Acta Physiol Scand 1991, 143, 223-224

Effect of hypo-osmolality on atrial natriuretic peptide gene expression in neonatal cultured card iomyocytes H. TOKOLA", P. A. U U S I M A A t , T. T A S K I N E N f , I. E. HASSINEN? and H. RUSKOAHO"

Departments of Pharmacology and Toxicology", - _ Medical Biochemistry? and Physiology 1, University of Oulu, Oulu, Finland

Atrial natriuretic peptide (ANP) is primarily synthesized in the atria of the heart, but biosynthesis of ANP also occurs in cardiac ventricles (Needleman et al. 1989). Stretching of the myocytes is the major stimulus for ANP secretion from cardiac atria (Lang et al. 1985). Further, pressure and volume overload in vivo, presumably leading to mechanical stretch, increase ANP biosynthesis in atrial cells and induce reexpression of ANP gene in hypertrophied ventricular myocytes (Needleman et al. 1989). However, it is not known whether mechanical stretch without humoral factors directly stimulates ANP gene expression in cardiac cells. In the present study we used the hypo-osmolalityinduced swelling ofthe cultured neonatal rat myocytes, known to stimulate ANP secretion in isolated cardiomyocytes (Greenwald et al. 1989), as an experimental model to examine whether cellular stretch affects ANP gene expression. Three hyponatraemic complete serum-free culture media (CSFM), which differed only in the concentration of mannitol and, thus, by their osmolality were used : 289 mosm kg-' H,O (control), 245 mosm kg-' H,O and 205 mosm kg-' H,O. Cells were prepared as described by Uusimaa et al. (1990) except that atrial auricles and ventricular apices were processed separately to establish pure atrial and ventricular cultures. The cells were placed into 35 mm plastic culture wells (Nunc, Roskilde, Denmark) and incubated in the RPMI 1640-medium supplemented with 10% fetal calf serum for 24 h before switching to hyponatraemic (2.98 g I-') 289 mosm kg-' H,O CSFM, either with or without 100 n M dexamethasone. After 24 h the latter medium was replaced with 289-CSFM, 245-CSFM or 205-

Received 12 March 1991, accepted 25 June 1991. Key r o o d : Atrial natriuretic peptide, cardiac myocytes, hypo-osmolality ; stretch. Correspondence : Heikki Ruskoaho, M.D., Department of Pharmacology and Toxicology, University of Oulu, Kajaanintie 52 D, SF-90220 Oulu, Finland.

CSFM for 24-h experiment. Finally, the cells were washed twice with PBS and quickly frozen at -70 "C. RNA was isolated by the guanidine thiocyanateLiCl method. Northern and dot blots were hybridized with full-length rat ANP cDNA probe Car-55 and oligonucleotide probe complementary to rat 18 S ribosomal RNA. ANP mRNA concentrations were estimated by ANP mRNA: 18 S ribosomal RNA ratio (Kinnunen et al. 1990). Northern blot analysis using Car-55 probe identified a single 0.9-kb mRNA species in the cultured myocytes. ANP mRNA levels in cultured atrial myocytes at the age of 3-5 days were over twofold higher than those in ventricular cells. Treatment of atrial cells with dexamethasone (100 nM) increased ANP mRNA levels 92% (1.00&0.21 us. 1.92k0.18 arbitrary densitometric units, n = 4, P < 0.02), showing the validity of our cell culture. Lowering the osmolality moderately from 289 to 245 mosm kg-' H,O for 24 h caused a 26% increase (P< 0.004) in ANP mRNA levels (Fig. 1). Thus, the osmotic stretch of plasma membrane of atrial myocytes appears to stimulate ANP gene expression under these experimental conditions. A further decrease in osmolality from 289 to 205 mosm kg-' H,O had no significant effect on ANP mRNA levels (Fig. 1). In hypotonic media, the cells initially swell but then, over several minutes, shrink back to their resting volumes (Hoffman & Simonsen 1989). This is caused by activation of ion transport pathways, and thus a sustained increase in intracellular ions including Ca2+could explain increase in ANP gene expression induced by hypo-osmolality. When intracellular Ca2+ was measured in perifused cultured rat cardiomyocytes by the method described by Uusimaa et al. (1990), a transient Ca2+ rise was observed (Uusimaa et al., unpublished observation). Thus the increase in ANP gene expression induced by hypo-osmotic stretch may be mediated through the increase in cytosolic Ca2+, which has been shown to have a significant effect on the concentration of the ANP transcripts in cultured neonatal rat myocytes (LaPointe et al. 1990). However, a number of factors in addition to cytosolic Ca2+

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present findings fit the model that cellular mechanisms of ANP secretion and synthesis differ in the neonatal atrial and ventricular cells. In conclusion, data obtained demonstrate that osmotic stretch of the plasma membrane of atrial but not ventricular myocytes stimulates ANP gene expression. Further characterization of biochemical mechanisms involved in these changes in response to hypo-osmotic swelling will be important in clarifying how pressure and volume overload increase ANP gene expression tn C I V O .

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We thank Ms Tuula Taskinen for expert technical assistance. This study was supported by the Academy of Finland and Sigrid Juselius Foundation.

REFERENCES BLOCK, K.D., SEIDMAN, J.G., NAFTILAN,J.D., FALLON,J.T. & SEIDMAN, C.E. 1986). Neonatal (5) 289

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245 205 Osrnololity (mosm k9-l H20)

Fig. 1. Effect of hypo-osmolal cell culture media on atrial natriuretic peptide (,4NP) gene expression in neonatal cultured myocytes. ,4NP mRNA : 18 S ribosomal RNL4 ratios are shown relatihe to control (289 mosm kg-’ H,O) in the atrial (A) and ventricular (B) cells Results are expressed as meanf SEM. Number of experiments in each group is shown in parenthesis * P < 0.05 ; ** P < 0.01 cs. control within each cell culture (Student’s t-test, paired). implicated in the volume regulatory responses to cell swelling (Hoffman & Simonsen 1989) may be responsible for increased ANP gene expression in response to hypo-osmotic swelling. In ventricular cell cultures, lowering the osmolality form from 289 to 245 or 205 mosm kg-’ h,O for 24 h resulted in a 43Yh ( P < 0.03) and 34?,6 decrease (n.s.) in ,4NP mRNA levels, respectively (Fig. 1). Also, the response to dexamethasone was only slightly positive (9 Oo) and statistically insignificant in ventricular cells. Neonatal atrial and ventricular cells secrete and synthesize ANP via different, tissue-specific pathways (Bloch et al. 1986). Accordingly, secretion of ,4NP from neonatal ventricular myocytes has been shown to be unresponsive to differenr stimuli, such as protein kinase C activation (Uusimaa et al. 1990). Thus, our

atria and ventricles secrete atrial natriuretic factor via tissue-specific secretory pathways. Cell 47, 693-702. GREENWALD, J.E., APKON, M., HRUSKA,K.A. & NEEDLEMAN, P. 1989. Stretch-induced atriopeptin secretion in the isolated rat myocyte and its negative modulation by calcium.3Clin Invest 83,1061-1065. L.O. 1989. Membrane HOFFMAN, E.K. & SIMONSEN, mechanisms in volume and pH regulation in vertebrate cells. Physiol Rev 69, 315-382. T., LEPPALUOTO, J. & KINNLNEN,P., TASKINEN, RUSKOAHO, H . 1990. Release of atrial natriuretic peptide from rat myocardium in vitro: effect of minoxidil-induced hypertrophy. Br 3 Pharmacol 99, 701-708. LANG,R.E., THOLKEN, H., GANTEN,D., LUFT,F.C., RUSKOAHO, H. & UNGER,T . 1985. Atrial natriuretic factor - a circulating hormone stimulated by volume loading. Nature 314, 264-266. C.F., Wu, J. & LAPOINTE,M.C., DESCHEPPER, GARDNER, D.G. 1990. Extracellular calcium regulates expression of the gene for atrial natriuretic factor. Hypertension 15, 2&28. J.E., NEEDLEMAN, P., BLAINE,E.H., GREENWALD, MICHENER, M.L., SAPER,C.B., STOCKMANN, P.T. & TOLUNAY, H.E. 1989. .The biochemical pharmacology of atrial peptides. Annu Rev Pharmacol T O W ~29, CO 23-54. ~ UUSIMAA, P.A., RUSKOAHO, H., LEPPALUOTO, J. & HASSINEN, I.E. 1990. Cytosolic Ca2+ during atrial natriuretic peptide secretion from cultured neonatal cardiomyocytes. Mol Cell Endocrinol 73, 153-163.