibitors destroyed by trypsin or exhibiting a sex difference in their levels. Untreated or trypsin-activated male or female anterior pituitary homogenates were mixed, preincubated and then assayed. Such experiments failed to provide evidence suggesting the presence of katlikrein inhibitors in pituita~ homogenates (data not shown). Hormonal regulation of rut anterior pituitary glandular kallikrein Ovariectomy decreased the total kallikrein activity of untreated and trypsin-activated female homogenates by 84% and 60%, respectively;

1

**.* Q"fTf P&f ++ r r 6P E EP S-2302 S-i 266

-f-t&! . L

.

I-z

P E EP

s-2222

Fig. 5. Effect of gonadectomy and hormone replacement therapy on proteolytic activity of the anterior pituitary. Panel u: proteolytic activity of untreated anterior pituitary homogenates from sham-operated or gonadectomized rats. Panel h: proteolytic activity of trypsin-activated homogenates from sham-operated or gonadectomized rats. Panel c: effect of hormone replacement therapy on proteolytic activity of trypsin-activated homogenates. E = estradiol benzoate treated; EP = estradiol benzoate plus progesterone treated: P = progesterone treated. Bar height represents the mean and the vertical line the SE of determinations on 4 pools of 2 lobes per pool. * P i 0.01 vs. sham operated; ** P < 0.01 vs. ovariectom~zed or sham operated.

171

orchidectomy did not affect the total kallikrein activity of male homogenates (Fig. 5a and b). Treatment of ovariectomized rats with estradiol benzoate markedly increased kallikrein activity in trypsin-activated homogenates: progesterone alone had no effect (Fig. 5~). The increase in kallikrein activity produced by estradiol benzoate alone was not different from that produced by estradiol benzoate plus progesterone. Untreated or trypsin-activated pituitary homogenates from sham-operated or gonadectomized male and female rats did not differ in their levels of S-2222 hydrolytic activity (Fig. 5a and b). However, treatment of ovariectomized rats with estradiol benzoate produced a slight increase (+40%) in S-2222 hydrolytic activity (Fig. 5~). Pituitary extracts from sham-operated, ovariectomized, and ovariectomized plus estradiol benzoate-treated female rats were fractionated on DEAE-Sephadex to examine the effects of such treatments on each of the kallikrein-like proteases. Ovariectomy or estradiol replacement therapy did not notably affect on the kallikrein activity eluting from DEAE-Sephadex with 230 mM NaCl (Fig. 6). In contrast, ovariectomy caused a 90% reduction in kallikrein activity eluting with 530 mM

FRACTION

NUMBER

Fig. 6. DEAE-Sephadex chromatography of trypsin-activated anterior pituitary homogenates from sham-operated ( 0 ), ovariectomized (d ), and ovariectomized plus estradiol benzoate-treated rats (d +E). Extracts were applied to columns equilibrated in 30 mM NaCl, pH 7.5. Arrows indicate the start of elution with 230 mM NaCl and 530 mM NaCI. Fractions cdllected were assayed for kallikrein activity using S-2302. Inset: kininogenase activity of fractions.

NaCl (glandular kallikrein) and estradiol benzoate caused a 60-fold increase in this protease to levels over 6 times greater than in extracts from shamoperated rats. The recovery of kallikrein activity during chromatography was between 85 and 90% for all groups. Discussion The rat anterior pituitary contains two kallikrein-like serine proteases which predominantly exist in latent forms that can be activated by trypsin. Sensitive and specific assay techniques and large amounts of undiluted homogenate are required to detect basal enzyme activity in untreated homogenates. This probably explains the failure of others to report kallikrein or other serine proteases in the pituitary (Chang et al., 1982; Loh and Gainer, 1982; Pelaprat et al., 1984) or to detect a major sex difference using nonspecific substrates for trypsin-like enzymes (Kenessey et al., 1979; McPartland et al., 1981a). Trypsin is well known to activate zymogens of glandular kallikrein as well as many other proteases and this is probably the mechanism of activation in the present study. Treatments capable of solubilizing proteases in particulate fractions or dissociating protein-protein complexes did not activate proteolytic activity. However, definitive identification of the nature of the latent forms must await their purification and detailed characterization. Nonetheless, the present findings demonstrate that the activities of these proteases are tightly regulated in the cell. Elucidation of the endogenous mechanisms regulating their activity should help illuminate their function. The pig pituitary also contains latent forms of a novel kallikrein-like protease (Powers and Nasjletti, 1984b). Further, the rat pituitary intermediate lobe contains a glandular kallikrein which predominantly exists in a latent form (Powers, 1986a), and its neuroendocrine regulation suggests a role in prohormone processing (Powers, 1985, 1986b). Such findings raise the possibility that other endocrine tissues may contain latent trypsin-like serine proteases. In this regard, it is noteworthy that studies seeking prohormone processing proteases in other endocrine tissues have often failed to detect trypsin-like serine pro-

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teases and have instead focused on other types of proteases (for review see Lazure et al.. 1983). However, such studies have not considered the possibility that processing proteases may exist in latent forms under tight regulation despite numerous precedents for such regulation in blood or nonendocrine tissues (i.e. serine proteases involved in peptide or zymogen processing in the kallikrein-kinin. growth factor, coagulation, fibrinolytic, and complement systems). Examination of other endocrine tissues for latent trypsin-like serine proteases may be illuminating. One of the pituitary kallikrein-like proteases was activated over ZO-fold by trypsin and was equally concentrated, in male and female lobes. This enzyme (hereafter designated pituitary kallikrein A) possessed weak but definite kininogenase activity and preferentially cleaved chromogenic substrates for kallikreins. Pituitary kallikrein A accounts for about 40% of the kininogenase activity, and 80% of the S-2302 hydrolytic activity of trypsin-activated homogenates of the male anterior pituitary (5% of the kininogenase activity and 20% of the S-2302 hydrolytic activity of female lobes). Pituitary kallikrein A is distinct from rat plasma kallikrein which is a much more potent kininogenase that does not bind to DEAE-Sephadex (Uchida et al., 1980; Johansen and Briseid, 1983). On the other hand, the properties of kallikrein A are suggestive of a relationship to a pig pituitary kininogenase which also predominantly exists in a latent form (Powers and Nasjletti, 1982, 1984b). The second anterior pituitary kallikrein is a potent kininogenase which exhibits enzymatic and immunological properties characteristic of glandular kallikreins (Fiedler, 1979; Schachter, 1980; Uchida et al., 1980; McPartland et al., 1981b: Hofmann and Geiger, 1983), and is 20 times more concentrated in female than in male lobes. This protease shall be referred to as glandular kallikrein; however, definitive identification will require further structural analysis. About 80% of the glandular kallikrein in the female anterior pituitary is latent whereas over 95% of pituitary kallikrein A appears to be latent. This explains why the sex difference in total kallikrein activity is greater in untreated homogenates than in trypsinactivated homogenates.

It should be noted that glandular kal~ikrein-like mRNA has recently been detected in the rat anterior pituitary (Fuller et al., 1985). This mRNA was suggested to code for a ‘glandular kallikrein’ lacking kininogenase activity on the basis of my earlier report that the male anterior pituitary lacks notable kininogenase activity (Powers and Nasjletti, 1983). However, the report of Fuller et al. used only female rats and failed to consider the possibility of a sex difference in glandular kallikrein levels or the existence of latent forms. The results presented here as well as elsewhere (Powers and Nasjletti, 1984a) clearly demonstrate that glandular kallikrein in the female anterior lobe possesses kininogenase activity. The 20-fold sex difference in glandular kallikrein is the largest reported in the anterior pituitary. This sex difference is due to regulation by ovarian estrogens since it was almost abolished by ovariectomy and estradiol benzoate massively increased glandular kallikrein levels. Prolactin has long been considered to be the major estrogen-induced protein of the rat pituitary. However, estrogen doses similar to those used here increase prolactin synthesis in ovariectomized rats by no more than lo-fold (MacLeod et al., 1969; Yamamoto et al., 1975). Also, pituitary levels of prolactin are only 2 to 3 times higher in females than in males (Doehler et al., 1977; Lewis et al., 1969). Clearly, glandular kallikrein is also one of the major estrogen-regulated proteins of the rat pituitary; this suggests that it may play an important role in estrogen effects on the pituitary and female reproductive biology. Glandular kallikrein in the rat kidney, urine, submandibular gland, pancreas. and neurointermediate lobe of the pituitary either does not exhibit a sex difference in its tissue levels or is slightly elevated in males (McPartland et al., 1981a,b; Chao and Margolius, 1983; Powers and Nasjletti, 1984a; Powers, 1986a). Thus, estrogen regulation of glandular kallikrein appears to be highly specific for the anterior pituitary in the rat. Furthermore, no other members of the glandular kallikrein family have been reported to be under estrogen regulation; those which are strongly sex dependent are influenced by androgens (see below). Glandular kallikreins are prototypical members

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of a large subfamily of serine proteases which exhibit extensive sequence homologies (Fiedler and Fritz, 1981; Mason et al., 1983; Ashley and MacDonald, 1985). Two members of this enzyme family, the y-subunit of nerve growth factor (yNGF) and the epidermal growth factor binding protein (EGF-BP), possess only weak kininogenase activity (Bothwell et al., 1979) but appear to process growth factor precursors in the mouse submandibular gland (Server and Shooter, 1977; Frey et al., 1979; Thomas et al., 1981). It is noteworthy that the levels of NGF, y-NGF, EGF and EGF-BP are lo- to 20-fold higher in male than in female submandibular glands due to hormonal induction by androgens (Server and Shooter, 1977; Hirata and Orth, 1979; Mowry et al., 1984). Thus, other members of the glandular kallikrein family which are strongly regulated by sex steroids appear to function in the processing of sex-dependent growth factors. In view of the above, the estrogen-regulated glandular kallikrein may conceivably function in the processing of a peptide linked to female reproductive biology. The only pituitary hormone whose synthesis is markedly stimulated by estrogens is prolactin (MacLeod et al., 1969; Yamamoto et al., 1975). The prolactin precursor does not require trypsin-like processing to generate the lactogenic peptide (Cooke et al., 1980). However, a cleaved prolactin with novel mitogenic activity has been reported in rat, mouse, and human pituitaries (Mittra, 1980a, b; Sinha and Gilligan, 1984; Sinha et al., 1985) and the cleaved prolactin is induced by estrogen in the rat (Mittra, 1980a). The possibility that the estrogen-regulated protease generates a mitogenic peptide from prolactin deserves attention. Acknowledgements This work was supported by NIH grant AM32783, New York Medical College intramural research grant 49-146-1 and a Sinsheimer Scholar Award to C. Andrew Powers. I gratefully acknowledge the gift of antiserum from Drs. O.A. Carretero, A.G. Scicli, J. Chao and H.S. Margolius, and thank Pam Blank for preparing the manuscript.

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