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Renin activity and angiotensin I concentration in genetically selective inbred line of hypertensive mice
BBRC Biochemical and Biophysical Research Communications 316 (2004) 842–844 www.elsevier.com/locate/ybbrc
Renin activity and angiotensin I concentration in genetically selective inbred line of hypertensive mice Mukarram Uddin* and N. Harris-Nelson Department of Pathology, Anatomy, and Cell Biology, Meharry Medical College, 1005 D.B. Todd Blvd., Nashville, TN 37208, USA Received 5 February 2004
Abstract Blood pressure lowering kallikrein–kinin and blood pressure raising renin–angiotensin systems play a major role in the maintenance of normal blood pressure. In a previous study, we have shown that a kallikrein-like prorenin converting enzyme (PRCE C) is elevated in the submandibular gland tissue of a mouse line (BPH) that was genetically selected and inbred for high blood pressure in comparison to normotensive line (BPN) that was derived from the ancestors of BPH line. In the present investigation we wanted to find out if elevated levels of PRCE C were involved in the modulation of tissue (local) renin–angiotensin system in the submandibular gland tissue. Results indicate significantly high renin activity but low angiotensin I level in the tissue of BPH mouse model. These results tend to suggest PRCE C’s involvement in tissue (local) renin–angiotensin system. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Prorenin converting enzyme; Submandibular gland; Renin; Angiotensin I; BPH mice; BPN mice
Blood pressure lowering kallikrein–kinin and blood pressure raising renin–angiotensin systems, notwithstanding other related systems, play a major role in the maintenance of normal blood pressure. Abnormal/subnormal behavior of key regulatory enzymes of the two systems would result in an imbalance of the two systems consequently resulting in the genesis of hypertension. Although kallikrein–kinin and renin–angiotensin systems are considered independent systems, evidence in rats [1] and in mice [2–5] indicates that some kallikreinlike enzymes, in addition to possessing kallikrein activity also, exhibit prorenin converting activity. In our previous study, we have demonstrated that a kallikrein-like prorenin converting enzyme (PRCE C) was elevated in the submandibular gland tissue [6] of a mice line (BPH) that was genetically selected and inbred for high blood pressure in comparison to normotensive line (BPN) that was derived from the ancestors of BPH line [7]. Since renin is a key regulatory enzyme of the renin– angiotensin system and kallikrein-like PRCE C converts inactive prorenin into biologically active renin, the pre* Corresponding author. Fax: 1-615-327-6655. E-mail address: [email protected] (M. Uddin).
0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.02.128
mise of the present investigation is that elevation of PRCE C in genetically selective hypertensive mice line could be a contributing factor in the overall genesis of high blood pressure by way of tissue (local) renin–angiotensin system modulation. To test this hypothesis, we measured renin activity and angiotensin I levels from the aqueous extracts of the submandibular gland tissue of BPH and BPN mice lines by radioimmunoassay. We chose submandibular gland for this investigation because renin is synthesized in this gland [8,9] and there is evidence in the literature of inactive renin secretion into the plasma and interstitial tissue [10,11]. We also determined protein concentration of the aqueous extract by BSA method. Our results indicate significantly high renin activity but low angiotensin I level in the tissue of BPH line. These results tend to suggest PRCE C’s involvement in the generation of active renin and consequently in the modulation of (local) tissue renin–angiotensin system in BPH mouse line. Methods Animals and tissue extraction. Six BPH and six BPN strains of adult male mice were used in this investigation. In the morning 2 h before
M. Uddin, N. Harris-Nelson / Biochemical and Biophysical Research Communications 316 (2004) 842–844 sacrifice food and water were taken away. Animals were taken out of the cage one by one and quickly decapitated. Tissue was taken out, weighed, and homogenized in 2 ml distilled water containing 4 ll cathepsin D. Following homogenization the solution was centrifuged at 5000g for 6 min. Clear aqueous extract was saved for further experiments and the precipitate was discarded. Renin activity. Renin activity was measured by the Gamma Coat Plasma Renin Activity 125 I RIA Kit (DiaSorin, USA). One milliliter of BPH and BPN submandibular gland aqueous extract was transferred to uncoated plastic tubes. Ten microliters of phenylmethylsulfonyl fluoride (PMSF), 100 ll maleate generation buffer, and 20 ll nephrectomized rat plasma were added to the extract. The extract was the divided into two parts. One part was incubated at 37 °C for 2 h whereas the other part was incubated at 4 °C for 2 h. Standard contained 0.0, 0.2, 0.8, 3.0, 10, and 50 ng/ml angiotensin I. To the standard, 37 and 4 °C incubated BPH and BPN extracts, 1 ml of tracer-buffer reagent was added. This was followed by 3 h of incubation at room temperature. At the end of this incubation, all tubes were decanted and were subjected to gamma counter for 1 min with the window suitably adjusted for 125 I. Renin activity was calculated from the standard graph by subtracting 4 °C incubated samples from 37 °C incubated samples and expressed as pg/mg protein/2 h. Angiotensin I concentration of BPH and BPN submandibular gland is calculated from the standard graph using 4 °C incubated samples and expressed as picogram/milligram protein. Protein determinations. Protein was determined by bovine serum albumin method. Statistics. Computer program statix was used for statistical analysis.
Results Fig. 1 is a bar graph showing renin activity in pg/mg protein/2 h in BPN and BPH lines of genetically selective inbred normotensive and hypertensive mice. The sample size for BPH line is 30 and for BPN is 33. Number of animals in each group is six. This figure shows that renin activity in the submandibular gland tissue of BPH mouse model is significantly higher in comparison to BPN line ðP < 0:009Þ. Fig. 2 is a bar graph showing angiotensin I levels in picogram/milligram protein from BPH and BPN lines of mice following 2 h of incubation at 4 °C. The sample size in each group is 27 and number of animals in each group is 6. Angiotensin I level in BPH mouse model is significantly lower in comparison to BPN line ðP < 0:03Þ.
Fig. 1. Submandibular gland renin activity. N, normotensive BPN mouse line; H, hypertensive BPH mouse line; n, number of animals. Incubation time 2 h. SE, standard error of the mean.
843
Fig. 2. Submandibular gland rangiotensin I levels. N, normotensive BPN mouse line; H, hypertensive BPH mouse line; n, number of animals. Incubation 2 h. SE, standard error of the mean. Table 1 Renin activity and angiotensin I levels in submandibular gland tissue of BPH and BPN mice lines
BPN strain BPH strain
Renin activity (pg/mg protein/2 h)
Angiotensin I level (pg/mg protein)
98 67 793 239 ðP 6 0:009Þ
1995 360 996 270 ðP 6 0:03Þ
Table 1 below shows renin activity and angiotensin I levels from submandibular gland tissue of BPH and BPN lines of mice. Renin activity in BPN mice line is 98 pg/mg protein/2 h 67 whereas in BPH mouse model it is 793 pg/mg protein/2 h 239. Group t test indicates significantly higher activity in BPH submandibular gland tissue ðP < 0:009Þ. On the other hand, angiotensin I level in BPH mice line is 1995 pg/mg protein/2 h 360 whereas in BPN line it is 996 pg/mg protein/2 h 270. Group t test indicates significantly lower levels of angiotensin I in BPH mouse model ðP < 0:03Þ.
Discussion Results of the present investigation clearly demonstrate that renin activity in the submandibular gland tissue of hypertensive BPH line of mice is significantly higher than in the normotensive BPN line of mice that are derived from the ancestors of BPH line ðP < 0:009Þ. In a previous study [6] we had shown that mRNA and protein levels of a kallikrein-like prorenin converting enzyme C (PRCE C) were also elevated in the submandibular gland tissue of BPH mouse model. The results of the present investigation indicating high renin activity in BPH strain demonstrate a definite relationship to PRCE C elevation of BPH strain. The release of submandibular gland renin into the interstitium surrounding the blood vessels in hypertensive mice has been well established. It has been suggested that interstitial renin release is responsible for local (tissue) renin–angiotensin effect. Our result of high renin
844
M. Uddin, N. Harris-Nelson / Biochemical and Biophysical Research Communications 316 (2004) 842–844
activity in the submandibular gland tissue of BPH strain is consistent with the hypothesis of local (tissue) renin– angiotensin regulation. The other possible mechanism is that increase in the renin activity in BPH mouse line may result in more renin secretion into the bloodstream and exert its effect on the generation of plasma angiotensin I from the substrate angiotensinogen. High levels of plasma renin have been reported in spontaneously hypertensive rats [12]. In our laboratory, we have also found increased levels of plasma levels in BPH strains (unpublished results). However, these results are contrary to published results by Iwao et al. [13] who have demonstrated a decrease in plasma renin in BPH strain. Mouse submandibular gland contains two renin genes [14]. Some strains of mice have one renin gene (Ren-1). Mice with Ren-1 gene produce low or normal renin [15]. Other strains of mice have two renin genes (Ren-1 and Ren-2). Mice with Ren-1 and Ren-2 genes produce high renin. At present, it is not known whether BPH strain of mice contain one or both renin genes. Since BPN strain is derived from the ancestors of BPH strain, it is highly unlikely that these strains have different renin genes (Ren). Angiotensin I concentration in levels in the submandibular gland tissue of hypertensive BPH mouse model is significantly lower in comparison to BPN line of mice ðP < 0:03Þ. Low levels of angiotensin I levels in hypertensive line of mice, in the first instance, are hard to reconcile in the light of high renin activity in BPH mice. One possibility is that angiotensin I in the submandibular gland of BPH mouse strain is metabolized at a faster rate than in BPN mouse line. It has been demonstrated that there is an altered metabolism of angiotensin I by endopeptidase/aminopeptidase in spontaneously hypertensive rats [16,17]. Whether a similar mechanism exists in BPH mouse model has not been studied and needs to be explored further. The rate of angiotensin I conversion to angiotensin II by angiotensin converting enzyme is also not clear in BPH mouse model. In summary, submandibular gland tissue of BPH line of mouse model shows higher renin activity in comparison to BPN line of mice. On the other hand, angiotensin I concentration and levels in hypertensive BPH mice are significantly lower in comparison to normotensive BPN mice.
Acknowledgments We thank Dr. T. Inagami, Biochemistry Department, Vanderbilt University, for providing nephrectomized rat plasma for renin assays. We also thank Dr. J.T. Clark and Mike Greenfield, Department of Physiology, Meharry Medical College, for their help in nephrectom-
izing male adult rats. This investigation is supported by the NIHNHLB Grant K01 HL04038 to M.U.
References [1] T. Moreau, M. Brillard-Bourdet, J. Bouhnik, F. Gauthier, Protein products of the rat kallikrein family. Substrate specificities of kallikrein rK2(tonin) and kallikrein rK9, J. Biol. Chem. 267 (1992) 10045–10051. [2] Y. Kikkawa, N. Yamanaka, J. Tada, N. Kanamori, K. Tsumura, K. Hosoi, Prorenin processing and restricted endoproteolysis by mouse tissue kallikrein family enzymes(mKI, mK9, mK13 and mK22), Biochem. Biophys. Acta 1382 (1998) 55–64. [3] W.S. Kim, K. Hatsuzawa, Y. Ishizuka, K. Hashiba, K. Murakami, K. Nakayama, A processing enzyme for prorenin in mouse submandibular gland. Purification and characterization, J. Biol. Chem. 265 (1990) 5930–5933. [4] W.S. Kim, K. Nakayama, K. Murakami, Presence of two types of prorenin converting enzymes in the mouse submandibular gland, FASEB Lett. 293 (1990) 142–144. [5] W.S. Kim, K. Nakayama, T. Nakagawa, Y. Kawamura, Mouse submandibular gland prorenin-converting enzyme is a member of glandular kallikrein family, J. Biol. Chem. 266 (1991) 19283– 19287. [6] M. Uddin, M. Polley-Mandal, O.U. Beg, Kallikrein-like prorenin converting enzymes in inbred hypertensive mice, Biochem. Biophys. Res. Commun. 304 (2003) 724–728. [7] G. Schlager, Genetic hypertension in mice, in: D. Gantor and W. de Jong (Eds.), Handbook of Hypertension. Vol. 16: Experimental and Genetic Model for Hypertension (second ed.), Elsevier Science, Amesterdam, pp. 158–172. [8] E. Gresik, A. Michelakis, T. Barka, T. Ross, Immunocytochemical localization of renin in the submandibular gland of the mouse, J. Histochem. Cytochem. 26 (1978) 855–861. [9] J.W. Menzie, L.H. Hoffman, A.M. Michelakis, Immunofluorescent localization of renin in mouse submaxillary gland and kidney, Am. J. Physiol. 234 (1978) E480–E483. [10] S. Miyazaki, A. Sakanaka, K. Chimori, J. Kosaka, R. Goi, S. Dodo, K. Miura, F. Suzuki, Reno-submandibular axis controls release of extrarenal inactive renin, J. Hypertens. 6 (1988) 30–40. [11] A. Johannessen, A.H. Nielsen, K. Poulsen, Measurement of inactive renin in rat plasma: effect of nephrectomy and sialoadenectomy on the plasma concentration, J. Hypertens. 8 (1990) 34–39. [12] W. DeJong, W. Lovenberg, A. Sjoerdsma, Increase plasma renin activity in the spontaneously hypertensive rats, Proc. Soc. Exp. Biol. Med. 139 (1972) 1213–1216. [13] H. Iwao, N. Nakamura, S. Kim, F. Ikemoto, K. Yamamoto, G. Schlager, Renin angiotensin system in genetically hypertensive mice, Jpn. Circ. J. 48 (1984) 1270–1279. [14] S.H. Kim, N. Maeda, G.T. Oh, L.G. Fernandez, R.A. Gomes, O. Smothies, Homeostasis in mice with genetically decreased angiotensinogen is primarily controlled by an increased number of renin producing cells, J. Biol. Chem. 274 (1999) 4210–4217. [15] F. Bertaux, W.H. Colledge, S.E. Smith, M. Evans, N.J. Samani, C.C.T. Miller, Normotensive blood pressure in mice with a disrupted renin Ren-1d gene, Transgenic Res. 6 (1997) 191–196. [16] J. Baner, H. Berthold, F. Schaefer, H. Ehmke, N. Parekh, Quantification of conversion and degradation of circulating angiotensin in rats, Am. J. Physiol. 277 (1999) R412–R418. [17] M.K. Sim, X.S. Qiu, Formation of des-Asp-angiotensin I in the hypothalamic extract of normo- and hypertensive rats, Blood Press. 3 (1994) 260–264.
Renin activity and angiotensin I concentration in genetically selective inbred line of hypertensive mice Mukarram Uddin* and N. Harris-Nelson Department of Pathology, Anatomy, and Cell Biology, Meharry Medical College, 1005 D.B. Todd Blvd., Nashville, TN 37208, USA Received 5 February 2004
Abstract Blood pressure lowering kallikrein–kinin and blood pressure raising renin–angiotensin systems play a major role in the maintenance of normal blood pressure. In a previous study, we have shown that a kallikrein-like prorenin converting enzyme (PRCE C) is elevated in the submandibular gland tissue of a mouse line (BPH) that was genetically selected and inbred for high blood pressure in comparison to normotensive line (BPN) that was derived from the ancestors of BPH line. In the present investigation we wanted to find out if elevated levels of PRCE C were involved in the modulation of tissue (local) renin–angiotensin system in the submandibular gland tissue. Results indicate significantly high renin activity but low angiotensin I level in the tissue of BPH mouse model. These results tend to suggest PRCE C’s involvement in tissue (local) renin–angiotensin system. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Prorenin converting enzyme; Submandibular gland; Renin; Angiotensin I; BPH mice; BPN mice
Blood pressure lowering kallikrein–kinin and blood pressure raising renin–angiotensin systems, notwithstanding other related systems, play a major role in the maintenance of normal blood pressure. Abnormal/subnormal behavior of key regulatory enzymes of the two systems would result in an imbalance of the two systems consequently resulting in the genesis of hypertension. Although kallikrein–kinin and renin–angiotensin systems are considered independent systems, evidence in rats [1] and in mice [2–5] indicates that some kallikreinlike enzymes, in addition to possessing kallikrein activity also, exhibit prorenin converting activity. In our previous study, we have demonstrated that a kallikrein-like prorenin converting enzyme (PRCE C) was elevated in the submandibular gland tissue [6] of a mice line (BPH) that was genetically selected and inbred for high blood pressure in comparison to normotensive line (BPN) that was derived from the ancestors of BPH line [7]. Since renin is a key regulatory enzyme of the renin– angiotensin system and kallikrein-like PRCE C converts inactive prorenin into biologically active renin, the pre* Corresponding author. Fax: 1-615-327-6655. E-mail address: [email protected] (M. Uddin).
0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.02.128
mise of the present investigation is that elevation of PRCE C in genetically selective hypertensive mice line could be a contributing factor in the overall genesis of high blood pressure by way of tissue (local) renin–angiotensin system modulation. To test this hypothesis, we measured renin activity and angiotensin I levels from the aqueous extracts of the submandibular gland tissue of BPH and BPN mice lines by radioimmunoassay. We chose submandibular gland for this investigation because renin is synthesized in this gland [8,9] and there is evidence in the literature of inactive renin secretion into the plasma and interstitial tissue [10,11]. We also determined protein concentration of the aqueous extract by BSA method. Our results indicate significantly high renin activity but low angiotensin I level in the tissue of BPH line. These results tend to suggest PRCE C’s involvement in the generation of active renin and consequently in the modulation of (local) tissue renin–angiotensin system in BPH mouse line. Methods Animals and tissue extraction. Six BPH and six BPN strains of adult male mice were used in this investigation. In the morning 2 h before
M. Uddin, N. Harris-Nelson / Biochemical and Biophysical Research Communications 316 (2004) 842–844 sacrifice food and water were taken away. Animals were taken out of the cage one by one and quickly decapitated. Tissue was taken out, weighed, and homogenized in 2 ml distilled water containing 4 ll cathepsin D. Following homogenization the solution was centrifuged at 5000g for 6 min. Clear aqueous extract was saved for further experiments and the precipitate was discarded. Renin activity. Renin activity was measured by the Gamma Coat Plasma Renin Activity 125 I RIA Kit (DiaSorin, USA). One milliliter of BPH and BPN submandibular gland aqueous extract was transferred to uncoated plastic tubes. Ten microliters of phenylmethylsulfonyl fluoride (PMSF), 100 ll maleate generation buffer, and 20 ll nephrectomized rat plasma were added to the extract. The extract was the divided into two parts. One part was incubated at 37 °C for 2 h whereas the other part was incubated at 4 °C for 2 h. Standard contained 0.0, 0.2, 0.8, 3.0, 10, and 50 ng/ml angiotensin I. To the standard, 37 and 4 °C incubated BPH and BPN extracts, 1 ml of tracer-buffer reagent was added. This was followed by 3 h of incubation at room temperature. At the end of this incubation, all tubes were decanted and were subjected to gamma counter for 1 min with the window suitably adjusted for 125 I. Renin activity was calculated from the standard graph by subtracting 4 °C incubated samples from 37 °C incubated samples and expressed as pg/mg protein/2 h. Angiotensin I concentration of BPH and BPN submandibular gland is calculated from the standard graph using 4 °C incubated samples and expressed as picogram/milligram protein. Protein determinations. Protein was determined by bovine serum albumin method. Statistics. Computer program statix was used for statistical analysis.
Results Fig. 1 is a bar graph showing renin activity in pg/mg protein/2 h in BPN and BPH lines of genetically selective inbred normotensive and hypertensive mice. The sample size for BPH line is 30 and for BPN is 33. Number of animals in each group is six. This figure shows that renin activity in the submandibular gland tissue of BPH mouse model is significantly higher in comparison to BPN line ðP < 0:009Þ. Fig. 2 is a bar graph showing angiotensin I levels in picogram/milligram protein from BPH and BPN lines of mice following 2 h of incubation at 4 °C. The sample size in each group is 27 and number of animals in each group is 6. Angiotensin I level in BPH mouse model is significantly lower in comparison to BPN line ðP < 0:03Þ.
Fig. 1. Submandibular gland renin activity. N, normotensive BPN mouse line; H, hypertensive BPH mouse line; n, number of animals. Incubation time 2 h. SE, standard error of the mean.
843
Fig. 2. Submandibular gland rangiotensin I levels. N, normotensive BPN mouse line; H, hypertensive BPH mouse line; n, number of animals. Incubation 2 h. SE, standard error of the mean. Table 1 Renin activity and angiotensin I levels in submandibular gland tissue of BPH and BPN mice lines
BPN strain BPH strain
Renin activity (pg/mg protein/2 h)
Angiotensin I level (pg/mg protein)
98 67 793 239 ðP 6 0:009Þ
1995 360 996 270 ðP 6 0:03Þ
Table 1 below shows renin activity and angiotensin I levels from submandibular gland tissue of BPH and BPN lines of mice. Renin activity in BPN mice line is 98 pg/mg protein/2 h 67 whereas in BPH mouse model it is 793 pg/mg protein/2 h 239. Group t test indicates significantly higher activity in BPH submandibular gland tissue ðP < 0:009Þ. On the other hand, angiotensin I level in BPH mice line is 1995 pg/mg protein/2 h 360 whereas in BPN line it is 996 pg/mg protein/2 h 270. Group t test indicates significantly lower levels of angiotensin I in BPH mouse model ðP < 0:03Þ.
Discussion Results of the present investigation clearly demonstrate that renin activity in the submandibular gland tissue of hypertensive BPH line of mice is significantly higher than in the normotensive BPN line of mice that are derived from the ancestors of BPH line ðP < 0:009Þ. In a previous study [6] we had shown that mRNA and protein levels of a kallikrein-like prorenin converting enzyme C (PRCE C) were also elevated in the submandibular gland tissue of BPH mouse model. The results of the present investigation indicating high renin activity in BPH strain demonstrate a definite relationship to PRCE C elevation of BPH strain. The release of submandibular gland renin into the interstitium surrounding the blood vessels in hypertensive mice has been well established. It has been suggested that interstitial renin release is responsible for local (tissue) renin–angiotensin effect. Our result of high renin
844
M. Uddin, N. Harris-Nelson / Biochemical and Biophysical Research Communications 316 (2004) 842–844
activity in the submandibular gland tissue of BPH strain is consistent with the hypothesis of local (tissue) renin– angiotensin regulation. The other possible mechanism is that increase in the renin activity in BPH mouse line may result in more renin secretion into the bloodstream and exert its effect on the generation of plasma angiotensin I from the substrate angiotensinogen. High levels of plasma renin have been reported in spontaneously hypertensive rats [12]. In our laboratory, we have also found increased levels of plasma levels in BPH strains (unpublished results). However, these results are contrary to published results by Iwao et al. [13] who have demonstrated a decrease in plasma renin in BPH strain. Mouse submandibular gland contains two renin genes [14]. Some strains of mice have one renin gene (Ren-1). Mice with Ren-1 gene produce low or normal renin [15]. Other strains of mice have two renin genes (Ren-1 and Ren-2). Mice with Ren-1 and Ren-2 genes produce high renin. At present, it is not known whether BPH strain of mice contain one or both renin genes. Since BPN strain is derived from the ancestors of BPH strain, it is highly unlikely that these strains have different renin genes (Ren). Angiotensin I concentration in levels in the submandibular gland tissue of hypertensive BPH mouse model is significantly lower in comparison to BPN line of mice ðP < 0:03Þ. Low levels of angiotensin I levels in hypertensive line of mice, in the first instance, are hard to reconcile in the light of high renin activity in BPH mice. One possibility is that angiotensin I in the submandibular gland of BPH mouse strain is metabolized at a faster rate than in BPN mouse line. It has been demonstrated that there is an altered metabolism of angiotensin I by endopeptidase/aminopeptidase in spontaneously hypertensive rats [16,17]. Whether a similar mechanism exists in BPH mouse model has not been studied and needs to be explored further. The rate of angiotensin I conversion to angiotensin II by angiotensin converting enzyme is also not clear in BPH mouse model. In summary, submandibular gland tissue of BPH line of mouse model shows higher renin activity in comparison to BPN line of mice. On the other hand, angiotensin I concentration and levels in hypertensive BPH mice are significantly lower in comparison to normotensive BPN mice.
Acknowledgments We thank Dr. T. Inagami, Biochemistry Department, Vanderbilt University, for providing nephrectomized rat plasma for renin assays. We also thank Dr. J.T. Clark and Mike Greenfield, Department of Physiology, Meharry Medical College, for their help in nephrectom-
izing male adult rats. This investigation is supported by the NIHNHLB Grant K01 HL04038 to M.U.
References [1] T. Moreau, M. Brillard-Bourdet, J. Bouhnik, F. Gauthier, Protein products of the rat kallikrein family. Substrate specificities of kallikrein rK2(tonin) and kallikrein rK9, J. Biol. Chem. 267 (1992) 10045–10051. [2] Y. Kikkawa, N. Yamanaka, J. Tada, N. Kanamori, K. Tsumura, K. Hosoi, Prorenin processing and restricted endoproteolysis by mouse tissue kallikrein family enzymes(mKI, mK9, mK13 and mK22), Biochem. Biophys. Acta 1382 (1998) 55–64. [3] W.S. Kim, K. Hatsuzawa, Y. Ishizuka, K. Hashiba, K. Murakami, K. Nakayama, A processing enzyme for prorenin in mouse submandibular gland. Purification and characterization, J. Biol. Chem. 265 (1990) 5930–5933. [4] W.S. Kim, K. Nakayama, K. Murakami, Presence of two types of prorenin converting enzymes in the mouse submandibular gland, FASEB Lett. 293 (1990) 142–144. [5] W.S. Kim, K. Nakayama, T. Nakagawa, Y. Kawamura, Mouse submandibular gland prorenin-converting enzyme is a member of glandular kallikrein family, J. Biol. Chem. 266 (1991) 19283– 19287. [6] M. Uddin, M. Polley-Mandal, O.U. Beg, Kallikrein-like prorenin converting enzymes in inbred hypertensive mice, Biochem. Biophys. Res. Commun. 304 (2003) 724–728. [7] G. Schlager, Genetic hypertension in mice, in: D. Gantor and W. de Jong (Eds.), Handbook of Hypertension. Vol. 16: Experimental and Genetic Model for Hypertension (second ed.), Elsevier Science, Amesterdam, pp. 158–172. [8] E. Gresik, A. Michelakis, T. Barka, T. Ross, Immunocytochemical localization of renin in the submandibular gland of the mouse, J. Histochem. Cytochem. 26 (1978) 855–861. [9] J.W. Menzie, L.H. Hoffman, A.M. Michelakis, Immunofluorescent localization of renin in mouse submaxillary gland and kidney, Am. J. Physiol. 234 (1978) E480–E483. [10] S. Miyazaki, A. Sakanaka, K. Chimori, J. Kosaka, R. Goi, S. Dodo, K. Miura, F. Suzuki, Reno-submandibular axis controls release of extrarenal inactive renin, J. Hypertens. 6 (1988) 30–40. [11] A. Johannessen, A.H. Nielsen, K. Poulsen, Measurement of inactive renin in rat plasma: effect of nephrectomy and sialoadenectomy on the plasma concentration, J. Hypertens. 8 (1990) 34–39. [12] W. DeJong, W. Lovenberg, A. Sjoerdsma, Increase plasma renin activity in the spontaneously hypertensive rats, Proc. Soc. Exp. Biol. Med. 139 (1972) 1213–1216. [13] H. Iwao, N. Nakamura, S. Kim, F. Ikemoto, K. Yamamoto, G. Schlager, Renin angiotensin system in genetically hypertensive mice, Jpn. Circ. J. 48 (1984) 1270–1279. [14] S.H. Kim, N. Maeda, G.T. Oh, L.G. Fernandez, R.A. Gomes, O. Smothies, Homeostasis in mice with genetically decreased angiotensinogen is primarily controlled by an increased number of renin producing cells, J. Biol. Chem. 274 (1999) 4210–4217. [15] F. Bertaux, W.H. Colledge, S.E. Smith, M. Evans, N.J. Samani, C.C.T. Miller, Normotensive blood pressure in mice with a disrupted renin Ren-1d gene, Transgenic Res. 6 (1997) 191–196. [16] J. Baner, H. Berthold, F. Schaefer, H. Ehmke, N. Parekh, Quantification of conversion and degradation of circulating angiotensin in rats, Am. J. Physiol. 277 (1999) R412–R418. [17] M.K. Sim, X.S. Qiu, Formation of des-Asp-angiotensin I in the hypothalamic extract of normo- and hypertensive rats, Blood Press. 3 (1994) 260–264.