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THE BLADDER ANGIOTENSIN SYSTEM IN FEMALE RATS: PONSE TO INFUSIONS OF ANGIOTENSIN I AND THE ANGIOTENSIN CONVERTING ENZYME INHIBITOR ENALAPRILAT
0022-5347/01/1655-1735/0 THE JOURNAL OF UROLOGY® Copyright © 2001 by AMERICAN UROLOGICAL ASSOCIATION, INC.®
Vol. 165, 1735–1738, May 2001 Printed in U.S.A.
THE BLADDER ANGIOTENSIN SYSTEM IN FEMALE RATS: RESPONSE TO INFUSIONS OF ANGIOTENSIN I AND THE ANGIOTENSIN CONVERTING ENZYME INHIBITOR ENALAPRILAT DANA WEAVER-OSTERHOLTZ, GARRY REAMS, CRISTELA FARIA DE VERGEL AND JOHN H. BAUER From the Departments of Surgery and Medicine, University of Missouri and Harry S. Truman Memorial Veterans Hospital, Columbia, Missouri
ABSTRACT
Purpose: A local renin angiotensin (ANG) system has been identified in the bladder. To our knowledge little is known about this system. To define further the physiology of this system we performed this study. Materials and Methods: The circulating and bladder tissue concentrations of ANG I and ANG II were examined in anesthetized Sprague-Dawley female rats in estrus, diestrus or pregnancy. Each was given an intravenous bolus infusion of ANG I, the ANG converting enzyme inhibitor enalaprilat or saline. Results: The mean concentrations of ANG I and ANG II were markedly higher in bladder tissue than in whole blood at the highest levels in pregnancy. The concentration of ANG I and ANG II increased significantly in the bladder tissue and circulation after the ANG I infusion in estrus and diestrus. In pregnancy only circulatory ANG I increased, while circulatory ANG II, tissue ANG I and ANG II remained unchanged. Enalaprilat infusion was associated with an increased concentration of whole blood ANG I in all groups and decreased plasma ANG II in estrus and diestrus but not in pregnancy. The bladder tissue ANG I response was unchanged in all groups. Bladder tissue ANG II was decreased in estrus and diestrus but unchanged in pregnancy. Conclusions: These data support the hypothesis that ANG converting enzyme is deactivated and an alternate pathway is activated in pregnancy. The data also demonstrate that tissue absorption of ANG peptide is diminished in pregnancy. Compared with responses in similarly studied male rats the circulating conversion of ANG I to ANG II is delayed in female rats and responses to ANG converting enzyme inhibition are enhanced, thus, suggesting subtle differences in the sexes in the renin ANG system. Enalapril appears to have more effect at the tissue level in nonpregnant females than in pregnant females and male rats. KEY WORDS: angiotensins; bladder; estrus; pregnancy, animal; rats, Sprague-Dawley
Interstitial cystitis is a disease of the bladder with an unknown etiology. Several causes have been suggested, including ischemia. The renin angiotensin (ANG) system has long been known to be aberrant systemically due to ischemia of the kidney, that is Goldblatt kidney. Evidence has accumulated for the existence of a local renin ANG system in tissues, such as the kidney, heart, vascular tissue, adrenal glands, brain, testes, ovaries, uterus and placenta. It is known to be locally aberrant in the heart when ischemia is present with coronary artery disease.1, 2 Because to date the data suggest that a tissue renin ANG system may be altered in ischemia, we have been interested in the bladder renin ANG system as a possible etiology for interstitial cystitis. However, information is limited on the role of the renin ANG system in bladder physiology.3 ANG II receptors have been identified within the bladder4 – 6 and are modulated by dietary sodium and calcium.7–10 ANG II in the bladder has been found to cause smooth muscle contractility, as demonstrated in in vitro studies in several animal species,4, 11–13 including humans.3, 5, 14 ANG I-type receptors have been found in rats to mediate the contractile response within the bladder.11 ANG II is also thought to have a role in the regulation of smooth muscle growth and collagen production in the bladder response to outlet obstruction.15 ANG converting enzyme has a relatively low level in the bladder comAccepted for publication November 17, 2000.
pared with other genitourinary tissues.16 ANG converting enzyme is considered to be the major enzyme responsible for the conversion of ANG I to ANG II, although various tissues, including the bladder, have been suggested to have alternative pathways for this conversion.17 We have reported confirmation of the autocrine-paracrine function of the tissue specific renin ANG system in the bladder.18 Therefore, sufficient data exist to hypothesize that interstitial cystitis may be affected by the tissue renin ANG system in the bladder. Interstitial cystitis has a 10:1 female-to-male ratio. In women its symptoms are exacerbated during premenstruation and ameliorated during pregnancy.19 The renin ANG system is linked to reproductive function. Circulatory levels are known to be cyclical in women and increased in pregancy.2 Likewise the local renin ANG system in the ovaries in humans has been identified to be regulated by gonadotropins.2 Because there is no reliable animal model of interstitial cystitis, we investigated the bladder response to pharmacological maneuvers in the estrus, diestrus and pregnant states in rats to determine whether they parallel clinical observations. Baseline concentrations of circulating and bladder tissue ANG I and ANG II octapeptide were directly measured in normal anesthetized female Sprague-Dawley rats in estrus, diestrus or pregnancy. In addition, circulating and bladder ANG I and ANG II responses to an acute bolus infusion of ANG I, the ANG converting enzyme inhibitor enalaprilat or saline were examined.
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BLADDER ANGIOTENSIN SYSTEM IN FEMALE RATS MATERIALS AND METHODS
Animal preparation. Female Sprague-Dawley rats (Harlan Sprague Dawley, Inc., Indianapolis, Indiana) weighing between 250 and 500 gm. were housed in individual cages and maintained on regular rat chow (Purina, St. Louis, Missouri) for at least 7 days before the study. Tap water was freely available. All experiments were performed with subjects in the post-absorptive state. All animal care provided during these studies met institutional guidelines. Anesthesia was induced intraperitoneally with 100 mg./kg. Inactin (Andrew Lockwood and Associates, Sturtevant, Wisconsin). Tracheostomy was performed and PE-50 polyethylene catheters were inserted by cutdown into the carotid artery and jugular vein. The arterial catheter was connected by a P23Db strain gauge pressure transducer (Statham, Oxnard, California) to a 7714 to 041A recorder (Hewlett Packard, St. Louis, Missouri) for continuous mean arterial blood pressure monitoring. Immediately after the surgical procedures a sustained intravenous infusion of isotonic saline at a rate of 100 l. per minute was started via an infusion pump (Sage Instruments, Boston, Massachusetts). All animals entered the experimental phase of the study. Experimental design. The experimental design consisted of 2 groups of female rats. In each group rats in estrus, diestrus and pregnancy were studied. Estrus and diestrus was determined by the vaginal mucous test and pregnancy was determined by uterine size. In group 1 after a 30-minute equilibration delay 0.5 g. of ANG I (Sigma Chemical Co., St. Louis, Missouri) dissolved in 1 ml. of isotonic saline was infused for 1 minute into the jugular vein. A saline vehicle (1 ml.) served as the control. Arterial blood (6 to 8 ml.) and whole bladder tissue samples were collected to measure ANG I and ANG II 5 minutes after the initiation of the ANG Isaline infusion. All arterial blood samples were collected in 60 seconds. In group 2 after a 30-minute equilibration delay 5 g./gm. of the ANG converting enzyme inhibitor enalaprilat was dissolved in 1 ml. of isotonic saline (Merck, Sharp and Dohme, Blue Bell, Pennsylvania) and infused for 1 minute into the jugular vein. A saline vehicle (1 ml.) served as the control. Arterial blood (6 to 8 ml.) and whole bladder tissue samples were collected to measure ANG I and ANG II 30 minutes after completing the ANG converting enzyme inhibitor-saline infusion. Blood and tissue were processed as previously described.18, 20, 21 Recovery, sensitivity and reproducibility of ANG I and ANG II assays. Plasma: The mean recovery of unlabeled ANG I in whole blood in 8 experiments was 90.4% (range 83.6% to 100.6%). The lower limit of detection of ANG I in whole blood was 1 fmol./ml. In 8 experiments the intra-assay coefficient of variation for ANG I was 8.8% and the interassay coefficient of variation was 9.4%.
The approximate mean recovery of 2,500 cpm/ml. 3H-ANG II (130 cpm approximately equal to 1 pg.) added to plasma was 65% (range 63.8% to 65.7%) in 8 experiments. The recovery of unlabeled ANG II in plasma was similar to the recovery of radiolabeled ANG II. The lower limit of detection of ANG II in plasma was 0.5 fmol./ml. In 8 experiments the intra-assay coefficient of variation for ANG II was 9.5% and the interassay coefficient of variation was 10.7%. No 125IANG II was generated when 2,000 cpm/ml. 125I-ANG I were added to plasma and incubated at 4C for 30 minutes. Bladder Tissue: The approximate mean recovery of 2,000 cpm/ml. 125I-ANG I added to a bladder tissue homogenate in 8 experiments was 64.4% (range 63.5% to 66.2%). In 8 experiments the intra-assay coefficient of variation for ANG I in tissue was 2.5% and the interassay coefficient of variation was 3.8%. The approximate mean recovery of 2,000 cpm/ml. 125I-ANG II added to a bladder tissue homogenate in 8 experiments was 74.8% (range 73.6% to 76.9%). In 8 experiments the intra-assay coefficient of variation for ANG II in tissue was 3.9% and the interassay coefficient of variation was 4.2%. No 125 I-ANG II was generated when 2,000 cpm/ml. 125I-ANG I were added to a tissue homogenate and incubated at 4C for 30 minutes. Data analysis. The values of ANG I and ANG II in plasma and bladder tissue were corrected for the recovery of each throughout the whole procedure (solid phase extraction, high performance liquid chromatography and radioimmunoassay). Group data are expressed as the mean plus or minus standard error of mean. Data were analyzed using nonparametric analysis of variance. The least significant difference test was used when appropriate for post hoc analysis. Differences were considered statistically significant at p ⬍0.05.22 RESULTS
The mean arterial pressure response to the infusion of ANG I increased by 40% in all cycles after the bolus infusion of ANG I (p ⬍0.01, table 1). Mean arterial pressure was not significantly altered by the saline vehicle. Baseline bladder tissue ANG I was 20-fold higher than circulating ANG I. Baseline ANG II in the circulation and tissue was higher in pregnancy than in estrus and diestrus. Circulatory ANG I levels were significantly increased in estrus, diestrus and pregnancy after completion of the ANG I infusion. In contrast to circulatory values, an increase in tissue ANG I was observed in estrus and diestrus after completion of the ANG I infusion but not in pregnancy. The level of baseline bladder tissue ANG II was significantly 2-fold higher than the circulating level of ANG II in all cycles. Baseline circulatory and tissue ANG II were elevated in pregnancy compared to estrus
TABLE 1. Circulatory and tissue levels of Ang I, Ang II and mean arterial pressure during estrus, diestrus and pregnancy before and after Ang I infusion Estrus (mean cpm/ml. ⫾ SEM) Control mean arterial pressure After infusion Control: Circulatory Ang I After infusion Control: Circulatory Ang II After infusion Control tissue Ang I After infusion Control tissue Ang II After infusion * Compared to control p ⱕ0.05. † Compared to diestrus p ⱕ0.05. ‡ Compared to estrus p ⱕ0.05. § Compared to pregnancy p ⱕ0.05.
Diestrus (mean cpm/ml. ⫾ SEM)
Pregnancy (mean cpm/ml. ⫾ SEM)
142 ⫾ 5/144 ⫾ 5 143 ⫾ 4/189 ⫾ 3*
137 ⫾ 3/14 ⫾ 2 142 ⫾ 3/186 ⫾ 3*
134 ⫾ 4/134 ⫾ 4 133 ⫾ 5/182 ⫾ 7
75 ⫾ 22 514 ⫾ 151*
63 ⫾ 17 938 ⫾ 271*
74 ⫾ 35 351 ⫾ 83*,†
76 ⫾ 15 1,074 ⫾ 217* 2,324 ⫾ 125† 2,744 ⫾ 150*,† 154 ⫾ 22 2,217 ⫾ 195*
61 ⫾ 10§ 824 ⫾ 137*,§ 1,034 ⫾ 69§ 1,617 ⫾ 85*,§ 149 ⫾ 10§ 2,012 ⫾ 79*,§
145 ⫾ 46‡ 105 ⫾ 18‡ 1,300 ⫾ 87‡ 1,086 ⫾ 90‡ 301 ⫾ 14‡ 248 ⫾ 15*,‡
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BLADDER ANGIOTENSIN SYSTEM IN FEMALE RATS
TABLE 2. Circulatory and tissue levels of Ang I, Ang II and mean arterial pressure during estrus, diestrus and pregnancy before and after ANG converting enzyme infusion Estrus (mean cpm/ml. ⫾ SEM)
Diestrus (mean cpm/ml. ⫾ SEM)
144 ⫾ 5/138 ⫾ 5 143 ⫾ 4/129 ⫾ 5*
Control mean arterial pressure After infusion Control: Circulatory Ang I After infusion Control: Circulatory Ang II After infusion Control tissue Ang I After infusion Control tissue Ang II After infusion * Compared to control p ⱕ0.05. † Compared to diestrus p ⱕ0.05. ‡ Compared to estrus p ⱕ0.05. § Compared to pregnancy p ⱕ0.05.
142 ⫾ 3/138 ⫾ 3 146 ⫾ 2/136 ⫾ 4*
Pregnancy (mean cpm/ml. ⫾ SEM) 125 ⫾ 9/123 ⫾ 8 134 ⫾ 4/118 ⫾ 5*
61 ⫾ 21 1,606 ⫾ 277*
27 ⫾ 12 1,327 ⫾ 294*
82 ⫾ 29 2,602 ⫾ 502*
52 ⫾ 8† 26 ⫾ 3* 1,192 ⫾ 127 856 ⫾ 139† 146 ⫾ 16 86 ⫾ 10*
95 ⫾ 16 44 ⫾ 19*,§ 942 ⫾ 123§ 1,305 ⫾ 134 120 ⫾ 4§ 85 ⫾ 3*,§
124 ⫾ 26‡ 229 ⫾ 39*,‡ 1,703 ⫾ 115‡ 1,364 ⫾ 84*,‡ 492 ⫾ 93‡ 491 ⫾ 85‡
and diestrus. After ANG I infusion circulatory ANG II was significantly elevated in estrus and diestrus but unchanged in pregnancy. Similarly tissue ANG II was elevated in estrus and diestrus but in pregnancy there was a significant decrease. The administration of enalaprilat decreased mean arterial pressure by 10% in all cycles studied (p ⬍0.05). Mean arterial pressure was not significantly altered by the saline vehicle (table 2). Enalaprilat administration was associated with a greater than 20-fold increase in circulatory ANG I in all cycle phases studied. There was no significant change in bladder tissue ANG I in estrus and diestrus but a significant decrease was evident in pregnancy. The infusion of enalaprilat was associated with decreased circulatory ANG II in estrus and diestrus but an increase in pregnancy. Tissue ANG II responses were similar with a reduction in the circulatory and tissue levels in estrus and diestrus but no change in pregnancy. DISCUSSION
Our results demonstrate that in anesthetized female Sprague-Dawley rats the concentration of bladder tissue ANG I is more than 10-fold that of the circulatory level of ANG I and bladder tissue ANG II is twice that of circulatory ANG II. These values are higher than those explained by the simple tissue uptake of ANG peptide from the circulation. This finding is compatible with similar observations in male rats (table 3)18 and in other tissues, including the heart23 and kidney,24 in which local autocrine paracrine renin ANG system systems have been described. Angiotensin I with a half-life of 30 seconds was rapidly metabolized to ANG II in the circulation of the rats in estrus and diestrus (a 10-fold increase) but not in the pregnant population. Bladder tissue ANG I was markedly increased after ANG I infusion compared with the saline control and it was rapidly converted to ANG II in estrus and diestrus but not in pregnancy. Compared with previously performed studies in male rats18 responses during estrus and diestrus in females were similar to the responses in males, although the conversion of ANG I to ANG II was slower in the females
(table 3.). On the other hand, the response in pregnancy was dissimilar, as it was to the estrus and diestrus studies. The absence of conversion of circulatory ANG I to ANG II in pregnancy with increased circulatory ANG I may be explained by diminished ANG converting enzyme activity. Decreased ANG converting enzyme has been measured in normal unchallenged human pregnancies.25 The unchanged bladder tissue level of ANG I in pregnancy despite high circulatory ANG I supports impeded tissue uptake in pregnancy compared with nonpregnant states in females as well as in males. It has been observed that pregnancy is less responsive to peptide administration25 and adversely affected by ANG converting enzyme inhibitors with resulting fetal distress and death. Receptor levels have been evaluated in rat smooth muscle and have not been found to change in response to chronic ANG II infusion.26 If ANG converting enzyme activity and tissue uptake of peptide are diminished, as indicated by these studies, these alterations would explain the response to peptide infusion observed in pregnancy. The circulating ANG peptide response to ANG converting enzyme inhibition was similar in estrus and diestrus but not in pregnancy. Circulating ANG I was increased more than 10-fold, while circulating ANG II decreased by 50% in estrus and diestrus, which was an exaggerated response compared with that in previously studied male rats (table 4). On the other hand, in pregnancy a doubling of circulatory ANG II was noted with elevated circulatory ANG I. Others have suggested that ANG converting enzyme inhibition is not effective for blocking ANG I induced bladder smooth muscle contraction or the conversion of ANG I to ANG II, suggesting alternative pathways for ANG II generation.3, 9, 16 The inability of enalaprilat to block circulatory ANG II production suggests that an alternate circulatory pathway is activated in pregnancy. The bladder ANG I and ANG II response to ANG converting enzyme inhibitor was similar to that observed in the circulation in estrus and diestrus but again not in pregnancy. Enalaprilat was associated with a significant decrease in tissue ANG I but no change in tissue ANG II in pregnancy.
TABLE 3. Observed response to Ang I infusion Male p Value 2 Mins. Mean arterial pressure ⱕ0.01 (increase) Circulating Ang I No change Circulating Ang II ⱕ0.01 (increase) Tissue Ang I No change Tissue Ang II ⱕ0.01 (increase) In females in diestrus all p ⱕ0.01 (increase).
Female p Value 5 Mins. ⱕ0.01 (increase) No change No change ⱕ0.05 (increase) No change
Estrus ⱕ0.01 ⱕ0.01 ⱕ0.01 ⱕ0.05 ⱕ0.01
(increase) (increase) (increase) (increase) (increase)
Pregnancy ⱕ0.01 (increase) ⱕ0.05 (increase) No change No change ⬉0.05 (decrease)
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BLADDER ANGIOTENSIN SYSTEM IN FEMALE RATS TABLE 4. Observed response to ANG converting enzyme infusion Female p Value
Male p Value Mean arterial pressure Circulating Ang I Circulating Ang II Tissue Ang I Tissue Ang II
ⱕ0.05 (decrease) ⱕ0.05 (increase) ⱕ0.05 (decrease) No change No change
Estrus
Diestrus
Pregnancy
ⱕ0.05 (decrease) ⱕ0.01 (increase) ⱕ0.05 (decrease) No change ⱕ0.01 (decrease)
ⱕ0.05 (decrease) ⱕ0.01 (increase) ⱕ0.05 (decrease) No change ⱕ0.01 (decrease)
ⱕ0.05 (decrease) ⱕ0.01 (increase) ⱕ0.05 (increase) ⱕ0.05 (decrease) No change
Variability in the effectiveness of ANG converting enzyme inhibitors has been reported in male rat bladder tissue.18 It would appear from these studies that enalaprilat has diminished bladder effectiveness in pregnancy as well as in males compared with females in estrus and diestrus. To our knowledge abnormalities of the renin ANG system have not been assessed in interstitial cystitis. However, the bladder is known to respond in vitro to dietary changes, low Na and low Ca with a decrease in ANG receptors.7–10 Patients with interstitial cystitis are well documented to be diet sensitive, although to our knowledge the mechanism is unknown. Sodium bicarbonate given orally and intravesically provides relief of symptoms. On immunohistochemical studies we have found that dietary changes in patients with interstitial cystitis also change AT1 and AT2 receptors (unpublished data). Interstitial cystitis symptoms are diminished in pregnancy.19 In this study the renin ANG system was also dramatically altered in pregnancy. Although there is no known direct association of interstitial cystitis with the renin ANG system system, these observations suggest that the renin ANG system of the bladder deserves a further in-depth evaluation. CONCLUSIONS
Our current study supports the deactivation of ANG converting enzyme and the activation of an alternate pathway in pregnancy. It also supports the diminished tissue absorption of ANG peptide in pregnancy. Compared with similarly studied male rats the circulating conversion of ANG I to ANG II is delayed and there are enhanced responses to ANG converting enzyme inhibition, supporting subtle differences in the renin ANG system in the sexes. Under the current experimental conditions enalapril appears to have improved effectiveness in bladder tissue in nonpregnant females than in pregnant females and male rats. In addition to the pharmacological stimuli studied, other factors undoubtedly influence the renin ANG system systems. Given the role of ANG II as a mediator of cellular proliferation, angiogenesis, glycosaminoglycan synthesis and fibrosis the renin ANG system may have a role in various disease states affecting the bladder. Further studies are necessary to investigate the significance of the renin ANG system in bladder pathophysiology. REFERENCES
1. Weber, K. T.: Fibrosis, a common pathway to organ failure: angiotensin II and tissue repair. Semin Nephrol, 17: 467, 1997 2. Nielsin, A. H., Hagemann, A. and Poulsen, K.: The tissue reninangiotensin system in the female reproductive tissues. In: Tissue Renin-Angiotensin System. Edited by A. K. Mukhopadhyay and M. D. Raizada. New York: Plenum Press, 1995 3. Andersson, K.-E., Hedlund, H. and Stahl, M.: Contractions induced by angiotensin I, angiotensin II and bradykinin in isolated smooth muscle from the human detrusor. Acta Physiol Scand, 145: 253, 1992 4. Anderson, G. F., Barraco, R. A., Normile, H. J. et al: Evidence for angiotensin II receptors in the urinary bladder of the rabbit. Can J Physiol Pharmacol, 62: 390, 1984 5. Saito, M., Kondo, A., Kato, T. et al: Response of the human urinary bladder to angiotensin: a comparison between neuro-
genic and control bladders. J Urol, 149: 408, 1993 6. Herblin, W. F., Chiu, A. T., McCall, D. E. et al: Angiotensin II receptor heterogeneity. Am J Hypertension, 4: 299s, 1991 7. Aguilera, G. and Catt, K.: Regulation of vascular angiotensin II receptors in the rat during altered sodium intake. Circ Res, 49: 751, 1981 8. Speth, R. C., Singh, R., Smeby, R. R. et al: Restricted dietary sodium intake alters peripheral but not central angiotensin II receptors. Neuroendocrinology, 38: 387, 1984 9. Baksi, S. N., Abhold, R. H. and Speth, R. C.: Low-calcium diet increases blood pressure and alters peripheral but not central angiotensin II binding sites in rats. J Hypertens, 7: 423, 1989 10. Platiam, M. P., Catt, C. J., Hodges, G. D. et al: Regulation of primate angiotensin II receptors during altered sodium intake. Hypertension, 8: 1121, 1986 11. Tanabe, N., Ueno, A. and Tsujimoto, G.: Angiotensin II receptors in the rat urinary bladder smooth muscle: type 1 subtype receptors mediate contractile responses. J Urol, 150: 1056, 1993 12. Falconieri-Erspamer, G., Negri, L. and Piccinelli, D.: The use of preparations of urinary bladder smooth muscle for bioassay of and discrimination between polypeptides. NaunynSchmiedeberg’s Arch Pharmacol, 279: 61, 1973 13. Steidle, C. P., Cohen, M. L. and Neubauer, B. L.: Bradykinininduced contractions of canine prostate and bladder: effect of angiotensin-converting enzyme inhibition. J Urol, 144: 390, 1990 14. Erspamer, V., Ronzoni, G. and Falconieri-Erspamer, G.: Effects of active peptides on the isolated muscle of the human urinary bladder. Invest Urol, 18: 302, 1981 15. van Sande, M., Inokuchi, J.-I., Nagamatsu, A. et al: Tripeptidyl carboxypeptidase activity of angiotensin-converting enzyme in human tissues of the urogenital tract. Urol Int, 40: 100, 1985 16. Cheng, E. Y., Decker, R. S. and Lee, C.: Role of angiotensin II in bladder smooth muscle growth and function. Adv Exp Med Biol, 462: 183–91, 1999 17. Lindberg, B. F., Nilsson, L.-G., Hedlund, H. et al: Angiotensin I is converted to angiotensin II by a serine protease in human detrusor smooth muscle. Am J Physiol, 266: R1861, 1994 18. Weaver-Osterholtz, D. J., Reams, G., Shen, W. et al: The urinary bladder angiotensin system: Response to infusions of angiotensin I and angiotensin converting enzyme inhibitors. Am J Kidney Dis, 28: 603, 1996 19. Singh, G. S., Whitmore, L. E., Gordon, D. A. et al: Interstitial cystitis and pregnancy. Presented at annual meeting of American Urogynecology Society, Tucson, Arizona, September 1997 20. Souther, M. E., Lumpkin, R. H., Luo, K. C. et al: High performance liquid chromatographic-radioimmunoassay method for the measurement of angiotensin II peptides in human plasma. J Chromatogr, 417: 27, 1987 21. Reams, G. P., Souther, M., Parisi, M. et al: Arterial-venous determinations of the “immunoreactive” angiotensin peptides in human subjects. J Lab Clin Med, 113: 749, 1989 22. Conover, W. J.: Contingency tables. In: Practical Nonparametric Statistics. New York: Wiley, p. 203, 1971 23. Danser, A. H., van Kats, J. P., Admiraal, P. J. et al: Cardiac renin and angiotensins. Hypertension, 24: 37, 1994 24. Reams, G., Villarreal, D., Wu, Z. et al: Renal tissue angiotensin II: Response to infusion of angiotensin I and an angiotensin converting enzyme inhibitor. Am J Kidney Dis, 22: 851, 1993 25. de Jong, C. L., Dekker, G. A. and Sibai, B. M.: The reninangiotensin-aldosterone system in preeclampsia: a review. Clin Perinatol, 18: 683, 1991 26. Paller, M. S.: Mechanism of decreased pressor responsiveness to ang II, NE, and vasopressin in pregnant rats. Am J Physiol, 247: 100, 1984
Vol. 165, 1735–1738, May 2001 Printed in U.S.A.
THE BLADDER ANGIOTENSIN SYSTEM IN FEMALE RATS: RESPONSE TO INFUSIONS OF ANGIOTENSIN I AND THE ANGIOTENSIN CONVERTING ENZYME INHIBITOR ENALAPRILAT DANA WEAVER-OSTERHOLTZ, GARRY REAMS, CRISTELA FARIA DE VERGEL AND JOHN H. BAUER From the Departments of Surgery and Medicine, University of Missouri and Harry S. Truman Memorial Veterans Hospital, Columbia, Missouri
ABSTRACT
Purpose: A local renin angiotensin (ANG) system has been identified in the bladder. To our knowledge little is known about this system. To define further the physiology of this system we performed this study. Materials and Methods: The circulating and bladder tissue concentrations of ANG I and ANG II were examined in anesthetized Sprague-Dawley female rats in estrus, diestrus or pregnancy. Each was given an intravenous bolus infusion of ANG I, the ANG converting enzyme inhibitor enalaprilat or saline. Results: The mean concentrations of ANG I and ANG II were markedly higher in bladder tissue than in whole blood at the highest levels in pregnancy. The concentration of ANG I and ANG II increased significantly in the bladder tissue and circulation after the ANG I infusion in estrus and diestrus. In pregnancy only circulatory ANG I increased, while circulatory ANG II, tissue ANG I and ANG II remained unchanged. Enalaprilat infusion was associated with an increased concentration of whole blood ANG I in all groups and decreased plasma ANG II in estrus and diestrus but not in pregnancy. The bladder tissue ANG I response was unchanged in all groups. Bladder tissue ANG II was decreased in estrus and diestrus but unchanged in pregnancy. Conclusions: These data support the hypothesis that ANG converting enzyme is deactivated and an alternate pathway is activated in pregnancy. The data also demonstrate that tissue absorption of ANG peptide is diminished in pregnancy. Compared with responses in similarly studied male rats the circulating conversion of ANG I to ANG II is delayed in female rats and responses to ANG converting enzyme inhibition are enhanced, thus, suggesting subtle differences in the sexes in the renin ANG system. Enalapril appears to have more effect at the tissue level in nonpregnant females than in pregnant females and male rats. KEY WORDS: angiotensins; bladder; estrus; pregnancy, animal; rats, Sprague-Dawley
Interstitial cystitis is a disease of the bladder with an unknown etiology. Several causes have been suggested, including ischemia. The renin angiotensin (ANG) system has long been known to be aberrant systemically due to ischemia of the kidney, that is Goldblatt kidney. Evidence has accumulated for the existence of a local renin ANG system in tissues, such as the kidney, heart, vascular tissue, adrenal glands, brain, testes, ovaries, uterus and placenta. It is known to be locally aberrant in the heart when ischemia is present with coronary artery disease.1, 2 Because to date the data suggest that a tissue renin ANG system may be altered in ischemia, we have been interested in the bladder renin ANG system as a possible etiology for interstitial cystitis. However, information is limited on the role of the renin ANG system in bladder physiology.3 ANG II receptors have been identified within the bladder4 – 6 and are modulated by dietary sodium and calcium.7–10 ANG II in the bladder has been found to cause smooth muscle contractility, as demonstrated in in vitro studies in several animal species,4, 11–13 including humans.3, 5, 14 ANG I-type receptors have been found in rats to mediate the contractile response within the bladder.11 ANG II is also thought to have a role in the regulation of smooth muscle growth and collagen production in the bladder response to outlet obstruction.15 ANG converting enzyme has a relatively low level in the bladder comAccepted for publication November 17, 2000.
pared with other genitourinary tissues.16 ANG converting enzyme is considered to be the major enzyme responsible for the conversion of ANG I to ANG II, although various tissues, including the bladder, have been suggested to have alternative pathways for this conversion.17 We have reported confirmation of the autocrine-paracrine function of the tissue specific renin ANG system in the bladder.18 Therefore, sufficient data exist to hypothesize that interstitial cystitis may be affected by the tissue renin ANG system in the bladder. Interstitial cystitis has a 10:1 female-to-male ratio. In women its symptoms are exacerbated during premenstruation and ameliorated during pregnancy.19 The renin ANG system is linked to reproductive function. Circulatory levels are known to be cyclical in women and increased in pregancy.2 Likewise the local renin ANG system in the ovaries in humans has been identified to be regulated by gonadotropins.2 Because there is no reliable animal model of interstitial cystitis, we investigated the bladder response to pharmacological maneuvers in the estrus, diestrus and pregnant states in rats to determine whether they parallel clinical observations. Baseline concentrations of circulating and bladder tissue ANG I and ANG II octapeptide were directly measured in normal anesthetized female Sprague-Dawley rats in estrus, diestrus or pregnancy. In addition, circulating and bladder ANG I and ANG II responses to an acute bolus infusion of ANG I, the ANG converting enzyme inhibitor enalaprilat or saline were examined.
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1736
BLADDER ANGIOTENSIN SYSTEM IN FEMALE RATS MATERIALS AND METHODS
Animal preparation. Female Sprague-Dawley rats (Harlan Sprague Dawley, Inc., Indianapolis, Indiana) weighing between 250 and 500 gm. were housed in individual cages and maintained on regular rat chow (Purina, St. Louis, Missouri) for at least 7 days before the study. Tap water was freely available. All experiments were performed with subjects in the post-absorptive state. All animal care provided during these studies met institutional guidelines. Anesthesia was induced intraperitoneally with 100 mg./kg. Inactin (Andrew Lockwood and Associates, Sturtevant, Wisconsin). Tracheostomy was performed and PE-50 polyethylene catheters were inserted by cutdown into the carotid artery and jugular vein. The arterial catheter was connected by a P23Db strain gauge pressure transducer (Statham, Oxnard, California) to a 7714 to 041A recorder (Hewlett Packard, St. Louis, Missouri) for continuous mean arterial blood pressure monitoring. Immediately after the surgical procedures a sustained intravenous infusion of isotonic saline at a rate of 100 l. per minute was started via an infusion pump (Sage Instruments, Boston, Massachusetts). All animals entered the experimental phase of the study. Experimental design. The experimental design consisted of 2 groups of female rats. In each group rats in estrus, diestrus and pregnancy were studied. Estrus and diestrus was determined by the vaginal mucous test and pregnancy was determined by uterine size. In group 1 after a 30-minute equilibration delay 0.5 g. of ANG I (Sigma Chemical Co., St. Louis, Missouri) dissolved in 1 ml. of isotonic saline was infused for 1 minute into the jugular vein. A saline vehicle (1 ml.) served as the control. Arterial blood (6 to 8 ml.) and whole bladder tissue samples were collected to measure ANG I and ANG II 5 minutes after the initiation of the ANG Isaline infusion. All arterial blood samples were collected in 60 seconds. In group 2 after a 30-minute equilibration delay 5 g./gm. of the ANG converting enzyme inhibitor enalaprilat was dissolved in 1 ml. of isotonic saline (Merck, Sharp and Dohme, Blue Bell, Pennsylvania) and infused for 1 minute into the jugular vein. A saline vehicle (1 ml.) served as the control. Arterial blood (6 to 8 ml.) and whole bladder tissue samples were collected to measure ANG I and ANG II 30 minutes after completing the ANG converting enzyme inhibitor-saline infusion. Blood and tissue were processed as previously described.18, 20, 21 Recovery, sensitivity and reproducibility of ANG I and ANG II assays. Plasma: The mean recovery of unlabeled ANG I in whole blood in 8 experiments was 90.4% (range 83.6% to 100.6%). The lower limit of detection of ANG I in whole blood was 1 fmol./ml. In 8 experiments the intra-assay coefficient of variation for ANG I was 8.8% and the interassay coefficient of variation was 9.4%.
The approximate mean recovery of 2,500 cpm/ml. 3H-ANG II (130 cpm approximately equal to 1 pg.) added to plasma was 65% (range 63.8% to 65.7%) in 8 experiments. The recovery of unlabeled ANG II in plasma was similar to the recovery of radiolabeled ANG II. The lower limit of detection of ANG II in plasma was 0.5 fmol./ml. In 8 experiments the intra-assay coefficient of variation for ANG II was 9.5% and the interassay coefficient of variation was 10.7%. No 125IANG II was generated when 2,000 cpm/ml. 125I-ANG I were added to plasma and incubated at 4C for 30 minutes. Bladder Tissue: The approximate mean recovery of 2,000 cpm/ml. 125I-ANG I added to a bladder tissue homogenate in 8 experiments was 64.4% (range 63.5% to 66.2%). In 8 experiments the intra-assay coefficient of variation for ANG I in tissue was 2.5% and the interassay coefficient of variation was 3.8%. The approximate mean recovery of 2,000 cpm/ml. 125I-ANG II added to a bladder tissue homogenate in 8 experiments was 74.8% (range 73.6% to 76.9%). In 8 experiments the intra-assay coefficient of variation for ANG II in tissue was 3.9% and the interassay coefficient of variation was 4.2%. No 125 I-ANG II was generated when 2,000 cpm/ml. 125I-ANG I were added to a tissue homogenate and incubated at 4C for 30 minutes. Data analysis. The values of ANG I and ANG II in plasma and bladder tissue were corrected for the recovery of each throughout the whole procedure (solid phase extraction, high performance liquid chromatography and radioimmunoassay). Group data are expressed as the mean plus or minus standard error of mean. Data were analyzed using nonparametric analysis of variance. The least significant difference test was used when appropriate for post hoc analysis. Differences were considered statistically significant at p ⬍0.05.22 RESULTS
The mean arterial pressure response to the infusion of ANG I increased by 40% in all cycles after the bolus infusion of ANG I (p ⬍0.01, table 1). Mean arterial pressure was not significantly altered by the saline vehicle. Baseline bladder tissue ANG I was 20-fold higher than circulating ANG I. Baseline ANG II in the circulation and tissue was higher in pregnancy than in estrus and diestrus. Circulatory ANG I levels were significantly increased in estrus, diestrus and pregnancy after completion of the ANG I infusion. In contrast to circulatory values, an increase in tissue ANG I was observed in estrus and diestrus after completion of the ANG I infusion but not in pregnancy. The level of baseline bladder tissue ANG II was significantly 2-fold higher than the circulating level of ANG II in all cycles. Baseline circulatory and tissue ANG II were elevated in pregnancy compared to estrus
TABLE 1. Circulatory and tissue levels of Ang I, Ang II and mean arterial pressure during estrus, diestrus and pregnancy before and after Ang I infusion Estrus (mean cpm/ml. ⫾ SEM) Control mean arterial pressure After infusion Control: Circulatory Ang I After infusion Control: Circulatory Ang II After infusion Control tissue Ang I After infusion Control tissue Ang II After infusion * Compared to control p ⱕ0.05. † Compared to diestrus p ⱕ0.05. ‡ Compared to estrus p ⱕ0.05. § Compared to pregnancy p ⱕ0.05.
Diestrus (mean cpm/ml. ⫾ SEM)
Pregnancy (mean cpm/ml. ⫾ SEM)
142 ⫾ 5/144 ⫾ 5 143 ⫾ 4/189 ⫾ 3*
137 ⫾ 3/14 ⫾ 2 142 ⫾ 3/186 ⫾ 3*
134 ⫾ 4/134 ⫾ 4 133 ⫾ 5/182 ⫾ 7
75 ⫾ 22 514 ⫾ 151*
63 ⫾ 17 938 ⫾ 271*
74 ⫾ 35 351 ⫾ 83*,†
76 ⫾ 15 1,074 ⫾ 217* 2,324 ⫾ 125† 2,744 ⫾ 150*,† 154 ⫾ 22 2,217 ⫾ 195*
61 ⫾ 10§ 824 ⫾ 137*,§ 1,034 ⫾ 69§ 1,617 ⫾ 85*,§ 149 ⫾ 10§ 2,012 ⫾ 79*,§
145 ⫾ 46‡ 105 ⫾ 18‡ 1,300 ⫾ 87‡ 1,086 ⫾ 90‡ 301 ⫾ 14‡ 248 ⫾ 15*,‡
1737
BLADDER ANGIOTENSIN SYSTEM IN FEMALE RATS
TABLE 2. Circulatory and tissue levels of Ang I, Ang II and mean arterial pressure during estrus, diestrus and pregnancy before and after ANG converting enzyme infusion Estrus (mean cpm/ml. ⫾ SEM)
Diestrus (mean cpm/ml. ⫾ SEM)
144 ⫾ 5/138 ⫾ 5 143 ⫾ 4/129 ⫾ 5*
Control mean arterial pressure After infusion Control: Circulatory Ang I After infusion Control: Circulatory Ang II After infusion Control tissue Ang I After infusion Control tissue Ang II After infusion * Compared to control p ⱕ0.05. † Compared to diestrus p ⱕ0.05. ‡ Compared to estrus p ⱕ0.05. § Compared to pregnancy p ⱕ0.05.
142 ⫾ 3/138 ⫾ 3 146 ⫾ 2/136 ⫾ 4*
Pregnancy (mean cpm/ml. ⫾ SEM) 125 ⫾ 9/123 ⫾ 8 134 ⫾ 4/118 ⫾ 5*
61 ⫾ 21 1,606 ⫾ 277*
27 ⫾ 12 1,327 ⫾ 294*
82 ⫾ 29 2,602 ⫾ 502*
52 ⫾ 8† 26 ⫾ 3* 1,192 ⫾ 127 856 ⫾ 139† 146 ⫾ 16 86 ⫾ 10*
95 ⫾ 16 44 ⫾ 19*,§ 942 ⫾ 123§ 1,305 ⫾ 134 120 ⫾ 4§ 85 ⫾ 3*,§
124 ⫾ 26‡ 229 ⫾ 39*,‡ 1,703 ⫾ 115‡ 1,364 ⫾ 84*,‡ 492 ⫾ 93‡ 491 ⫾ 85‡
and diestrus. After ANG I infusion circulatory ANG II was significantly elevated in estrus and diestrus but unchanged in pregnancy. Similarly tissue ANG II was elevated in estrus and diestrus but in pregnancy there was a significant decrease. The administration of enalaprilat decreased mean arterial pressure by 10% in all cycles studied (p ⬍0.05). Mean arterial pressure was not significantly altered by the saline vehicle (table 2). Enalaprilat administration was associated with a greater than 20-fold increase in circulatory ANG I in all cycle phases studied. There was no significant change in bladder tissue ANG I in estrus and diestrus but a significant decrease was evident in pregnancy. The infusion of enalaprilat was associated with decreased circulatory ANG II in estrus and diestrus but an increase in pregnancy. Tissue ANG II responses were similar with a reduction in the circulatory and tissue levels in estrus and diestrus but no change in pregnancy. DISCUSSION
Our results demonstrate that in anesthetized female Sprague-Dawley rats the concentration of bladder tissue ANG I is more than 10-fold that of the circulatory level of ANG I and bladder tissue ANG II is twice that of circulatory ANG II. These values are higher than those explained by the simple tissue uptake of ANG peptide from the circulation. This finding is compatible with similar observations in male rats (table 3)18 and in other tissues, including the heart23 and kidney,24 in which local autocrine paracrine renin ANG system systems have been described. Angiotensin I with a half-life of 30 seconds was rapidly metabolized to ANG II in the circulation of the rats in estrus and diestrus (a 10-fold increase) but not in the pregnant population. Bladder tissue ANG I was markedly increased after ANG I infusion compared with the saline control and it was rapidly converted to ANG II in estrus and diestrus but not in pregnancy. Compared with previously performed studies in male rats18 responses during estrus and diestrus in females were similar to the responses in males, although the conversion of ANG I to ANG II was slower in the females
(table 3.). On the other hand, the response in pregnancy was dissimilar, as it was to the estrus and diestrus studies. The absence of conversion of circulatory ANG I to ANG II in pregnancy with increased circulatory ANG I may be explained by diminished ANG converting enzyme activity. Decreased ANG converting enzyme has been measured in normal unchallenged human pregnancies.25 The unchanged bladder tissue level of ANG I in pregnancy despite high circulatory ANG I supports impeded tissue uptake in pregnancy compared with nonpregnant states in females as well as in males. It has been observed that pregnancy is less responsive to peptide administration25 and adversely affected by ANG converting enzyme inhibitors with resulting fetal distress and death. Receptor levels have been evaluated in rat smooth muscle and have not been found to change in response to chronic ANG II infusion.26 If ANG converting enzyme activity and tissue uptake of peptide are diminished, as indicated by these studies, these alterations would explain the response to peptide infusion observed in pregnancy. The circulating ANG peptide response to ANG converting enzyme inhibition was similar in estrus and diestrus but not in pregnancy. Circulating ANG I was increased more than 10-fold, while circulating ANG II decreased by 50% in estrus and diestrus, which was an exaggerated response compared with that in previously studied male rats (table 4). On the other hand, in pregnancy a doubling of circulatory ANG II was noted with elevated circulatory ANG I. Others have suggested that ANG converting enzyme inhibition is not effective for blocking ANG I induced bladder smooth muscle contraction or the conversion of ANG I to ANG II, suggesting alternative pathways for ANG II generation.3, 9, 16 The inability of enalaprilat to block circulatory ANG II production suggests that an alternate circulatory pathway is activated in pregnancy. The bladder ANG I and ANG II response to ANG converting enzyme inhibitor was similar to that observed in the circulation in estrus and diestrus but again not in pregnancy. Enalaprilat was associated with a significant decrease in tissue ANG I but no change in tissue ANG II in pregnancy.
TABLE 3. Observed response to Ang I infusion Male p Value 2 Mins. Mean arterial pressure ⱕ0.01 (increase) Circulating Ang I No change Circulating Ang II ⱕ0.01 (increase) Tissue Ang I No change Tissue Ang II ⱕ0.01 (increase) In females in diestrus all p ⱕ0.01 (increase).
Female p Value 5 Mins. ⱕ0.01 (increase) No change No change ⱕ0.05 (increase) No change
Estrus ⱕ0.01 ⱕ0.01 ⱕ0.01 ⱕ0.05 ⱕ0.01
(increase) (increase) (increase) (increase) (increase)
Pregnancy ⱕ0.01 (increase) ⱕ0.05 (increase) No change No change ⬉0.05 (decrease)
1738
BLADDER ANGIOTENSIN SYSTEM IN FEMALE RATS TABLE 4. Observed response to ANG converting enzyme infusion Female p Value
Male p Value Mean arterial pressure Circulating Ang I Circulating Ang II Tissue Ang I Tissue Ang II
ⱕ0.05 (decrease) ⱕ0.05 (increase) ⱕ0.05 (decrease) No change No change
Estrus
Diestrus
Pregnancy
ⱕ0.05 (decrease) ⱕ0.01 (increase) ⱕ0.05 (decrease) No change ⱕ0.01 (decrease)
ⱕ0.05 (decrease) ⱕ0.01 (increase) ⱕ0.05 (decrease) No change ⱕ0.01 (decrease)
ⱕ0.05 (decrease) ⱕ0.01 (increase) ⱕ0.05 (increase) ⱕ0.05 (decrease) No change
Variability in the effectiveness of ANG converting enzyme inhibitors has been reported in male rat bladder tissue.18 It would appear from these studies that enalaprilat has diminished bladder effectiveness in pregnancy as well as in males compared with females in estrus and diestrus. To our knowledge abnormalities of the renin ANG system have not been assessed in interstitial cystitis. However, the bladder is known to respond in vitro to dietary changes, low Na and low Ca with a decrease in ANG receptors.7–10 Patients with interstitial cystitis are well documented to be diet sensitive, although to our knowledge the mechanism is unknown. Sodium bicarbonate given orally and intravesically provides relief of symptoms. On immunohistochemical studies we have found that dietary changes in patients with interstitial cystitis also change AT1 and AT2 receptors (unpublished data). Interstitial cystitis symptoms are diminished in pregnancy.19 In this study the renin ANG system was also dramatically altered in pregnancy. Although there is no known direct association of interstitial cystitis with the renin ANG system system, these observations suggest that the renin ANG system of the bladder deserves a further in-depth evaluation. CONCLUSIONS
Our current study supports the deactivation of ANG converting enzyme and the activation of an alternate pathway in pregnancy. It also supports the diminished tissue absorption of ANG peptide in pregnancy. Compared with similarly studied male rats the circulating conversion of ANG I to ANG II is delayed and there are enhanced responses to ANG converting enzyme inhibition, supporting subtle differences in the renin ANG system in the sexes. Under the current experimental conditions enalapril appears to have improved effectiveness in bladder tissue in nonpregnant females than in pregnant females and male rats. In addition to the pharmacological stimuli studied, other factors undoubtedly influence the renin ANG system systems. Given the role of ANG II as a mediator of cellular proliferation, angiogenesis, glycosaminoglycan synthesis and fibrosis the renin ANG system may have a role in various disease states affecting the bladder. Further studies are necessary to investigate the significance of the renin ANG system in bladder pathophysiology. REFERENCES
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