Regulatory Peptides, 37 (1992)227-236
227
© 1992 Elsevier Science Publishers B.V. All rights reserved. 0167-0115/92/$05.00 REGPEP 01139
Inhibition of polyamine synthesis impairs the secretion of atrial natriuretic peptide Ulka R. Tipnis and Paul J. Boor Department of Pathology, University of Texas Medical Branch, Galveston, TX (U.S.A.)
(Received 6 August 1991; revisedversionreceived25 October 1991; accepted28 October 1991) Key words: Polyamine; DFMO; Atrial natriuretic peptide
Summary The aim of the present study was to evaluate in vivo the role of polyamines in the secretion of atrial natriuretic peptide (ANP). a-Difluoromethylornithine (DFMO) which inhibits ornithine decarboxylase activity and polyamine synthesis was given in drinking water and through intraperitoneal administration to Sprague-Dawley rats. Carotid artery was cannulated for collection of blood samples and measurement of blood pressure following the administration of arginine-vasopressin (AVP). Analysis of polyamines in cardiac tissue indicated that DFMO treatment decreased contents of putrescine and spermidine in cardiac tissue by 80 ~o and 48 ~ , respectively. Quantitation of ANP in plasma by radioimmunoassay indicated that both basal and stimulated levels of ANP in DFMO-treated animals were 21.5~o and 50~o of those in control rats. The administration of putrescine restored the levels of basal and AVP-stimulated levels of ANP in plasma which confirmed that DFMO effect on ANP secretion occurred specifically through the polyamine pathway.
Introduction Atrial natriuretic peptide (ANP) is synthesized as a pre-prohormone in the atrial myocyte and stored as the pro-hormone in the secretory granules (reviewed in Ref. 1). Atria secrete ANP into plasma in response to several stimuli which include vasocon-
Correspondence: U.R. Tipnis, Department of Pathology,Keiller Building,Rt. F-05, Universityof Texas Medical Branch, Galveston,TX 77550, U.S.A.
228 strictor agents, morphine or anesthetics, exercise etc. [2-7]. The cleavage product ANP99_126 released into plasma inhibits the secretion of aldosterone [8] and renin [9], causes relaxation of blood vessels [10] and induces natriuresis and diuresis resulting in hypotension [ 1]. Polyamines, putrescine, spermidine and spermine are ubiquitous aliphatic cations in mammalian cells and are necessary for growth and differentiation [ 11,12]. In a previous study, we reported that ornithine decarboxylase (ODC), the initial rate-limiting enzyme in the biosynthesis of polyamines, is present predominantly in the atrial granules [ 13]. In view of this finding and that of others [14] reporting a high concentration of polyamines in the secretory granules, we have investigated if polyamines are of functional importance in the secretion of ANP. In order to assess the significance of polyamines in ANP secretion, we used a-difluoromethylornithine (DFMO) which is an inhibitor of ODC activity and polyamine synthesis [ 11 ]. We have examined the changes in secretion of ANP and in the blood pressure following the administration of argininevasopressin (AVP), which is a vasopressor agent and a potent inducer of ANP secretion [2].
Materials and Methods
Materials. a-Difluoromethylornithine (DFMO) was obtained as a gift from Merrell
Dow Research Institute, Cincinnati, OH. Arginine-vasopressin (pitressin) was from Park Davis, Inc., NJ. Sep-Pak C-18 columns and the radioimmunoassay kit for rat ANP were purchased from Peninsula Laboratories. DFMO treatment. Male Sprague-Dawley rats weighing 250-300 g were used for all the studies. The animals were housed in wire-bottom cages and were given Purina Rat Chow and water ad libitum. The animals were divided into two groups: the first group (control) received plain drinking water while the second group (DFMO) received DFMO (2~o) in drinking water for 10 days. Control and DFMO group received intraperitoneal administration of saline (0.2 ml, i.p.) and DFMO (200 mg/kg, 0.2 ml, i.p.), respectively, 24 h, 18 h and 1 h prior to experiment. Animal experiment. The animals were anesthetized with diethyl ether and maintained
under it until the completion of the experiment. The carotid artery was cannulated with polyethylene catheter (PE 50) for measurement of blood pressure and removal of blood samples from 0 to 30 min. The animals were allowed to stabilize, then AVP (1 #g/kg) was injected through the subclavian vein. The blood pressure was monitored from 0-30 min after AVP injection by connecting the cannula to a transducer and using Narco-Biosystems Physiography Model (DMP-LB). Blood was sampled at intervals from 0 to 30 min after AVP administration. 0.3 ml of blood was collected in tubes containing 2.5 mM EDTA, chymostatin (2/~M), leupeptin (2/2M) and phenylmethylsulfonylfluoride (2mM). The samples were immediately centrifuged at 4000 rpm at 4 °C and plasma was removed and frozen at -70°C.
229
Analysisofpolyamines.Analysis of polyamines was done as previously described [ 13]. The hearts were homogenized in 5 volumes of 10~o trichloroacetic acid. After centrifugation at 1200 g, the distilled ether was added to the supernatant to remove acid and the samples were made basic by addition of saturated solution of sodium bicarbonate. The polyamines were dansylated by addition of dansyl chloride (10 mg/ml) and the •dansyl derivatives were extracted into toluene, centrifuged and evaporated under nitrogen gas and reconstituted in methanol (0.5 M). The efficiency of this procedure was assessed by determining the recovery of polyamines after addition of [3H]putrescine (10,000 cpm) prior to dansylation. The polyamines were separated through high-pressure liquid chromatography on a C-18 chromatography on a C-18 reverse phase column using a linear gradient of 15-90~o acetonitrile in water as the mobile phase at a flow rate of 2 ml/min. 200 pmol each of putrescine, spermidine and spermine were chromatographed before each group of samples to serve as a reference for retention times and quantitation. The polyamines were detected by fluorospectrophotometer using wavelength at 342 and 512 nm for activation and emission, respectively. Extraction ofANPfrom plasma. The plasma samples were acidified with two volumes of 0.1 ~o trifluoroacetic acid (TFA) and centrifuged at 4000 rpm for 15 min. Sep-Pak column 18 was activated with 60~o acetonitrile followed by equilibration with 0.1 TFA. Plasma was loaded onto the column and washed slowly with 0.1 ~ TFA. ANP was eluted with 60~ acetonitrile. Extraction efficiency as assessed by passing [125I]ANP (Peninsula Laboratories) through Sep-Pak column was 90 ~ . The samples were lyophilized in a Jouan System 2 Centrifugal vacuum concentrator and stored at 70 °C until assayed. -
RadioimmunoassayofANP. ANP in plasma samples was assayed using RIA kit from Peninsula Laboratories. Briefly, 100#1 of ANP standard ranging from 1 pg to 128 pg/tube or 100 #1 of sample was incubated overnight with 100 #1 of rabbit antiserum against rat ANP. 100 #1 of [ ~25I]ANP (10,000 cpm/tube) was added and contents were incubated at 4 °C overnight. Free ANP from bound ANP was separated by incubating with 100 #1 each of goat anti-rabbit IgG serum and normal rabbit serum. Finally, 0.5 ml of RIA buffer was added and samples were centrifuged at 3000 rpm for 20 min, supernatants were removed and pellets were counted in a gamma counter (RIA star, Packard Instrument). The dose-response curve for synthetic ANP in Fig. 1 showed that the half maximal binding occurred at 12 pg and usable linear portion of this curve was between 4 and 100 pg. Statisticalanalysis. The results are expressed as means + S.D. The significance of the difference between control and DFMO group was tested by t-test. P values < 0.05 were considered significant.
230 100,
90i 80, 70, o gO
~. m
60. 50 40 30 20 10 I
!
10
100
ANP (Pg/tube) Fig. 1. Dose-response curve showing the displacement of the [~25I]ANP from binding to the specific ANP antibody by the synthetic rat ANP standard. The curve was constructed by assaying ANP standards from 1 to 128 pg as described in Materials and Methods.
Results
Changes in polyamine content The efficacy of DFMO treatment was determined by analysis of polyamine contents in the heart. The results shown in Table I indicated that DFMO treatment reduced the putrescine, spermidine and spermine contents by 80, 48 and 17~, respectively. Changes in ANP levels in plasma Immunoreactive ANP levels in control rats ranged from 50 pg/ml to 200 pg/ml while those in DFMO treated rats were from 6 to 78 pg/ml. The basal levels ofimmunoreactive (ir) ANP in control rats averaged about 116 + 27 pg/ml while those in DFMO-treated group were significantly lower than the controls and averaged only about 25 + 12 pg/ml (Fig. 2A). These results suggested that inhibition of polyamine synthesis partially impaired the basal secretion of ANP into plasma.
TABLE I Cardiac polyamine contents in control and DFMO-treated rat Polyamines (nmol/g)
Control DFMO
putrescine
spermidine
spermine
7.4 + 2 1.5 +0.5
122 + 10 64+ 7
110 + 9 92+8
Polyamines were analysed as described before [13]. Hearts were homogenized in 10~o trichloracetic acid, extracted with diethyl ether and dansylated. The dansylated derivatives were resolved by HPLC using a C-18 column with a liner gradient of 5-85~o acetonitrile in water as the mobile phase at a flow rate of 2 ml/min. The results (mean + S.D.) are based on analysis ofpolyamines in cardiac tissue from three control and three DFMO rats.
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Fig. 2. (A) Secretion of ANP in control and DFMO-treated rats following the administration of AVP. The rats were treated with DFMO as described in Materials and Methods. AVP was injected (1/Jg i.v.) to stimulate the secretion of ANP. Acidified plasma samples were passed through Sep-Pak column and ANP was eluted with 60% acetonitrile in 0.1% trifluoroacetic acid and assayed by radioimmuno-assay as described in Materials and Methods. (B) Restoration of ANP secretion by putrescine. Rats were given DFMO as described in A. Putrescine (80/~M/kg, i.p.) was given i h prior to AVP administration. Number of animals used: control n = 6, DFMO n = 6, con + put n = 4, DFMO + put n = 7. The error bars represent mean + S.D. (*P value < 0.05, **