Ontogeny of Cyclic AMP and Cyclic GMP Phosphodiesterase Activities and of Calmodulin Levels in Guinea Pig Ureter

0022-534 7/88/1395-1095$2.00/0

Vol. 139, May Printed in U.S.A.

THE JOURNAL OF UROLOGY

Copyright© 1988 by The Williams & Wilkins Co.

ONTOGENY OF CYCLIC AMP AND CYCLIC GMP PHOSPHODIESTERASE ACTIVITIES AND OF CALMODULIN LEVELS IN GUINEA PIG URETER YONG H. CHO, MARCIA A. WHEELER AND ROBERT M. WEISS* From the Section of Urology, Yale University School of Medicine, New Haven, Connecticut

ABSTRACT

Cyclic AMP and cyclic GMP phosphodiesterase (PDE) activities and calmodulin levels were determined in ureters from guinea pigs of the following ages: 50 and 56 days fetuses, three, 10, 21, 50, and 90 days, and three years old. While there is little change in ureteral cyclic AMP-PDE with age, cyclic GMP-PDE increases with age. Activity of cyclic GMP-PDE in supernatants prepared from three-year-old guinea pig ureter homogenates is 462% and 216% higher than that from 50-day fetus and three-day animals, respectively. Calmodulin levels have a bimodal distribution with age; values are highest in supernatants from 10 and 21 day ureters, but also increase in the three-year ureters when compared to 50 and 90-day values. (J. Ural., 139: 1095-1098, 1988) Although isoproterenol decreases contractile force of neonatal guinea pig ureters to a greater extent than that of ureters from old animals, the effects of dibutyryl cyclic AMP on ureteral contractility are age independent. 1 · 2 These data are consistent with the age dependent actions of isoproterenol being due to changes in the functional cascade that regulates cellular AMP levels. Changes in the enzymatic activities involved in the synthesis and degradation of cyclic AMP, in effect adenylate cyclase and phosphodiesterase, could alter the levels of cyclic nucleotides. Using a guinea pig ureter model, we recently have shown that basal and agonist stimulated adenylate cyclase activities decrease with age. 3 Endogenous and beta agonist stimulated cyclic AMP levels also decrease with age. 4 There is a 56% decline in the endogenous ureteral cyclic AMP levels between two weeks and three years of age. Isoproterenol, 10- 5 M, causes an 84 % increase in the cyclic AMP levels in ureters from two-week guinea pigs and only a 10% increase in levels in ureters from the three-year animals. Endogenous cyclic GMP levels also decrease with age but at a slower rate than AMP levels. Values from three-year guinea pig ureters are 71 % of two-week values.4 In the present study, we investigated the effects of age on cyclic AMP and cyclic GMP phosphodiesterase activities. Since calmodulin is a potent activator ofphosphodiesterase, 5 we also measured changes in calmodulin levels with age. MATERIALS AND METHODS

Chemicals and reagents. Adenosine-3',5' cyclic monophos[2,8-3HJ (specific activity 33.5 Ci/mmol.) and [8- 3H] guanosine-3',5' cyclic-monophosphate (34.6 Cijmmol.) were purchased from New England Nuclear Co. Cyclic AMP, cyclic GMP, theophylline, adenosine, guanosine, dithiothreitol bovine serum albumin (BSA), crotalus atrox venom, ethyleneglycol-bis (beta-aminoethyl ether) N, N, N', N'-tetraacetic acid (EG TA), N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid (TES), isobutylmethylxanthine (MIX), ethylenediaminetetraacetate (EDT A), trifluoperazine (TFP), calmodulin, and calmodulin-deficient phosphodiesterase were obtained from Sigma Chemical Co. QAE Sephadex A-25 (Pharmacia Fine Chemicals) was equilibrated with the column buffer Accepted for publication January 18, 1988. * Requests for reprints: Section of Urology, Yale University School of Medicine, 789 Howard Ave., New Haven, CT 06504. Supported by PHS grants AG 00112 from the National Institute on Aging and DK 38311 from the National Institute of Diabetes and Digestive and Kidney Diseases.

prior to use. All other chemicals were reagent grade or the best commercially available. Preparation of ureter whole homogenates. Ureters were obtained from Hartley guinea pigs of both sexes of the following ages: 50 and 56-day fetuses, three, 10, 21, 50 and 90 days, and three years old (10 animals/age group). Animals were killed by decapitation and ureters were removed immediately. Following removal of surrounding connective tissue, fat, and blood vessels, the ureters were frozen in liquid nitrogen. After thawing, ureters were weighed and homogenized (10% w/v) in a solution containing 20 mM Tris-HCl buffer (pH 7.5), two mM MgCb, and one mM DTT with a Polytron (Brinkman Inst.) using three 30 sec. pulses at speed 7 and 4C. Preparation of supernatant fraction. A supernatant fraction is prepared by centrifugation of whole homogenate at 100,000 g, 30 min. (4C). Prior to the determination of enzymatic activity the supernatant is diluted with homogenization buffer so that it hydrolyzes five to 30% of the cAMP. Assay of phosphodiesterase activity. Phosphodiesterase activity is measured using the method of Wells et al. 6 The assay mixture consists of one µM cyclic AMP or cyclic GMP, containing 106 cpm of 3 H substrate, two mM MgCb, and 40 mM Tris-HCl buffer (pH 7.5) in a volume of 200 µl. The reaction is started by the addition of 50 µl. of enzyme containing supernatant. After 30 min. (30C) the reaction is terminated by the addition of 25 µl. of a 50 mM EDTA-30 mM theophylline solution. Crotalus atrox venom (25 µl.) is then added and the incubation continued (30C, 10 min.). Then 0. 75 ml. of 0.1 mM adenosine and guanosine is added and the product eluted from an anion exchange resin column (QEA Sephadex, A-25, 0.7 X 2.0 cm. column). The amount of [3 H] adenosine or [3 HJ-guanosine is determined in a liquid scintillation counter. Product accumulation is linear with time and with enzyme dilution under all conditions studied. All values are corrected for that obtained in the absence of the added enzyme. The results are expressed as pmoles of cyclic AMP or cyclic GMP hydrolyzed per milligram of supernatant protein per 30 minutes. Assay for calmodulin content. The assay of calmodulin content is based on the ability to activate a calmodulin-deficient brain phosphodiesterase as described by Wallace et al. 7 and Kakiuchi et al. 8 The 100,000 g supernatant is boiled for five minutes, and cooled on ice. The heat denatured protein is then removed by centrifugation at 5,000 g for five min. Standard or unknown calmodulin are added to a mixture containing 40 µM CaCb, 20 µM EGTA, 125 mM TES, three mM MgClz, BSA

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CHO, WHEELER AND WEISS

(0.8 mg./ml.), 0.4 µM cyclic GMP containing 15,000 cmp of 3 H substrate, and 20 µl. calmodulin-deficient phosphodiesterase (0.2 µg. protein) in a final volume 200 µl. (pH 7.5). After 30 min. incubation at 30C, the reaction is stopped with 25 µl. of a solution containing 50 mM EDTA, 15 mM cyclic GMP, five mM 5'GMP, and five mM MIX. Crotalus atrox snake venom, 10 mg./ml. is then added in a volume of 25 µl. After the addition of 0. 75 ml. of unlabeled guanosine, the 3H guanosine is isolated using an ion exchange resin column as described above. All assays are performed in duplicate and the results are expressed as µg. of calmodulin per mg. of supernatant protein. Protein is determined by the method of Lowry et al. 9 using bovine serum albumin as the standard. Statistics. Statistical analysis of age trends in PDE activities and calmodulin levels is performed with a one-way analysis of variance, and Scheffe criteria are used in the determination of significance between age groups.

increase when compared to the three-day value. Since cyclic AMP-PDE varies little with age while cGMP-PDE increases, the ratio of cyclic AMP-PDE to cyclic GMP-PDE declines with age (fig. 2). The cyclic AMP /cyclic GMP ratio for the 13-day prenatal ureter is 0.458 while the ratio in the three-year ureter is 0.150. Calcium (10-5 M), calmodulin (five µg./ml.), EGTA (two mM) or calcium plus calmodulin when added to supernatant fractions from three day and three year old guinea pig ureters had little effect on cyclic AMP or cyclic GMP PDE activities. The effect of age on calmodulin levels is shown in figure 3. Calmodulin levels range from 4.3-13.8 µg./mg. protein, with the highest amount found in 10 and 21 day guinea pig ureter supernatants. A small increase in calmodulin levels is found in the retired breeder when compared to other adult levels. Average value for calmodulin summed over all ages is 8.0 ± 1.1 µg. calmodulin/mg. supernatant protein. Only three to 5% of the

RESULTS

Cyclic AMP and cyclic GMP phosphodiesterase (PDE) activities are measured in supernatant fractions from guinea pig ureters of eight age groups, ranging from 13 days prior to birth (50-day fetus) to three years of age in order to assess both developmental and senescent changes. When all age groups are taken into consideration, an average of 88% of cyclic AMPPDE and 96% of cyclic GMP-PDE activity is found in the supernatant fractions. The range of PDE activity in ureteral particulate fraction as a function of total protein varied from two to 21 % for cAMP PDE and from three to 5% for cGMP PDE. Ureters from guinea pigs 10 days old and younger had an average of 10.3% of the cAMP PDE activity in their particulate fraction; those 50 days and older had an average of 14.2% of the cAMP PDE activity in the particulate fraction. For cGMP PDE, the percentage activity in the pellet was 4.7% for ureters from guinea pigs 10 days old and younger and 3.3% for ureters from guinea pigs 50 days and older. Thus, there is no significant change in particulate PDE activity with age. There is little change in cyclic AMP-PDE activity with age in supernatant fractions (fig. lA). The average value for cyclic AMP-PDE activity for all ages is 5659 ± 391 pm cyclic AMP hydrolyzed/mg. protein/30 min. The average value for cyclic GMP-PDE activity for all ages is 24,922 ± 4339 pm cGMP hydrolyzed/mg. protein/30 min. Cyclic GMP-PDE activity is greater than cyclic AMP-PDE activity in all age groups. Ureteral cGMP-PDE activity is highest in the three-year animal (fig. lB). There is a 462% increase in cGMP-PDE in the threeyear animal when compared to the 50-day fetus and a 216%

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ONTOGENY OF PHOSPHODIESTERASE AND CALMODULIN IN GUINEA PIG URETER

total calmodulin content was found in the pellet fraction of ureters obtained from any age group. DISCUSSION

The present study shows that there is little change in ureteral cyclic AMP-PDE activity with age while cyclic GMP-PDE increases with age, reaching its highest level in the three-year guinea pig ureter. These relationships are based on data expressed as enzyme activity per mg. protein. The relationships would be similar if the data were expressed as enzyme activity per mg. of wet weight since although the weight of the ureter increases 10-fold from the fetus to the three-year guinea pig, the protein content per mg. of wet weight of ureter does not change with age. Cyclic AMP and cyclic GMP PDE activities and calmodulin levels are low in the particulate fractions of these guinea pig ureters. Disruption of smooth muscle tissue requires homogenization techniques that may lead to proteolytic cleavage of particulate low Km PDEs 10 and migration of membrane bound PDEs and calmodulin to the soluble fraction. The effect of age on PDE activity varies from tissue to tissue and from species to species. Smoake et al. 11 examined eight rat tissues for cyclic AMP-PDE activity and found that brain had forty times more PDE than thymus. Although brain cyclic AMP-PDE activity increased steadily with age, activity in testis, thymus, and liver showed a decline in the adult. The of decline in activity were not linear, however, but wc,uv,u,~ with the highest levels of activity occurring at different ages in the various tissues examined. Davis and Kuo 12 measuring a low Km cyclic AMP-PDE in cytosol of guinea pig lung, liver, heart, and brain found increased cyclic AMP-PDE in brain with aging, and decreased activity in lung, heart, and livel'. Mersmann et al. 13 found complex developmental patterns in the cAMP-PDE activities in four tissues from swine (adipose, heart, skeletal muscle, and liver). They did, however, see a trend to lower values in older (150 day) animals. Stancel et al. 14 examined a low Km cyclic AMP-PDE in rat uterus, and found that specific activity was markedly reduced when 40 to 50 day animals were compared with five to 30 day animals. Total cyclic AMP-PDE was also decreased signifithough not as substantially as the low Km cyclic AMPPDE. In rabbit renal cortex, however, cyclic AMP-PDE activity was similar in the newborn and adult; 15 a finding that agrees ,,vith our data in the guinea pig ureter. Less information is available on the effect of ontogeny on cyclic GMP-PDE activity. With aging of the guinea pig, cyclic GMP-PDE activity decreases in lung and liver, increases in and remains the same in heart. 12 Cyclic GMP-PDE decreases with age in the rat liver 16 and kidney. 17 In rat uterus, cyclic GMP-PDE appears to decrease with age when data are expressed per mg. protein. 14 Sicard and Aprille 16 found higher levels of cyclic AMP-PDE than cyclic GMP-PDE in rat liver and a similar relationship was observed by Davis and Kuo 12 in the fetal guinea pig lung. In most tissues, however, GMP-PDE activity is higher than cyclic AMP-PDE 12 There appears to be more cGMP-PDE activity relative to cAMP-PDE activity in the ureter than in other tissues. variation of PDE activity has also been found between pig and rat ileum, 18 and in various regions of brain. 19 decline in the ratio of hydrolysis of cyclic AMP to cyclic GMP by ureteral phosphodiesterases with age (fig. 1) is similar to that seen by Davis and Kuo in lung and liver. 12 In contrast, cAMP-PDE/cGMP-PDE ratios in heart and brain remain almost constant with age. 12 Although ureteral adenylate cyclase activity 3 (basal and and forskolin stimulated) and ureteral cyclic levels 4 ( endogenous and beta agonist stimulated) decrease with aging, cyclic AMP-PDE activity does not change with age. This could indicate that the decrease in cyclic AMP content and in isoproterenol stimulation of ureteral relaxation with

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aging may be due to decreases in the amount of or function of the adenylate cyclase enzyme, rather than to changes in PDE. Palmer18 similarly concluded that the aging profiles of cyclic nucleotide PDE are not associated with changes in receptor mediated adenylate cyclase systems in brain and peripheral tissues. On the other hand, the decrease in ureteral cyclic GMP content with aging4 may be dependent on the increases seen in cyclic GMP-PDE activity. In general, calmodulin levels decline in guinea pig ureter supernatants when adult values are compared to neonatal values. In the rat cerebellum, Tallant and Cheung20 found no change in levels between O and 30 days of age. An earlier study by this group showed little change in calmodulin levels in brain, thymus, and liver in neonate and adult. 11 Hoskins and Scott, 21 however, showed a 32% decline in calmodulin levels between three weeks and one year in rat whole brain preparations, although this decline appears to be species dependent. 22 A large increase with aging (five-fold) in calmodulin levels was noted in testis preparations which correlates with the onset of spermatogenesis. 11 •23 The pronounced increase in ureteral calmodulin levels seen between three and 21 days parallels the rise in ureteral contractility seen in the neonate. 24 The smaller increase in calmodulin levels seen in ureters from three-year old guinea pigs is coincident with increases in cyclic GMP-PDE activity. These correlative changes do not necessarily imply cause and effect.

REFERENCES 1. Weiss, R. M., Wheeler, M.A. and Biancani, P.: Age related changes in the response ofureteral smooth muscle to autonomic agonists. Fed. Proc., 44: 506, 1985. 2. Cho, Y. H., Biancani, P. and Weiss, R. M.: Adenyl and guanyl nucleotide induced relaxation of ureter smooth muscle. Fed. Proc., 43: 353, 1984. 3. Wheeler, M.A., Housman, A., Cho, Y. H. and Weiss, R. M.: Age dependence of adenylate cyclase in guinea pig ureter homogenate. J. Pharmacol. Exp. Ther. 239: 99, 1986. 4. Weiss, R. M., Cho, Y. H., Wheeler, M. A. and Latifpour, J.: Correlation of cyclic nucleotide levels with age dependent functional responses of ureteral smooth muscle to beta agonist. Proceedings of the International Union of Physiological Sciences (XXX Congress), 16: 148, 1986. 5. Cheung, W. Y.: Calmodulin plays a pivotal role in cellular regulation. Science, 207: 19, 1980. 6. Wells, J. N., Baird, C. E., Wu, Y. J. and Hardman, J. G.: Cyclic nucleotide phosphodiesterase activities of pig coronary arteries. Biochem. Biophys. Acta, 384: 430, 1975. 7. Wallace, R. W., Tallant, E. A. and Cheung, W. Y.: Assay of calmodulin by Ca++_dependent phosphodiesterase. Methods Enzymol., 102: 39, 1983. 8. Kakiuchi, S., Yasuda, S., Yamazaki, R., Teshima, Y., Kanda, K., Kakiuchi, R. and Sobue, H.: Quantitative determination of calmodulin in the supernatant and particulate fractions of mammalian tissue. J. Biochem., 92: 1041, 1982. 9. Lowry, 0. H., Rosebrough, N. J., Farr, A. and Randall, R. J.: Protein measurement with Falin-phenol reagent. J. Biol. Chem. 193: 265, 1951. 10. Loten, E. G., Francis, S. H. and Corbin, J. D.: Proteolytic solubilization and modification of hormone-sensitive cyclic nucleotide phosphodiesterase. J. Biol. Chem., 256: 7838, 1980. 11. Smoake, J. A., Song, S. Y. and Cheung, W. Y.: Cyclic 3'5' nucleotide phosphodiesterase. Distribution and developmental changes of the enzyme and its protein activator in mammalian tissues and cells. Biochem. Biophys. Acta, 341: 402, 1974. 12. Davis, C. W. and Kuo, J. F.: Ontogenetic changes in levels of phosphodiesterase for adenosine 3'5'-monophosphate and guanosine 3'5'-monophosphate in the lung, liver, brain and heart from guinea pigs. Biochem. Biophys. Acta, 444: 554, 1976. 13. Mersmann, H. J., Phinney, G., Brown, L. J. and Steffen, D. G.: Ontogency of adenylate cyclase and phosphodiesterase activities in swine tissues. Biol. Neonate, 32: 266, 1977. 14. Stance!, G. M., Thompson, W. J. and Strada, S. J.: Cyclic nucleo-

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tide phosphodiesterase in uterine development. Mol. Cell. Endocrinol., 3: 283, 1975. Linarelli, L. G., Bobik, J. and Bobik, C.: The effect of parathyroid hormone on rabbit renal cortex adenyl cyclase during development. Pediatr. Res., 7: 878, 1973. Sicard, R. E. and Aprille, J. R.: Adenylate cyclase and cyclic nucleotide phosphodiesterase in developing rat liver. Biochem. Biophys. Acta, 500: 235, 1977. Schlondorff, D. and Trizna, W.: Increases of guanosine 3'5' -monophosphate-related enzymes in kidneys of developing rats. Pediatr. Res., 12: 882, 1978. Stancheva, V., Petkov, V. and Uzonov, P.: Ontogenetic development of 3'5' adenosine monophosphate phosphodiesterase in guinea pig and rat ileum. Acta Biol. Med. Ger., 38: 959, 1979. Palmer, G. C.: Significance of phosphodiesterase in the brain. Life

Sci., 28: 2785, 1971. 20. Tallant, E. A. and Cheung, W. Y.: Calmodulin-dependent protein phosphatases: a developmental study. Biochem., 22: 3630, 1983. 21. Hoskins, B. and Scott, J. M.: Changes in activities of calmodulinmediated enzymes in rat brain during aging. Mech. Aging Develop., 26: 231, 1984. 22. Hoskins, B., Ho, J. K. and Meydrech, E. F.: Effect of morphine and aging on brain calmodulin levels in mice. Exp. Aging Res., 11: 143, 1985. 23. Feinberg, J., Pariset, C. and Weinman, S.: Calmodulin level and cAMP dependent protein kinase activity in rat spermatogenic cells and hormonal control of spermatogeneses. Dev. Biol., l 08: 179, 1985. 24. Hong, K. W., Biancani, P. and Weiss, R. M.: Effect of age on contractility of guinea pig ureter. Invest. Urol., 17: 459, 1980.