Acta Physiol Scand 1990, 139, 355-360
Adrenal cortex contributes t o the regulation of NaCI-stimulated ANP release in the rat 0. ARJAMAA, 0. V U O L T E E N A H O , T. T A S K I N E N , M. T U O M I N E N and J. L E P P A L U O T O Department of Physiology, University of Oulu, Finland ARJAMAA, O., VUOLTEENAHO, O., TASKINEN, T., TUOMINEN, M. & LEPPALUOTO, J. 1990. Adrenal cortex contributes to the regulation of NaC1-stimulated ANP release in the rat. Acta Physiol Scand 139,3559360. Received 12October 1989, accepted 22 January 1990. ISSN 0001-6772. Department of Physiology, University of Oulu, Finland. T h e present study documents the effects of hypophysectomy and the effects of dexamethasone substitution on the NaC1-stimulated release and on the basal secretion rates of ANP from the rat atria in vitro. We also measured the concentration of mRNA in the atria after hypophysectomy. Rats (n = 12) were subjected to hypophysectomy by a parapharyngeal approach. One group of rats (n = 6) received dexamethasone 0.2 mg S.C.daily for 4 weeks, while the other group was left unsubstituted. After 4 weeks, the atrial block (n = 10) was excised, placed in an organ bath (field stimulation 4 s ~ ' 20 , V, I ms; resting tension = 5 mN) and superfused (7 ml min-') either with a physiological buffer solution (295 mosmol kg-') or with a hyperosmotic NaCl solution (330 mosmol kg-'). T h e atria from the hypophysectomized rats did not respond to the stimulus: the concentration of ANP in the I-min samples of the perfusate was under IOO pg ml-'. Dexamethasone treatment significantly (P < 0.05) increased the ANP concentration to a maximum of 165 f 17 (mean fSEM) pg ml-' during the superfusion while the control concentration was I I O 19 ~ pg ml-'. T h e ANP mRNAII8 S RNA ratios did not differ between the atria of hypophysectomized and control rats. In conclusion, glucocorticoids are required in the stimulus-induced release of ANP and the impaired release of ANP after hypophysectomy does not depend on an impaired synthesis of ANP. Key words; atrial natriuretic peptide (ANP), dexamethasone, hypophysectomy, osmolality, superfusion.
Atrial natriuretic peptide (ANP) is a hormone, released from cardiac atria, acting on kidneys and vasculature to regulate volume and blood pressure (for review, see deBold 1985). Little is known about the mechanisms that adjust the release of ANP. Atrial stretch, without any central regulation, is thought to be a major stimulus for the release of ANP (Ruskoaho et al., 1986). We have shown previously that increased osmolality augments the release of ANP from isolated rat atria (Arjamaa & Vuolteenaho 1985, Arjamaa 1989). Gibbs (1987), using dispersed atrial myocytes, was also able to show that the Correspondence : Olli Arjamaa, Department of Physiology, University of Oulu, Kajaanintie 52A, 90220 Oulu, Finland.
secretion of ANP was markedly stimulated by increase in extracellular osmolality, regardless of the solute. Pithing and hypophysectomy, which remove neurogenic influences and humoral communication of the brain and peripheral organs, completely blocked stimulus-induced release of ANP (Eskay et al. 1986, Zamir et al. 1987, Ruskoaho et al. 1989). In hypophysectomized rats, the response to a volume load was attenuated (Dietz & Nazian 1988), which did not result from a decrease in the right atrial pressure (Arjamaa et al. 1988). These findings led us to study also the effects of hypophysectomy on NaC1-stimulated release of ANP from isolated rat atria. We showed that hypophysectomy greatly attenuated the response and decreased
355 12-2
356
0. Arjamaa et al.
the basal secretion as well (Arjamaa 1989). As there was evidence that glucocorticoids regulate the synthesis, natriuretic effect, release (Garcia et al. 1985) and the expression of the gene for ANP (Gardner et al. 1988), we tested the hypothesis that a glucocorticoid substitution could restore the responsiveness to a n N a C l stimulus in hypophysectomized rats in vitro. We also measured the concentration of mRNA i n atria of hypophysectomized rats t o find o u t whether the ANP gene expression was impaired. We report that dexamethasone substitution partially restores the response to a NaCl stimulus a n d that hypophysectomy does not have any effect on the level of ANP m R N A .
the perfusion rate constant (7 ml min-'). After an equilibration period of 60 min the perfusate was collected in I-min fractions for 5 min and then the bath was superfused with a buffer"so1ution of 330 mosmol kgF' for 10 min (the increase in osmolality was produced with NaCI), which was followed by a superfusion with PBS for 15 min. Solutions were gassed with I O O ~0, / ; , and kept at 37 "C throughout experiments. A N P RIA. The ANP content of samples was determined with a radioimmunoassay using antiserum '7-44', which was raised in rabbits against rat ANP,-,,. This antiserum was specific for the midportion of rat ANP and the sensitivity of the assay was 0.5 pg per tube (Vuolteenaho et al. 1985). Radioiodinated rat ANP was purchased from Amersham International PLC. A N P mRNA determination. RNA was isolated from atrial tissue of two control rats and of two hypoMATERIALS AND METHODS physectomized rats using the guanidine thiocyanateAnimals. Male rats of the Sprague-Dawley strain LiCl method (Cathala et ul. 1983). For the RNA blot were used. They had been bred at ZCFZZ "C and had analysis, I-pg samples of RNA from auricles were free access to tap water and standard pellets. T h e light dotted onto Zeta-Probe blotting membrane (Bio-Rad cycle was 1 2h light/Iz h dark starting from 08.00 h. Laboratories, Richmond, CA, USA) and hybridized At the age of about 1 2 weeks, rats ( n = 12) were with a full-length rat ANl' cDNA probe CAR-5s anaesthetized with Hypnorm (10 mg ml-l fluanisone (Flynn et al. 1985) (a gift from Dr Peter L. Davies, and 0 . 2 mg ml-' fentanylcitrate; Duphar BV, Amster- Queen's University, Kingston, Canada). The probe dam, Holland; 0.07 ml IOO gF' i m . ) and hypo- was labelled with [32P]dCTP (Amersham) using a physectomized by a parapharyngeal approach. After random primed labelling kit (Boehringer-Mannheim). the surgical operation, the animals were allowed to Hybridization was carried out overnight at 65 "C recover at an ambient temperature of 28 "C for about according to the method of Maniatis et al. (1982). The 4 weeks. Six rats received daily dexamethasone membrane was exposed to XAR-5 X-ray film (Eastman-Kodak) at -70 "C with a Cronex Lightning (Oradexon, NV Organon, OSS, Holland) 0.2 ml ( = Plus intensifying screen (DuPont). T o quantify the 0.2 mg) S.C.during a period of 4 weeks, while four rats received dexamethasone for the first 2 days to speed amount of RNA, the membrane was rehybridized up the recovery from the surgical operation. These after extensive strip-wash with a 32P-labelled oligofour rats received no further dexamethasone treatment. nucleotide complementary to rat 18 S ribosomal RNA The hypophysectomy was considered successful if the (Lee et al. 1988, Albretsen et al. 1988). The ANP animal lost weight after the operative procedure and if mRNA/18 S RNA ratios were obtained by densithe weights of testes were about 25 yo of age-matched tometry analysis of the membrane analysed with the controls. T h e weight of testes and the body weight of two different probes. Determination of osmolality. Osmolalities of perhypophysectomized rats are shown in Table I. Experimental design. The in-nitro experimental fusates and buffer solutions were determined by an procedure has previously been described in detail osmometer (Advanced Wide-Range Osmometer 3W2, (Arjamaa 1989). Briefly, a rat was decapitated and the Advanced Instruments, Inc.) based on the freezingatrial block was excised and mounted in a vertical point method. Statistical analyses. Mean SEM are given organ bath of 8 ml. Two hooks kept the block between the bottom of the bath and a force transducer which throughout. I n statistical analyses of the results which monitored preset tension (= 5 mN) and contractions. were repeated measures over time a paired t-test was By means of two platinium plate electrodes, the block used as suggested by O'Brien & Shampo (1988). was electrically stimulated (supramaximal field stimulation 4 IIz, 20 V, I ms). The atrial block was R E S U L T S superfused with physiological buffer solution (PBS) (NaCI 137 mmol IF', KCI 5.6 mmol I - l , CaCI, 2.2 Figure I shows the secretory responses of heart atria from either hypophysectomized (a) or mmol I-', MgCI, 0.12 mmol I-', and Hepes "-2hydroxyethyl-piperazine-"-2-ethanesulphonic acid] hypophysectomized a n d dexamethasone-treated 1 5 mmol IF' in I litre of distilled water at p H 7.40; (b) rats to a hyperosmotic (330 mosmol kg-') osmolality 295 mosmol kgF1). A perfusion pump kept stimulus. Basal ANP secretion in the non-
ANP and adrenal cortex in the rat
357
h
7- 150E m a
Y
a
z
a 100-
1
5
10
15 20 TIME ( m i d
25
30
5
10
15 20 TIME ( m i d
25
30
Fig. I . Responses of rat atria in nitro after hypophysectomy (a) and after hypophysectomy followed by dexamethasone treatment (b) before, during, and after hyperosmotic (NaCI) superfusion. Lower panels show the changes in osmolalities. Data were collected in I-min fractions of superfusion. Means fSEM. Hypophysectomy, n = 4;hypophysectomy dexamethasone, n = 6. Paired t-test, P < 0 . 0 5 .
+
substituted hypophysectomized group was under IOO pg ml-', while it was higher, I 10 f 19 (n = 9) pg ml-', in the group substituted with dexamethasone. The. atria from the non-substituted, hypophysectomized rats did not respond to the stimulus and the secretion of ANP in the I-min samples of the perfusate remained under IOO pg ml-' during the whole period of osmotic stimulation. T h e atria from hypophysectomized rats receiving dexamethasone responded to the same stimulus by an increase in ANP secretion. During the hyperosmotic superfusion, the concentration of ANP in perfusates increased, and at 8 min (165 p g 4 17 ml-l) it was significantly (P< 0.05) higher compared with the control values of the prestimulation period. However, in spite of a continuous osmotic stimulus, the ANP secretion tended to diminish, reaching control values before the hyperosmotic superfusion was terminated. Using the "P-labelled oligonucleotide which was complementary to rat 18 S ribosomal RNA, we found no difference in concentrations of mRNA in hypophysectomized and in control (no
a
b
e d Fig. 2. ANP mRNA dot blot analysis of heart atria from hypophysectomized and control rats. I pg of total right auricular ANP (3-4 dots per sample) was dotted onto nylon membrane which was hybridized against 32P-labelled rat ANP cDNA probe CAR 55. (a) and (b) Hypophysectomized rats, mixed RNA samples of left and right auricles. (c) Control rat, left auricular RNA. (d) Control rat, right auricular RNA.
358
0. Arjamaa et al.
I . Weight of testes and body weight (means, n in parentheses) after dexamethasone treatment in hypophysectomized rats
Table
Testes (mg)
Body weight (g) At
Treatment
Right
Left
operation
Hypophysectomy Hypophysectomy + dexamethasone
404 (4) 420 (4)
389 (4) 423 (4)
360 (4) 365 (4)
After operation
m8 (4) 235
(4)
Weighing took place 4 weeks after the surgical operation. hypophysectomy) rats. The ANP mRNA/18 S RNA ratios (a densitometry analysis with two different probes) were between 0.18 and 0.25. Figure z shows the blot micrographs from control and from hypophysectomized rats. The weight of testes and body weights of the rats used in the experiments are shown in Table I . DISCUSSION
We have shown in the present experiments that dexamethasone substitution treatment of hypophysectomized rats partially restored the secretory ANP response to a hyperosmotic NaCl perfusion in isolated atria. Measurements of the mRNA contents of atria also showed that hypophysectomy appeared to have no effect on the expression of the ANP gene. As a model for st6dies on ANP release we have used isolated atria in an organ bath in which atria can be electrically stimulated (field stimulation) and resting tension can be kept constant. Both pacing of atria in vitro (Schiebinger & Linden 1986a) and changes in atrial tension in vitro (Schiebinger & Linden 1986b) may influence the ANP release. Dietz (1984) and Lang et al. (1985) have shown that atrial stretch is a major stimulus for the release of ANP from heart atria. However, in addition to these mechanical stimuli, we have been able to show that increased osmolality in vitro (Arjamaa & Vuolteenaho 1985, Arjamaa 1989) acts as a stimulus for the ANP release from atrial myocytes. This is also the case in enzymatically dispersed atrial myocytes as shown by Gibbs (1987). The present study, demonstrating increased ANP release from heart atria as a response to increased osmolality, confirms our previous findings, Although the osmotic stimulus
is relatively strong (330 mosmol kg-'), the atria release exclusively ANP 1-28 (Arjamaa 1989) indicating that no damage occurs in myocytes during the hyperosmotic superfusion. Hypophysectomy greatly attenuates stimulusinduced ANP release from heart atria in vitro to a hyperosmotic NaCl perfusion, and the basal secretion rate is significantly decreased as well (Arjamaa 1989). Our previous studies (Arjamaa et al. 1988) have also shown that hypophysectomy reduces the weight of heart atria. In present studies, dexamethasone treatment after hypophysectomy restored the responsiveness of atria to the NaCl stimulus and also increases the basal ANP secretion rate although the treatment does not increase the body weight. Therefore the decreased secretion rate of ANP after hypophysectomy cannot only be explained by the decreased number of secreting myocytes as previously suggested (Arjamaa 1989). The release of ANP as a response to a volume load appears to be under multifactorial control. Zamir et al. (1987) have suggested that the anterior pituitary is a prerequisite for the ANP release, while Ruskoaho et al. (1989) have concluded that pressor hormones, especially vasopressin, are of importance in regulating the ANP release. These in-vivo studies must, however, be interpreted cautiously, as pithed or hypophysectomized experimental animals may have abnormal circulatory volume and cardiac and renal haemodynamics. Garcia et al. (1985) have found that simultaneous administration of dexamethasone and deoxycorticosterone acetate to adrenalectomized rats produces a marked increase in ANP concentration in atrial tissue and plasma ; they conclude that glucocorticoids may regulate the synthesis and release of ANP, and mineralocorticoids may have a permissive role. Our present zn-vitro findings in the isolated
ANP and adrenal cortex in the rat atrial preparation therefore support the notion that glucocorticoids contribute to the regulation of ANP release. T h e recovery of the ANP response in the present study is, however, only partial when compared with our previous results (Arjamaa 1989), which are compatible with the results of Garcia et al. (1985). We have earlier reported (Arjamaa et al. 1988) that hypophysectomy does not change the concentration of A N P in the atrial tissue of the rat. Zamir et al. (1987) have shown that after hypophysectomy the A N P concentration is significantly increased in the right atrium while in the left atrium the concentration remains unchanged. Dietz & Nazian (1988) have found a significant decrease in the ANP concentration in both the left and right atrium. To explain these discrepancies between previous studies we have measured the concentration of m R N A in the rat atria. I t is evident from these experiments that hypophysectomy does not decrease the expression of the ANP gene. T o conclude, we have shown that glucocorticoids are required for the A N P release in response to a hyperosmotic superfusion and that the impaired release of A N P after hypophysectomy does not depend on an impaired ANP gene expression. We wish to thank Ms Tuula Lumijarvi for excellent technical assistance and Ms Liisa Asikkala for taking care of the experimental animals. Financial support was obtained from the Einnish Cultural Foundation and Academy of Finland.
REFERENCES ALBRETSEN, C., HAUKANES, B.-J., ASSLAND, R. & KLEPPE, K. 1988. Optimal conditions for hybridization with oligonucleotides: a study with myc-oncogene DNA probes. A n a l Biochem 170, 193-202. ARJAMAA, 0. 1989. Atrial natriuretic peptide (ANP) : response to NaCl is attenuated in rat atria in vitro after hypophysectomy. Acta Physiol Scand 136, 499-505. ARJAMAA, 0. & VUOLTEENAHO, 0. 1985. Sodium ion stimulates the release of atrial natriuretic polypeptides (ANP) from rat atria. Biochem Biophys Res Commun 132, 375-381. ARJAMAA, O., RUSKOAHO,H., TUOMINEN, M., 0. & LEPPALUOTO, J, 1988. The conVAKKURI, centration of atrial natriuretic peptide (ANP) is decreased in plasma but not in atria in hypophysectomized rats. Life Sci 42, 589-596.
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DEBOLD,A. 1985. Atrial natriuretic factor: A new hormone produced by the heart. Science 230, 767-770. CATHALA, G., SAVOURET, J.F., MENDEX, B., WEST, B.L., KARIN,M., MARTIAL, J.A. & BAXTER, J.D. 1983. A method for isolation of intact, translationally active ribonucleic acid. D N A z,329-335. DIETZ,J.R. 1984. Release of natriuretic factor from rat heart-lung preparation by atrial distension. A m 3 Physiol 247, R I 093-R I 096. DIETZ,J.R. & NAZIAN,S.J. 1988. Release of atrial natriuretic factor in hypophysectomized rats. A m 3 Physiol 255, R534-R538. ESKAY,R., ZUKOWSKA-GROJEC, Z., HAASS, M., DAVE, J.R. & ZAMIR, N. 1986. Circulating atrial natriuretic peptides in conscious rats: Regulation of release by multiple factors. Science 232, 636-639. FLYNN, T.G., DAVIES, P.L., KENNEDY, B.P., DEBOLD, M.I. & DEBOLD,A.J. 1985. Alignment of rat cardionatrin sequences with the preprocardionatrin sequence from complementary DNA. Science zz8, 323-325. GARCIA, R., DEBRINSKI, W., GUTKOWSKA, J., KUCHEL, O., THIBAULT, G., GENEST, J. & CANTIN,M. 1985. Gluco- and mineralocorticoids may regulate the natriuretic effect and the synthesis and release of natriuretic factor by the rat atria in vivo. Biochem Biophys Res Commun 131, 806-814. GARDNER, D.G., GERTZ,B.J., DESCHEPPER, C.F. & KIM, D.Y. 1988. Gene for the rat atrial natriuretic peptide is regulated by glucocorticoids in vitro. 3 Clin Invest 82, 1275-1281. GIBBS,D.M. 1987. Noncalcium-dependent modulation of in vitro atrial natriuretic factor release by extracellular osmolality. Endocrinology 120, 194'97. LANG,R.E., THOLKEN, H., GANTEN, D., LUFT,F.C., RUSKOAHO, H. & UNGER,TH. 1985. Atrial natriuretic factor - a circulating hormone stimulated by volume loading. Nature 314, 264-266. LEE, R.T., BLOCH,K.D., PFEFFER, M.A., NEER,J. & SEIDMAN, C.E. 1988. Atrial natriuretic factor gene expression in ventricles of rat with spontaneous biventricular hypertrophy. 3 Clin Invest 81, 43 '-434. MANIATIS, T., FRITSCH, E.F. & SAMBROOKE, J. 1982. Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory. O'BRIEN,P.C. & SHAMPO,M.A. 1988. Statistical considerations for performing multiple tests in a single experiment. 3 . Repeated measures over time. Mayo Clin Proc 63, 918-920. RUSKOAHO, H., THOLKEN, H. & LANG,R. 1986. Increase in atrial pressure releases atrial natriuretic peptide from isolated perfused rat hearts. PJiigevs Arch 407, 170-174. RUSKOAHO, H., VAKKURI, O., ARJAMAA,0. VUOLTEENAHO, 0. & LEPPALUOTO, J. 1989. Pressor
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0.Arjamaa
et
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hormones regulate atrial-stretch-induced release of atrial natriuretic peptide in the pithed rat. Circ Res 64,482-492. SCHIEBINGER, R.J. & LINDEN, J. 1986a. Effect of atrial contraction frequency on atrial natriuretic peptide secretion. Am 3 Ph,ysiol 251, H ~ o g g - H ~ o g g . SCHIEBINGER, R.J. & LINDEN, J. 1985b. T h e influence of resting tension on immunoreactive atrial natriuretic peptide secretion by rat atria superfused in vitro. Circ Res 59, 105-109.
VUOLTEENAHO, O., ARJAMAA, 0. & LING,N. 1985. Atrial natriuretic polypeptides : Rat atria store high molecular weight precursor but secrete processed peptides of 25-35 amino acids. Biochem Biophys Res Commun 129,82-88. ZAMIR,N, HAASS,M., DAVE,J.R. & ZUKOWSKAGROJEC, Z. 1987. Anterior pituitary gland modulates the release of atrial natriuretic peptides from cardiac atria. Proc Natl Acad Sci 84, 541-545.
Adrenal cortex contributes t o the regulation of NaCI-stimulated ANP release in the rat 0. ARJAMAA, 0. V U O L T E E N A H O , T. T A S K I N E N , M. T U O M I N E N and J. L E P P A L U O T O Department of Physiology, University of Oulu, Finland ARJAMAA, O., VUOLTEENAHO, O., TASKINEN, T., TUOMINEN, M. & LEPPALUOTO, J. 1990. Adrenal cortex contributes to the regulation of NaC1-stimulated ANP release in the rat. Acta Physiol Scand 139,3559360. Received 12October 1989, accepted 22 January 1990. ISSN 0001-6772. Department of Physiology, University of Oulu, Finland. T h e present study documents the effects of hypophysectomy and the effects of dexamethasone substitution on the NaC1-stimulated release and on the basal secretion rates of ANP from the rat atria in vitro. We also measured the concentration of mRNA in the atria after hypophysectomy. Rats (n = 12) were subjected to hypophysectomy by a parapharyngeal approach. One group of rats (n = 6) received dexamethasone 0.2 mg S.C.daily for 4 weeks, while the other group was left unsubstituted. After 4 weeks, the atrial block (n = 10) was excised, placed in an organ bath (field stimulation 4 s ~ ' 20 , V, I ms; resting tension = 5 mN) and superfused (7 ml min-') either with a physiological buffer solution (295 mosmol kg-') or with a hyperosmotic NaCl solution (330 mosmol kg-'). T h e atria from the hypophysectomized rats did not respond to the stimulus: the concentration of ANP in the I-min samples of the perfusate was under IOO pg ml-'. Dexamethasone treatment significantly (P < 0.05) increased the ANP concentration to a maximum of 165 f 17 (mean fSEM) pg ml-' during the superfusion while the control concentration was I I O 19 ~ pg ml-'. T h e ANP mRNAII8 S RNA ratios did not differ between the atria of hypophysectomized and control rats. In conclusion, glucocorticoids are required in the stimulus-induced release of ANP and the impaired release of ANP after hypophysectomy does not depend on an impaired synthesis of ANP. Key words; atrial natriuretic peptide (ANP), dexamethasone, hypophysectomy, osmolality, superfusion.
Atrial natriuretic peptide (ANP) is a hormone, released from cardiac atria, acting on kidneys and vasculature to regulate volume and blood pressure (for review, see deBold 1985). Little is known about the mechanisms that adjust the release of ANP. Atrial stretch, without any central regulation, is thought to be a major stimulus for the release of ANP (Ruskoaho et al., 1986). We have shown previously that increased osmolality augments the release of ANP from isolated rat atria (Arjamaa & Vuolteenaho 1985, Arjamaa 1989). Gibbs (1987), using dispersed atrial myocytes, was also able to show that the Correspondence : Olli Arjamaa, Department of Physiology, University of Oulu, Kajaanintie 52A, 90220 Oulu, Finland.
secretion of ANP was markedly stimulated by increase in extracellular osmolality, regardless of the solute. Pithing and hypophysectomy, which remove neurogenic influences and humoral communication of the brain and peripheral organs, completely blocked stimulus-induced release of ANP (Eskay et al. 1986, Zamir et al. 1987, Ruskoaho et al. 1989). In hypophysectomized rats, the response to a volume load was attenuated (Dietz & Nazian 1988), which did not result from a decrease in the right atrial pressure (Arjamaa et al. 1988). These findings led us to study also the effects of hypophysectomy on NaC1-stimulated release of ANP from isolated rat atria. We showed that hypophysectomy greatly attenuated the response and decreased
355 12-2
356
0. Arjamaa et al.
the basal secretion as well (Arjamaa 1989). As there was evidence that glucocorticoids regulate the synthesis, natriuretic effect, release (Garcia et al. 1985) and the expression of the gene for ANP (Gardner et al. 1988), we tested the hypothesis that a glucocorticoid substitution could restore the responsiveness to a n N a C l stimulus in hypophysectomized rats in vitro. We also measured the concentration of mRNA i n atria of hypophysectomized rats t o find o u t whether the ANP gene expression was impaired. We report that dexamethasone substitution partially restores the response to a NaCl stimulus a n d that hypophysectomy does not have any effect on the level of ANP m R N A .
the perfusion rate constant (7 ml min-'). After an equilibration period of 60 min the perfusate was collected in I-min fractions for 5 min and then the bath was superfused with a buffer"so1ution of 330 mosmol kgF' for 10 min (the increase in osmolality was produced with NaCI), which was followed by a superfusion with PBS for 15 min. Solutions were gassed with I O O ~0, / ; , and kept at 37 "C throughout experiments. A N P RIA. The ANP content of samples was determined with a radioimmunoassay using antiserum '7-44', which was raised in rabbits against rat ANP,-,,. This antiserum was specific for the midportion of rat ANP and the sensitivity of the assay was 0.5 pg per tube (Vuolteenaho et al. 1985). Radioiodinated rat ANP was purchased from Amersham International PLC. A N P mRNA determination. RNA was isolated from atrial tissue of two control rats and of two hypoMATERIALS AND METHODS physectomized rats using the guanidine thiocyanateAnimals. Male rats of the Sprague-Dawley strain LiCl method (Cathala et ul. 1983). For the RNA blot were used. They had been bred at ZCFZZ "C and had analysis, I-pg samples of RNA from auricles were free access to tap water and standard pellets. T h e light dotted onto Zeta-Probe blotting membrane (Bio-Rad cycle was 1 2h light/Iz h dark starting from 08.00 h. Laboratories, Richmond, CA, USA) and hybridized At the age of about 1 2 weeks, rats ( n = 12) were with a full-length rat ANl' cDNA probe CAR-5s anaesthetized with Hypnorm (10 mg ml-l fluanisone (Flynn et al. 1985) (a gift from Dr Peter L. Davies, and 0 . 2 mg ml-' fentanylcitrate; Duphar BV, Amster- Queen's University, Kingston, Canada). The probe dam, Holland; 0.07 ml IOO gF' i m . ) and hypo- was labelled with [32P]dCTP (Amersham) using a physectomized by a parapharyngeal approach. After random primed labelling kit (Boehringer-Mannheim). the surgical operation, the animals were allowed to Hybridization was carried out overnight at 65 "C recover at an ambient temperature of 28 "C for about according to the method of Maniatis et al. (1982). The 4 weeks. Six rats received daily dexamethasone membrane was exposed to XAR-5 X-ray film (Eastman-Kodak) at -70 "C with a Cronex Lightning (Oradexon, NV Organon, OSS, Holland) 0.2 ml ( = Plus intensifying screen (DuPont). T o quantify the 0.2 mg) S.C.during a period of 4 weeks, while four rats received dexamethasone for the first 2 days to speed amount of RNA, the membrane was rehybridized up the recovery from the surgical operation. These after extensive strip-wash with a 32P-labelled oligofour rats received no further dexamethasone treatment. nucleotide complementary to rat 18 S ribosomal RNA The hypophysectomy was considered successful if the (Lee et al. 1988, Albretsen et al. 1988). The ANP animal lost weight after the operative procedure and if mRNA/18 S RNA ratios were obtained by densithe weights of testes were about 25 yo of age-matched tometry analysis of the membrane analysed with the controls. T h e weight of testes and the body weight of two different probes. Determination of osmolality. Osmolalities of perhypophysectomized rats are shown in Table I. Experimental design. The in-nitro experimental fusates and buffer solutions were determined by an procedure has previously been described in detail osmometer (Advanced Wide-Range Osmometer 3W2, (Arjamaa 1989). Briefly, a rat was decapitated and the Advanced Instruments, Inc.) based on the freezingatrial block was excised and mounted in a vertical point method. Statistical analyses. Mean SEM are given organ bath of 8 ml. Two hooks kept the block between the bottom of the bath and a force transducer which throughout. I n statistical analyses of the results which monitored preset tension (= 5 mN) and contractions. were repeated measures over time a paired t-test was By means of two platinium plate electrodes, the block used as suggested by O'Brien & Shampo (1988). was electrically stimulated (supramaximal field stimulation 4 IIz, 20 V, I ms). The atrial block was R E S U L T S superfused with physiological buffer solution (PBS) (NaCI 137 mmol IF', KCI 5.6 mmol I - l , CaCI, 2.2 Figure I shows the secretory responses of heart atria from either hypophysectomized (a) or mmol I-', MgCI, 0.12 mmol I-', and Hepes "-2hydroxyethyl-piperazine-"-2-ethanesulphonic acid] hypophysectomized a n d dexamethasone-treated 1 5 mmol IF' in I litre of distilled water at p H 7.40; (b) rats to a hyperosmotic (330 mosmol kg-') osmolality 295 mosmol kgF1). A perfusion pump kept stimulus. Basal ANP secretion in the non-
ANP and adrenal cortex in the rat
357
h
7- 150E m a
Y
a
z
a 100-
1
5
10
15 20 TIME ( m i d
25
30
5
10
15 20 TIME ( m i d
25
30
Fig. I . Responses of rat atria in nitro after hypophysectomy (a) and after hypophysectomy followed by dexamethasone treatment (b) before, during, and after hyperosmotic (NaCI) superfusion. Lower panels show the changes in osmolalities. Data were collected in I-min fractions of superfusion. Means fSEM. Hypophysectomy, n = 4;hypophysectomy dexamethasone, n = 6. Paired t-test, P < 0 . 0 5 .
+
substituted hypophysectomized group was under IOO pg ml-', while it was higher, I 10 f 19 (n = 9) pg ml-', in the group substituted with dexamethasone. The. atria from the non-substituted, hypophysectomized rats did not respond to the stimulus and the secretion of ANP in the I-min samples of the perfusate remained under IOO pg ml-' during the whole period of osmotic stimulation. T h e atria from hypophysectomized rats receiving dexamethasone responded to the same stimulus by an increase in ANP secretion. During the hyperosmotic superfusion, the concentration of ANP in perfusates increased, and at 8 min (165 p g 4 17 ml-l) it was significantly (P< 0.05) higher compared with the control values of the prestimulation period. However, in spite of a continuous osmotic stimulus, the ANP secretion tended to diminish, reaching control values before the hyperosmotic superfusion was terminated. Using the "P-labelled oligonucleotide which was complementary to rat 18 S ribosomal RNA, we found no difference in concentrations of mRNA in hypophysectomized and in control (no
a
b
e d Fig. 2. ANP mRNA dot blot analysis of heart atria from hypophysectomized and control rats. I pg of total right auricular ANP (3-4 dots per sample) was dotted onto nylon membrane which was hybridized against 32P-labelled rat ANP cDNA probe CAR 55. (a) and (b) Hypophysectomized rats, mixed RNA samples of left and right auricles. (c) Control rat, left auricular RNA. (d) Control rat, right auricular RNA.
358
0. Arjamaa et al.
I . Weight of testes and body weight (means, n in parentheses) after dexamethasone treatment in hypophysectomized rats
Table
Testes (mg)
Body weight (g) At
Treatment
Right
Left
operation
Hypophysectomy Hypophysectomy + dexamethasone
404 (4) 420 (4)
389 (4) 423 (4)
360 (4) 365 (4)
After operation
m8 (4) 235
(4)
Weighing took place 4 weeks after the surgical operation. hypophysectomy) rats. The ANP mRNA/18 S RNA ratios (a densitometry analysis with two different probes) were between 0.18 and 0.25. Figure z shows the blot micrographs from control and from hypophysectomized rats. The weight of testes and body weights of the rats used in the experiments are shown in Table I . DISCUSSION
We have shown in the present experiments that dexamethasone substitution treatment of hypophysectomized rats partially restored the secretory ANP response to a hyperosmotic NaCl perfusion in isolated atria. Measurements of the mRNA contents of atria also showed that hypophysectomy appeared to have no effect on the expression of the ANP gene. As a model for st6dies on ANP release we have used isolated atria in an organ bath in which atria can be electrically stimulated (field stimulation) and resting tension can be kept constant. Both pacing of atria in vitro (Schiebinger & Linden 1986a) and changes in atrial tension in vitro (Schiebinger & Linden 1986b) may influence the ANP release. Dietz (1984) and Lang et al. (1985) have shown that atrial stretch is a major stimulus for the release of ANP from heart atria. However, in addition to these mechanical stimuli, we have been able to show that increased osmolality in vitro (Arjamaa & Vuolteenaho 1985, Arjamaa 1989) acts as a stimulus for the ANP release from atrial myocytes. This is also the case in enzymatically dispersed atrial myocytes as shown by Gibbs (1987). The present study, demonstrating increased ANP release from heart atria as a response to increased osmolality, confirms our previous findings, Although the osmotic stimulus
is relatively strong (330 mosmol kg-'), the atria release exclusively ANP 1-28 (Arjamaa 1989) indicating that no damage occurs in myocytes during the hyperosmotic superfusion. Hypophysectomy greatly attenuates stimulusinduced ANP release from heart atria in vitro to a hyperosmotic NaCl perfusion, and the basal secretion rate is significantly decreased as well (Arjamaa 1989). Our previous studies (Arjamaa et al. 1988) have also shown that hypophysectomy reduces the weight of heart atria. In present studies, dexamethasone treatment after hypophysectomy restored the responsiveness of atria to the NaCl stimulus and also increases the basal ANP secretion rate although the treatment does not increase the body weight. Therefore the decreased secretion rate of ANP after hypophysectomy cannot only be explained by the decreased number of secreting myocytes as previously suggested (Arjamaa 1989). The release of ANP as a response to a volume load appears to be under multifactorial control. Zamir et al. (1987) have suggested that the anterior pituitary is a prerequisite for the ANP release, while Ruskoaho et al. (1989) have concluded that pressor hormones, especially vasopressin, are of importance in regulating the ANP release. These in-vivo studies must, however, be interpreted cautiously, as pithed or hypophysectomized experimental animals may have abnormal circulatory volume and cardiac and renal haemodynamics. Garcia et al. (1985) have found that simultaneous administration of dexamethasone and deoxycorticosterone acetate to adrenalectomized rats produces a marked increase in ANP concentration in atrial tissue and plasma ; they conclude that glucocorticoids may regulate the synthesis and release of ANP, and mineralocorticoids may have a permissive role. Our present zn-vitro findings in the isolated
ANP and adrenal cortex in the rat atrial preparation therefore support the notion that glucocorticoids contribute to the regulation of ANP release. T h e recovery of the ANP response in the present study is, however, only partial when compared with our previous results (Arjamaa 1989), which are compatible with the results of Garcia et al. (1985). We have earlier reported (Arjamaa et al. 1988) that hypophysectomy does not change the concentration of A N P in the atrial tissue of the rat. Zamir et al. (1987) have shown that after hypophysectomy the A N P concentration is significantly increased in the right atrium while in the left atrium the concentration remains unchanged. Dietz & Nazian (1988) have found a significant decrease in the ANP concentration in both the left and right atrium. To explain these discrepancies between previous studies we have measured the concentration of m R N A in the rat atria. I t is evident from these experiments that hypophysectomy does not decrease the expression of the ANP gene. T o conclude, we have shown that glucocorticoids are required for the A N P release in response to a hyperosmotic superfusion and that the impaired release of A N P after hypophysectomy does not depend on an impaired ANP gene expression. We wish to thank Ms Tuula Lumijarvi for excellent technical assistance and Ms Liisa Asikkala for taking care of the experimental animals. Financial support was obtained from the Einnish Cultural Foundation and Academy of Finland.
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