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The responses of atrial natriuretic factor concentrations to acute volume changes in conscious rats
Life Sciences, Vol. 41, pp. 2339-2347 Printed in the U.S.A.
Pergamon Journals
THE RESPONSES OF ATRIAL NATRIURETIC FACTOR CONCENTRATIONS TO ACUTE VOLUME CHANGES IN CONSCIOUSRATS Peter J.S. Chiu, Subbarao Vemulapalli, Mark Policelli, Imre Kifor*, Edmund J. Sybertz and Victor J. Dzau* Department of Pharmacology, Schering-Plough Corporation Research Division, Bloomfield, New Jersey 07003 and Molecular and Cellular Vascular Research Laboratory, Division of Vascular Medicine and Atherosclerosis, Brigham and Women's Hospital Harvard Medical School 02115 Boston, Massachusettes (Received in final form September 25, 1987) Summary A rapid and sensitive radioimmunoassay has been developed for measurements of atria1 natriuretic factor (ANF) in rat plasma. The antiserum, raised to rat ANF (99-126), cross-reacts with rat ANF (103-123), ANF (103-125), ANF (103-126) but not with smaller fragments, human ANF (99-126), angiotensin II, bradykinin or vasopressin. The plasma ANF concentration is 181+24 pg/ml (N=24) in the unstressed conscious rats (CharTes River CD, male). The ANF imunoreactivity in the plasma extracts was verified by HPLC analysis, which displayed one major imunoreactive peak of ANF corresponding to rat ANF (99-126) and some smaller fragments. Intravenous injection of saline elevated circulating ANF, whereas acute volume depletion by hemorrhage, water deprivation and furosemide diuresis greatly lowered plasma ANF. The prompt response of plasma ANF levels to acute changes in volume status is consistent with the proposed role of ANF as a volume-regulatory hormone. The myocytes of mammalian atria contain atria1 natriuretic factors (ANF) which have potent natriuretic, diuretic and vasorelaxant properties (1). Release of ANF from isolated atria has been demonstrated (2,3). ANF levels in coronary sinus blood are several-fold higher than in samples from peripheral blood in man (4,5) and dogs (6,7), further suggesting active secretion. A 28-amino acid polypeptide ANF (99-126) (Ser-Leu-Arg-Argatriopeptin III) has been identified as the major circulating form of ANF in rats (&rANF) and man (d-hANF) (8,9,10). In addition to its direct effects on renal excretion of sodium and water (11,12,13), ANF inhibits secretion of renin and other volume regulatory hormones including aldosterone (1,14). Consequently it is proposed that ANF plays a role in the control and regulation of extracellular fluid (ECF) volume (1,14). An increase in circulating ANF usually follows acute expansion of the ECF volume with saline, blood or high sodium diet in animals (15-17) and man (5,10,18,19), presumably mediating in part the accompanying natriuresis in order to 0024-3205/87 $3.00 + .oo Copyright (c) 1987 Pergamon Journals Ltd.
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ANF Responses to Acute Volume Changes
Vol. 41, No. 21, 1987
restore body fluid and electrolyte balance (20-22). In the present study we have developed a radioimmunoassay of ANF in rat plasma and determined the responsiveness of plasma immunoreactive ANF concentrations to acute volume depletion due to hemorrhage, water deprivation and furosemide-induced diuresis in conscious rats.
Methods
Preparation of plasma for radioimmunoassay (RIA). Male Charles River CD rats (300-350 g) were used. Blood samples (6 ml) were usually collected from conscious rats by decapitation into chilled Vacutainer tubes containing a mixture of EDTA (1.5 mg/ml), aprotinin (200 kallikrein inhibitor unit/ml) and soybean trypsin inhibitor (20 No(-benzoyl-L-arginine ethyl ester unit/ml), with 100 pl each. For the purpose of characterizing the RIA methodology, blood samples (10 ml) were drawn from the abdominal aorta of rats anesthetized with Inactin to obtain "pooled plasma". Samples were immediately centrifuged at 3,000 rpm for 10 min. The pooled plasma was divided into 1 ml aliquots and stored at -2O'C whereas the experimental plasma samples were continously processed through extraction and the entire RIA procedures. Extraction of plasma samples. A mixture of 1 ml plasma, 0.6 ml 8 M urea and 20 ml 1.5 M NaCl in disposable syringes was passed slowly through octadecylsilane Sep-Pak Cl8 cartridges (Waters Assoc., Milford, MA) which were previously activated by passing 5 ml methanol and 5 ml 8 M urea and followed by washing with 10 ml water. The use of high urea was intended to disrupt hydrogen bonding between ANF and large proteins. The sample-loaded cartridges were washed with 10 ml water and eluted with 8 ml 4% acetic acid in 90% ethanol. The eluants were collected in 50-ml polypropylene centrifuge tubes (Corning) each containing 100 pl 1% glycerol in water and were blown under a stream of N to near dryness. The samples were dissolved in 0.4 ml 0.02 M ace z.ic acid and 0.6 ml assay buffer after The assay buffer was composed of undergoing lyophilization to dry powder. each of the following: O.OlM K HP04, 0.03M EDTA, 0.015 mM 8-hydroxyquinoline (a fungistatic), 0.02% neomycin sulfate and 0.25% bovine serum albumin; the mixture was adjusted to pH 7.4 and piaced in a water bath at 56 C for 30 min. The assay buffer is stored at 4 C. Preparation of antiserum. Rat ANF 99-126 (d rat ANF) was covalently conjugated by carbodiimiae to bovine thyroglobulin (23). The material was kept frozen after dialysis against 0.15 M NaCl. To prepare for immunization, the ANF-protein conjugate was emulsified with three volumes of complete Freund's adjuvant and a 2 ml emulsion was injected into the dorsal skin of rabbits at multiple sites. Booster injections, in which incomplete Freund's adjuvant was used instead, were performed after 4-6 weeks on the four legs im. Four to five weeks following booster, serum was collected fro8 one rabbit via the central ear artery, aliquoted and kept frozen at -20 C. Radioimmunoassay for ANF. The extracted plasma samples (100 pl) or standards (rat ANF 99-126, 4-100P2Jg/tube) were incubated with 100 pl antiserum 11:24.000) and 100 ul I-labelled rat ANF 99-126 f3.500-4.500 cpm) in triplicate at-4'C forr24 h using polypropylene culture tubes . Free and bound ANF were separated using 0.5 ml dextran-coated (Fisher). charcoal (DCC). The DCC stock solution was prepared by adding 5 g Norit
Vol. 41, No. 21, 1987
ANF Responses to Acute Volume Changes
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charcoal and 0.59 dextran t8 200 ml O.lM tris buffer at pH 7.4, stirred overnight and was kept at 4 c. A 1:40 dilution of the DCC stock solution was used. Upog addition of DCC, the individual samples were vortexed and incubated at 4 C for 15 min, followed by centrifugation at 3,000 rpm for 15 min. The supernatants were decanted into 12x75 mm polystyrene tubes (Fisher) for counting in an automatic gamna counter (Micromedic Systems, Horsham, PA; efficiency, >75%). High performance liquid chromatography (HPLC) identification of immunoreactive ANF in rat plasma. HPLC analysis of pooled plasma was performed on a Varian 5560 Liquid Chromatograph, using a Beckman Altex Ultrapore RPSC column (pore size, 300 particle size, 5 pm; column size, 0.46 x 15 cm). A linear gradient elution system was used from 0 to 90% over 35 min; solvent A, O.Olm trifluoroacetic acid (TFA) in water; solvent B, 0.01 M TFA in 90% acetonitrile; flow rate, 1 ml/min. Fractions of 1 ml were collected, lyophilized and quantitated for immunoreactive ANF.
A;
Under ether anesthesia the Acute volume expansion with saline. vein of rats was cannulated for administration of isotonic saline. 2.5 h after recovery from anesthesia, 4 or B ml of saline was given min; the control animals received no saline challenge. The animals immediately sacrificed and trunk blood was collected for ANF RIA to with the sham operated animals.
jugular Two to in 1 were compare
Water deprivation and furosemide treatment. Rats, deprived of food and water overnight, were divided into two groups. One group was sacrificed immediately and the other sacrificed 1 hr following treatment with furosemide 40 mg/kg po. The ANF levels of the treated animals were compared with those of the control animals. Rat ANF 99-126 (rANP) ANF 103-123 (atriopeptin I), ANF Reagents. 103-125 (atriopeptin II), ANF 103-126 (atriopeptin III), ANF 92-126, ANF 99-119, ANF 116-{$5 and h uman ANF 99-126 (o(hANP) were purchased from Peninsula Labs; I-rat ANF 99-126, specific activity: 2000-2200 pCi/mmole (Amersham or New England Nuclear); carbodiimide, bovine thyroglobulin, aprotinin, soybean trypsin inhibitor, &hydroxyquinoline, angiotensin II, bradykinin and vasopressin were purchased from Sigma; Norit charcoal, urea, NaCl, EDTA and K HP0 from Fisher; neomycin sulfate (Gibco Labs); bovine serum albumin (IZN B!omedicals). Statistical analysis. All data are presented as mean+S.E.M. unless specified otherwise. Student's t-test or Duncan's multiple range test was used to ascertain statistical significance (PcO.05).
Results Characterization of radioimmunoassay for ANF. Total binding of the tracer to antiserum was 49.4+0.7% (N=8); nonspecific binding was 5%. The 50% displacement point on a standard curve (EC ) was 73.9t6.9 pg/tube (N=8); the detection limit (10% displacement) was s'pg/tube. The antiserum demonstrated cross-reactivity with rat ANF 103-123 (74%), ANF 103-125 (75%), ANF 103-126 (63%) and ANF 92-126 (100%) but did not cross-react with rat ANF 99-109, 116-126 and human ANF 99-126 or with angiotensin II, Recovery of rat ANF 99-126, added to arginine vasopressin and bradykinin. pooled plasma to yield a concentration of 250 pg/ml, is 63+9% (N=7). All ANF values reported subsequently are not corrected for recovery. The
2342
ANF Responses to Acute Volume Changes
Vol. 41, No. 21, 1987
interassay variation was 9% (N=3) and the intra-assay variation 8% (N=3). Trunk plasma ANF concentrations in unstressed conscious and anesthetized rats are summarized in Table 1. The rats anesthetized with Inactin showed significantly higher plasma ANF levels. Table 1.
ANF concentrations in rats
Method of collection
N
ANF (pg/ml)
Conscious, via decapitation
24
181~24 (34-632)
Anesthetized, via aortic puncture
24
337~15" (223-524)
*P
Vol. 41, No. 21, 1987
ANF Responses to Acute Volume Changes
2343
.
+ P-zO.05, VS CONTROL AND 0 TIME
.
100
0
I CONTROL
Figure 1.
I
HEMORRHAGE (0 TIME)
.
I
HEMORRHAGE (+ 15 MIN)
Changes in plasma ANF levels following acute hemorrhage in conscious rats. Hemorrhage was performed by drawing 6 ml of blood in 2 min via the arterial cannula. Blood collection for ANF RIA was performed by decapitation immediately (0 time) and 15 min after hemorrhage, respectively. *P
2344
Vol. 41, No. 21, 1987
ANF Responses to Acute Volume Changes
700 * P< 0.05, VS CONTROL + Pi 0.05, VS WATER DEPRIVATION
600
500 = E 5 II 400 Q Y Y :
. .
300
.
.
f
. -*
200
0
0
-*t
100
: 0 .
I
0 NORMAL CONTROL
I WATER DEPRIVATION
WATER DEPRIVATION FUROSEMI&
Figure
2.
40 mglkg po
Changes in plasma ANF levels following water deprivation and furosemide treatment in conscious rats. Two groups of rats of 6 each were deprived of water and food overnight. On the following day, one group was sacrificed inmediately and the other 1 hr after receiving additional treatment with furosemide, 40 mg/kg po.
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ANF Responses to Acute Volume Changes
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fragments of ANF 99-126 (ANF 99-109 and ANF 116-126 respectively) are biologically inactive and also do not cross-react with the antiserum (24,25). To obviate the interference of plasma proteins, which would otherwise yield spuriously high levels of ANF immunoreactivity in RIA (6,8,26), it was necessary to use Sep-Pak C cartridges to extract ANF from the plasma. HPLC analysis of the extracted Y at plasma indicated that the circulating ANF consists of mainly ANF 99-126 and possibly some low-molecular-weight fragments. On the basis of data from immunoreactivity, chromatographic migration, bioassay and sequence analysis, Schwartz et al. (9) reported that ANF 99-126 is the major species of ANF in the rat circulation and atriopeptin III a minor component. Other groups demonstrated the presence of low-molecular-weight forms of ANF other than ANF 99-126 in rat plasma (15,27). ANF 99-126 is believed to be the product of selective cleavage of the high molecular weight precursor in the atrium upon release (1). ANF concentrations in the plasma samples from anesthetized animals are higher than those from conscious, unstressed animals. The discrepancy can also be ascribed to differences in the method of collection, namely decapitation vs aortic puncture. Gutkowska et al. (6) reported on the influence of anesthetics, route of collection, drugs, etc. on the plasma ANF in rats. The following rat plasma ANF concentrations using RIA quantitation have been reported: 478 pg/ml (27); 58 pg/ml (15); 800 pg/ml (9); 80-125 pg/ml (17); 82-94 pg/ml (6). Hence it becomes evident that the plasma ANF values we obtained in the conscious and anesthetized rats fall within the range of reported values. We have demonstrated a marked increase in circulating ANF in response to volume expansion with saline in rats, which is the most consistent way of stimulating ANF release (10,15,17). The results indicate that our RIA technique is associated with adequate sensitivity to detect this challenge. A 5-day water deprivation caused a great fall in rat plasma ANF (28). We showed that the rats responded to even an overnight water deprivation with a fall in plasma ANF. Furthermore, a supramaximal diuretic dose of furosemide promptly caused a further decline in plasma ANF. The rapid fall in plasma ANF following hemorrhage further indicates that plasma ANF is greatly sensitive to acute alterations in extracellular fluid volume. Inhibition of atria1 secretion in response to volume depletion, in conjunction with the rather short half-life of ANF (~1 min) may account for the rapid fall in circulating ANF (29-31). In conclusion, the prompt response of plasma ANF levels to acute changes in volume status in an appropriate manner is consistent with the hypothesis that ANF plays a role in the control and regulation of body fluid and electrolyte balance. Acknowledgements The authors thank W. Baginsky and Dr. C. Foster for providing the HPLC data. This study was supported in part by NIH grants HL 35610, HL 35793, HL 19259, HL 35252 and NIH specialized Center of Research HL 36568. V.J. Dzau is an Established Investigator of the American Heart Association.
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ANF Responses to Acute Volume Changes
vol. 41, No. 21, 1987
References 1. 2. 3. 4. 5. 6.
7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.
P. NEEDLEMAN and J.E. GREENWALD, N. Eng. J. Med. 314:828-834 (1986). M.G. CURRIE, D.SUKIN, D.M. GELLER, B.R. COLE and P. NEEDLEMAN, Biochem. Biophys. Res. Comm. 124:711-717 (1984). 0. VUOLTEENAHO, 0. ARJAMAA and N. LING, Biochim. Biophys. Res. Comn. 129:82-88 (1985). I.G. CROZIER, M.G. NICHOLLS, H. IKRAM, E.A. ESPINER, T.G. YANDLE and S. JANS, Hypertension 8 (Suppl. II): 11-11-11-15 (1986). M. NARUSE, K. NARUSE, K. OBANA, F. KURIMOTO, H. SAKURAI, T. HONDA, T. HIGASHIDA, H. DEMURA, T. INAGAMI and K. SHIZUMI, Peptides 7:141-145 (1986). J. GUTKOWSKA, K. HORKY, E.L. SCHIFFRIN, G. THIBAULT, R. GARCIA, A. DELEAN, P. HAMET, J. TREMBLAY, M.B. ANAND-SRIVASTAVA, P. JANUSZEWICZ, J. GENEST and M. CANTIN, Fed. Proc,.45:2101-2105 (1986). N. WILSON, J.R. LEDSOME, R. KEELER, A.J. RANKIN, J.P. WADE and C.A. COURNEYA, J. Immunoassay 7:73-96 (1986). R.M. ARENDT, E. STANGL, J. JAHRINGER, D.C. LIEBISCH and A. HERZ, Febs Letters 189:57-61 (1985). D. SCHWARTZ, D.M. GELLER, P.T. MANNING, N.R. SIEGEL, K.F. FOK, C.E. SMITH and P. NEEDLEMAN, Science 229:397-400 (1985). T. YAMAJI, M. ISHIBASHI and F. TAKAKU, J. Clin. Invest. 76:1705-1709 (1985). A.J. DEBOLD, H.B. BORENSTEIN, A.T. VERESS AND and SONNENBERG, Life Sci. 28:89-94 (1981). M.J.F. CAMARGO, H.D. KLEINERT, S.A. ATLAS, J.E. SEALEY, J.H. LARAGH and T. MAACK, Am. J. Physiol. 246:F447-F456 (1984). A.A. SEYMOUR, Clin. Exp. Hypertens. Theory and Practice A7:887-904 (1985). J.H. LARAGH, N. Eng. J. Med. 313:1330-1340 (1985). R.E. LANG, H. THOLKEN, D. GANTEN, F.C. LUFT, H. RUSKOAHO and T. UNGER, Nature (London) 314:264-266 (1985). R. ESKAY, Z. ZUKOWSKA, M. HAOSS, J.R. DAVE and N. ZAMIR, Science 323:636-639 (1986). A. PETTERSSON, J. HEDNER, S.-E. RICKSTEN, A.C. TOWLE and T. HEDNER, Life Sci. 38:1127-1133 (1986). G.A. SAGNELLA, N.D. MARKANDU, A.C. SHORE and G.A. MACGREGOR, Lancet ii:1208-1211 (1985). Y. SHENKER, R.S. SIDER, E.A. OSTAFIN and R.J. GREKIN, J. Clin. Invest. 76:1684-1687 (1985). A.T. VERESS and H. SONNENBERG, Am. J. Physiol. 247:R610-R613 (1984). I. KOBRIN, M.B. KARDON, N.C. TRIPPODO, B.L. PEGRAM and E.D. FROHLICH, J. Hypertens. 3:145-148 (1985). T.R. SCHWAB, B.S. EDWARDS, D.M. HEUBLEIN and J.C. BURNETT, Jr., Am. J. Physiol. 251:R310-R313 (1986). B.M. JAFFE and H.R. BEHRMAN, Methods of Hormone Radioimmunoassay, 2nd Ed, Academic Press, New York (1919). K.S. MISONO, H.FUKUMI, R.T. GRAMMER and T. INAGAMI, Biochem. Biophys. Res. Comn. 119:524-529 (1984). T. MAACK, M.J. CAMARGO, H.D. KLEINERT, J.H. LARAGH and S.A. ATLAS, Kidney Int. 27:607-615 (1985). J. GUTKOWSKA, K. HORKY, G. THIBAULT, P. JANUSZEWICZ, M. CANTIN and J. GENEST, Biochem. Biophys. Res. Comm. 125:315-323 (1984). I. TANAKA, K.S. MISONO and T. INAGAMI, Biochem. Biophys. Res. Comm. 124:663-668 (1986). R. TAKAYANAGI, I. TANAKA, M. MAKI and T. INAGAMI, Life Sci. 36:1843-1848 (1985).
Vol. 41, No. 21, 1987
29. 30. 31.
ANF Responses to Acute Volume Changes
J. TANG, R.J. WEBBER, D. CHANG, J.K. CHANG, K. KIANG and E.T. WEI, Reg. Peptides 9:53-59 (1984). F.C. LUFT, E.E. LANG, G.R. ARONOFF, H. RUSKOAHO, M. TOTH, D. GANTEN, R.B. STERZEL and T. UNGER, J. Pharmacol. Exp. Ther. 236:416-418 (1986). K.K. MURTHY, G. THIBAULT, E.L. SCHIFFRIN, R. GARCIA, L. CHARTIER, J. GUTKOWSKA, J. GENEST and M. CANTIN, Peptides 7:241-246 (1986).
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Pergamon Journals
THE RESPONSES OF ATRIAL NATRIURETIC FACTOR CONCENTRATIONS TO ACUTE VOLUME CHANGES IN CONSCIOUSRATS Peter J.S. Chiu, Subbarao Vemulapalli, Mark Policelli, Imre Kifor*, Edmund J. Sybertz and Victor J. Dzau* Department of Pharmacology, Schering-Plough Corporation Research Division, Bloomfield, New Jersey 07003 and Molecular and Cellular Vascular Research Laboratory, Division of Vascular Medicine and Atherosclerosis, Brigham and Women's Hospital Harvard Medical School 02115 Boston, Massachusettes (Received in final form September 25, 1987) Summary A rapid and sensitive radioimmunoassay has been developed for measurements of atria1 natriuretic factor (ANF) in rat plasma. The antiserum, raised to rat ANF (99-126), cross-reacts with rat ANF (103-123), ANF (103-125), ANF (103-126) but not with smaller fragments, human ANF (99-126), angiotensin II, bradykinin or vasopressin. The plasma ANF concentration is 181+24 pg/ml (N=24) in the unstressed conscious rats (CharTes River CD, male). The ANF imunoreactivity in the plasma extracts was verified by HPLC analysis, which displayed one major imunoreactive peak of ANF corresponding to rat ANF (99-126) and some smaller fragments. Intravenous injection of saline elevated circulating ANF, whereas acute volume depletion by hemorrhage, water deprivation and furosemide diuresis greatly lowered plasma ANF. The prompt response of plasma ANF levels to acute changes in volume status is consistent with the proposed role of ANF as a volume-regulatory hormone. The myocytes of mammalian atria contain atria1 natriuretic factors (ANF) which have potent natriuretic, diuretic and vasorelaxant properties (1). Release of ANF from isolated atria has been demonstrated (2,3). ANF levels in coronary sinus blood are several-fold higher than in samples from peripheral blood in man (4,5) and dogs (6,7), further suggesting active secretion. A 28-amino acid polypeptide ANF (99-126) (Ser-Leu-Arg-Argatriopeptin III) has been identified as the major circulating form of ANF in rats (&rANF) and man (d-hANF) (8,9,10). In addition to its direct effects on renal excretion of sodium and water (11,12,13), ANF inhibits secretion of renin and other volume regulatory hormones including aldosterone (1,14). Consequently it is proposed that ANF plays a role in the control and regulation of extracellular fluid (ECF) volume (1,14). An increase in circulating ANF usually follows acute expansion of the ECF volume with saline, blood or high sodium diet in animals (15-17) and man (5,10,18,19), presumably mediating in part the accompanying natriuresis in order to 0024-3205/87 $3.00 + .oo Copyright (c) 1987 Pergamon Journals Ltd.
2340
ANF Responses to Acute Volume Changes
Vol. 41, No. 21, 1987
restore body fluid and electrolyte balance (20-22). In the present study we have developed a radioimmunoassay of ANF in rat plasma and determined the responsiveness of plasma immunoreactive ANF concentrations to acute volume depletion due to hemorrhage, water deprivation and furosemide-induced diuresis in conscious rats.
Methods
Preparation of plasma for radioimmunoassay (RIA). Male Charles River CD rats (300-350 g) were used. Blood samples (6 ml) were usually collected from conscious rats by decapitation into chilled Vacutainer tubes containing a mixture of EDTA (1.5 mg/ml), aprotinin (200 kallikrein inhibitor unit/ml) and soybean trypsin inhibitor (20 No(-benzoyl-L-arginine ethyl ester unit/ml), with 100 pl each. For the purpose of characterizing the RIA methodology, blood samples (10 ml) were drawn from the abdominal aorta of rats anesthetized with Inactin to obtain "pooled plasma". Samples were immediately centrifuged at 3,000 rpm for 10 min. The pooled plasma was divided into 1 ml aliquots and stored at -2O'C whereas the experimental plasma samples were continously processed through extraction and the entire RIA procedures. Extraction of plasma samples. A mixture of 1 ml plasma, 0.6 ml 8 M urea and 20 ml 1.5 M NaCl in disposable syringes was passed slowly through octadecylsilane Sep-Pak Cl8 cartridges (Waters Assoc., Milford, MA) which were previously activated by passing 5 ml methanol and 5 ml 8 M urea and followed by washing with 10 ml water. The use of high urea was intended to disrupt hydrogen bonding between ANF and large proteins. The sample-loaded cartridges were washed with 10 ml water and eluted with 8 ml 4% acetic acid in 90% ethanol. The eluants were collected in 50-ml polypropylene centrifuge tubes (Corning) each containing 100 pl 1% glycerol in water and were blown under a stream of N to near dryness. The samples were dissolved in 0.4 ml 0.02 M ace z.ic acid and 0.6 ml assay buffer after The assay buffer was composed of undergoing lyophilization to dry powder. each of the following: O.OlM K HP04, 0.03M EDTA, 0.015 mM 8-hydroxyquinoline (a fungistatic), 0.02% neomycin sulfate and 0.25% bovine serum albumin; the mixture was adjusted to pH 7.4 and piaced in a water bath at 56 C for 30 min. The assay buffer is stored at 4 C. Preparation of antiserum. Rat ANF 99-126 (d rat ANF) was covalently conjugated by carbodiimiae to bovine thyroglobulin (23). The material was kept frozen after dialysis against 0.15 M NaCl. To prepare for immunization, the ANF-protein conjugate was emulsified with three volumes of complete Freund's adjuvant and a 2 ml emulsion was injected into the dorsal skin of rabbits at multiple sites. Booster injections, in which incomplete Freund's adjuvant was used instead, were performed after 4-6 weeks on the four legs im. Four to five weeks following booster, serum was collected fro8 one rabbit via the central ear artery, aliquoted and kept frozen at -20 C. Radioimmunoassay for ANF. The extracted plasma samples (100 pl) or standards (rat ANF 99-126, 4-100P2Jg/tube) were incubated with 100 pl antiserum 11:24.000) and 100 ul I-labelled rat ANF 99-126 f3.500-4.500 cpm) in triplicate at-4'C forr24 h using polypropylene culture tubes . Free and bound ANF were separated using 0.5 ml dextran-coated (Fisher). charcoal (DCC). The DCC stock solution was prepared by adding 5 g Norit
Vol. 41, No. 21, 1987
ANF Responses to Acute Volume Changes
2341
charcoal and 0.59 dextran t8 200 ml O.lM tris buffer at pH 7.4, stirred overnight and was kept at 4 c. A 1:40 dilution of the DCC stock solution was used. Upog addition of DCC, the individual samples were vortexed and incubated at 4 C for 15 min, followed by centrifugation at 3,000 rpm for 15 min. The supernatants were decanted into 12x75 mm polystyrene tubes (Fisher) for counting in an automatic gamna counter (Micromedic Systems, Horsham, PA; efficiency, >75%). High performance liquid chromatography (HPLC) identification of immunoreactive ANF in rat plasma. HPLC analysis of pooled plasma was performed on a Varian 5560 Liquid Chromatograph, using a Beckman Altex Ultrapore RPSC column (pore size, 300 particle size, 5 pm; column size, 0.46 x 15 cm). A linear gradient elution system was used from 0 to 90% over 35 min; solvent A, O.Olm trifluoroacetic acid (TFA) in water; solvent B, 0.01 M TFA in 90% acetonitrile; flow rate, 1 ml/min. Fractions of 1 ml were collected, lyophilized and quantitated for immunoreactive ANF.
A;
Under ether anesthesia the Acute volume expansion with saline. vein of rats was cannulated for administration of isotonic saline. 2.5 h after recovery from anesthesia, 4 or B ml of saline was given min; the control animals received no saline challenge. The animals immediately sacrificed and trunk blood was collected for ANF RIA to with the sham operated animals.
jugular Two to in 1 were compare
Water deprivation and furosemide treatment. Rats, deprived of food and water overnight, were divided into two groups. One group was sacrificed immediately and the other sacrificed 1 hr following treatment with furosemide 40 mg/kg po. The ANF levels of the treated animals were compared with those of the control animals. Rat ANF 99-126 (rANP) ANF 103-123 (atriopeptin I), ANF Reagents. 103-125 (atriopeptin II), ANF 103-126 (atriopeptin III), ANF 92-126, ANF 99-119, ANF 116-{$5 and h uman ANF 99-126 (o(hANP) were purchased from Peninsula Labs; I-rat ANF 99-126, specific activity: 2000-2200 pCi/mmole (Amersham or New England Nuclear); carbodiimide, bovine thyroglobulin, aprotinin, soybean trypsin inhibitor, &hydroxyquinoline, angiotensin II, bradykinin and vasopressin were purchased from Sigma; Norit charcoal, urea, NaCl, EDTA and K HP0 from Fisher; neomycin sulfate (Gibco Labs); bovine serum albumin (IZN B!omedicals). Statistical analysis. All data are presented as mean+S.E.M. unless specified otherwise. Student's t-test or Duncan's multiple range test was used to ascertain statistical significance (PcO.05).
Results Characterization of radioimmunoassay for ANF. Total binding of the tracer to antiserum was 49.4+0.7% (N=8); nonspecific binding was 5%. The 50% displacement point on a standard curve (EC ) was 73.9t6.9 pg/tube (N=8); the detection limit (10% displacement) was s'pg/tube. The antiserum demonstrated cross-reactivity with rat ANF 103-123 (74%), ANF 103-125 (75%), ANF 103-126 (63%) and ANF 92-126 (100%) but did not cross-react with rat ANF 99-109, 116-126 and human ANF 99-126 or with angiotensin II, Recovery of rat ANF 99-126, added to arginine vasopressin and bradykinin. pooled plasma to yield a concentration of 250 pg/ml, is 63+9% (N=7). All ANF values reported subsequently are not corrected for recovery. The
2342
ANF Responses to Acute Volume Changes
Vol. 41, No. 21, 1987
interassay variation was 9% (N=3) and the intra-assay variation 8% (N=3). Trunk plasma ANF concentrations in unstressed conscious and anesthetized rats are summarized in Table 1. The rats anesthetized with Inactin showed significantly higher plasma ANF levels. Table 1.
ANF concentrations in rats
Method of collection
N
ANF (pg/ml)
Conscious, via decapitation
24
181~24 (34-632)
Anesthetized, via aortic puncture
24
337~15" (223-524)
*P
Vol. 41, No. 21, 1987
ANF Responses to Acute Volume Changes
2343
.
+ P-zO.05, VS CONTROL AND 0 TIME
.
100
0
I CONTROL
Figure 1.
I
HEMORRHAGE (0 TIME)
.
I
HEMORRHAGE (+ 15 MIN)
Changes in plasma ANF levels following acute hemorrhage in conscious rats. Hemorrhage was performed by drawing 6 ml of blood in 2 min via the arterial cannula. Blood collection for ANF RIA was performed by decapitation immediately (0 time) and 15 min after hemorrhage, respectively. *P
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Vol. 41, No. 21, 1987
ANF Responses to Acute Volume Changes
700 * P< 0.05, VS CONTROL + Pi 0.05, VS WATER DEPRIVATION
600
500 = E 5 II 400 Q Y Y :
. .
300
.
.
f
. -*
200
0
0
-*t
100
: 0 .
I
0 NORMAL CONTROL
I WATER DEPRIVATION
WATER DEPRIVATION FUROSEMI&
Figure
2.
40 mglkg po
Changes in plasma ANF levels following water deprivation and furosemide treatment in conscious rats. Two groups of rats of 6 each were deprived of water and food overnight. On the following day, one group was sacrificed inmediately and the other 1 hr after receiving additional treatment with furosemide, 40 mg/kg po.
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ANF Responses to Acute Volume Changes
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fragments of ANF 99-126 (ANF 99-109 and ANF 116-126 respectively) are biologically inactive and also do not cross-react with the antiserum (24,25). To obviate the interference of plasma proteins, which would otherwise yield spuriously high levels of ANF immunoreactivity in RIA (6,8,26), it was necessary to use Sep-Pak C cartridges to extract ANF from the plasma. HPLC analysis of the extracted Y at plasma indicated that the circulating ANF consists of mainly ANF 99-126 and possibly some low-molecular-weight fragments. On the basis of data from immunoreactivity, chromatographic migration, bioassay and sequence analysis, Schwartz et al. (9) reported that ANF 99-126 is the major species of ANF in the rat circulation and atriopeptin III a minor component. Other groups demonstrated the presence of low-molecular-weight forms of ANF other than ANF 99-126 in rat plasma (15,27). ANF 99-126 is believed to be the product of selective cleavage of the high molecular weight precursor in the atrium upon release (1). ANF concentrations in the plasma samples from anesthetized animals are higher than those from conscious, unstressed animals. The discrepancy can also be ascribed to differences in the method of collection, namely decapitation vs aortic puncture. Gutkowska et al. (6) reported on the influence of anesthetics, route of collection, drugs, etc. on the plasma ANF in rats. The following rat plasma ANF concentrations using RIA quantitation have been reported: 478 pg/ml (27); 58 pg/ml (15); 800 pg/ml (9); 80-125 pg/ml (17); 82-94 pg/ml (6). Hence it becomes evident that the plasma ANF values we obtained in the conscious and anesthetized rats fall within the range of reported values. We have demonstrated a marked increase in circulating ANF in response to volume expansion with saline in rats, which is the most consistent way of stimulating ANF release (10,15,17). The results indicate that our RIA technique is associated with adequate sensitivity to detect this challenge. A 5-day water deprivation caused a great fall in rat plasma ANF (28). We showed that the rats responded to even an overnight water deprivation with a fall in plasma ANF. Furthermore, a supramaximal diuretic dose of furosemide promptly caused a further decline in plasma ANF. The rapid fall in plasma ANF following hemorrhage further indicates that plasma ANF is greatly sensitive to acute alterations in extracellular fluid volume. Inhibition of atria1 secretion in response to volume depletion, in conjunction with the rather short half-life of ANF (~1 min) may account for the rapid fall in circulating ANF (29-31). In conclusion, the prompt response of plasma ANF levels to acute changes in volume status in an appropriate manner is consistent with the hypothesis that ANF plays a role in the control and regulation of body fluid and electrolyte balance. Acknowledgements The authors thank W. Baginsky and Dr. C. Foster for providing the HPLC data. This study was supported in part by NIH grants HL 35610, HL 35793, HL 19259, HL 35252 and NIH specialized Center of Research HL 36568. V.J. Dzau is an Established Investigator of the American Heart Association.
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