Pepndes, Vol 11, pp 33-37 ©Pergamon Press plc, 1990 Pnntedm the U S A
0196-9781/90 $3 00 + 00
The Rat Thymus A Site of Atrial Natriuretic Peptide Synthesis A N G E L I K A M . V O L L M A R , * R U D O L F E. L A N G , t AND RLIDIGER SCHULZ*
J O R G H,g, N Z E t
*Institute of Pharmacology, Toxicology and Pharmacy, University of Munich KOniginstrafle 16, D-8000 Miinchen, FRG "PGerman Institute for High Blood Pressure Research and Department of Pharmacology University of Heidelberg, Im Neuenheimer Feld, D-6900 Heidelberg, FRG R e c e i v e d 5 July 1989
VOLLMAR, A M , R E LANG, J. HANZE AND R SCHULZ The rat thymus--A site of atrial natrturettc pepnde synthests PEPTIDES 11(1) 33-37, 1990 --The amum of the heart has been demonstrated to represent the major s~te of synthesis of atrial natnuretlc pepude (ANP), a potent namureUc, dmretlc and vasoactwe hormone. Our recent studies revealed ANP-hke material outside the heart, namely, m lympho,d folhcles of the intestine and m the thymus, and now we report data demonstrating the thymus as a sxte of synthesis for ANP The experimental evidence is as follows" firstly, the lmmunoreactlve material detected corresponds chromatographically with the precursor of ANP Secondly, the thymus contains mRNA for ANP Thirdly, ~mmunohlstochem~stry locates ANP-hke material to cortical thymocytes with pamcularly dense staining m the subcapsular areas of the thymus Interestmgly, both ANP-hke matenal and the mRNA coding for ANP were expressed to a larger extent m newborn rats as compared to adult ammals, suggesting that ANP may be revolved m the development and/or functmn of T-cells. Atrial natnuretlc peptlde
Rat thymus
mRNA
Immunoh~stochemlstry
ATRIAL natriuretic peptlde (ANP) has been shown to affect kadney, adrenal, vascular and certain brain functions, leadmg to the concensus that the peptide is a major regulator of fired and electrolyte homeostasis (4, 8, 12, 13). However, ANP might serve physiological roles other than that implied by its name. Our recent finding of lmmunoreactlve ANP (IR-ANP) material in lymphoid follicles of the guinea pig intestine (18), as well as in the rat thymus (19), suggests a link between ANP and the immune system. In the present study we have investigated whether the IR-ANP previously detected originates from within the thymus itself. This was achieved by demonstrating the presence of mRNA coding for ANP and the distinct anatomical locahzation of the IR-ANP within the tissue. In additmn, we compared ANP synthesis and content in thymus glands from newborn and adult rats m an attempt to address the proposal that this peptide may play a role in the immune system. The thymus shows major morphological and functmnal changes during development (6, 10, 16), and thus served this purpose well.
4°C) and the clear supernatant was submitted to activated Amberlite XAD-4 adsorbent resin (Serva, Heidelberg, FRG) ANP-like material was eluted with 80% acetomtnle in 0.1% TFA. The lyophllized material was dissolved in 0.1 M phosphate buffer (pH 7.4), containing I mM EDTA and 0.5 mM phenylmethylsulfonylfluonde, and applied to an ANP-immunoaffimty column. The gel matrix consisted of protein A purified anti-rat ANP(99-126) immunoglobuhns of antiserum " L o l s l " (19) bound to activated CH-Sepharose-4B gel (protein A and Sepharose obtained from Pharmacla, Frelburg, FRG). The bound ANP lmmunoreactive matenal was eluted with 1 M acetic acid and immediately lyophdlzed An aliquot thereof was subjected to HPLC, using a C18 ODS Ultrasphere TM column (5 Ixm, 4 . 6 × 2 5 0 0 ram, Beckman Instruments, San Remo, USA) previously calibrated with rat ANP fragments (103-123), (103-126), (99-126) and (2-126). Elution was carried out with a linear gradient of acetomtnle (20-55%, 55 min) m 0.1% TFA (flow rate. 1.5 ml/mm). Fractions (2 2 ml) were assayed for ANP immunoreactivity, using the N-terminal directed ANP antiserum " G T - 2 3 " and the Cterminal recognizing antibody " T o n i III" as previously described (19).
METHOD
Extractton and Charactertzanon of ANP-Ltke Material
Quantification of 1R-ANP Material
Thymus glands from male Sprague-Dawley rats (1- or 75day-old) were excised under a dissection microscope and immediately transferred to 0.1 M HC1, 4°C. After homogenization (glassground homogenizer) and boding for 60 sec in a microwave oven, the tissue suspension was centrifuged (20,000 × g, 20 mln,
Acidic extracts of thymus glands from either I- or 75-day-old rats, containing identical amounts of protein (Bradford method), were preextracted by Amberlite XAD-4 adsorption The lyophlhzed extracts were chromatographed on Sephadex G-50 columns (19) and fractions were analyzed for ANP by RIA using
33
34
VOLLMAR, LANG, HANZE AND SCHULZ
in 0.1 × SSC and 0 1% SDS at room temperature and exposed to an X-ray film and intensifying screens at - 7 0 ° C
T-- ,r" 0
Ez
0196-9781/90 $3 00 + 00
The Rat Thymus A Site of Atrial Natriuretic Peptide Synthesis A N G E L I K A M . V O L L M A R , * R U D O L F E. L A N G , t AND RLIDIGER SCHULZ*
J O R G H,g, N Z E t
*Institute of Pharmacology, Toxicology and Pharmacy, University of Munich KOniginstrafle 16, D-8000 Miinchen, FRG "PGerman Institute for High Blood Pressure Research and Department of Pharmacology University of Heidelberg, Im Neuenheimer Feld, D-6900 Heidelberg, FRG R e c e i v e d 5 July 1989
VOLLMAR, A M , R E LANG, J. HANZE AND R SCHULZ The rat thymus--A site of atrial natrturettc pepnde synthests PEPTIDES 11(1) 33-37, 1990 --The amum of the heart has been demonstrated to represent the major s~te of synthesis of atrial natnuretlc pepude (ANP), a potent namureUc, dmretlc and vasoactwe hormone. Our recent studies revealed ANP-hke material outside the heart, namely, m lympho,d folhcles of the intestine and m the thymus, and now we report data demonstrating the thymus as a sxte of synthesis for ANP The experimental evidence is as follows" firstly, the lmmunoreactlve material detected corresponds chromatographically with the precursor of ANP Secondly, the thymus contains mRNA for ANP Thirdly, ~mmunohlstochem~stry locates ANP-hke material to cortical thymocytes with pamcularly dense staining m the subcapsular areas of the thymus Interestmgly, both ANP-hke matenal and the mRNA coding for ANP were expressed to a larger extent m newborn rats as compared to adult ammals, suggesting that ANP may be revolved m the development and/or functmn of T-cells. Atrial natnuretlc peptlde
Rat thymus
mRNA
Immunoh~stochemlstry
ATRIAL natriuretic peptlde (ANP) has been shown to affect kadney, adrenal, vascular and certain brain functions, leadmg to the concensus that the peptide is a major regulator of fired and electrolyte homeostasis (4, 8, 12, 13). However, ANP might serve physiological roles other than that implied by its name. Our recent finding of lmmunoreactlve ANP (IR-ANP) material in lymphoid follicles of the guinea pig intestine (18), as well as in the rat thymus (19), suggests a link between ANP and the immune system. In the present study we have investigated whether the IR-ANP previously detected originates from within the thymus itself. This was achieved by demonstrating the presence of mRNA coding for ANP and the distinct anatomical locahzation of the IR-ANP within the tissue. In additmn, we compared ANP synthesis and content in thymus glands from newborn and adult rats m an attempt to address the proposal that this peptide may play a role in the immune system. The thymus shows major morphological and functmnal changes during development (6, 10, 16), and thus served this purpose well.
4°C) and the clear supernatant was submitted to activated Amberlite XAD-4 adsorbent resin (Serva, Heidelberg, FRG) ANP-like material was eluted with 80% acetomtnle in 0.1% TFA. The lyophllized material was dissolved in 0.1 M phosphate buffer (pH 7.4), containing I mM EDTA and 0.5 mM phenylmethylsulfonylfluonde, and applied to an ANP-immunoaffimty column. The gel matrix consisted of protein A purified anti-rat ANP(99-126) immunoglobuhns of antiserum " L o l s l " (19) bound to activated CH-Sepharose-4B gel (protein A and Sepharose obtained from Pharmacla, Frelburg, FRG). The bound ANP lmmunoreactive matenal was eluted with 1 M acetic acid and immediately lyophdlzed An aliquot thereof was subjected to HPLC, using a C18 ODS Ultrasphere TM column (5 Ixm, 4 . 6 × 2 5 0 0 ram, Beckman Instruments, San Remo, USA) previously calibrated with rat ANP fragments (103-123), (103-126), (99-126) and (2-126). Elution was carried out with a linear gradient of acetomtnle (20-55%, 55 min) m 0.1% TFA (flow rate. 1.5 ml/mm). Fractions (2 2 ml) were assayed for ANP immunoreactivity, using the N-terminal directed ANP antiserum " G T - 2 3 " and the Cterminal recognizing antibody " T o n i III" as previously described (19).
METHOD
Extractton and Charactertzanon of ANP-Ltke Material
Quantification of 1R-ANP Material
Thymus glands from male Sprague-Dawley rats (1- or 75day-old) were excised under a dissection microscope and immediately transferred to 0.1 M HC1, 4°C. After homogenization (glassground homogenizer) and boding for 60 sec in a microwave oven, the tissue suspension was centrifuged (20,000 × g, 20 mln,
Acidic extracts of thymus glands from either I- or 75-day-old rats, containing identical amounts of protein (Bradford method), were preextracted by Amberlite XAD-4 adsorption The lyophlhzed extracts were chromatographed on Sephadex G-50 columns (19) and fractions were analyzed for ANP by RIA using
33
34
VOLLMAR, LANG, HANZE AND SCHULZ
in 0.1 × SSC and 0 1% SDS at room temperature and exposed to an X-ray film and intensifying screens at - 7 0 ° C
T-- ,r" 0
Ez
