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Local renin–angiotensin system in the pineal gland
Molecular Brain Research 54 Ž1998. 237–242
Research report
Local renin–angiotensin system in the pineal gland Ovidiu Baltatu a , Andrea Lippoldt a , Anita Hansson a , Detlev Ganten a
a,b
, Michael Bader
a,)
Hypertension Research, Max-Delbruck-Center for Molecular Medicine (MDC), D-13122 Berlin-Buch, Germany ¨ b Institute for Clinical Pharmacology, Free UniÕersity, D-12200 Berlin, Germany Accepted 14 October 1997
Abstract Besides the classical endocrine renin–angiotensin system ŽRAS., a local RAS has been described also in the brain. We attempted to clarify the existence of a local RAS in the pineal gland. Through the use of a ribonuclease protection assay, it proved possible to detect the mRNA for angiotensinogen ŽAOGEN., for the angiotensin receptor type 1A ŽAT1a . and 1B ŽAT1b . and for the angiotensin-converting enzyme ŽACE. in pineal glands from rats. Renin mRNA, however, could not be found by this method. By in situ hybridization and immunocytochemistry, AOGEN mRNA was co-localized with the astrocyte marker glial fibrillary acidic protein. AT1b mRNA expression exceeded the expression of AT1a mRNA and was co-localized with the pinealocyte-specific tryptophan hydroxylase. Thus, in the mammalian pineal gland there is a local formation of the components of the RAS. The presence of angiotensin II receptors further substantiates a role for angiotensins and the pineal RAS in the physiology of this gland. q 1998 Elsevier Science B.V. Keywords: Brain; Pineal gland; Angiotensinogen; Angiotensin II receptor; Renin; ACE; Renin–angiotensin system
1. Introduction The mammalian pineal gland is phylogenetically derived from the caudal diencephalic roof as an unpaired structure of the epithalamus, comprising one of the neuroendocrine glands present within the brain. The gland is considered to transduce neural signals generated from photoreceptors into an endocrine signal and to regulate the reproductive activity of mammals via secretion of melatonin. Moreover, accumulating evidence demonstrates additional functions of the pineal gland and melatonin such as the modulation of the neuro–immune axis, of aging processes and biological rhythms. It has been established that the predominantly sympathetic innervation of the pineal gland modulates the basic circadian pattern of melatonin production via noradrenergic fibers originating in the superior cervical ganglion. This circadian rhythm of melatonin synthesis and secretion appears to be an essential feature of most mammals w20,24x. In addition to melatonin and its indole derivatives, the
) Corresponding author. Hypertension Research, Max-Delbruck-Center ¨ for Molecular Medicine ŽMDC., Robert-Rosslestr. 10, D-13122 Berlin¨ Buch, Germany. Fax: q49-30-9406-2110
0169-328Xr98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 9 - 3 2 8 X Ž 9 7 . 0 0 3 3 9 - 2
pineal gland has been reported to contain several classical transmitters, such as norepinephrine, dopamine and serotonin and also peptide hormones, such as neuropeptide Y, vasopressin, substance P, enkephalins and vasoactive intestinal peptide Žreviewed in Refs. w7,17,23x.. The biochemical complexity of the gland, moreover, is not limited to this array of transmitters and hormones. Haulica et al. w9x and Mizuno et al. w16x for example, have detected in the pineal gland high renin activity which is one of the enzymes involved in angiotensin II ŽAng II. formation. Additional studies have confirmed the presence of enzymatic pathways for Ang II formation in the pineal gland and have even suggested role for this peptide in gland function w8,10,12,18x. More widely known as the active peptide of the renin–angiotensin system ŽRAS., Ang II is classically involved in the regulation of cardiovascular homeostasis. Besides its synthesis in plasma from circulating angiotensinogen ŽAOGEN., Ang II is also produced in tissues from locally synthesized AOGEN w3x. However, the presence of a local RAS in the pineal gland has not previously been reported. In the present study, we present evidence for a local RAS in the pineal gland by describing the corresponding presence and localization of mRNAs that encode the components of a RAS.
O. Baltatu et al.r Molecular Brain Research 54 (1998) 237–242
238
2. Materials and methods 2.1. Animals Male Sprague–Dawley rats, adult weighing 250 to 400 g, were obtained from the animal breeding units of the Max-Delbruck-Center for Molecular Medicine. The rats ¨ were housed under a 12 h lightr12 h dark schedule Žlight on at 0600., at 24 " 28C and given free access to a standard rat diet and tap water. The rats were sacrificed by decapitation under ether anesthesia, at 1000–1100. For experiments that required RNA isolation, pineal glands or other brain regions were quickly dissected on dry ice and stored at y808C. For experiments requiring tissue preservation, brains containing the pineal gland were immediately snap-frozen in Isopentane at y308C and than stored at y808C. 2.2. Probes used for mRNA detection The cDNA sequences used as probes for the mRNAs of RAS components were obtained by RT-PCR and cloned into various vectors ŽTable 1.. Labelled probes were prepared by linearization and transcription of the plasmids. Linearization was achieved by overnight incubation at 378C with the appropriate restriction enzyme Žsee Table 1.. Linearized plasmids were purified following the procedure of Sambrook et al. w22x. The labelled RNA probes were synthesized in the presence of 32 P–UTP or digoxygenin ŽDIG. –UTP by using an RNA transcription kit ŽStratagene, Heidelberg, Germany. and purified on a 5% acrylamider8 M urea gel. 2.3. Ribonuclease protection assay Total RNA was isolated from tissues by using the TRIzole reagent ŽLife Technologies, Eggenstein, Germany. followed by chloroform–isopropanol extraction, according to the protocol of the manufacturer. To obtain an appropriate amount of RNA, five to six pineals or one to two pituitary glands were pooled into a common tube. Identification of mRNA specific for rat AOGEN, renin, ACE, AT1a and AT1b receptors was effected by ribonuclease protection assay ŽRPA., using the Ambion RPA II kit ŽAMS Biotechnology, Witney, UK.. 20 m g Žfor AOGEN and renin. or 60 m g Žfor AT1a and AT1b recep-
tors. of total RNA per sample were hybridized with approximately 30 000 cpm of the radiolabelled antisense probe. The b-actin probe was used as control for the amount of RNA used for the RPA. The hybridized fragments protected from RNAse A q T1 digestion were separated by electrophoresis on a denaturing gel Ž5% polyacrylamide, 8 M urea. and analyzed using a FUJIX BAS 2000 Phospho-Imager system ŽFuji, Dusseldorf, Germany.. The ¨ assays were repeated at least three times with identical results. 2.4. In situ hybridization In situ hybridization was performed as described by Lippoldt et al. w15x. Thick sections of 12 m m were cut using a cryostat ŽLeica Jung CM 3000. and mounted onto poly-L-lysine ŽPLL. coated slides. Brain sections were fixed with 3% buffered paraformaldehyde and deproteinized with 0.2 M HCl. To decrease background binding, the sections were acetylated. After incubation in a prehybridization mix Ž0.05 M tris–HCl wpH 7.6x, 0.025 M EDTA wpH 8x, 2.5 = Denhardt’s solution, 0.25 mgrml yeast tRNA, 0.02 M NaCl and 50% deionized formamide., the sections were hybridized with DIG-labelled antisense and sense RNA, Žused as negative control. specific for AOGEN, ACE, AT1a or AT1b . The DIG-labelled probes were obtained by transcription of cDNA, using the same plasmids used in the RPA ŽTable 1.. The labelled probes were diluted in a solution containing 0.02 M tris–HCl wpH 7.6x, 0.001 M EDTA wpH 8x, 1 = Denhardt’s solution, 0.5 mgrml yeast tRNA, 0.33 M NaCl, 0.1 M DTT, 10% dextran sulfate and 50% deionized formamide. After hybridization, the sections were washed in 1 = SSC at 488C for 30 min and then in 0.5 = SSCr50% formamide at 488C for 4 h. After washing, the sections were incubated with blocking buffer Ž0.1 M Tris–HCl wpH 7.6x, 0.15 M NaCl, 1% normal sheep serum.. Immunological detection was then effected by incubating with alkaline phosphatase-conjugated anti-DIG antibody Ž1:300 dilution in blocking buffer.. Following subsequent washes, an alkaline phosphatase dependent color reaction was developed by incubating the sections with detection buffer Ž0.375 mgrml NBT, 0.19 mgrml BCIP, 0.1 Tris–HCl wpH 8x, 0.1 M NaCl, 0.02 M MgCl 2 .. To identify the cell type expressing mRNA for AOGEN or AT1b receptor, immunocytochemistry ŽICC. for glial
Table 1 AOGEN ŽRef. w14x. AT1B AT1A Renin ŽRef. w6x. ACE ŽRef. w4x. b-actin ŽRef. w6x.
Length of protected sequence
Vector
cRNA synthesis
mRNA synthesis
290 bp 299 bp 230 bp 300 bp 375 bp 150 bp
pGEM4 pCReII pCReII pGEM3 PBluescript IIks q Bluescript SKII q
EcoRI cut and T7 RNA poly BamHI cut and T7 RNA poly XbaI cut and SP6 RNA poly HindIII cut and T7 RNA poly BamHI cut and T3 RNA poly XbaI cut and T7 RNA poly
BamHI cut and SP6 RNA poly XbaI cut and SP6 RNA poly BamHI cut and T7 RNA poly KpnI cut and SP6 RNA poly XbaI cut and T7 RNA poly Sal I cut and SP6 poly
O. Baltatu et al.r Molecular Brain Research 54 (1998) 237–242
fibrillary acidic protein ŽGFAP. and tryptophan hydroxylase ŽTPH. was performed after the in situ hybridization. GFAP is a known marker for glial cells, while TPH is a recognized marker for pinealocytes. Polyclonal antibodies against GFAP ŽBoehringer Mannheim, Mannheim, Germany. and TPH ŽBiotrend, Koln, ¨ Germany. were detected by using Vectastain w ABC kit ŽSERVA, Heidelberg, Germany. according to the manufacturer instructions. Incubations only with the secondary antibodies were used as negative controls. The assays were repeated at least three times with identical results.
239
3. Results 3.1. Ribonuclease protection assay To determine the local synthesis of RAS components in the pineal gland, cRNA riboprobes complementary to the mRNAs for AOGEN, renin, ACE, AT1a and AT1b receptors were used. In the RPA with total RNA from pineal gland and other brain regions for comparison, mRNA specific for AOGEN was detected in high amounts in the pineal gland when normalized to the b-actin expression
Fig. 1. AOGEN mRNA in the pineal gland and various brain regions. Twenty m g of total RNA were used for the RPA. Pin, pineal gland; Pit, pituitary gland; Die, diencephalon; BrSt, brain stem; Cor, cortex; Cer, cerebellum.
Fig. 2. AT1b and AT1a mRNA expression in pineal, pituitary and adrenal gland. Sixty m g of pineal gland RNA and 70 m g of pituitary and adrenal gland RNA were used for RPA. Pin, pineal gland; Pit, pituitary gland; Adr, adrenal gland.
Fig. 3. ACE mRNA expression in the pineal gland and various brain regions. Twenty m g of total RNA was used for RPA. Pin, pineal gland; Pit, pituitary gland; Die, diencephalum; BrSt, brain stem; Cor, cortex; Cer, cerebellum.
240
O. Baltatu et al.r Molecular Brain Research 54 (1998) 237–242
ŽFig. 1.. The presence of mRNAs for the two subtypes of angiotensin receptor type 1 could also be demonstrated ŽFig. 2., with predominance of the AT1b type. Investigation of the local synthesis of renin and ACE, the classical
enzymes of RAS, showed that the pineal gland contains high amounts of mRNA for ACE ŽFig. 3., while the mRNA for renin was undetectable with our probe Ždata not shown..
Fig. 4. In situ hybridization of AOGEN and AT1b mRNA in the pineal gland. Tissue sections were hybridized with DIG-labeled AOGEN ŽA, B. or AT1b ŽC, D. probes. Detection of hybridized probe was achieved with alkaline phosphatase-conjugated anti-DIG antibody Ždark violet.. Localization of cells containing GFAP ŽB. or TPH ŽD. was done by using polyclonal antibodies; positive immunoreactivity was visualized by peroxidase reaction Žbrown color.. In B, arrows indicate astroglial prolongations. In D, arrows indicate parenchymal cells positive for TPH.
O. Baltatu et al.r Molecular Brain Research 54 (1998) 237–242
3.2. In situ hybridization AOGEN and AT1b mRNAs were also detected in frozen tissue sections by in situ hybridization ŽFig. 4A–D, respectively.. For distinction of the expressing cell type, the sections were counterstained for GFAP or TPH after in situ hybridization. AOGEN mRNA appears to be in the same cell type that stained for GFAP ŽFig. 4B.. TPH is the enzyme involved in the first step of melatonin synthesis in pinealocytes, the neurosecretory cells of the pineal gland. Positive hybridization signals for AT1b mRNA were detected on the plasma membrane of TPH-positive cells ŽFig. 4D..
4. Discussion The present study demonstrates the existence of a local RAS in the rat pineal gland. The mRNA for AOGEN was detected in this tissue, thereby revealing the local synthesis of the precursor molecule of Ang II. The detection of the mRNA for the Ang II receptor AT1b , furthermore, indicates a local action of this peptide on the pineal. Since the first report describing the possible action of Ang II on the pineal gland w19x, additional observations have suggested the existence of a local formation and action of angiotensins. Immunocytochemical studies of RAS components in the pineal gland, however, have given contradictory results w21x. Renin has been reported to be present in the mammalian pineal gland w1,9,11,12x on the basis of biochemical and immunohistochemical methods. Enzyme activity measurements, in fact, showed that the pineal gland is the brain structure containing the highest renin-like activity w9x. ACE, moreover, was detectable in rat w18x and human w2x pineal glands. Indeed, we recently demonstrated that the human pineal tissue converts angiotensin I into Ang II in vitro more readily than any other brain region studied: pituitary gland, diencephalon, cortex, mesencephalon and cerebellum w2x. We also found, by using biochemical and immunohistochemical approaches, high levels of ACE as well as chymase, a potent enzyme in alternative generation of Ang II w25x in the human pineal. These observations have indicated that the mammalian pineal gland contains an extensive enzymatic system for the production of the active peptides of the RAS. In this study, we have further elaborated the biochemistry of angiotensin production and action in the pineal gland. By using specific cRNA riboprobes, we detected the mRNA for ACE. We were unable, however, to detect renin mRNA by RPA. The following hypotheses may explain this inability: Ž1. Renin mRNA is present at levels in the pineal that are too low to be detected by RPA. Ž2. Renin is recruited by the gland from the circulation, since at the pineal level the blood–brain barrier is poorly developed. Ž3. There are other enzymes in the gland that can produce angiotensins from AOGEN.
241
In order to establish the existence of a local pineal RAS, it is necessary to identify both the synthesis of a pineal AOGEN as the only source of Ang II in the system, as well as the existence of Ang II receptors as target molecules of the system. In the present study, we demonstrate that AOGEN can indeed be produced in the pineal. AOGEN mRNA was detected in the pineal by RPA and localized to astroglial cells by counterstaining with GFAP antibodies, as has been described in other brain regions w5x. Receptor-binding assays and autoradiographic studies have indicated that the mammalian pineal contains saturable Ang II-binding sites specific for AT1-type Ang II receptors w2x. Two subtypes of the AT-1 receptor have been described in rodents: AT1a and AT1b . While AT1a has been localized in brain areas involved in the control of blood pressure, fluid homeostasis and neurosecretion, AT1b subtype was originally described in glandular tissues, such as anterior pituitary and adrenal gland w13x. In the present study, we have used RPA to detect the mRNA for both AT-1 receptor subtypes in the pineal gland. While the mRNA for the AT1a receptor is poorly expressed, the AT1b receptor predominates. In situ hybridization and immunocytochemistry additionally shows that the AT1b mRNA occurs in association with TPH immunoreactivity. Such localization of AT1b mRNA in pinealocytes suggests a role for angiotensins in the neurosecretory functions of the pineal gland. In addition, the predominance of the AT1b subtype over AT1a in the pineal, pituitary and adrenal glands indicates that the stimulation of the AT1b receptor subtype is specifically involved in neuroendocrine processes. Possible effects of Ang II on the pineal gland, in turn, are: stimulation of serotonin synthesis w10x, increase in vitro release of noradrenaline and the production of hydroxy- and methoxyindoles w8x. In any case, the localized production of RAS components as established in the present study suggests the existence of a pineal RAS, most likely involved in the neurosecretory functions of this pivotal gland.
Acknowledgements This work was partially funded by the Bundesministerium fur ¨ Bildung und Forschung. References w1x A. de Agostini, A. Reinharz, M.B. Vallotton, Renin-like and cathepsin D activities in bovine pineal glands, Neuroendocrinology 35 Ž1982. 457–463. w2x O. Baltatu, H. Nishimura, S. Hoffmann, G. Stoltenburg, I. Haulica, A. Lippoldt, D. Ganten, H. Urata, High levels of human chymase expression in the pineal and pituitary glands, Brain Res. 759 Ž1997. 269–278. w3x B. Bunnemann, K. Fuxe, D. Ganten, The brain renin–angiotensin system: Localization and general significance, J. Cardiovasc. Pharmacol. 19 Ž1992. S51–S62.
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w4x O. Costerousse, J. Allegrini, H. Huang, J. Bounhik, F. Alhenc Gelas, Regulation of ACE gene expression and plasma levels during rat postnatal development, Am. J. Physiol. 267 Ž1994. E745–E753. w5x C.F. Deschepper, J. Bouhnik, W.F. Ganong, Co-localization of angiotensinogen and glial fibrillary acidic protein in astrocytes in rat brain, Brain Res. 374 Ž1986. 195–198. w6x B. Djavidani, M. Sander, R. Kreutz, K. Zeh, M. Bader, S.H. Mellon, P. Vecsei, J. Peters, D. Ganten, Chronic dexamethasone treatment suppresses hypertension development in the transgenic rat TGRŽmREN2.27, J. Hypertens. 13 Ž1995. 637–645. w7x M. Ebadi, R.F. Hexum, R.F. Pfeiffer, P. Govitrapong, The pineal and retinal peptides and their receptors, Pineal Res. Rev. 7 Ž1989. 1–156. w8x L.M. Finocchiaro, D.J. Goldstein, S. Finkielman, V.E. Nahmod, Interaction of angiotensin II with the cholinergic and noradrenergic systems in the rat pineal gland: Regulation of indole metabolism, J. Endocrinol. 126 Ž1990. 59–66. w9x I. Haulica, D.D. Branisteanu, V. Rosca, A. Stratone, V. Berbeleu, G. Balan, L. Ionescu, A renin-like activity in pineal gland and hypophysis, Endocrinology 96 Ž1975. 508–510. w10x I. Haulica, G. Petrescu, M. Uluitu, V. Rosca, S. Slatineanu, Influence of angiotensin II on dog pineal serotonin content, Neurosci. Lett. 18 Ž1980. 329–332. w11x S. Hirose, H. Yokosawa, T. Inagami, R.J. Workman, Renin and prorenin in hog brain: Ubiquitous distribution and high concentration in the pituitary and pineal, Brain Res. 19 Ž1980. 489–499. w12x T. Inagami, M.R. Celio, D.L. Clemens, D. Lau, Y. Takii, A.G. Kasselberg, S. Hirose, Renin in rat and mouse brain: Immunohistochemical identification and localization, Clin. Sci. 59 Ž1980. 49s– 51s, Suppl. 6. w13x S.S. Kakar, J.C. Sellers, D.C. Devor, L.C. Musgrove, J.D. Neill, Angiotensin II type-1 receptor subtype cDNAs: Differential tissue expression and hormonal regulation, Biochem. Biophys. Res. Commun. 183 Ž1992. 1090–1096. w14x S. Kimura, J.J. Mullins, B. Bunnemann, R. Metzger, U. Hilgenfeldt,
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w23x w24x w25x
F. Zimmermann, H. Jacob, K. Fuxe, D. Ganten, M. Kaling, High blood pressure in transgenic mice carrying the rat angiotensinogen gene, EMBO J. 11 Ž1992. 821–827. A. Lippoldt, B. Andbjer, H. Gerst, D. Ganten, K. Fuxe, Basic fibroblast growth factor expression and tenascin C immunoreactivity after partial unilateral hemitransection of the rat brain, Brain Res. 730 Ž1996. 1–16. K. Mizuno, S. Hashimoto, M. Ojima, N. Kunii, M. Tani, S. Niimura, H. Watari, S. Fukuchi, Immunoreactive renin in human brain: Distribution and properties, Jpn. Circ. J. 49 Ž1985. 1005–1011. M. Moller, J.P. Ravault, B. Cozzi, The chemical neuroanatomy of the mammalian pineal gland: Neuropeptides, Neurochem. Int. 28 Ž1996. 23–33. V.E. Nahmod, M.S. Balda, C.J. Pirola, S. Finkielman, P.V. Gejman, D.P. Cardinali, Circadian rhythm and neural regulation of rat pineal angiotensin-converting enzyme, Brain Res. 236 Ž1982. 216–220. N.M. Panagiotis, G.F. Hungerford, Response of pineal sympathetic nerve processes and endings to angiotensin, Nature 211 Ž1966. 374–376. J.P. Preslock, The pineal gland: basic implications and clinical correlations, Endocr. Rev. 5 Ž1984. 282–308. E. Rix, E. Hackenthal, U. Hilgenfeldt, R. Taugner, Neuropeptides in the pineal gland? A critical immunocytochemical study, Histochemistry 72 Ž1981. 33–38. J. Sambrook J., E.F. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989. V. Simonneaux, Neuropeptides of the mammalian pineal gland, Neuroendocrinol. Lett. 2 Ž1995. 115–130. L. Tamarkin, C.J. Baird, O.F.X. Almeida, Melatonin: A coordinating signal for mammalian reproduction?, Science 227 Ž1985. 714–719. H. Urata, A. Kinoshita, K.S. Misono, F.M. Bumpus, A. Husain, Identification of a highly specific chymase as the major angiotensin II-forming enzyme in the human heart, J. Biol. Chem. 265 Ž1990. 22348–22357.
Research report
Local renin–angiotensin system in the pineal gland Ovidiu Baltatu a , Andrea Lippoldt a , Anita Hansson a , Detlev Ganten a
a,b
, Michael Bader
a,)
Hypertension Research, Max-Delbruck-Center for Molecular Medicine (MDC), D-13122 Berlin-Buch, Germany ¨ b Institute for Clinical Pharmacology, Free UniÕersity, D-12200 Berlin, Germany Accepted 14 October 1997
Abstract Besides the classical endocrine renin–angiotensin system ŽRAS., a local RAS has been described also in the brain. We attempted to clarify the existence of a local RAS in the pineal gland. Through the use of a ribonuclease protection assay, it proved possible to detect the mRNA for angiotensinogen ŽAOGEN., for the angiotensin receptor type 1A ŽAT1a . and 1B ŽAT1b . and for the angiotensin-converting enzyme ŽACE. in pineal glands from rats. Renin mRNA, however, could not be found by this method. By in situ hybridization and immunocytochemistry, AOGEN mRNA was co-localized with the astrocyte marker glial fibrillary acidic protein. AT1b mRNA expression exceeded the expression of AT1a mRNA and was co-localized with the pinealocyte-specific tryptophan hydroxylase. Thus, in the mammalian pineal gland there is a local formation of the components of the RAS. The presence of angiotensin II receptors further substantiates a role for angiotensins and the pineal RAS in the physiology of this gland. q 1998 Elsevier Science B.V. Keywords: Brain; Pineal gland; Angiotensinogen; Angiotensin II receptor; Renin; ACE; Renin–angiotensin system
1. Introduction The mammalian pineal gland is phylogenetically derived from the caudal diencephalic roof as an unpaired structure of the epithalamus, comprising one of the neuroendocrine glands present within the brain. The gland is considered to transduce neural signals generated from photoreceptors into an endocrine signal and to regulate the reproductive activity of mammals via secretion of melatonin. Moreover, accumulating evidence demonstrates additional functions of the pineal gland and melatonin such as the modulation of the neuro–immune axis, of aging processes and biological rhythms. It has been established that the predominantly sympathetic innervation of the pineal gland modulates the basic circadian pattern of melatonin production via noradrenergic fibers originating in the superior cervical ganglion. This circadian rhythm of melatonin synthesis and secretion appears to be an essential feature of most mammals w20,24x. In addition to melatonin and its indole derivatives, the
) Corresponding author. Hypertension Research, Max-Delbruck-Center ¨ for Molecular Medicine ŽMDC., Robert-Rosslestr. 10, D-13122 Berlin¨ Buch, Germany. Fax: q49-30-9406-2110
0169-328Xr98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 9 - 3 2 8 X Ž 9 7 . 0 0 3 3 9 - 2
pineal gland has been reported to contain several classical transmitters, such as norepinephrine, dopamine and serotonin and also peptide hormones, such as neuropeptide Y, vasopressin, substance P, enkephalins and vasoactive intestinal peptide Žreviewed in Refs. w7,17,23x.. The biochemical complexity of the gland, moreover, is not limited to this array of transmitters and hormones. Haulica et al. w9x and Mizuno et al. w16x for example, have detected in the pineal gland high renin activity which is one of the enzymes involved in angiotensin II ŽAng II. formation. Additional studies have confirmed the presence of enzymatic pathways for Ang II formation in the pineal gland and have even suggested role for this peptide in gland function w8,10,12,18x. More widely known as the active peptide of the renin–angiotensin system ŽRAS., Ang II is classically involved in the regulation of cardiovascular homeostasis. Besides its synthesis in plasma from circulating angiotensinogen ŽAOGEN., Ang II is also produced in tissues from locally synthesized AOGEN w3x. However, the presence of a local RAS in the pineal gland has not previously been reported. In the present study, we present evidence for a local RAS in the pineal gland by describing the corresponding presence and localization of mRNAs that encode the components of a RAS.
O. Baltatu et al.r Molecular Brain Research 54 (1998) 237–242
238
2. Materials and methods 2.1. Animals Male Sprague–Dawley rats, adult weighing 250 to 400 g, were obtained from the animal breeding units of the Max-Delbruck-Center for Molecular Medicine. The rats ¨ were housed under a 12 h lightr12 h dark schedule Žlight on at 0600., at 24 " 28C and given free access to a standard rat diet and tap water. The rats were sacrificed by decapitation under ether anesthesia, at 1000–1100. For experiments that required RNA isolation, pineal glands or other brain regions were quickly dissected on dry ice and stored at y808C. For experiments requiring tissue preservation, brains containing the pineal gland were immediately snap-frozen in Isopentane at y308C and than stored at y808C. 2.2. Probes used for mRNA detection The cDNA sequences used as probes for the mRNAs of RAS components were obtained by RT-PCR and cloned into various vectors ŽTable 1.. Labelled probes were prepared by linearization and transcription of the plasmids. Linearization was achieved by overnight incubation at 378C with the appropriate restriction enzyme Žsee Table 1.. Linearized plasmids were purified following the procedure of Sambrook et al. w22x. The labelled RNA probes were synthesized in the presence of 32 P–UTP or digoxygenin ŽDIG. –UTP by using an RNA transcription kit ŽStratagene, Heidelberg, Germany. and purified on a 5% acrylamider8 M urea gel. 2.3. Ribonuclease protection assay Total RNA was isolated from tissues by using the TRIzole reagent ŽLife Technologies, Eggenstein, Germany. followed by chloroform–isopropanol extraction, according to the protocol of the manufacturer. To obtain an appropriate amount of RNA, five to six pineals or one to two pituitary glands were pooled into a common tube. Identification of mRNA specific for rat AOGEN, renin, ACE, AT1a and AT1b receptors was effected by ribonuclease protection assay ŽRPA., using the Ambion RPA II kit ŽAMS Biotechnology, Witney, UK.. 20 m g Žfor AOGEN and renin. or 60 m g Žfor AT1a and AT1b recep-
tors. of total RNA per sample were hybridized with approximately 30 000 cpm of the radiolabelled antisense probe. The b-actin probe was used as control for the amount of RNA used for the RPA. The hybridized fragments protected from RNAse A q T1 digestion were separated by electrophoresis on a denaturing gel Ž5% polyacrylamide, 8 M urea. and analyzed using a FUJIX BAS 2000 Phospho-Imager system ŽFuji, Dusseldorf, Germany.. The ¨ assays were repeated at least three times with identical results. 2.4. In situ hybridization In situ hybridization was performed as described by Lippoldt et al. w15x. Thick sections of 12 m m were cut using a cryostat ŽLeica Jung CM 3000. and mounted onto poly-L-lysine ŽPLL. coated slides. Brain sections were fixed with 3% buffered paraformaldehyde and deproteinized with 0.2 M HCl. To decrease background binding, the sections were acetylated. After incubation in a prehybridization mix Ž0.05 M tris–HCl wpH 7.6x, 0.025 M EDTA wpH 8x, 2.5 = Denhardt’s solution, 0.25 mgrml yeast tRNA, 0.02 M NaCl and 50% deionized formamide., the sections were hybridized with DIG-labelled antisense and sense RNA, Žused as negative control. specific for AOGEN, ACE, AT1a or AT1b . The DIG-labelled probes were obtained by transcription of cDNA, using the same plasmids used in the RPA ŽTable 1.. The labelled probes were diluted in a solution containing 0.02 M tris–HCl wpH 7.6x, 0.001 M EDTA wpH 8x, 1 = Denhardt’s solution, 0.5 mgrml yeast tRNA, 0.33 M NaCl, 0.1 M DTT, 10% dextran sulfate and 50% deionized formamide. After hybridization, the sections were washed in 1 = SSC at 488C for 30 min and then in 0.5 = SSCr50% formamide at 488C for 4 h. After washing, the sections were incubated with blocking buffer Ž0.1 M Tris–HCl wpH 7.6x, 0.15 M NaCl, 1% normal sheep serum.. Immunological detection was then effected by incubating with alkaline phosphatase-conjugated anti-DIG antibody Ž1:300 dilution in blocking buffer.. Following subsequent washes, an alkaline phosphatase dependent color reaction was developed by incubating the sections with detection buffer Ž0.375 mgrml NBT, 0.19 mgrml BCIP, 0.1 Tris–HCl wpH 8x, 0.1 M NaCl, 0.02 M MgCl 2 .. To identify the cell type expressing mRNA for AOGEN or AT1b receptor, immunocytochemistry ŽICC. for glial
Table 1 AOGEN ŽRef. w14x. AT1B AT1A Renin ŽRef. w6x. ACE ŽRef. w4x. b-actin ŽRef. w6x.
Length of protected sequence
Vector
cRNA synthesis
mRNA synthesis
290 bp 299 bp 230 bp 300 bp 375 bp 150 bp
pGEM4 pCReII pCReII pGEM3 PBluescript IIks q Bluescript SKII q
EcoRI cut and T7 RNA poly BamHI cut and T7 RNA poly XbaI cut and SP6 RNA poly HindIII cut and T7 RNA poly BamHI cut and T3 RNA poly XbaI cut and T7 RNA poly
BamHI cut and SP6 RNA poly XbaI cut and SP6 RNA poly BamHI cut and T7 RNA poly KpnI cut and SP6 RNA poly XbaI cut and T7 RNA poly Sal I cut and SP6 poly
O. Baltatu et al.r Molecular Brain Research 54 (1998) 237–242
fibrillary acidic protein ŽGFAP. and tryptophan hydroxylase ŽTPH. was performed after the in situ hybridization. GFAP is a known marker for glial cells, while TPH is a recognized marker for pinealocytes. Polyclonal antibodies against GFAP ŽBoehringer Mannheim, Mannheim, Germany. and TPH ŽBiotrend, Koln, ¨ Germany. were detected by using Vectastain w ABC kit ŽSERVA, Heidelberg, Germany. according to the manufacturer instructions. Incubations only with the secondary antibodies were used as negative controls. The assays were repeated at least three times with identical results.
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3. Results 3.1. Ribonuclease protection assay To determine the local synthesis of RAS components in the pineal gland, cRNA riboprobes complementary to the mRNAs for AOGEN, renin, ACE, AT1a and AT1b receptors were used. In the RPA with total RNA from pineal gland and other brain regions for comparison, mRNA specific for AOGEN was detected in high amounts in the pineal gland when normalized to the b-actin expression
Fig. 1. AOGEN mRNA in the pineal gland and various brain regions. Twenty m g of total RNA were used for the RPA. Pin, pineal gland; Pit, pituitary gland; Die, diencephalon; BrSt, brain stem; Cor, cortex; Cer, cerebellum.
Fig. 2. AT1b and AT1a mRNA expression in pineal, pituitary and adrenal gland. Sixty m g of pineal gland RNA and 70 m g of pituitary and adrenal gland RNA were used for RPA. Pin, pineal gland; Pit, pituitary gland; Adr, adrenal gland.
Fig. 3. ACE mRNA expression in the pineal gland and various brain regions. Twenty m g of total RNA was used for RPA. Pin, pineal gland; Pit, pituitary gland; Die, diencephalum; BrSt, brain stem; Cor, cortex; Cer, cerebellum.
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ŽFig. 1.. The presence of mRNAs for the two subtypes of angiotensin receptor type 1 could also be demonstrated ŽFig. 2., with predominance of the AT1b type. Investigation of the local synthesis of renin and ACE, the classical
enzymes of RAS, showed that the pineal gland contains high amounts of mRNA for ACE ŽFig. 3., while the mRNA for renin was undetectable with our probe Ždata not shown..
Fig. 4. In situ hybridization of AOGEN and AT1b mRNA in the pineal gland. Tissue sections were hybridized with DIG-labeled AOGEN ŽA, B. or AT1b ŽC, D. probes. Detection of hybridized probe was achieved with alkaline phosphatase-conjugated anti-DIG antibody Ždark violet.. Localization of cells containing GFAP ŽB. or TPH ŽD. was done by using polyclonal antibodies; positive immunoreactivity was visualized by peroxidase reaction Žbrown color.. In B, arrows indicate astroglial prolongations. In D, arrows indicate parenchymal cells positive for TPH.
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3.2. In situ hybridization AOGEN and AT1b mRNAs were also detected in frozen tissue sections by in situ hybridization ŽFig. 4A–D, respectively.. For distinction of the expressing cell type, the sections were counterstained for GFAP or TPH after in situ hybridization. AOGEN mRNA appears to be in the same cell type that stained for GFAP ŽFig. 4B.. TPH is the enzyme involved in the first step of melatonin synthesis in pinealocytes, the neurosecretory cells of the pineal gland. Positive hybridization signals for AT1b mRNA were detected on the plasma membrane of TPH-positive cells ŽFig. 4D..
4. Discussion The present study demonstrates the existence of a local RAS in the rat pineal gland. The mRNA for AOGEN was detected in this tissue, thereby revealing the local synthesis of the precursor molecule of Ang II. The detection of the mRNA for the Ang II receptor AT1b , furthermore, indicates a local action of this peptide on the pineal. Since the first report describing the possible action of Ang II on the pineal gland w19x, additional observations have suggested the existence of a local formation and action of angiotensins. Immunocytochemical studies of RAS components in the pineal gland, however, have given contradictory results w21x. Renin has been reported to be present in the mammalian pineal gland w1,9,11,12x on the basis of biochemical and immunohistochemical methods. Enzyme activity measurements, in fact, showed that the pineal gland is the brain structure containing the highest renin-like activity w9x. ACE, moreover, was detectable in rat w18x and human w2x pineal glands. Indeed, we recently demonstrated that the human pineal tissue converts angiotensin I into Ang II in vitro more readily than any other brain region studied: pituitary gland, diencephalon, cortex, mesencephalon and cerebellum w2x. We also found, by using biochemical and immunohistochemical approaches, high levels of ACE as well as chymase, a potent enzyme in alternative generation of Ang II w25x in the human pineal. These observations have indicated that the mammalian pineal gland contains an extensive enzymatic system for the production of the active peptides of the RAS. In this study, we have further elaborated the biochemistry of angiotensin production and action in the pineal gland. By using specific cRNA riboprobes, we detected the mRNA for ACE. We were unable, however, to detect renin mRNA by RPA. The following hypotheses may explain this inability: Ž1. Renin mRNA is present at levels in the pineal that are too low to be detected by RPA. Ž2. Renin is recruited by the gland from the circulation, since at the pineal level the blood–brain barrier is poorly developed. Ž3. There are other enzymes in the gland that can produce angiotensins from AOGEN.
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In order to establish the existence of a local pineal RAS, it is necessary to identify both the synthesis of a pineal AOGEN as the only source of Ang II in the system, as well as the existence of Ang II receptors as target molecules of the system. In the present study, we demonstrate that AOGEN can indeed be produced in the pineal. AOGEN mRNA was detected in the pineal by RPA and localized to astroglial cells by counterstaining with GFAP antibodies, as has been described in other brain regions w5x. Receptor-binding assays and autoradiographic studies have indicated that the mammalian pineal contains saturable Ang II-binding sites specific for AT1-type Ang II receptors w2x. Two subtypes of the AT-1 receptor have been described in rodents: AT1a and AT1b . While AT1a has been localized in brain areas involved in the control of blood pressure, fluid homeostasis and neurosecretion, AT1b subtype was originally described in glandular tissues, such as anterior pituitary and adrenal gland w13x. In the present study, we have used RPA to detect the mRNA for both AT-1 receptor subtypes in the pineal gland. While the mRNA for the AT1a receptor is poorly expressed, the AT1b receptor predominates. In situ hybridization and immunocytochemistry additionally shows that the AT1b mRNA occurs in association with TPH immunoreactivity. Such localization of AT1b mRNA in pinealocytes suggests a role for angiotensins in the neurosecretory functions of the pineal gland. In addition, the predominance of the AT1b subtype over AT1a in the pineal, pituitary and adrenal glands indicates that the stimulation of the AT1b receptor subtype is specifically involved in neuroendocrine processes. Possible effects of Ang II on the pineal gland, in turn, are: stimulation of serotonin synthesis w10x, increase in vitro release of noradrenaline and the production of hydroxy- and methoxyindoles w8x. In any case, the localized production of RAS components as established in the present study suggests the existence of a pineal RAS, most likely involved in the neurosecretory functions of this pivotal gland.
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