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Aminopeptidase activities after water deprivation in male and female rats
Regulatory Peptides 101 Ž2001. 189–194 www.elsevier.comrlocaterregpep
Aminopeptidase activities after water deprivation in male and female rats I. Prieto a , J.M. Martınez ´ a, M.J. Ramırez ´ a, G. Arechaga a, F. Alba b, M. De Gasparo c,1, F. Vargas b, A.B. Segarra a , M. Ramırez ´ a,) a
b
Unit of Physiology, UniÕersity of Jaen, ´ Bldg B-3, 23071 Jaen, ´ Spain Department of Physiology, UniÕersity of Granada, 18071, Granada, Spain c NoÕartis Pharma, CH-4002 Basel, Switzerland
Received 14 February 2001; received in revised form 22 May 2001; accepted 7 June 2001
Abstract Aminopeptidases ŽAPs. play a major role in the metabolism of circulating and local peptides, such as angiotensins and vasopressin, substances involved in the control of blood pressure and water balance. In the present work, we studied the influence of dehydration on angiotensinases and vasopressin-degrading activity. Since sex differences may exist in the regulation of water balance by angiotensin II and differential sexual steroid modulation of vasopressin secretion, in response to osmotic stimulation have been reported, gonadotropin releasing hormone ŽGnRH.-degrading activity was also analysed in serum, neurohypophysis and adrenal glands of male and female rats. Our results did not suggest sex differences in the response to changes in osmolality. GnRH-degrading activity decreased in serum of dehydrated males and females, which suggests a longer action of the peptide under these conditions. In neurohypophysis, there was an increase in the activity of aminopeptidase A ŽAPA., the enzyme responsible for the metabolism of angiotensin II to angiotensin III. This occurs with a decrease in alanyl aminopeptidase activity, which would lead to a prolonged action of angiotensin III by reduction of its metabolism. In adrenals of dehydrated animals, the results would imply a high degree of metabolism of angiotensin III and vasopressin. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Dehydration; Sex differences; Neurohypophysis; Adrenal gland
1. Introduction The hormonal renin–angiotensin–aldosterone system ŽRAAS. plays a major role in the control of blood pressure and water balance w1x. In addition, there are local tissue renin–angiotensin systems ŽRAS. in several organs whose components possess additional autocrine or paracrine actions w2x, complementary to the systemic RAAS. Thus, local RAS, including angiotensinase activities, have been described especially in hypothalamus–neurohypophyseal neurons w3x and adrenal glands w3,4x, which are involved in the regulation of blood pressure and water balance. Aminopeptidase ŽAP. activities play a major role in the metabolism of circulating and local peptides involved in blood pressure and water balance. Glutamyl aminopeptidase ŽGluAP. Žaminopeptidase A., by removing the Asp
)
Corresponding author. Tel.: q34-953-012302; fax: q34-953-012141. E-mail address: [email protected] ŽM. Ramırez ´ .. 1 Present address: MGConsulting, rue es Planches 123, 2842 Rossemaison, Switzerland.
residue, is the enzyme responsible for the rapid metabolism of Ang II w5x. In addition, Aspartate aminopeptidase ŽAspAP. also cleaves Asp residues but this enzyme is distinct from GluAP. This new enzyme is also probably responsible for the metabolism of Ang I and Ang II w6x. Angiotensin III, produced from Ang II by GluAP or AspAP, is further converted to Ang IV by AP B ŽArgAP. or AP M ŽAlaAP.. Therefore, GluAP, AspAP, AlaAP and ArgAP are considered as angiotensinases. In addition, it should be taken into account that other vasoactive peptides such as neurokinin, substance P or kinins are susceptible substrates of AlaAP and ArgAP w7,8x. On the other hand, CysAP has been reported to hydrolyse oxytocin and vasopressin but, while its functional role as oxytocinase is well known, the actual role as vasopressin-degrading activity is not well defined w3x. Aminopeptidase activity is modified in several experimental models of hypertension, hydroelectrolytic unbalance, and after AT1 receptor blockade w3,9,10x. Sex differences in AP activities w11,12x and in the regulation of water balance have been reported w13x. Sex differences Žor similar differences. have also been advocated for the regu-
0167-0115r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 0 1 1 5 Ž 0 1 . 0 0 2 8 8 - 9
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I. Prieto et al.r Regulatory Peptides 101 (2001) 189–194
lation of water balance by angiotensin II w14x and sexual steroids were claimed to differentially modulate vasopressin secretion in response to osmotic stimulation w15x. Consequently, depending on the osmotic status of the animal, we could suspect sex differences in the metabolism of gonadotropin releasing hormone ŽGnRH.. The experiments reported here were performed to determine the influence of dehydration on angiotensinases, vasopressindegrading activity and GnRH-degrading activity Žpyroglutamyl aminopeptidase, pGluAP. w16x in serum, neurohypophysis and the adrenal gland of male and female rats, as locations directly involved in the neuroendocrine response to changes in the osmotic status.
2. Materials and methods 2.1. Animals Twelve male and 20 female Sprague–Dawley rats weighing 150–200 g were used in this study. Males and females were randomly divided into two groups: control male ŽC-M, n s 6. and female ŽC-F, n s 10. and dehydrated male ŽD-M, n s 6. and female ŽD-F, n s 10.. The animals were kept in a temperature-controlled room Ž24 " 1 8C. with a 12r12-h lightrdark schedule and housed in standard laboratory cages. Laboratory food and water were provided ad libitum to control groups, whereas only food was provided to the dehydrated groups. The dehydrated groups were water deprived for 5 days. After this time, females were selected only at proestrous, verified by daily vaginal smears, resulting: C-F, n s 6 and D-F, n s 5. The rest of the females were discarded. Then, all the selected animals were killed under equithensin anesthesia Ž2 mlrkg body weight.. The experimental procedures for animal use and care were in accordance with European Communities Council Directive 86r609rEEC.
adsorbent polymeric Biobeads SM-2 Ž100 mgrml. ŽBioRad, Richmond, VA, USA. and shaking for 2 h at 4 8C. 2.3. Procedures for enzymatic assays AlaAP, ArgAP and CysAP were measured fluorometrically using as substrates the aminoacyl-b-naphthylamides ŽaaNNap.: AlaNNap, ArgNNap and CysNNap, according to the modified method of Greenberg w17x. Ten microliters of each supernatant and serum were incubated during 30 min at 25 8C with 1 ml of the substrate solution: 2.14 mgr100 ml AlaNNap or 3.35 mgr 100 ml ArgNNap or 5.63 mgr100 ml CysNNap, 10 mgr100 ml bovine serum albumin ŽBSA., and 10 mgr100 ml dithiothreitol ŽDTT. in 50 mM of phosphate buffer, pH 7.4, for AlaAP and ArgAP; and 50 mM HCl–Tris buffer, pH 6, for CysAP. pGluAP was measured in a fluorogenic assay using pGluNNap as the substrate, according to the modified method of Schwabe and McDonald w18x: 10 ml of each supernatant was incubated during 120 min at 37 8C with 1 ml of substrate solution Ž2.54 mgr100 ml pGluNNap, 10 mgr100 ml BSA, 10 mgr100 ml DTT, 37.8 mgr100 ml EDTA in 50 mmolrl of phosphate buffer, pH 7.4.. AspAp was determined fluorometrically with AspNNap as the substrate, according to the method of Cheung and Cushman w19x modified as follows: 10 ml of each supernatant was incubated for 120 min at 37 8C with 1 ml of the substrate solution Ž2.58 mgr100 ml AspNNap, 10 mgr100 ml BSA, 10 mgr100 ml DTT and 39.4 mgr100 ml MnCl 2 in 50 mmolrl HCl–Tris buffer, pH 7.4.. GluAP was also determined in a fluorometric assay using GluNNap as the substrate according to the method of Tobe et al. w20x modified as follows: 10 ml of each supernatant was incubated during 120 min at 37 8C with 1
2.2. Samples Blood samples were obtained through left cardiac ventricle puncture, and serum was isolated by centrifugation for 10 min at 2000 = g. The neurohypophysis and the whole right adrenal were quickly removed and frozen in dry ice. To obtain the soluble fraction, tissue samples were homogenized in 10 volumes of 10 mM HCl–Tris buffer ŽpH s 7.4. and ultracentrifugated at 100 000 = g for 30 min Ž4 8C.. The resulting supernatants were used to measure soluble enzymatic activity and protein content, assayed in triplicate. To solubilize membrane proteins, the pellets were rehomogenized in HCl–Tris buffer ŽpH s 7.4. plus 1% Triton X-100. After centrifugation Ž100 000 = g, 30 min, 4 8C., the supernatants were used to measure membrane-bound activity and proteins, also in triplicate. To ensure the complete recovery of activity, the detergent was removed from the medium by adding to the samples
Fig. 1. Total protein content in serum of control and dehydrated males and females. Values, expressed as mg protrml, represent means"S.E.M. levels of groups of 6–10 animals assayed individually. Žq. represents differences between males vs. females. Ž ) . represents differences between control vs. dehydrated. ) , q p- 0.05.
I. Prieto et al.r Regulatory Peptides 101 (2001) 189–194
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Fig. 2. Serum aminopeptidase activities in control male ŽC-M, n s 6. control female ŽC-F, n s 10., dehydrated male ŽD-M, n s 6. and dehydrated female ŽD-F, n s 10.. Values represent means" S.E.M. levels of groups of 6–10 animals assayed individually. Aminopeptidase ŽAP. activities are expressed as nmol of alanyl- ŽAlaAP., arginyl- ŽArgAP. and cystinyl- ŽCysAP. b-naphthylamide hydrolysed per min per mg of protein or pmol of pyroglutamylŽpGluAP., glutamyl- ŽGluAP. and aspartyl- ŽAspAP. b-naphthylamide hydrolysed per min per mg of protein. Žq. represents differences between males vs. females. Ž ) . represents differences between control vs. dehydrated. ) , qp - 0.05; qq p - 0.01; qq p - 0.001.
ml of the substrate solution Ž2.72 mgr100 ml GluNNap, 10 mgr100 ml BSA, 10 mgr100 ml DTT and 0.555 gr100 ml CaCl 2 in 50 mmolrl HCl–Tris, pH 7.4.. All the reactions were stopped by adding 1 ml of 0.1 molrl of acetate buffer, pH 4.2. The amount of b-naph-
thylamine released as a result of the enzymatic activity was measured fluorometrically at 412-nm emission wavelength with an excitation wavelength of 345 nm. Proteins were quantified in triplicate by the method of Bradford w21x, using BSA as a standard. Depending on the substrate
Fig. 3. Soluble ŽSOL. and membrane-bound ŽM-B. aminopeptidase activities in the neurohypophysis of control male ŽC-M, n s 6. control female ŽC-F, n s 10., dehydrated male ŽD-M, n s 6. and dehydrated female ŽD-F, n s 10.. Values represent means" S.E.M. levels of groups of 6–10 animals assayed individually. Aminopeptidase ŽAP. activities are expressed as pmol of alanyl- ŽAlaAP., arginyl- ŽArgAP., cystinyl- ŽCysAP., pyroglutamyl- ŽpGluAP., glutamyl- ŽGluAP. and aspartyl- ŽAspAP. b-naphthylamide hydrolysed per min per mg of protein. Žq. represents differences between males vs. females. Ž ) . represents differences between control vs. dehydrated. ) p - 0.05; ) ) , qp - 0.01.
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and the tissue studied, specific soluble and membranebound aminopeptidase activities were expressed as nmol or pmol of AlaNNap, ArgNNap, CysNNap, AspNNap, pGluNNap or GluNNap hydrolyzed per min per mg of protein. Fluorogenic assays were linear with respect to time of hydrolysis and protein content. 2.4. Statistical analysis The data were analysed by two-way analysis of variance ŽANOVA.. For gender, two factors were considered: male and female. For water consumption, two factors were considered: control and dehydrated. Since a significant interaction between gender and water consumption was observed, the two variables were analysed separately by the one-way ANOVA. Data were processed with the statistical graphics system ŽSTATGRAPHICS.. Post-hoc comparisons were made using Tukey’s test, and P values below 0.05 were considered significant.
3. Results In serum of control as well as in dehydrated rats, the total protein content was higher in females than in males
Ž p - 0.05.. Dehydration increased significantly in a similar proportion the protein content in males Ž8.2%. and females Ž7.5%.. Therefore, the difference between dehydrated males and females remains as in the controls Ž9%. ŽFig. 1.. With regards to the effect of dehydration, there was a significant Ž p - 0.05. decrease of pGluAP activity in serum of dehydrated males and females. There was no other change in aminopeptidase activities in serum after dehydration. Except for GluAP, the other aminopeptidase activities were significantly lower in females than in males Ž p - 0.001 for AlaAP, ArgAP, and CysAP; p - 0.01 for pGluAP and p - 0.05 for AspAP.. In control, as well as in dehydrated animals, the highest activity levels were observed for AlaAP in both male and female rats ŽFig. 2.. In the tissues analysed, with the exception of membrane-bound AspAP activity in neurohypophysis, the changes produced after water deprivation were observed essentially in the soluble fraction. In the neurohypophysis, while soluble AlaAP activity decreased significantly Ž p 0.01 in males and p - 0.05 in females., soluble GluAP activity increased Ž p - 0.05. in both dehydrated males and females. In contrast, membrane-bound AspAP activity increased significantly Ž P - 0.05. after dehydration, in males, but not in females. Soluble CysAP activity was also significantly higher in females than in males in neurohypophysis. In this location, control and dehydrated male and
Fig. 4. Soluble ŽSOL. and membrane-bound ŽM-B. aminopeptidase activities in the adrenal gland of control male ŽC-M, n s 6. control female ŽC-F, n s 10., dehydrated male ŽD-M, n s 6. and dehydrated female ŽD-F, n s 10.. Values represent means" S.E.M. levels of groups of 6–10 animals assayed individually. Aminopeptidase activities are expressed as nmol of alanyl- ŽAlaAP., arginyl- ŽArgAP. and cystinyl- ŽCysAP. b-naphthylamide hydrolysed per min per mg of protein or pmol of pyroglutamyl- ŽpGluAP., glutamyl- ŽGluAP. and aspartyl- ŽAspAP. b-naphthylamide hydrolysed per min per mg of protein. Žq. represents differences between males vs. females. Ž ) . represents differences between control vs. dehydrated. qp - 0.05; ) ) , qp - 0.01; ))) p - 0.001.
I. Prieto et al.r Regulatory Peptides 101 (2001) 189–194
female rats exhibited their highest levels for soluble ArgAP and membrane-bound CysAP activities ŽFig. 3.. In the adrenal gland, soluble AlaAP Ž p - 0.01., ArgAP Ž p - 0.01. and CysAP Ž p - 0.001. activities increased significantly after dehydration in males and females. Comparing sex, membrane-bound AlaAP and ArgAP activities were higher Ž p - 0.01. in females than in males. However, soluble AspAP was higher Ž p - 0.01. in males. The adrenal gland showed its highest levels for soluble and membrane-bound AlaAP and ArgAP activities in all the different biological preparations ŽFig. 4..
4. Discussion Angiotensin II increases drinking and blood pressure when administered intracerebroventricularly. Intracerebroventricular injections of antiserum directed against aminopeptidase A ŽAPA., the most important enzyme that degrades Ang II to Ang III, reduced the drinking and blood pressure responses to 10 pmol Ang II by 73% and 59%, respectively. APA antiserum had no effect on responses to Ang III administered intracerebroventricularly. A Glu-thiol inhibitor of APA also reduced Ang II-induced drinking. These results suggest that the metabolism of Ang II to Ang III is an obligatory activation step for the brain angiotensin system w22x. Angiotensin IIrIII and vasopressin coexist in neurones as well as in fibres of the hypothalamo-neurohypophyseal system w23x. In addition, it has been conclusively demonstrated that the action of Ang II on vasopressin release depends upon the prior conversion of Ang II to Ang III by aminopeptidase A or A-like activity w24x. The further inhibition of AlaAP, which metabolises Ang III, induces vasopressin release by increasing the half-life of Ang III w25x. Therefore, Ang III is one of the main stimulatory peptides in the control of vasopressin release. Thus, the AP response to dehydration in neurohypophysis, reported for the first time in the present work, physiologically confirms this pattern: an increase in AP A ŽGluAP. and A-like activity ŽAspAP. leads to an increased metabolism of Ang II to Ang III. This occurs with a decrease in AlaAP activity, which will prolong the action of Ang III by the reduction of its metabolism. Interestingly, the response in adrenals is highly similar to that described in the reduced renal mass model of arterial hypertension, the so-called polydipsia–polyuria syndrome. It is characterised by the production of increased urine volume with reduced osmolality, despite an increased secretion of vasopressin w3x. The latter may be secondary to the increased plasma osmolality produced by the hypernatremia in these hypertensive animals. The present results imply a high degree of metabolism of Ang III and vasopressin in the adrenals of dehydrated animals and suggest that locally, the adrenal gland responds in the same way to an increase in plasmatic osmolality.
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Most of the changes observed in AP activities after water deprivation occurred in the soluble components of the neurohypophysis and the adrenal gland. Since activation of cell membrane-bound receptors is crucial for the physiological actions of Ang II, it is unclear how those changes may contribute to angiotensin metabolism. However, there is growing evidence that receptor-mediated endocytosis of Ang II is important for some physiological responses to Ang II and its subsequent degradation in lysosomes may also serve an important function in the disposal of this peptide w26x. Differences in the response of soluble or membrane-bound components of AP activities may be tissue-dependent. Previous studies on AP activities in a rat saline model of hypertension Žreduced renal mass, RRM., also modified specifically the soluble components of AP in neurohypophysis and adrenal gland w3x. However, there were important changes, essentially in membranebound activity, in the renal tissue of two animal models of hypertension, RRM and Goldblatt two-kidney one clip w9x. These changes were also selective for membrane-bound AP activities in the renal tissue of water deprived rats Ždata not shown.. On the other hand, it is unclear whether there is gender differences in the control of water balance. It has been reported that plasma osmolality was higher in dehydrated females than in dehydrated males w13x. However, other authors did not find any sex difference in plasma osmolality after water deprivation w27x. It has also been described that central angiotensinergic mechanisms controlling vasopressin release are differently influenced by gender w14x. Gonadectomy prevented the increase in hypothalamic vasopressin mRNA in response to increased osmolality in both males and females w28x, but there is a differential effect of estradiol and dihydrotestosterone on vasopressin mRNA, which imply distinct actions for these steroids w15x. We have previously reported gender differences in AP activities. There was dissociation between the results obtained in serum and tissues w11,29x. This is confirmed in the present results. While male AP activities were higher in serum, the tissue levels were higher in females. In addition, we have also reported significant sex differences and an influence of cholesterol and steroid hormones on AP activities. Depending on the nature of the AP, these enzymes responded in different ways to the presence of these substances and also responded differently to gonadectomy w12x. After dehydration, we found a similar increase in protein content in males Ž8.2%. and females Ž7.5%.. Therefore, the sex difference is proportionally the same than in controls, suggesting that there is no difference between sexes in the change in osmolality after dehydration. This hypothesis could be supported by the fact that pGluAP did not change after dehydration in neurohypophysis and adrenal gland of males and females, or that changes in serum occur in the same direction and intensity in males and females. On the other hand, this
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decrease in the metabolism of GnRH could permit a longer effect of this peptide, thus increasing the stimulation of gonadotropin release and enhancing the stimulatory effect of gonadal steroids on vasopressin secretion in response to hyperosmolality. In conclusion, according to the present results, there is no argument in favor of a sex difference in response to changes in osmolality. However, in water balance, a target organ for Ang II and vasopressin is the kidney. In this tissue, we have observed important changes in membrane-bound activities and gender differences in the response to water deprivation Ždata not shown.. References w1x De Gasparo M, Bottari S, Levens NR. Characteristics of angiotensin II receptors and their role in cell and organ physiology. In: Laragh JH, Brenner BM, editors. Hypertension: Pathophysiology, Diagnosis, and Management. New York: Raven Press 1995:1695–720. w2x Bottari SP, De Gasparo M, Steckelings UM, Levens NR. Angiotensin II receptor subtypes: characterization, signalling mechanisms, and possible physiological implications. Front Neuroendocrinol 1993;14:123–71. w3x Prieto I, Martinez A, Martinez JM, Ramirez MJ, Vargas F, Alba F, et al. Activities of aminopeptidases in a rat saline model of volume hypertension. Horm Metab Res 1998;30:246–8. w4x Murlow PJ. Renin–angiotensin system in the adrenal. Horm Metab Res 1998;30:346–9. w5x Wright JW, Harding JW. Brain angiotensin receptor subtypes AT1, AT2, and AT4 and their functions. Regul Pept 1995;59:269–95. w6x Wilk S, Wilk E, Magnusson RP. Purification, characterization, and cloning of a cytosolic aspartyl aminopeptidase. J Biol Chem 1998; 273:15961–70. w7x Russel JS, Chi H, Lantry LE, Stephens RE, Ward PE. Substance P and neurokinin A metabolism by cultured human skeletal muscle myocytes and fibroblasts. Peptides 1996;17:1397–403. w8x Ward PE, Benter IF, Dick L, Wilk S. Metabolism of vasoactive peptides by plasma and purified renal aminopeptidase M. Biochem Pharmacol 1990;40:1725–32. w9x Ramirez M, Prieto I, Martinez JM, Vargas F, Alba F. Renal aminopeptidase activities in animal models of hypertension. Regul Pept 1997;72:155–9. w10x Prieto I, Martınez JM, Hermoso F, Ramırez ´ ´ MJ, Vargas F, De Gasparo M, et al. Oral administration of losartan influences aminopeptidase activity in the frontal cortex. Eur Neuropsychopharmacol 2000;10:279–82. w11x Martinez JM, Prieto I, Ramirez MJ, De Gasparo M, Hermoso F, Arias JM, et al. Sex differences and age-related changes in human serum aminopeptidase A activity. Clin Chim Acta 1998;274:53–61. w12x Martınez JM, Ramirez MJ, Prieto I, Alba F, Ramirez M. Sex ´ differences and in vitro effects of steroids on serum aminopeptidase activities. Peptides 1998;19:1637–40.
w13x Dai WJ, Yao T. Effects of dehydration and salt-loading on hypothalamic vasopressin mRNA level in male and female rats. Brain Res 1995;676:178–82. w14x Stone JD, Crofton JT, Share L. Sex differences in central cholinergic and angiotensinergic control of vasopressin release. Am J Physiol 1992;263:R1030–4. w15x Swenson KL, Sladex CD. Gonadal steroid modulation of vasopressin secretion in response to osmotic stimulation. Endocrinology 1997;138:2089–97. w16x Martinez JM, Ramirez MJ, Prieto I, Petzelt C, Hermoso F, Alba F, et al. Human serum pyroglutamyl-b-naphthylamide hydrolyzing activity during development and aging. Arch Gerontol Geriatr 1999; 28:31–6. w17x Greenberg LJ. Fluorometric measurement of alkaline phosphatase and aminopeptidase activities in the order of 10y1 4 mole. Biochem Biophys Res Commun 1962;9:430–5. w18x Schwabe C, McDonald JK. Demonstration of a pyroglutamyl residue at the N terminus of the B-chain of porcine relaxin. Biochem Biophys Res Commun 1977;74:1501–4. w19x Cheung HS, Cushman DW. A soluble aspartate aminopeptidase from dog kidney. Biochim Biophys Acta 1971;242:190–3. w20x Tobe H, Kojima F, Aoyagi T, Umezawa H. Purification by affinity chromatography using amastatin and properties of aminopeptidase A from pig kidney. Biochim Biophys Acta 1980;613:459–68. w21x Bradford MM. A rapid and sensitive method for quantification of microgram quantities of protein-dye binding. Anal Biochem 1976;72: 248–54. w22x Song L, Wilk S, Healy DP. Aminopeptidase A antiserum inhibits intracerebroventricular angiotensin II-induced dipsogenic and pressor responses. Brain Res 1997;744:1–6. w23x Imboden H, Felix D. An immunocytochemical comparison of the angiotensin and vasopressin hypothalamo-neurohypophysial systems in normotensive rats. Regul Pept 1991;36:197–218. w24x Zini S, Demassey Y, Fournie-Zaluski MC, Bischoff L, Corvol P, ´ Llorens-Cortes C, et al. Inhibition of vasopressinergic neurons by central injection of a specific aminopeptidase A inhibitor. NeuroReport 1998;9:825–8. w25x Reaux A, de Mota N, Zini S, Cadel S, Fournie-Zaluski MC, Roques BP, et al. PC18, a specific aminopeptidase N inhibitor, induces vasopressin release by increasing the half-life of brain angiotensin III. Neuroendocrinology 1999;69:370–6. w26x Van Kats JP, de Lannoy LM, Danser AHJ, van Meegen JR, Verdouw PD, Schalekamp MAD. Angiotensin II type 1 ŽAT1. receptor-mediated accumulation of angiotensin II in tissues and its intracellular half-life in vivo. Hypertension 1997;30:42–9. w27x Wang YX, Crofton JT, Miller J, Sigman CJ, Liu H, Huber JM, et al. Sex difference in urinary concentrating ability of rats with water deprivation. Am J Physiol 1996;270:R550–5. w28x Crowley RS, Amico JA. Gonadal steroid modulation of oxytocin and vasopressin gene expression in the hypothalamus of the osmotically stimulated rat. Endocrinology 1993;133:2711–8. w29x De Gandarias JM, Ramirez M, Zulaica J, Casis L. Aminopeptidase Žarylamidase. activity in discrete areas of the rat brain: sex differences. Horm Metab Res 1989;21:285–6.
Aminopeptidase activities after water deprivation in male and female rats I. Prieto a , J.M. Martınez ´ a, M.J. Ramırez ´ a, G. Arechaga a, F. Alba b, M. De Gasparo c,1, F. Vargas b, A.B. Segarra a , M. Ramırez ´ a,) a
b
Unit of Physiology, UniÕersity of Jaen, ´ Bldg B-3, 23071 Jaen, ´ Spain Department of Physiology, UniÕersity of Granada, 18071, Granada, Spain c NoÕartis Pharma, CH-4002 Basel, Switzerland
Received 14 February 2001; received in revised form 22 May 2001; accepted 7 June 2001
Abstract Aminopeptidases ŽAPs. play a major role in the metabolism of circulating and local peptides, such as angiotensins and vasopressin, substances involved in the control of blood pressure and water balance. In the present work, we studied the influence of dehydration on angiotensinases and vasopressin-degrading activity. Since sex differences may exist in the regulation of water balance by angiotensin II and differential sexual steroid modulation of vasopressin secretion, in response to osmotic stimulation have been reported, gonadotropin releasing hormone ŽGnRH.-degrading activity was also analysed in serum, neurohypophysis and adrenal glands of male and female rats. Our results did not suggest sex differences in the response to changes in osmolality. GnRH-degrading activity decreased in serum of dehydrated males and females, which suggests a longer action of the peptide under these conditions. In neurohypophysis, there was an increase in the activity of aminopeptidase A ŽAPA., the enzyme responsible for the metabolism of angiotensin II to angiotensin III. This occurs with a decrease in alanyl aminopeptidase activity, which would lead to a prolonged action of angiotensin III by reduction of its metabolism. In adrenals of dehydrated animals, the results would imply a high degree of metabolism of angiotensin III and vasopressin. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Dehydration; Sex differences; Neurohypophysis; Adrenal gland
1. Introduction The hormonal renin–angiotensin–aldosterone system ŽRAAS. plays a major role in the control of blood pressure and water balance w1x. In addition, there are local tissue renin–angiotensin systems ŽRAS. in several organs whose components possess additional autocrine or paracrine actions w2x, complementary to the systemic RAAS. Thus, local RAS, including angiotensinase activities, have been described especially in hypothalamus–neurohypophyseal neurons w3x and adrenal glands w3,4x, which are involved in the regulation of blood pressure and water balance. Aminopeptidase ŽAP. activities play a major role in the metabolism of circulating and local peptides involved in blood pressure and water balance. Glutamyl aminopeptidase ŽGluAP. Žaminopeptidase A., by removing the Asp
)
Corresponding author. Tel.: q34-953-012302; fax: q34-953-012141. E-mail address: [email protected] ŽM. Ramırez ´ .. 1 Present address: MGConsulting, rue es Planches 123, 2842 Rossemaison, Switzerland.
residue, is the enzyme responsible for the rapid metabolism of Ang II w5x. In addition, Aspartate aminopeptidase ŽAspAP. also cleaves Asp residues but this enzyme is distinct from GluAP. This new enzyme is also probably responsible for the metabolism of Ang I and Ang II w6x. Angiotensin III, produced from Ang II by GluAP or AspAP, is further converted to Ang IV by AP B ŽArgAP. or AP M ŽAlaAP.. Therefore, GluAP, AspAP, AlaAP and ArgAP are considered as angiotensinases. In addition, it should be taken into account that other vasoactive peptides such as neurokinin, substance P or kinins are susceptible substrates of AlaAP and ArgAP w7,8x. On the other hand, CysAP has been reported to hydrolyse oxytocin and vasopressin but, while its functional role as oxytocinase is well known, the actual role as vasopressin-degrading activity is not well defined w3x. Aminopeptidase activity is modified in several experimental models of hypertension, hydroelectrolytic unbalance, and after AT1 receptor blockade w3,9,10x. Sex differences in AP activities w11,12x and in the regulation of water balance have been reported w13x. Sex differences Žor similar differences. have also been advocated for the regu-
0167-0115r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 0 1 1 5 Ž 0 1 . 0 0 2 8 8 - 9
190
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lation of water balance by angiotensin II w14x and sexual steroids were claimed to differentially modulate vasopressin secretion in response to osmotic stimulation w15x. Consequently, depending on the osmotic status of the animal, we could suspect sex differences in the metabolism of gonadotropin releasing hormone ŽGnRH.. The experiments reported here were performed to determine the influence of dehydration on angiotensinases, vasopressindegrading activity and GnRH-degrading activity Žpyroglutamyl aminopeptidase, pGluAP. w16x in serum, neurohypophysis and the adrenal gland of male and female rats, as locations directly involved in the neuroendocrine response to changes in the osmotic status.
2. Materials and methods 2.1. Animals Twelve male and 20 female Sprague–Dawley rats weighing 150–200 g were used in this study. Males and females were randomly divided into two groups: control male ŽC-M, n s 6. and female ŽC-F, n s 10. and dehydrated male ŽD-M, n s 6. and female ŽD-F, n s 10.. The animals were kept in a temperature-controlled room Ž24 " 1 8C. with a 12r12-h lightrdark schedule and housed in standard laboratory cages. Laboratory food and water were provided ad libitum to control groups, whereas only food was provided to the dehydrated groups. The dehydrated groups were water deprived for 5 days. After this time, females were selected only at proestrous, verified by daily vaginal smears, resulting: C-F, n s 6 and D-F, n s 5. The rest of the females were discarded. Then, all the selected animals were killed under equithensin anesthesia Ž2 mlrkg body weight.. The experimental procedures for animal use and care were in accordance with European Communities Council Directive 86r609rEEC.
adsorbent polymeric Biobeads SM-2 Ž100 mgrml. ŽBioRad, Richmond, VA, USA. and shaking for 2 h at 4 8C. 2.3. Procedures for enzymatic assays AlaAP, ArgAP and CysAP were measured fluorometrically using as substrates the aminoacyl-b-naphthylamides ŽaaNNap.: AlaNNap, ArgNNap and CysNNap, according to the modified method of Greenberg w17x. Ten microliters of each supernatant and serum were incubated during 30 min at 25 8C with 1 ml of the substrate solution: 2.14 mgr100 ml AlaNNap or 3.35 mgr 100 ml ArgNNap or 5.63 mgr100 ml CysNNap, 10 mgr100 ml bovine serum albumin ŽBSA., and 10 mgr100 ml dithiothreitol ŽDTT. in 50 mM of phosphate buffer, pH 7.4, for AlaAP and ArgAP; and 50 mM HCl–Tris buffer, pH 6, for CysAP. pGluAP was measured in a fluorogenic assay using pGluNNap as the substrate, according to the modified method of Schwabe and McDonald w18x: 10 ml of each supernatant was incubated during 120 min at 37 8C with 1 ml of substrate solution Ž2.54 mgr100 ml pGluNNap, 10 mgr100 ml BSA, 10 mgr100 ml DTT, 37.8 mgr100 ml EDTA in 50 mmolrl of phosphate buffer, pH 7.4.. AspAp was determined fluorometrically with AspNNap as the substrate, according to the method of Cheung and Cushman w19x modified as follows: 10 ml of each supernatant was incubated for 120 min at 37 8C with 1 ml of the substrate solution Ž2.58 mgr100 ml AspNNap, 10 mgr100 ml BSA, 10 mgr100 ml DTT and 39.4 mgr100 ml MnCl 2 in 50 mmolrl HCl–Tris buffer, pH 7.4.. GluAP was also determined in a fluorometric assay using GluNNap as the substrate according to the method of Tobe et al. w20x modified as follows: 10 ml of each supernatant was incubated during 120 min at 37 8C with 1
2.2. Samples Blood samples were obtained through left cardiac ventricle puncture, and serum was isolated by centrifugation for 10 min at 2000 = g. The neurohypophysis and the whole right adrenal were quickly removed and frozen in dry ice. To obtain the soluble fraction, tissue samples were homogenized in 10 volumes of 10 mM HCl–Tris buffer ŽpH s 7.4. and ultracentrifugated at 100 000 = g for 30 min Ž4 8C.. The resulting supernatants were used to measure soluble enzymatic activity and protein content, assayed in triplicate. To solubilize membrane proteins, the pellets were rehomogenized in HCl–Tris buffer ŽpH s 7.4. plus 1% Triton X-100. After centrifugation Ž100 000 = g, 30 min, 4 8C., the supernatants were used to measure membrane-bound activity and proteins, also in triplicate. To ensure the complete recovery of activity, the detergent was removed from the medium by adding to the samples
Fig. 1. Total protein content in serum of control and dehydrated males and females. Values, expressed as mg protrml, represent means"S.E.M. levels of groups of 6–10 animals assayed individually. Žq. represents differences between males vs. females. Ž ) . represents differences between control vs. dehydrated. ) , q p- 0.05.
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Fig. 2. Serum aminopeptidase activities in control male ŽC-M, n s 6. control female ŽC-F, n s 10., dehydrated male ŽD-M, n s 6. and dehydrated female ŽD-F, n s 10.. Values represent means" S.E.M. levels of groups of 6–10 animals assayed individually. Aminopeptidase ŽAP. activities are expressed as nmol of alanyl- ŽAlaAP., arginyl- ŽArgAP. and cystinyl- ŽCysAP. b-naphthylamide hydrolysed per min per mg of protein or pmol of pyroglutamylŽpGluAP., glutamyl- ŽGluAP. and aspartyl- ŽAspAP. b-naphthylamide hydrolysed per min per mg of protein. Žq. represents differences between males vs. females. Ž ) . represents differences between control vs. dehydrated. ) , qp - 0.05; qq p - 0.01; qq p - 0.001.
ml of the substrate solution Ž2.72 mgr100 ml GluNNap, 10 mgr100 ml BSA, 10 mgr100 ml DTT and 0.555 gr100 ml CaCl 2 in 50 mmolrl HCl–Tris, pH 7.4.. All the reactions were stopped by adding 1 ml of 0.1 molrl of acetate buffer, pH 4.2. The amount of b-naph-
thylamine released as a result of the enzymatic activity was measured fluorometrically at 412-nm emission wavelength with an excitation wavelength of 345 nm. Proteins were quantified in triplicate by the method of Bradford w21x, using BSA as a standard. Depending on the substrate
Fig. 3. Soluble ŽSOL. and membrane-bound ŽM-B. aminopeptidase activities in the neurohypophysis of control male ŽC-M, n s 6. control female ŽC-F, n s 10., dehydrated male ŽD-M, n s 6. and dehydrated female ŽD-F, n s 10.. Values represent means" S.E.M. levels of groups of 6–10 animals assayed individually. Aminopeptidase ŽAP. activities are expressed as pmol of alanyl- ŽAlaAP., arginyl- ŽArgAP., cystinyl- ŽCysAP., pyroglutamyl- ŽpGluAP., glutamyl- ŽGluAP. and aspartyl- ŽAspAP. b-naphthylamide hydrolysed per min per mg of protein. Žq. represents differences between males vs. females. Ž ) . represents differences between control vs. dehydrated. ) p - 0.05; ) ) , qp - 0.01.
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and the tissue studied, specific soluble and membranebound aminopeptidase activities were expressed as nmol or pmol of AlaNNap, ArgNNap, CysNNap, AspNNap, pGluNNap or GluNNap hydrolyzed per min per mg of protein. Fluorogenic assays were linear with respect to time of hydrolysis and protein content. 2.4. Statistical analysis The data were analysed by two-way analysis of variance ŽANOVA.. For gender, two factors were considered: male and female. For water consumption, two factors were considered: control and dehydrated. Since a significant interaction between gender and water consumption was observed, the two variables were analysed separately by the one-way ANOVA. Data were processed with the statistical graphics system ŽSTATGRAPHICS.. Post-hoc comparisons were made using Tukey’s test, and P values below 0.05 were considered significant.
3. Results In serum of control as well as in dehydrated rats, the total protein content was higher in females than in males
Ž p - 0.05.. Dehydration increased significantly in a similar proportion the protein content in males Ž8.2%. and females Ž7.5%.. Therefore, the difference between dehydrated males and females remains as in the controls Ž9%. ŽFig. 1.. With regards to the effect of dehydration, there was a significant Ž p - 0.05. decrease of pGluAP activity in serum of dehydrated males and females. There was no other change in aminopeptidase activities in serum after dehydration. Except for GluAP, the other aminopeptidase activities were significantly lower in females than in males Ž p - 0.001 for AlaAP, ArgAP, and CysAP; p - 0.01 for pGluAP and p - 0.05 for AspAP.. In control, as well as in dehydrated animals, the highest activity levels were observed for AlaAP in both male and female rats ŽFig. 2.. In the tissues analysed, with the exception of membrane-bound AspAP activity in neurohypophysis, the changes produced after water deprivation were observed essentially in the soluble fraction. In the neurohypophysis, while soluble AlaAP activity decreased significantly Ž p 0.01 in males and p - 0.05 in females., soluble GluAP activity increased Ž p - 0.05. in both dehydrated males and females. In contrast, membrane-bound AspAP activity increased significantly Ž P - 0.05. after dehydration, in males, but not in females. Soluble CysAP activity was also significantly higher in females than in males in neurohypophysis. In this location, control and dehydrated male and
Fig. 4. Soluble ŽSOL. and membrane-bound ŽM-B. aminopeptidase activities in the adrenal gland of control male ŽC-M, n s 6. control female ŽC-F, n s 10., dehydrated male ŽD-M, n s 6. and dehydrated female ŽD-F, n s 10.. Values represent means" S.E.M. levels of groups of 6–10 animals assayed individually. Aminopeptidase activities are expressed as nmol of alanyl- ŽAlaAP., arginyl- ŽArgAP. and cystinyl- ŽCysAP. b-naphthylamide hydrolysed per min per mg of protein or pmol of pyroglutamyl- ŽpGluAP., glutamyl- ŽGluAP. and aspartyl- ŽAspAP. b-naphthylamide hydrolysed per min per mg of protein. Žq. represents differences between males vs. females. Ž ) . represents differences between control vs. dehydrated. qp - 0.05; ) ) , qp - 0.01; ))) p - 0.001.
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female rats exhibited their highest levels for soluble ArgAP and membrane-bound CysAP activities ŽFig. 3.. In the adrenal gland, soluble AlaAP Ž p - 0.01., ArgAP Ž p - 0.01. and CysAP Ž p - 0.001. activities increased significantly after dehydration in males and females. Comparing sex, membrane-bound AlaAP and ArgAP activities were higher Ž p - 0.01. in females than in males. However, soluble AspAP was higher Ž p - 0.01. in males. The adrenal gland showed its highest levels for soluble and membrane-bound AlaAP and ArgAP activities in all the different biological preparations ŽFig. 4..
4. Discussion Angiotensin II increases drinking and blood pressure when administered intracerebroventricularly. Intracerebroventricular injections of antiserum directed against aminopeptidase A ŽAPA., the most important enzyme that degrades Ang II to Ang III, reduced the drinking and blood pressure responses to 10 pmol Ang II by 73% and 59%, respectively. APA antiserum had no effect on responses to Ang III administered intracerebroventricularly. A Glu-thiol inhibitor of APA also reduced Ang II-induced drinking. These results suggest that the metabolism of Ang II to Ang III is an obligatory activation step for the brain angiotensin system w22x. Angiotensin IIrIII and vasopressin coexist in neurones as well as in fibres of the hypothalamo-neurohypophyseal system w23x. In addition, it has been conclusively demonstrated that the action of Ang II on vasopressin release depends upon the prior conversion of Ang II to Ang III by aminopeptidase A or A-like activity w24x. The further inhibition of AlaAP, which metabolises Ang III, induces vasopressin release by increasing the half-life of Ang III w25x. Therefore, Ang III is one of the main stimulatory peptides in the control of vasopressin release. Thus, the AP response to dehydration in neurohypophysis, reported for the first time in the present work, physiologically confirms this pattern: an increase in AP A ŽGluAP. and A-like activity ŽAspAP. leads to an increased metabolism of Ang II to Ang III. This occurs with a decrease in AlaAP activity, which will prolong the action of Ang III by the reduction of its metabolism. Interestingly, the response in adrenals is highly similar to that described in the reduced renal mass model of arterial hypertension, the so-called polydipsia–polyuria syndrome. It is characterised by the production of increased urine volume with reduced osmolality, despite an increased secretion of vasopressin w3x. The latter may be secondary to the increased plasma osmolality produced by the hypernatremia in these hypertensive animals. The present results imply a high degree of metabolism of Ang III and vasopressin in the adrenals of dehydrated animals and suggest that locally, the adrenal gland responds in the same way to an increase in plasmatic osmolality.
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Most of the changes observed in AP activities after water deprivation occurred in the soluble components of the neurohypophysis and the adrenal gland. Since activation of cell membrane-bound receptors is crucial for the physiological actions of Ang II, it is unclear how those changes may contribute to angiotensin metabolism. However, there is growing evidence that receptor-mediated endocytosis of Ang II is important for some physiological responses to Ang II and its subsequent degradation in lysosomes may also serve an important function in the disposal of this peptide w26x. Differences in the response of soluble or membrane-bound components of AP activities may be tissue-dependent. Previous studies on AP activities in a rat saline model of hypertension Žreduced renal mass, RRM., also modified specifically the soluble components of AP in neurohypophysis and adrenal gland w3x. However, there were important changes, essentially in membranebound activity, in the renal tissue of two animal models of hypertension, RRM and Goldblatt two-kidney one clip w9x. These changes were also selective for membrane-bound AP activities in the renal tissue of water deprived rats Ždata not shown.. On the other hand, it is unclear whether there is gender differences in the control of water balance. It has been reported that plasma osmolality was higher in dehydrated females than in dehydrated males w13x. However, other authors did not find any sex difference in plasma osmolality after water deprivation w27x. It has also been described that central angiotensinergic mechanisms controlling vasopressin release are differently influenced by gender w14x. Gonadectomy prevented the increase in hypothalamic vasopressin mRNA in response to increased osmolality in both males and females w28x, but there is a differential effect of estradiol and dihydrotestosterone on vasopressin mRNA, which imply distinct actions for these steroids w15x. We have previously reported gender differences in AP activities. There was dissociation between the results obtained in serum and tissues w11,29x. This is confirmed in the present results. While male AP activities were higher in serum, the tissue levels were higher in females. In addition, we have also reported significant sex differences and an influence of cholesterol and steroid hormones on AP activities. Depending on the nature of the AP, these enzymes responded in different ways to the presence of these substances and also responded differently to gonadectomy w12x. After dehydration, we found a similar increase in protein content in males Ž8.2%. and females Ž7.5%.. Therefore, the sex difference is proportionally the same than in controls, suggesting that there is no difference between sexes in the change in osmolality after dehydration. This hypothesis could be supported by the fact that pGluAP did not change after dehydration in neurohypophysis and adrenal gland of males and females, or that changes in serum occur in the same direction and intensity in males and females. On the other hand, this
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decrease in the metabolism of GnRH could permit a longer effect of this peptide, thus increasing the stimulation of gonadotropin release and enhancing the stimulatory effect of gonadal steroids on vasopressin secretion in response to hyperosmolality. In conclusion, according to the present results, there is no argument in favor of a sex difference in response to changes in osmolality. However, in water balance, a target organ for Ang II and vasopressin is the kidney. In this tissue, we have observed important changes in membrane-bound activities and gender differences in the response to water deprivation Ždata not shown.. References w1x De Gasparo M, Bottari S, Levens NR. Characteristics of angiotensin II receptors and their role in cell and organ physiology. In: Laragh JH, Brenner BM, editors. Hypertension: Pathophysiology, Diagnosis, and Management. New York: Raven Press 1995:1695–720. w2x Bottari SP, De Gasparo M, Steckelings UM, Levens NR. Angiotensin II receptor subtypes: characterization, signalling mechanisms, and possible physiological implications. Front Neuroendocrinol 1993;14:123–71. w3x Prieto I, Martinez A, Martinez JM, Ramirez MJ, Vargas F, Alba F, et al. Activities of aminopeptidases in a rat saline model of volume hypertension. Horm Metab Res 1998;30:246–8. w4x Murlow PJ. Renin–angiotensin system in the adrenal. Horm Metab Res 1998;30:346–9. w5x Wright JW, Harding JW. Brain angiotensin receptor subtypes AT1, AT2, and AT4 and their functions. Regul Pept 1995;59:269–95. w6x Wilk S, Wilk E, Magnusson RP. Purification, characterization, and cloning of a cytosolic aspartyl aminopeptidase. J Biol Chem 1998; 273:15961–70. w7x Russel JS, Chi H, Lantry LE, Stephens RE, Ward PE. Substance P and neurokinin A metabolism by cultured human skeletal muscle myocytes and fibroblasts. Peptides 1996;17:1397–403. w8x Ward PE, Benter IF, Dick L, Wilk S. Metabolism of vasoactive peptides by plasma and purified renal aminopeptidase M. Biochem Pharmacol 1990;40:1725–32. w9x Ramirez M, Prieto I, Martinez JM, Vargas F, Alba F. Renal aminopeptidase activities in animal models of hypertension. Regul Pept 1997;72:155–9. w10x Prieto I, Martınez JM, Hermoso F, Ramırez ´ ´ MJ, Vargas F, De Gasparo M, et al. Oral administration of losartan influences aminopeptidase activity in the frontal cortex. Eur Neuropsychopharmacol 2000;10:279–82. w11x Martinez JM, Prieto I, Ramirez MJ, De Gasparo M, Hermoso F, Arias JM, et al. Sex differences and age-related changes in human serum aminopeptidase A activity. Clin Chim Acta 1998;274:53–61. w12x Martınez JM, Ramirez MJ, Prieto I, Alba F, Ramirez M. Sex ´ differences and in vitro effects of steroids on serum aminopeptidase activities. Peptides 1998;19:1637–40.
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