- Email: [email protected]
Phenylethanolamine N-methyltransferase mRNA in rat hypothalamus and cerebellum
Brain Research 779 Ž1998. 289–291
Short communication
Phenylethanolamine N-methyltransferase mRNA in rat hypothalamus and cerebellum John L. Andreassi II, William B. Eggleston, Guillian Fu, Jennifer K. Stewart
)
Department of Biology, Virginia Commonwealth UniÕersity, 816 Park AÕe, Richmond, VA 23284-2012, USA Accepted 9 September 1997
Abstract Using the reverse transcription polymerase chain reaction, we detected a single form of phenylethanolamine N-methyltransferase ŽPNMT. mRNA in hypothalamus and medullarpons and two forms in cerebellum. These findings indicate that the PNMT gene is expressed in these brain areas and suggest that tissue specific splicing of PNMT mRNA may occur. q 1998 Elsevier Science B.V. Keywords: PNMT; Catecholamine; Hypothalamus; Rat; mRNA; RT-PCR
Hokfelt and colleagues first suggested that ¨ phenylethanolamine N-methyltransferase ŽPNMT, EC 2.1.1.29. is synthesized in the brainstem and transported within axons to other brain areas w11x. Although PNMT mRNA has been detected in the brainstem w7,22x and at low levels in the amygdala and stria terminalis w14x, it is unclear whether the PNMT gene is expressed in other brain regions. Evidence that PNMT is synthesized in the rat hypothalamus is controversial. Two laboratories have detected immunoreactive PNMT in hypothalamic cells w8,17x; however, most antibodies to PNMT recognize the protein only in medullary cell bodies and hypothalamic nerve terminals w1,3,12,20x. Also, PNMT-like activity is present in the surgically isolated hypothalamus w4,19x and declines after destruction of hypothalamic cells w13x, but the activity could reflect a less specific N-methyltransferase w18x. Finally, although PNMT activity and immunofluorescence have been detected in endothelial cells isolated from rat brain w21x, immuno-electron microscopy has not revealed PNMT in vascular cells in the hypothalamus w3,12x. We used the reverse transcription polymerase chain reaction ŽRT-PCR. to test for PNMT mRNA in rat hypothalamus. Because a small amount of PNMT activity is found in the rat cerebellum w9,23x, and almost no activity is present in liver and kidney w16x, we assayed these tissues to detect low levels of PNMT gene expression that may be
)
Corresponding author. Fax: q1 Ž804. 828-0503; E-mail: [email protected] 0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 1 1 7 0 - 0
present in many tissues. The medullarpons was assayed as a positive control. Adult male Sprague–Dawley rats, 270–360 g from Harlan were killed with CO 2 , and brains were dissected according to Glowinski and Iverson w10x. Medial and lateral areas of the hypothalamus were dissected under 8 = magnification. Hypothalamic areas 0.5 mm lateral to the ventricle were defined as medial, and the remaining hypothalamus was considered to be lateral. Clean instruments were used for each tissue dissection to prevent cross-contamination. Tissues were frozen on dry ice and stored at y708C. RNA was extracted with TRI Reagent and bromochloropropane ŽMolecular Research Center. according to the manufacturer’s protocol, as modified from Chomczynski and Sacchi w5x. Total RNA Ž3 m g. was reverse transcribed with oligo Ždt. primers and the SuperScriptTM Preamplification System for First Strand cDNA Synthesis ŽGibcoBRL.. Reactions without reverse transcriptase were performed as negative controls for contamination with previous amplification products and for cross contamination of samples. Pre- and post-PCR samples were handled with dedicated pipettes and aerosol resistant barrier tips in separate areas. Oligonucleotide primers for PNMT and the endogenous positive control, b-actin, were synthesized by Integrated DNA Technologies from previously reported sequences w15,22x. Primers for PNMT Žexons 1 and 2. were 5XCAACAACTACGCGCCTCCTC-3X , corresponding to nucleotides 134–153, and 5X-TGAGGCAGACATGCTGGCTAT-3X , complimentary to nucleotides 913–893.
290
J.L. Andreassi II et al.r Brain Research 779 (1998) 289–291
Primers for b-actin Žexons 2 and 3. were 5X-TGGGTCAGAAGGACTCCTAC-3X , corresponding to nucleotides 1467–1487 and 5X-CTTCATGAGGTAGTCTGTCAGGT-3X , complimentary to nucleotides 2366–2344. The predicted PCR products from PNMT and b-actin spliced mRNA were 288 and 434 base pairs Žbp., respectively. If genomic DNA were present in the samples, each amplification product would include an intron and result in a 780 bp fragment for PNMT and a 898 bp fragment for b-actin. A 2 m l aliquot of the reverse transcription reaction and 0.2 m M of the 5X and 3X primers were added to PCR reagents from GibcoBRL Ž20 mM Tris-HCl ŽpH 8.4., 50 mM KCl, 1.5 mM MgCl 2 , 0.2 mM each dATP, dCTP, dGTP, dTTP and 5 U of Taq DNA polymerase. in a final reaction volume of 100 m l. PCR amplification Ž30–35 cycles of 948C, 1 min; 59.58C, 1 min; 728C, 1 min. was performed in a Coy Tempcycler. Negative controls from the RT reactions were amplified simultaneously. PCR fragments were size fractionated, Southern blotted, hybridized to a 32 P-labeled PNMT cDNA probe, and autoradiographed as described in Eggleston et al. w6x. The rat PNMT cDNA clone was provided by Dr. Barry Kaplan, University of Pittsburgh, Pittsburgh, PA. Ethidium bromide-stained gels and autoradiograms were photographed with a Stratagene EagleEye II image analyzer. RT-PCR products from medullarpons, cerebellum, medial and lateral hypothalamus, and kidney were detected on ethidium-bromide stained gels ŽFig. 1.. The expected size fragments for PNMT Ž288 bp. and b-actin Ž434 bp. were observed in all tissues, but an additional fragment Žapproximately 246 bp. was present in cerebellum and kidney. The 288 bp fragment also was faintly visible in liver samples Žnot shown.. No amplified fragment was detected in samples in which the reverse transcriptase was omitted from the reaction. Amplified fragments larger than the b-actin fragment occasionally were observed but were not consistently detected in any sample.
Fig. 2. Southern blot of RT-PCR products from kidney ŽK., liver ŽL., medullarpons ŽM., whole hypothalamus ŽH. and cerebellum ŽC.. Each RT reaction Ž2 m l. was amplified with PNMT primers for 33 cycles, and products were electrophoresed on a 1.5% gel, blotted, and hybridized with a 32 P-labeled PNMT cDNA probe. Base pairs of the DNA size markers are shown at right.
Southern blot hybridizations with the PNMT cDNA probe indicated that both the 288 and 246 bp amplified fragments from cerebellum and kidney were derived from the PNMT transcript ŽFig. 2.. A single fragment Ž288 bp. from medullarpons, hypothalamus, and liver hybridized to the PNMT cDNA probe. No hybridization signal was observed in negative control samples Žnot shown.. This study demonstrates that the PNMT gene is expressed in the rat hypothalamus and suggests two forms of PNMT mRNA are present in cerebellum and kidney. The findings are consistent with the hypothesis that PNMT is synthesized in the hypothalamus w4,13,19x and with observations that immunoreactive PNMT is present in hypothalamic cell bodies w8,17x. We cannot exclude the possibility that the PNMT mRNA in hypothalamus is located in extra-neuronal cells or dendrites, as found with mRNA for certain neuropeptides w2x. Further investigation is needed to determine the cell types that express PNMT in brain and peripheral tissues and to clarify whether the two hybridization signals observed in cerebellum and kidney represent alternative splicing of PNMT mRNA.
Acknowledgements This research was supported in part by National Institute of Neurological Disorders and Stroke Grant NS-26992 and a faculty grant-in-aid from Virginia Commonwealth University. The authors thank Dr. Carolyn Conway for comments on the manuscript.
References
Fig. 1. RT-PCR products from rat medullarpons ŽMED., cerebellum ŽCER., medial hypothalamus ŽMHY., lateral hypothalamus ŽLHY. and kidney ŽKID.. The negative image of an ethidium-bromide stained gel Ž1.5% agarose. is shown. No RT ssamples lacking reverse transcriptase. Base pairs of the DNA size markers are shown at left. Each PCR reaction contained 2 m l of the RT reaction amplified with PNMT and b-actin primers for 33 cycles. Twelve microliters of PCR reactions were loaded on the gel. Expected sizes of the PNMT and b-actin products are 288 and 434 bp, respectively.
w1x C.R. Anderson, P.R.C. Howe, Is phenylethanolamne N-methyltransferase ŽPNMT. contained in rat hypothalamic neurons?, Neurosci. Lett. 93 Ž1988. 164–169. w2x B. Bloch, A.F. Guitteny, E. Normand, S. Chouham, Presence of neuropeptide messenger RNAs in neuronal processes, Neurosci. Lett. 109 Ž1990. 259–264. w3x O. Bosler, A. Beaudet, L. Denoroy, Electron-microscopic characterization of adrenergic axon terminals in the diencephalon of the rat, Cell Tissue Res. 248 Ž1987. 393–398. w4x M.J. Brownstein, M. Palkovits, M.L. Tappaz, J.M. Saavedra, J.S. Kizer, Effect of surgical isolation of the hypothalamus on its neurotransmitter content, Brain Res. 117 Ž1976. 287–295. w5x P. Chomczynski, N. Sacchi, Single step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction, Anal. Biochem. 162 Ž1987. 156–159.
J.L. Andreassi II et al.r Brain Research 779 (1998) 289–291 w6x W.B. Eggleston, M. Alleman, J.L. Kermicle, Molecular organization and germinal instability of R-stippled maize, Genetics 141 Ž1995. 347–360. w7x M.J. Evinger, A.C. Towle, D.H. Park, P. Lee, T.H. Joh, Glucocorticoids stimulate transcription of the rat phenylethanolamine N-methyltransferase ŽPNMT. gene in vivo and in vitro, Cell. Mol. Neurobiol. 12 Ž1992. 193–215. w8x G.A. Foster, T. Hokfelt, J.T. Coyle, M. Goldstein, Immunohisto¨ chemical evidence for phenylethanolamine-N-methyltransferasepositivertyrosine-hydroxylase-negative neurons in the retina and the posterior hypothalamus of the rat, Brain Res. 330 Ž1985. 183–188. w9x R.W. Fuller, S. Hemrick-Luecke, Methylation of norepinephrine and a-methylnorepinephrine in brain, Biochem. Pharmacol. 31 Ž1982. 3128–3130. w10x J. Glowinski, L.L. Iversen, Regional studies of catecholamines in the rat brain, J. Neurochem. 13 Ž1966. 655–699. w11x T. Hokfelt, K. Fuxe, M. Goldstein, O. Johansson, Immunohisto¨ chemical evidence for the existence of adrenaline neurons in rat brain, Brain Res. 66 Ž1974. 235–251. w12x Z. Liposits, C. Phelix, W.K. Paull, Electron microscopic analysis of ty ro sin e h y d ro x y la se , d o p a m in e -B -h y d ro x y la se a n d phenylethanolamine N-methyltransferase immunoreactive innervation of the hypothalamic paraventricular nucleus in the rat, Histochemistry 84 Ž1986. 105–120. w13x M. Masana, I.N. Mefford, Evidence for the presence of PNMT-containing cell bodies in the hypothalamus, Brain Res. Bull. 23 Ž1989. 477–482. w14x E. Mezey, Phenylethanolamine N-methyltransferase containing neurons in the limbic system of the young rat, Proc. Natl. Acad. Sci. USA 86 Ž1989. 347–351. w15x U. Nudel, R. Zakut, M. Shani, S. Neuman, Z. Levy, D. Yaffe, The nucleotide sequence of the rat cytoplasmic beta-actin gene, Nucleic Acids Res. 11 Ž1983. 1759–1771.
291
w16x R.G. Pendleton, G. Gessner, J. Sawyer, Studies on the distribution of phenylethanolamine N-methyltransferase and epinephrine in the rat, Res. Commun. Chem. Pathol. Pharmacol. 21 Ž1978. 315–325. w17x C.A. Ross, D.A. Rugggiero, M.P. Meeley, D.H. Park, T.H. Joh, D.J. Reis, A new group of neurons in hypothalamus containing phenylethanolamine N-methyltransferase ŽPNMT. but not tyrosine hydroxylase, Brain Res. 306 Ž1984. 349–353. w18x J.M. Saavedra, J.T. Coyle, J. Axelrod, The distribution and properties of the nonspecific N-methyltransferase in brain, J. Neurochem. 20 Ž1973. 743–752. w19x J.M. Saavedra, J. Fernandez-Pardal, C. Ross, D. Reis, Dissociation between hypothalamic catecholamine levels and epinephrine-forming enzyme activity after midbrain hemitransections in the rat, Brain Res. 276 Ž1983. 367–371. w20x P.E. Sawchenko, L.W. Swanson, R. Grzanna, P.R.C. Howe, S.R. Bloom, J.M. Polak, Colocalization of neuropeptide Y immunoreactivity in brainstem catecholaminergic neurons that project to the paraventricular nucleus of the hypothalamus, J. Comp. Neurol. 241 Ž1985. 138–153. w21x M. Spatz, I. Nagatsu, C. Maruki, M. Yoshida, Y. Kondo, J. Bembry, The presence of phenylethanolamine N-methyltransferase in cerebral microvessel and endothelial cultures, Brain Res. 240 Ž1982. 191– 194. w22x Y. Suh, Y. Chun, I.S. Lee, S. Kim, Choi, Y.H. Chong, L. Hong, C. Park, C. Kim, Complete nucleotide sequence and tissue-specific expression of the rat phenylethanolamine N-methyltransferase gene, J. Neurochem. 63 Ž1994. 1603–1608. w23x M.G. Ziegler, B. Kennedy, H. Elayan, A sensitive radioenzymatic assay for epinephrine forming enzymes, Life Sci. 43 Ž1988. 2117– 2122.
Short communication
Phenylethanolamine N-methyltransferase mRNA in rat hypothalamus and cerebellum John L. Andreassi II, William B. Eggleston, Guillian Fu, Jennifer K. Stewart
)
Department of Biology, Virginia Commonwealth UniÕersity, 816 Park AÕe, Richmond, VA 23284-2012, USA Accepted 9 September 1997
Abstract Using the reverse transcription polymerase chain reaction, we detected a single form of phenylethanolamine N-methyltransferase ŽPNMT. mRNA in hypothalamus and medullarpons and two forms in cerebellum. These findings indicate that the PNMT gene is expressed in these brain areas and suggest that tissue specific splicing of PNMT mRNA may occur. q 1998 Elsevier Science B.V. Keywords: PNMT; Catecholamine; Hypothalamus; Rat; mRNA; RT-PCR
Hokfelt and colleagues first suggested that ¨ phenylethanolamine N-methyltransferase ŽPNMT, EC 2.1.1.29. is synthesized in the brainstem and transported within axons to other brain areas w11x. Although PNMT mRNA has been detected in the brainstem w7,22x and at low levels in the amygdala and stria terminalis w14x, it is unclear whether the PNMT gene is expressed in other brain regions. Evidence that PNMT is synthesized in the rat hypothalamus is controversial. Two laboratories have detected immunoreactive PNMT in hypothalamic cells w8,17x; however, most antibodies to PNMT recognize the protein only in medullary cell bodies and hypothalamic nerve terminals w1,3,12,20x. Also, PNMT-like activity is present in the surgically isolated hypothalamus w4,19x and declines after destruction of hypothalamic cells w13x, but the activity could reflect a less specific N-methyltransferase w18x. Finally, although PNMT activity and immunofluorescence have been detected in endothelial cells isolated from rat brain w21x, immuno-electron microscopy has not revealed PNMT in vascular cells in the hypothalamus w3,12x. We used the reverse transcription polymerase chain reaction ŽRT-PCR. to test for PNMT mRNA in rat hypothalamus. Because a small amount of PNMT activity is found in the rat cerebellum w9,23x, and almost no activity is present in liver and kidney w16x, we assayed these tissues to detect low levels of PNMT gene expression that may be
)
Corresponding author. Fax: q1 Ž804. 828-0503; E-mail: [email protected] 0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 1 1 7 0 - 0
present in many tissues. The medullarpons was assayed as a positive control. Adult male Sprague–Dawley rats, 270–360 g from Harlan were killed with CO 2 , and brains were dissected according to Glowinski and Iverson w10x. Medial and lateral areas of the hypothalamus were dissected under 8 = magnification. Hypothalamic areas 0.5 mm lateral to the ventricle were defined as medial, and the remaining hypothalamus was considered to be lateral. Clean instruments were used for each tissue dissection to prevent cross-contamination. Tissues were frozen on dry ice and stored at y708C. RNA was extracted with TRI Reagent and bromochloropropane ŽMolecular Research Center. according to the manufacturer’s protocol, as modified from Chomczynski and Sacchi w5x. Total RNA Ž3 m g. was reverse transcribed with oligo Ždt. primers and the SuperScriptTM Preamplification System for First Strand cDNA Synthesis ŽGibcoBRL.. Reactions without reverse transcriptase were performed as negative controls for contamination with previous amplification products and for cross contamination of samples. Pre- and post-PCR samples were handled with dedicated pipettes and aerosol resistant barrier tips in separate areas. Oligonucleotide primers for PNMT and the endogenous positive control, b-actin, were synthesized by Integrated DNA Technologies from previously reported sequences w15,22x. Primers for PNMT Žexons 1 and 2. were 5XCAACAACTACGCGCCTCCTC-3X , corresponding to nucleotides 134–153, and 5X-TGAGGCAGACATGCTGGCTAT-3X , complimentary to nucleotides 913–893.
290
J.L. Andreassi II et al.r Brain Research 779 (1998) 289–291
Primers for b-actin Žexons 2 and 3. were 5X-TGGGTCAGAAGGACTCCTAC-3X , corresponding to nucleotides 1467–1487 and 5X-CTTCATGAGGTAGTCTGTCAGGT-3X , complimentary to nucleotides 2366–2344. The predicted PCR products from PNMT and b-actin spliced mRNA were 288 and 434 base pairs Žbp., respectively. If genomic DNA were present in the samples, each amplification product would include an intron and result in a 780 bp fragment for PNMT and a 898 bp fragment for b-actin. A 2 m l aliquot of the reverse transcription reaction and 0.2 m M of the 5X and 3X primers were added to PCR reagents from GibcoBRL Ž20 mM Tris-HCl ŽpH 8.4., 50 mM KCl, 1.5 mM MgCl 2 , 0.2 mM each dATP, dCTP, dGTP, dTTP and 5 U of Taq DNA polymerase. in a final reaction volume of 100 m l. PCR amplification Ž30–35 cycles of 948C, 1 min; 59.58C, 1 min; 728C, 1 min. was performed in a Coy Tempcycler. Negative controls from the RT reactions were amplified simultaneously. PCR fragments were size fractionated, Southern blotted, hybridized to a 32 P-labeled PNMT cDNA probe, and autoradiographed as described in Eggleston et al. w6x. The rat PNMT cDNA clone was provided by Dr. Barry Kaplan, University of Pittsburgh, Pittsburgh, PA. Ethidium bromide-stained gels and autoradiograms were photographed with a Stratagene EagleEye II image analyzer. RT-PCR products from medullarpons, cerebellum, medial and lateral hypothalamus, and kidney were detected on ethidium-bromide stained gels ŽFig. 1.. The expected size fragments for PNMT Ž288 bp. and b-actin Ž434 bp. were observed in all tissues, but an additional fragment Žapproximately 246 bp. was present in cerebellum and kidney. The 288 bp fragment also was faintly visible in liver samples Žnot shown.. No amplified fragment was detected in samples in which the reverse transcriptase was omitted from the reaction. Amplified fragments larger than the b-actin fragment occasionally were observed but were not consistently detected in any sample.
Fig. 2. Southern blot of RT-PCR products from kidney ŽK., liver ŽL., medullarpons ŽM., whole hypothalamus ŽH. and cerebellum ŽC.. Each RT reaction Ž2 m l. was amplified with PNMT primers for 33 cycles, and products were electrophoresed on a 1.5% gel, blotted, and hybridized with a 32 P-labeled PNMT cDNA probe. Base pairs of the DNA size markers are shown at right.
Southern blot hybridizations with the PNMT cDNA probe indicated that both the 288 and 246 bp amplified fragments from cerebellum and kidney were derived from the PNMT transcript ŽFig. 2.. A single fragment Ž288 bp. from medullarpons, hypothalamus, and liver hybridized to the PNMT cDNA probe. No hybridization signal was observed in negative control samples Žnot shown.. This study demonstrates that the PNMT gene is expressed in the rat hypothalamus and suggests two forms of PNMT mRNA are present in cerebellum and kidney. The findings are consistent with the hypothesis that PNMT is synthesized in the hypothalamus w4,13,19x and with observations that immunoreactive PNMT is present in hypothalamic cell bodies w8,17x. We cannot exclude the possibility that the PNMT mRNA in hypothalamus is located in extra-neuronal cells or dendrites, as found with mRNA for certain neuropeptides w2x. Further investigation is needed to determine the cell types that express PNMT in brain and peripheral tissues and to clarify whether the two hybridization signals observed in cerebellum and kidney represent alternative splicing of PNMT mRNA.
Acknowledgements This research was supported in part by National Institute of Neurological Disorders and Stroke Grant NS-26992 and a faculty grant-in-aid from Virginia Commonwealth University. The authors thank Dr. Carolyn Conway for comments on the manuscript.
References
Fig. 1. RT-PCR products from rat medullarpons ŽMED., cerebellum ŽCER., medial hypothalamus ŽMHY., lateral hypothalamus ŽLHY. and kidney ŽKID.. The negative image of an ethidium-bromide stained gel Ž1.5% agarose. is shown. No RT ssamples lacking reverse transcriptase. Base pairs of the DNA size markers are shown at left. Each PCR reaction contained 2 m l of the RT reaction amplified with PNMT and b-actin primers for 33 cycles. Twelve microliters of PCR reactions were loaded on the gel. Expected sizes of the PNMT and b-actin products are 288 and 434 bp, respectively.
w1x C.R. Anderson, P.R.C. Howe, Is phenylethanolamne N-methyltransferase ŽPNMT. contained in rat hypothalamic neurons?, Neurosci. Lett. 93 Ž1988. 164–169. w2x B. Bloch, A.F. Guitteny, E. Normand, S. Chouham, Presence of neuropeptide messenger RNAs in neuronal processes, Neurosci. Lett. 109 Ž1990. 259–264. w3x O. Bosler, A. Beaudet, L. Denoroy, Electron-microscopic characterization of adrenergic axon terminals in the diencephalon of the rat, Cell Tissue Res. 248 Ž1987. 393–398. w4x M.J. Brownstein, M. Palkovits, M.L. Tappaz, J.M. Saavedra, J.S. Kizer, Effect of surgical isolation of the hypothalamus on its neurotransmitter content, Brain Res. 117 Ž1976. 287–295. w5x P. Chomczynski, N. Sacchi, Single step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction, Anal. Biochem. 162 Ž1987. 156–159.
J.L. Andreassi II et al.r Brain Research 779 (1998) 289–291 w6x W.B. Eggleston, M. Alleman, J.L. Kermicle, Molecular organization and germinal instability of R-stippled maize, Genetics 141 Ž1995. 347–360. w7x M.J. Evinger, A.C. Towle, D.H. Park, P. Lee, T.H. Joh, Glucocorticoids stimulate transcription of the rat phenylethanolamine N-methyltransferase ŽPNMT. gene in vivo and in vitro, Cell. Mol. Neurobiol. 12 Ž1992. 193–215. w8x G.A. Foster, T. Hokfelt, J.T. Coyle, M. Goldstein, Immunohisto¨ chemical evidence for phenylethanolamine-N-methyltransferasepositivertyrosine-hydroxylase-negative neurons in the retina and the posterior hypothalamus of the rat, Brain Res. 330 Ž1985. 183–188. w9x R.W. Fuller, S. Hemrick-Luecke, Methylation of norepinephrine and a-methylnorepinephrine in brain, Biochem. Pharmacol. 31 Ž1982. 3128–3130. w10x J. Glowinski, L.L. Iversen, Regional studies of catecholamines in the rat brain, J. Neurochem. 13 Ž1966. 655–699. w11x T. Hokfelt, K. Fuxe, M. Goldstein, O. Johansson, Immunohisto¨ chemical evidence for the existence of adrenaline neurons in rat brain, Brain Res. 66 Ž1974. 235–251. w12x Z. Liposits, C. Phelix, W.K. Paull, Electron microscopic analysis of ty ro sin e h y d ro x y la se , d o p a m in e -B -h y d ro x y la se a n d phenylethanolamine N-methyltransferase immunoreactive innervation of the hypothalamic paraventricular nucleus in the rat, Histochemistry 84 Ž1986. 105–120. w13x M. Masana, I.N. Mefford, Evidence for the presence of PNMT-containing cell bodies in the hypothalamus, Brain Res. Bull. 23 Ž1989. 477–482. w14x E. Mezey, Phenylethanolamine N-methyltransferase containing neurons in the limbic system of the young rat, Proc. Natl. Acad. Sci. USA 86 Ž1989. 347–351. w15x U. Nudel, R. Zakut, M. Shani, S. Neuman, Z. Levy, D. Yaffe, The nucleotide sequence of the rat cytoplasmic beta-actin gene, Nucleic Acids Res. 11 Ž1983. 1759–1771.
291
w16x R.G. Pendleton, G. Gessner, J. Sawyer, Studies on the distribution of phenylethanolamine N-methyltransferase and epinephrine in the rat, Res. Commun. Chem. Pathol. Pharmacol. 21 Ž1978. 315–325. w17x C.A. Ross, D.A. Rugggiero, M.P. Meeley, D.H. Park, T.H. Joh, D.J. Reis, A new group of neurons in hypothalamus containing phenylethanolamine N-methyltransferase ŽPNMT. but not tyrosine hydroxylase, Brain Res. 306 Ž1984. 349–353. w18x J.M. Saavedra, J.T. Coyle, J. Axelrod, The distribution and properties of the nonspecific N-methyltransferase in brain, J. Neurochem. 20 Ž1973. 743–752. w19x J.M. Saavedra, J. Fernandez-Pardal, C. Ross, D. Reis, Dissociation between hypothalamic catecholamine levels and epinephrine-forming enzyme activity after midbrain hemitransections in the rat, Brain Res. 276 Ž1983. 367–371. w20x P.E. Sawchenko, L.W. Swanson, R. Grzanna, P.R.C. Howe, S.R. Bloom, J.M. Polak, Colocalization of neuropeptide Y immunoreactivity in brainstem catecholaminergic neurons that project to the paraventricular nucleus of the hypothalamus, J. Comp. Neurol. 241 Ž1985. 138–153. w21x M. Spatz, I. Nagatsu, C. Maruki, M. Yoshida, Y. Kondo, J. Bembry, The presence of phenylethanolamine N-methyltransferase in cerebral microvessel and endothelial cultures, Brain Res. 240 Ž1982. 191– 194. w22x Y. Suh, Y. Chun, I.S. Lee, S. Kim, Choi, Y.H. Chong, L. Hong, C. Park, C. Kim, Complete nucleotide sequence and tissue-specific expression of the rat phenylethanolamine N-methyltransferase gene, J. Neurochem. 63 Ž1994. 1603–1608. w23x M.G. Ziegler, B. Kennedy, H. Elayan, A sensitive radioenzymatic assay for epinephrine forming enzymes, Life Sci. 43 Ž1988. 2117– 2122.