Characterization of rat testicular guanylate cyclase during development

484

Biochimica et Biophysica Acta, 587 ( 1 9 7 9 ) 4 8 4 - - 4 9 4 © E l s e v i e r / N o r t h - H o l l a n d Biomedical Press

BBA 29048

CHARACTERIZATION OF RAT TESTICULAR GUANYLATE CYCLASE DURING DEVELOPMENT *

W. A U S T I N SPRUILL, A L T O N L. STEINER and H. S H E L T O N EARP, III

University of North Caroline School of Medicine, Chapel Hill,N C 27514 (U.S.A.) (Received March 8th, 1979)

Key words: Guanylate cyclase; Testicular development; Cyclic GMP level; (Rat)

Summary The biochemical characteristics of rat testicular guanylate cyclase were investigated and the activity and subcellular distribution of the enzyme was determined during testicular development. Examination of the effects of metal ions, nucleotides, detergents and other in vitro activators on the activity of guanylate cyclase revealed that the testicular enzyme is similar in most respects to guanylate cyclase isolated from other mammalian tissues. Changes in the total activity of guanylate cyclase during testicular development paralleled changes in the tissue concentration of cyclic GMP; i.e. guanylate cyclase activity and tissue cyclic GMP were highest during the early stages of development. Subcellular fractionation revealed that the activity of the soluble form of guanylate cyclase was best correlated with tissue cyclic GMP. Biochemical analysis of the soluble enzyme prepared from testes of neonatal and adult rats did not reveal any significant differences in the characteristics of the enzyme during ontogeny with the exception of a 2.5 fold increase in V noted in the neonatal testis. The results of this study are consistent with a molecular mechanism that allows independent regulation of the different forms of guanylate cyclase.

Introduction

Numerous studies have documented alterations in tissue cyclic GMP levels following hormonal stimulation and during processes such as growth and development [1,2]. However, no definitive relationship between hormone action and alterations in tissue cyclic GMP has been established, nor it there a * This work w a s p r e s e n t e d in part at t h e F e d e r a t i o n o f A m e r i c a n S o c i e t i e s for E x p e r i m e n t a l B i o l o g y m e e t i n g in April 1977, Chicago, IL.

485 consistent alteration in the direction and magnitude of tissue cyclic GMP levels during growth and development. In light of the difficulty in understanding the role of cyclic GMP some investigators have sought insight by studying the regulation of the activity and subcellular localization of guanylate cyclase (GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2) [3--6]. The control of guanylate cyclase activity during processes in which cyclic GMP might have functional significance has been the topic of several reports [7--20]. Data from this laboratory have demonstrated that in the rat testis cyclic GMP levels are elevated during early development and decline with maturation [21]. In an attempt to understand the regulation of these growth-related alterations in cyclic GMP concentration, a study of the activity of testicular guanylate cyclase during development was undertaken. In order to understand the control of guanylate cyclase activity a detailed analysis of the biochemical characteristics of testicular guanylate cyclase was performed. Knowledge concerning the effect of metal ions, nucleotides, detergents and other activators on the in vitro activity of the different forms of guanylate cyclase has allowed us to study the activity and subcellular localization of the enzyme during testicular development. The data reveal that the activity of guanylate cyclase was altered during testicular development in a fashion that paralleled the change in tissue cyclic GMP levels, i.e., guanylate cyclase activity measured in vitro was elevated early during development and decreased with maturation. Secondly, when subcellular fractionation was performed the activity of the soluble guanylate cyclase was most closely correlated with the tissue concentration of cyclic GMP. Analysis of the biochemical characteristics of the guanylate cyclase isolated from testes of 5~lay-old rats did not demonstrate any significant alterations between the enzymes with the exception of an increased V observed in the soluble guanylate cyclase prepared from 5
486 with nitrogen at 60°C. Dried samples were reconstituted in 1 ml of 0.05 M sodium acetate buffer, pH 6.2, and aliquots used directly in the radioimmunoassay for cyclic GMP [22]. Specificity and accuracy of the radioimmunoassay for testicular cyclic GMP has been described [21]. Guanylate cyclase assay. Fresh tissue samples (100--300 mg) obtained from decapsulated testes were homogenized in 20 vols. of 0.25 M sucrose, containing 10 mM Tris-HCl buffer (pH 7.6) at 4°C, using a Brinkmann Polytron (Brinkmann Instruments, Inc., Westbury, NY) equipped with a PT-10 Probe. Homogenization at a setting of 4 for 20 s produced complete cellular disruption as determined by light microscopic examination of homogenized samples. Soluble and particulate fractions were separated by centrifugation at 105 000 × g for 60 min. Supernates were decanted, and the pellets were washed and resuspended in the original volume of buffer. Resuspended pellets were then dispersed with the Polytron before assay (setting of 5 for 15 s). Guanylate cyclase activity was determined in the assay developed by Kimura and Murad [3] as described previously [16]. The reaction mixture (150 pl) contained 50 mM Tris-HC1 buffer (pH 7.8), 3 mM MnC12, 15 mM phosphocreatine, 20 gg creatine phosphokinase, 0.2 mM 3-isobutyl-l-methylxanthine and 30--100 pg protein from the enzyme preparation. Guanylate cyclase assays were initiated by addition of 1 mM GTP and incubated for 10 rain at 37°C. Assays were terminated by the addition of 0.9 ml of a 50 mM sodium acetate buffer (pH 4.0) and heating for 2 min at 90°C. The cyclic GMP generated was determined by radioimmunoassay. Addition of 0.2 mM 3-isobutyl-1methylxanthine to the assay mixture was sufficient to allow recovery of greater than 90% of exogenously added cyclic GMP. In the experiments testing the effects of Ca 2+ on guanylate cyclase activity, 3 mM CaC12 was added to the incubation mixture in the presence of 0.6--3 mM MnC12. Similarly, varying concentrations (0.1--1.0 mM) of ATP were added to the incubation mixture to test the effects of ATP on testicular guanylate cyclase activity. When designated, 1 mM NaN3 was added to the reaction mixture (in the presence of 5 pg bovine catalase) and preincubated with the enzyme preparation for 10 min at 37°C before the addition of GTP. The effect of Triton X-100 on guanylate cyclase activity was determined after the preincubation of sample fractions with 1% Triton X-100 for 1 h at 4°C. The activity of guanylate cyclase in all fractions was linear with respect to protein concentration (up to 150 ~g) and time (up to 20 min). Protein was determined by the method of Lowry et al. [23]. DNA determinations were made according to the method of Burton [24]. Statistical analysis was performed by the Student's t-test for unpaired observations. Results

Characterization of testicular guanylate cyclase Unlike the well-studied adenylate cyclase which is found in the particulate fraction of most tissues, the subcellular distribution of guanylate cyclase varies from tissue to tissue [6]. The partitioning of guanylate cyclase in untreated homogenates into soluble and particulate fractions has been characterized from a number of tissues and varies from 90% particulate in the rat intestine [25] to 80% soluble in the rat lung [4]. In the adult rat (60 days old) centrifugation of

487

testicular homogenates at 105 000 × g reveals that the guanylate cyclase activity is approximately equally distributed between the soluble and particulate fraction (13.23 pmol cyclic GMP/mg protein per min and 16.9 pmol cyclic GMP/mg protein per min, respectively). Experimental analysis o f the biochemical characteristics of soluble and particulate guanylate cyclase reveals the following: Metal ion dependence. As with guanylate cyclase preparations from other tissues, the activity in the presence of Mn 2÷ is greater than that observed with other divalent cations [3,4]. The activity with either Ca 2÷ or Mg 2÷ alone is less than 10% of that observed with Mn 2÷, with the optimal activity of both the soluble and particulate guanylate cyclase observed at 3 mM Mn 2÷ (Fig. 1). In other tissues Ca 2÷ stimulates the activity of soluble guanylate cyclases when suboptimal concentrations o f Mn 2÷ are used [3,4]. In experiments with the testicular soluble guanylate cyclase, this p h e n o m e n o n has also been observed. In the presence of 0.6 mM Mn 2÷ the activity of soluble guanylate cyclase is less than 20% of that seen with 3 mM Mn 2*. The addition of 3 mM Ca 2. produces a four-fold rise in the soluble guanylate cyclase activity. When the assay is performed with 1 mM Mn 2÷ the addition o f 3 mM Ca 2÷ produces a two-fold rise. Effect of nucleotide trophosphates. Fig. 2 demonstrates the kinetic analysis of soluble and particulate guanylate cyclase with respect to GTP in the presence of optimal Mn 2÷. The soluble guanylate cyclase exhibits hyperbolic saturation curves. The Lineweaver-Burk plot (insert, Fig. 2) is linear with an apparent Km for GTP of 60 gM. Particulate guanylate cyclase, assayed after solubilization in Triton X-100, exhibits a sigmoidal saturation curve. Positive cooperative behavior with a non-linear Lineweaver-Burk plot was obtained and the apparent Km for GTP was 200 pM (insert, Fig. 2). These kinetic characteristics are typical of those observed for guanylate cyclase from other sources [3,4]. Concentrations of 0.1--1 mM ATP inhibit guanylate cyclase activity in other

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i t y w a s m e a s u r e d as d e s c r i b e d in M e t h o d s in t h e p r e s e n c e o f 1 m M G T P and varying c o n c e n t r a t i o n s o f M n C I 2 . T h e e n z y m e p r e p a r a t i o n s ( s o l u b l e = 3 0 ~ g ; p a r t i c u l a t e = 6 0 / ~ i p r o t e i n ) w e r e i n c u b a t e d for 1 0 m i n at 3 7 ° C . T h e d a t a is e x p r e s s e d as c G M P f o r m e d and e a c h p o i n t r e p r e s e n t s t h e m e a n o f t r i p l i c a t e d e t e r minations.

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GTP (raM) Fig. 2. K i n e t i c analysis o f t e s t i c u l a r g u a n y l a t e c y c l a s e . S o l u b l e and p a r t i c u l a t e f r a c t i o n s o f testis h o m o g e n a t e s w e r e s e p a r a t e d as d e s c r i b e d in t h e M e t h o d s s e c t i o n . T h e p a r t i c u l a t e e n z y m e w a s a s s a y e d a f t e r s o l u b f l i z a t i o n in 1% T r i t o n X - 1 0 0 for 1 h a t 4 ° C . T h e a c t i v i t i e s o f t h e s o l u b l e and p a r t i c u l a t e g u a n y l a t e c y c l a s e s w e r e d e t e r m i n e d in t h e p r e s e n c e o f 3 m M MnC12 and v a r y i n g c o n c e n t r a t i o n s o f GTP as indic a t e d . Each p o i n t r e p r e s e n t s t h e m e a n o f triplicate d e t e r m i n a t i o n s . Inserts are d o u b l e - r e c i p r o c a l p l o t s o f c y c l i c GMP f o r m a t i o n as a f u n c t i o n o f t h e GTP c o n c e n t r a t i o n .

tissues [3,26]. Inhibition by ATP is a characteristic of the testicular soluble enzyme also. At a concentration of 0.3 mM ATP inhibition is half-maximal. Additional experiments with 5'-AMP (0.1--1.0 mM) have demonstrated inhibition of soluble guanylate cyclase activity by an adenine monophosphate. However, cyclic AMP does not inhibit the basal activity of the soluble enzyme (unpublished observations). The effect o f non-ionic detergents. The incubation of soluble and particulate guanylate cyclase with non-ionic detergents such as Triton X-100 results in differential activation of guanylate cyclase activity [3,4]. In the rat testis incubation of the particulate guanylate cyclase results in the solubilization of the enzyme and a marked increase in activity. In the adult rat, the activity is increased by six-fold (17.75 _+ 1.31 versus 107.12 + 16.16 pmol cyclic GMP/mg protein per min, n = 5). Incubation of the soluble enzyme in 1% Triton X-100 for 1 h results in a less than 50% stimulation. In vitro activation. Several investigators have uncovered experimental conditions or agents which result in a significant activation of gnanylate cyclase in vitro. The discovery that c o m p o u n d s containing or capable of conversion to nitroso groups can activate guanylate cyclase is an example of this type of activation [27,28]. Sodium azide (NAN3), a potent nucleophile, was observed by Murad and coworkers [29,30] to activate (10--30-fold) gnanylate cyclase from

489 some, but not all, tissues. Their findings indicate that NaN3 is converted to an active compound in the presence of a protein factor. In several tissues the factor has been demonstrated to be the enzyme catalase [31,32]. In the testis, preincubation of the enzyme with 1 mM NaN3 for 10 min prior to the initiation of the guanylate cyclase assay results in a small stimulation of both soluble and particulate guanylate cyclase (50% activation or less). In contrast, preincubation of testicular soluble guanylate cyclase with both NaN3 and 5 pg bovine catalase results in a 16-fold increase in activity, a stimulation comparable to that seen with liver tissue preincubated with NaN3 alone. The testicular particulate enzyme activity is increased by 250% in the presence of catalase and NAN3. Thus, the enzyme is capable of responding in vitro to active nitrosocontaining compounds. Guanylate cyclase from certain tissues undergo spontaneous activation when incubated at 37°C for 30 min prior to assay [33]. This phenomenon of autoactivation was not observed in preparations of testicular guanylate cyclase. Again, this may be a product of differences in tissue metabolism rather than a difference in the guanylate cyclase molecule.

Guanylate cyclase activity during testicular development The tissue concentration of cyclic GMP, measured at intervals during rat testicular development, is depicted in Fig. 3. The initial elevation persists from 5 to 20 days of age. By 30 days of age the tissue concentration of cyclic GMP characteristic of the adult rat is reached. To determine whether the capacity to synthesize cyclic GMP correlated with the tissue cyclic GMP concentration guanylate cyclase activity was assessed in rats of varying ages (5--60 days old). Fig. 3 demonstrates that the homogenate guanylate cyclase activity is highest on day 5; a subsequent decline is seen on day 10, and by day 20 the adult level is approximated. As discussed previously, guanylate cyclase is partitioned into at least two subcellular fractions, the soluble and particulate, the activity of which has been demonstrated to change independently during growth of certain tissues [7,8, 16]. Therefore, the distribution and activity of the guanylate cyclases was determined in homogenates centrifuged for 1 h at 105 000 × g. The activity of the particulate guanylate cyclase paralleled that observed in the homogenate, i.e. the highest activity found on day 5 followed by a decrease apparent by day 10. In contrast, the soluble guanylate cyclase activity remained elevated on days 5, 10 and 20. Only after day 20 did the guanylate cyclase level decline towards the adult level. From examination of Fig. 3 it can be seen that the levels of tissue cyclic GMP were best correlated with the activity or cyclic nucleotide level/mg tissue protein. When the DNA content of testis from 5 and 60
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AGE (DAYS) Fig. 3. G u a n y l a t e c y e l a ~ activity and tissue cyclic GMP levels during testicular d e v e l o p m e n t . G u a n y l a t e cyclase activities and tissue c o n c e n t r a t i o n s o f cyclic GMP were d e t e r m i n e d as described in Methods. Points represents t h e m e a n o f t h e activity o r level, and vertical lines the S.E. The n u m b e r o f determinat i o n s is s h o w n a b o v e vertical lines. Asterisks d e n o t e significant differences ( P < 0 . 0 1 ) b e t w e e n activities or levels in n e o n a t a l testis (5 d a y ) and s u b s e q u e n t t i m e points.

The alteration in the specific activity/mg protein may represent an alteration in the number of enzyme molecules present or a change in the catalytic activity/molecule (or both). This question has been approached indirectly by comparing the biochemical properties o f the soluble and particulate guanylate cyclase prepared from testes of 5 and 60~iay-old rats. Table I compares the effect of Ca 2÷ and varying Mn 2÷ concentrations on soluble guanylate cyclsse activity from testes of 5 and 6 0 ~ l a y ~ l d rats. The results demonstrate that the sample have a proportional decrease in activity whe incubated with suboptimal Mn 2÷. The subsequent stimulation by Ca 2÷ addition are equivalent in the enzyme preparation from 5 and 60~lay-old rats. This, there is little change in the metal ion response of the soluble form of the testicular enzyme during development. The kinetic behavior with respect to GTP was analyzed to test for a change in the activity state o f the enzyme. Fig. 4 demonstrates that the Km for GTP of soluble guanylate cyclases from both 5 and 60~lay-old rats was 60 ~M. The dif-

491 TABLE I E F F E C T S O F CaCI 2 O N S O L U B L E G U A N Y L A T E C Y C L A S E A C T I V I ' I ~ IN T E S T I S F R O M N E O N A TAL AND ADULT RATS N e o n a t a l a n d a d u l t rats w e r e 5 a n d 6 0 d a y s o f age, r e s p e c t i v e l y . Soluble p r e p a r a t i o n s w e r e a s s a y e d in the p r e s e n c e or a b s e n c e o f 3 m M CaCI 2 a n d the c o n c e n t r a t i o n o f MnCI2 w a s varied as i n d i c a t e d . T h e v a l u e s r e p r e s e n t t h e m e a n ± S.E. o f five e x p e r i m e n t s . Reaction mixture

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Mn 2+ Mn 2+ + 3.0 m M Ca 2+ Mn 2+ Mn 2+ + 3 . 0 m M Ca 2+ Mn 2+ Mn 2+ + 3 . 0 m M Ca 2+

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A d u l t testis

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ference in kinetic behavior noted was the 2.5-fold increase in the V exhibited by the enzyme from the 5~lay-old rat. This different kinetic behavior could be explained by an increase in the enzyme concentration or by the presence of an activator or an inhibitor that would alter the V but not the K m of the enzyme. To determine whether a transferable activator or inhibitor was present, mixing experiments were performed. In these experiments the enzymes prepared from testes of 5 and 6 0 < l a y ~ l d rats were incubated separately and together. In three experiments, no significant stimulation or loss of activity was observed in the mixed preparations, i.e. greater than 85% of the activity was present in the mixed enzyme incubations. This indicates that neither a transferable activator or inhibitor was present. However, activation of the enzyme could occur in vivo and the effect persist in vitro [35]. In order to approach this possibility, experiments were performed to assess the ability of guanylate cyclase from testes of 5 and 60~lay-

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492

old rats to respond to a known in vitro activator, NAN3. The conditions that produce a maximally activated enzyme in adult testis were used. When the soluble enzyme from 5~lay~)ld rats was preincubated with catalase and NAN3, a 15.9-fold activation was observed {52.9 -+ 10.3 vs. 841.3 + 78.6 pmol cyclic GMP/mg protein per min, n = 5). Similarly treated soluble enzyme from 60d a y ~ l d rats was stimulated b y 15.5-fold {21.9 -+ 1.4 vs. 341.1 +_16.7, n = 5). This proportionality would argue against an explanation that the enzyme from the 5
The biochemical characterization of the soluble and particulate testicular guanylate cyclase demonstrates that the testicular enzymes are very similar to those studied in other mammalian tissues. The lack of stimulation by NaN3 alone and the absence of autoactivation are probably a product of the difference in the enzyme profile of testicular tissue rather than any difference in the guanylate cyclase. During development the concentration of tissue cyclic GMP remains elevated through day 20 b u t decreases with maturation. Examination of guanylate cyclase activity in subcellular fractions revealed a correlation between tissue cyclic GMP levels and the soluble guanylate cyclase activity measured in vitro. However, the tissue level of cyclic GMP reflects a dynamic balance between synthesis and destruction. Others have demonstrated that an increase in cyclic nucleotide phosphodiesterase activity occurs during testicular development [36] and thus it is difficult to determine to what extent the observed elevation in the soluble guanylate cyclase contributes to the elevated cyclic GMP levels. We have examined the relationship between tissue cyclic GMP and guanylate cyclase activity in another testicular model, unilateral cryptorchidism [17]. In contrast to the reduction in soluble guanylate cyclase activity observed during maturation, an increase in soluble enzyme activity is observed in the testis rendered surgically cryptorchid. In these experiments, within 10 days postoperatively, the surgically placed abdominal testis exhibits a five-fold higher content of tissue cyclic GMP and a three-fold higher soluble guanylate cyclase

493

activity when compared to the scrotal testis from the same rat. In cryptorchidism the elevations in tussue cyclic GMP and the soluble guanylate cyclase activity are equivalent when expressed either as per mg protein or per mg DNA. Therefore, both during testicular development and atrophy a similar relationship is observed, a correlation between changes in tissue cyclic GMP and soluble guanylate cyclase activity: In the course of studies characterizing a soluble form of adenylate cyclase present in adult testis, Braun et al. [37] also measured guanylate cyclase activity during testicular development. Three time periods were examined (13, 27 and 90 days). The data presented from the one published experiment show a lower activity for cytosol guanylate cyclase (20 000 × g supernatant) in the 13day-old rat than the 27
494

References I 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37

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