Tonin and Kallikrein in the Brain of Transgenic Rat Line
Expressing Human Tissue Kallikrein
Eliane S.L. Lomez, Ronaldo C. Araujo, Michael Bader, Joao B. Pesquero, Jorge L. Pesquero
Abstract—A transgenic rat line harboring the human tissue kallikrein gene was investigated for expression and activity of
tonin and kallikrein in different regions of the brain. The introduction of the transgene into the rat genome produced a
significant augmentation of the expression levels and activity of rat tissue kallikrein. The possibility that human
kallikrein does not hydrolyze the rat substrate is probably responsible for the augmented expression of the rat enzyme.
On the other hand, although expression of tonin was significantly reduced, tonin activity was not altered in most brain
structures, except for cerebellum and neurohypophysis. (Hypertension. 2002;39:229-232.)
Key Words: tonin 䡲 kallikrein 䡲 angiotensin II 䡲 rats 䡲 brain
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T
homogeneous for other brain regions. In this study, we used
reverse transcription–polymerase chain reaction (RT-PCR) to
determine the regional expression pattern of tonin and kallikrein in the brain of the TGR(hKLK1). The activities of the
enzymes were also determined in the same tissues of the brain
and cerebellum of the rat.
he coexistence of angiotensin (Ang) II– generating and
kallikrein-kinin (KKS) systems in different sites suggests
diverse physiological effects for these systems in the local
control of blood flow and in the pathophysiology of hypertension and cardiovascular diseases. In the literature, there is
a growing body of evidence for alternative pathways in the
formation of Ang II in many tissues, including the brain. It
has been suggested that proteinases other than renin and ACE
may be involved in the local liberation of Ang II directly from
angiotensinogen.1– 4 An alternative route that produces Ang II
directly from angiotensinogen involves the enzyme tonin.5
Tonin activity has been found in several rat tissues, including
the brain.6 – 8 The liberation of Ang II by tonin in the brain
may be important for the control of blood pressure and body
fluid volumes. There is evidence that the KKS is also
involved with the modulation of the brain circulation and
central control of the blood pressure.9 To elucidate the
functions of the KKS, transgenic techniques have been
employed to either overexpress or ablate pertinent genes.10,11
This has led to new animal models and has substantially
increased our understanding of the physiological functions of
this peptide system.12
A transgenic rat (TGR) harboring the human kallikrein
gene (hKLK1) was recently established. The TGR(hKLK1)
model is hypotensive.13 Kallikreins are present in common
sites to tonin such as the submandibular gland, kidney, and
brain.14 Recently, we determined the regional distribution of
tonin- and kallikrein-simile activities in the brain of Wistar
rats.8 We showed that the highest values of tonin activity is in
the neurohypophysis and archicerebellum, whereas the kallikrein activity is not present in the neurohypophysis and is
Methods
The Methods section can be found in an online data supplement
available at http://www.hypertensionaha.org.15–19
Results
Our results show that the human transgene hKLK1 is
expressed in all tested brain structures from the TGR
(Figure 1) but is not expressed in the structures of the
control Sprague-Dawley rat (data not shown). Figures 2
and 3 show the results of expression of the rat kallikrein in
the Sprague-Dawley rat and TGR(hKLK1), respectively.
Low levels of rat kallikrein expression were observed in
the regions neurohypophysis, adenohypophysis, hypothalamus, and choroid plexus of the Sprague-Dawley rat. No
expression of the rat kallikrein gene was observed in the
other regions (Figure 2). Higher level expression of the rat
kallikrein gene was observed in the brain structures of the
TGR(hKLK1). Unlike the Sprague-Dawley rat, it was
possible to detect expression of the rat kallikrein gene in
almost all of the structures of TGR(hKLK1), except in the
medulla oblongata (Figure 3). When compared with
Sprague-Dawley rat, the expression levels in neurohypophysis and adenohypophysis are strongly higher.
Received July 25, 2001; first decision August 13, 2001; revision accepted November 23, 2001.
From the Departments of Physiology and Biophysics, Instituto de Ciencias Biologicas, Universidade Federal de Minas Gerais (E.S.L.L., J.L.P.),
University of Mogi das Cruzes (R.C.A.), and Universidade Federal de Sao Paulo (J.B.P.), Brazil; and Molecular Biology of Peptides Hormones,
Max-Delbruck Center for Molecular Medicine (M.B.), Berlin-Buch, Germany.
Corresponding author Dr Jorge Luiz Pesquero, Depto Fisiologia e Biofisica, ICB, Universidade Federal de Minas Gerais, Av. Antonio Carlos 6627,
31270-901, Belo Horizonte, MG, Brazil. E-mail [email protected]
© 2002 American Heart Association, Inc.
Hypertension is available at http://www.hypertensionaha.org
229
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February 2002
Figure 1. Human tissue kallikrein expression in TGR(hKLK1) as
determined by RT-PCR. Ac indicates archicerebellum; Ah, adenohypophysis; Cn, cerebellar nuclei; Co, cerebellar cortex; Cp,
choroid plexus; Hc, hippocampus; Ht, hypothalamus; Mb, midbrain; Mo, medulla oblongata; Nh, neurohypophysis; Po, pons;
Sg, submandibular gland; and Th, thalamus.
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In the Sprague-Dawley rat, tonin is expressed in all
structures tested with the highest level in the midbrain (Figure
4). In the transgenic rat, the expression levels are diminished
in all structures, becoming undetectable in the midbrain and
neurohypophysis (Figure 5). The results of ribonuclease
protection assay (RPA) for tonin expression in the TGR(hKLK1) are in agreement with those of RT-PCR. By RPA, it
was possible to detect tonin expression in the cerebellar
cortex, medulla oblongata, and thalamus (Figure 6). With
regard to the kallikrein activity, the results are in agreement to
the observed for gene expression as determined by RT-PCR.
Kallikrein activity is higher in several brain structures of the
TGR(hKLK1) than of the Sprague-Dawley rat (Figure 7A).
The most altered levels of kallikrein activity were observed in
the neurohypophysis, choroid plexus, medulla oblongata, and
archicerebellum. In general, the levels of tonin activity are
not significantly different for both animals. Significant augment of tonin activity by the introduction of the transgene
was observed for archicerebellum and cerebellar cortex and
the reduction of activity in neurohypophysis, cerebellar nuclei, and thalamus (Figure 7B).
Discussion
Recently we elucidated some aspects related to the distribution of the tonin- and kallikrein-like activities in the
brain and cerebellum of the Wistar rat. We verified that in
Figure 2. Rat kallikrein expression in Sprague-Dawley rats as
determined by RT-PCR. For brain region information, see legend
of Figure 1.
Figure 3. Rat kallikrein expression in TGR(hKLK1) as determined
by RT-PCR. For brain region information, see legend of Figure 1.
the rat, kallikrein activity is homogeneously distributed in
the brain, except for the neurohypophysis, where it is not
present. On the other hand, tonin activity is concentrated in
the neurohypophysis and archicerebellum. A probable
function of tonin in the hypophysis-hypothalamus axis is
to process precursor proteins into active forms and peptides, as it can do with ␤-lipoprotein and adrenocorticotropic hormone.20
In this study, we show the levels of these enzymes,
determined by quantitation of the specific mRNA and activity, in the same regions of the brain of a rat expressing human
kallikrein. Because this rat is hypotensive,13 we speculate that
tonin and kallikrein are involved in this phenotype. As we
determined the activity of the enzymes in crude extracts and
the finding that the D-Pro-Phe-Arg-p-nitroanilide is a selective substrate for kallikrein rather than a specific one, we
performed an assay in the presence of 5 ␮mol/L soybean
trypsin inhibitor (SBTI) in an attempt to selectively inhibit
interfering proteinases while still retaining tissue kallikrein
activity. From the kallikrein superfamily, tissue kallikrein is
the major proteinase that shows relative resistance to inhibition by SBTI.21 Our results showed that the introduction of
the human kallikrein gene into rat genome produced alterations in the activity and gene expression of the enzymes. For
kallikrein, in general, we observed augmented activity and
mRNA levels. In the presence of SBTI, the activity of
enzyme in the thalamus of TGR(hKLK1) was inhibited by
50%; however, no inhibition was observed for other regions.
This means that in the thalamus, plasma kallikrein may be
interfering in our assay.
Figure 4. Tonin expression determined by RT-PCR in SpragueDawley rats. For brain region information, see legend of Figure 1.
Lomez et al
Kallikrein and Tonin in the Brain
231
Figure 5. Tonin expression determined by RT-PCR in TGR(hKLK1). For brain region information, see legend of Figure 1.
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How can the expressed human transgene upregulate the
expression levels of the endogenous rat enzyme? One
possible explanation involves the specificity of the
substrate-enzyme interactions. We postulate that the product of the human transgene can interact with the rat
substrate but cannot hydrolyze it well. Indeed, we verified
that human urinary kallikrein is not able to hydrolyze
partially purified rat kininogen. In this way, less substrate
is available for the endogenous kallikrein, and as a result,
less kinin is produced. The lower levels of kinins may
represent a stimulus for rat kallikrein synthesis and release.
This higher level of kallikrein would be able to keep the
same levels or an increased level of kinins compared with
that of genetically unmodified rats. However, another
interesting aspect that could also explain the hypotensive
phenotype in the TGR(hKLK1) is the decrease in the levels
of activity and expression of tonin. The expression of tonin
diminished in all structures studied, and the activity
decreased in the neurohypophysis, cerebellar nuclei, and
thalamus. Even though we had been not able to detect tonin
mRNA in the midbrain and in the neurohypophysis of the
transgenic rat, tonin activity is present in these structures.
Perhaps the enzyme is made somewhere in the brain and
transported to these structures by a mechanism that, for
neurohypophysis, is facilitated in the transgenic animal. To
determine the specificity of our assay, tonin activity was
performed in the absence and in the presence of an
antitonin antibody. In the presence of the antibody, the
angiotensin-liberating activity was 100% inhibited. No
alteration of kallikrein activity was observed by antitonin
antibody. The distinct gene and tissue-specific expression
patterns presented in this study, in conjunction with the
regional activity levels of the enzymes strongly suggest
that tonin and kallikrein are involved with the hypotensive
phenotype of the TGR(hKLK1).
Figure 6. Tonin expression determined by RPA in TGR(hKLK1).
For brain region information, see legend of Figure 1.
Figure 7. Kallikrein-specific (A) and tonin-specific (B) activities
(mean⫾SD) of the brain and cerebellum structures. #P⬍0.05;
*P⬍0.01. For brain region information, see legend of Figure 1.
Acknowledgments
This work was supported by grants from Coordenação de Aperfeiçoamento de Pessoal de Nı́vel Superior, Deutscher Akademischer
Austauschdienst, Fundação de Amparo a Pesquisa do Estado de São
Paulo, and Fundação de Amparo a Pesquisa do Estado de Minas
Gerais.
References
1. Yosipiv IV, El-Dahr SS. Activation of angiotensin-generating systems in
the developing rat kidney. Hypertension. 1996;27:281–286.
2. Baltatu O, Nishimura H, Hoffmann S, Stoltenburg G, Eaulica I, Lippoldt
A, Ganten D, Urata H. High levels of human chymase expression in the
pineal and pituitary glands. Brain Res. 1997;752:269 –278.
3. Rosenthal J, Thurnreiter M, Plasche M, Geyer M, Reiter W, Dahlheim H.
Renin-like enzymes in human vasculature. Hypertension. 1990;15:
848 – 853.
4. Saye JA, Ragsdale NV, Carey RM, Peach MJ. Localization of angiotensin
peptide-forming enzymes of 3T3-F442A adipocytes. Am J Physiol. 1993;
264:C1570 –C1576.
5. Grisé C, Boucher R, Thibault G, Genest J. Formation of angiotensin II by
tonin from partially purified human angiotensinogen. Can J Biochem.
1981;59:250 –255.
6. Araujo GW, Pesquero JB, Lindsey CJ, Paiva ACM, Pesquero JL.
Identification of serine proteinases with tonin-like activity in the rat
submandibular and prostate glands. Biochim Biophys Acta. 1991;1074:
167–171.
7. Johansen L, Berg T, Bergundhaugen H, Nustad K, Scicli AG, Carretero
OA. Excess antibody immunoassay for the measurement of tonin in rat
tissue and plasma. J Immunol Meth. 1987;98:257–265.
8. Lopes ES, Sumitani M, Juliano L, Beraldo WT, Pesquero JL. Distribution
of tonin- and kallikrein-like activities in the rat brain. Brain Res. 1997;
769:152–157.
9. Whal M, Gorlach C, Hortobagyi T, Benyo Z. Effects of bradykinin in the
central circulation. Acta Physiol Hung. 1999;86:155–160.
10. Wang J, Xiong W, Yang Z, Davis T, Dewey MJ, Chao J, Chao L. Human
tissue kallikrein induces hypotension in transgenic mice. Hypertension.
1994;23:236 –243.
11. Pesquero JB, Araujo RC, Heppenstall PA, Stucky CL, Silva JA Jr,
Walther T, Oliveira SM, Pesquero JL, Paiva ACM, Calixto JB, Lewin
GR, Bader M. Hypoalgesia and altered inflammatory responses in mice
lacking kinin B1 receptors. Proc Nat Acad Sci U S A. 2000;97:
8140 – 8145.
12. Pesquero JB, Bader M. Molecular biology of the kallikrein-kinin
system: from structure to function. Braz J Med Biol Res. 1998;31:
1197–1203.
232
Hypertension
February 2002
13. Silva JA Jr, Araujo RC, Baltatu O, Oliveira SM, Tschope C, Fink E,
Hoffmann S, Plehm R, Chai KX, Chao L, Chao J, Ganten D, Pesquero JB,
Bader M. Reduced cardiac hypertrophy and altered blood pressure control
in transgenic rats with the human tissue kallikrein gene. FASEB J.
2000;14:1858 –1860.
14. Zacharatos DT, Vasak EZ, Cheng ESP, van Lennep EW, Morris
BJ. Immunohistochemistry of tonin in rat submandibular gland during
development, lactation and secretion. Histochemistry. 1983;79:
433– 442.
15. Baltatu O, Lippoldt A, Hansson A, Ganten D, Bader M. Local renin-angiotensin system in the pineal gland. Mol Brain Res. 1997;54:237–242.
16. Gualberto MP, Nunes RL, Pesquero JL. Tonin-like activity present in
the human submandibular gland. Agents Actions Suppl. 1992;38:
392– 400.
17. Chagas JR, Hirata IY, Juliano MA, Xiong W, Wang C, Chao J, Juliano L,
Prado E. Substrates specificities of tissues kallikrein and T-kininogenase:
18.
19.
20.
21.
their possible role in kininogen processing. Biochemistry. 1992;31:
4969 – 4974.
Bradford MM. A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein-dye
binding. Anal Biochem. 1976;72:248 –254.
Juliano L, Paiva ACM. Conformation of angiotensin II in aqueous
solution. Titration of several peptide analogs and homologs. Biochemistry. 1974;13:2445–2450.
Seidah NG, Chan JSD, Mardini G, Benjannet S, Chrétien M, Boucher R,
Genest J. Specific cleavage of ␤-LPH and ACTH by tonin: release of an
opiate-like peptide ␤-LPH (61–78). Biochem Biophys Res Commun.
1979;86:1002–1013.
Shori DK, Proctor GB, Chao J, Chan KM, Garret JR. New specific assays
for tonin and tissue kallikrein activities in rat submandibular glands.
Biochem Pharmacol. 1992;43:1209 –1217.
Downloaded from http://hyper.ahajournals.org/ by guest on June 17, 2017
Tonin and Kallikrein in the Brain of Transgenic Rat Line Expressing Human Tissue
Kallikrein
Eliane S.L. Lomez, Ronaldo C. Araujo, Michael Bader, Joao B. Pesquero and Jorge L. Pesquero
Downloaded from http://hyper.ahajournals.org/ by guest on June 17, 2017
Hypertension. 2002;39:229-232
doi: 10.1161/hy0202.104264
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