Proc. Natl. Acad. Sci. USA Vol. 88, pp. 6496-6500, August 1991 Medical Sciences Natriuretic peptide receptors regulate endothelin synthesis and release from parathyroid cells (atrial natriuretic peptide/brain natriuretic peptide/peptide hormone receptor/hypertension) MARIA LAURA DE FEO*, OLGA BARTOLINI*, CLAUDIO ORLANDO*, MARIO MAGGI*, MARIO SERIO*, MARK PINESt, SHMUEL HURWITZt, YOSHIO FUJII*, KAZUSHIGE SAKAGUCHI4, GERALD D. AURBACHt, AND MARIA LUISA BRANDI*§ *Department of Clinical Physiopathology, University of Florence, Viale Pieraccini 6, 50139 Florence, Italy; tInstitute of Animal Science, Agricultural Research Organization, The Volcani Center, Bet Dagan 50250, Israel; and tMetabolic Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892 Contributed by Gerald D. Aurbach, April 4, 1991 ABSTRACT Cloned rat parathyroid cells (PTr cell line) MATERIALS AND METHODS that produce parathyroid hormone-related peptide plus endo- thelin 1 and primary cultures of human parathyroid cells were Cell Culture. Two systems, a parathyroid cell line obtained tested for growth and differentiation responses to atrial natri- from hyperplastic rat parathyroid glands (PTr cells; ref. 16) uretic peptide (ANP) and brain natriuretic peptide (BNP). and primary cultures of cells from human parathyroid tissue, High- and low-affinity binding sites for ANP were found on PTr were used to characterize receptor binding and biological cells; BNP appeared to bind to the same receptors with similar responses to the natriuretic hormones. PTr cells were cul- affinities. Either ANP or BNP stimulated production of cGMP tured in growth medium consisting of a 1:1 mixture of and caused a 30% decrease in Na'-K+-Cl cotransport. Each Dulbecco's modified Eagle's minimal essential medium peptide increased synthesis and secretion of endothelin 1 by (DMEM) and Coon's modified Ham's F12 with supplements PTr cells in a dose-dependent fashion, but cell growth was not as described (16). PTr cells were detached by 0.05% trypsin affected. Human parathyroid cells (normal and pathological) in phosphate-buffered saline (PBS)/2 mM EDTA and plated also responded to ANP or BNP with an increase in cGMP on 100-mm dishes. At confluence, growth medium was production. The finding of receptors for natriuretic hormones removed and replaced with identical medium but without on parathyroid cells with consequent effects on release of serum or serum substitutes (steady-state medium) and con- endothelin 1 might be ofrelevance in understanding the clinical taining aprotinin (500 kallikrein inhibitor units/ml; Sigma). association between hyperparathyroidism and hypertension. The medium was collected on ice at the indicated times after the stimulus, centrifuged at 40C, and stored at -20'C until Classical studies have established the physiological role of assayed. parathyroid hormone (PTH) in regulating calcium homeosta- Human parathyroid tissue was obtained from patients sis through effects on its recognized target tissues, bone and undergoing surgery for hyperparathyroidism (parathyroid kidney (1). However, the biological significance of other hyperplasia due to multiple endocrine neoplasia type I, n = actions of the parathyroid glands, particularly the inhibition 2; secondary hyperparathyroidism, n = 1; parathyroid ade- of smooth muscle contraction in blood vessels and other noma, n = 1). Parathyroid specimens were minced in small tissues, is not fully understood (2). Recent research has fragments with a scalpel and digested with a solution of PBS shown the parathyroids to be the source ofancillary peptides containing collagenase (1 mg/ml, type IV; Sigma) at 37TC in such as PTH-related peptide (PTHrP; refs. 3 and 4) and a humidified atmosphere containing 5% CO2. After 8-9 hr of endothelin 1 (ET-1; refs. 5-7). The latter peptides exert digestion, tissue fragments were washed twice with PBS and opposing biological effects on smooth muscle, PTHrP inhib- then mechanically dispersed by pipet. The cell suspension iting and ET-1 stimulating contraction (8, 9). Such observa- was centrifuged and then plated in Petri culture dishes with tions suggest that the parathyroid glands may physiologically Coon's modified Ham's F12 medium containing 20% fetal modulate vascular smooth muscle tone through the release of bovine serum. After 4-6 days of primary culture, cells were accessory peptides. Clinical findings supporting this hypoth- detached with trypsin and plated in 16-mm wells (multiwell esis are the hypertension that often accompanies primary plates) for experimental procedures. hyperparathyroidism and the pattern of altered calcium me- Binding Studies. PTr cells were grown to subconfluency in tabolism frequently observed in experimental and human 16-mm-well plates and then incubated in 20 mM Hepes/ hypertension (see ref. 10 for review). Note also the putative DMEM, pH 7.8/0.2% bovine serum albumin at 220C for 30 "parathyroid hypertensive factor" described in extracts of min with 1251I-labeled rat ANP (125I-rANP; specific activity, parathyroid tissue from the SHR (spontaneously hyperten- 2000 Ci/mmol; Amersham; 1 Ci = 37 GBq) plus unlabeled sive) rat (11). A functional interaction between PTH and rANP or porcine BNP (pBNP) (Peninsula Laboratories) at atrial natriuretic peptide (ANP) in modulating cyclic nucle- concentrations shown. The cells were washed with ice-cold otide accumulation and cell proliferation has been observed PBS/0.2% bovine serum albumin and solubilized in 1 M in chondrocytes and osteoblasts (12, 13). These observations, NaOH, and extracts were analyzed for radioactivity by plus the well-known hypotensive effect of ANP (14), led us crystal scintillation detection. Association kinetics were de- to evaluate the possible direct actions of the peptide and of termined from the rate of specific binding of0.1 nM (200,000 the recently characterized brain natriuretic peptide (BNP; cpm) 125I-rANP. The dissociation rate was determined after ref. 15) on parathyroid cell cultures. Abbreviations: ANP, atrial natriuretic peptide; hANP and rANP, The publication costs of this article were defrayed in part by page charge human and rat ANP; BNP, brain natriuretic peptide; pBNP, porcine payment. This article must therefore be hereby marked "advertisement" BNP; ET-1, endothelin 1; PTH, parathyroid hormone. in accordance with 18 U.S.C. §1734 solely to indicate this fact. §To whom reprint requests should be addressed. 6496 Downloaded by guest on September 28, 2021 Medical Sciences: De Feo et al. Proc. Natl. Acad. Sci. USA 88 (1991) 6497 addition of unlabeled rANP or pBNP (2 ,M) to the reaction (26) was used for the analysis of sigmoidal dose-response mixture at equilibrium. curves obtained in cGMP production studies. Statistical Cyclic Nucleotide Production. PTr or human parathyroid significance was evaluated by one-way analysis of variance cells were incubated for the indicated times at 370C with and by Duncan's new multiple range test (27). various concentrations of rANP, human ANP (hANP), or pBNP in steady-state medium containing 1 mM 3-isobutyl- 1-methylxanthine (Sigma). Total (cells and medium) cGMP RESULTS content was assayed by RIA (cGMP RIA kit; DuPont/NEN) Binding Studies. The binding of 125I-rANP to PTr cells at after acetylation of samples and standards. 220C was a linear function of the number of cells added Total cAMP was determined by RIA after ethanol extrac- between 2 x 105 and 9 x 105 cells (correlation coefficient r = tion (17) under basal conditions or upon addition of forskolin 0.986; n = 2); specific binding became maximal at 30-40 min (0.1 ,uM; Sigma) or pertussis toxin (100 ng/ml; List Biological (Fig. lA). The simultaneous computer analysis of three Laboratories, Campbell, CA) with or without hANP or pBNP dissociation curves with the program EXPFIT indicated that at 1-1000 nM. the introduction of a second exponential function signifi- Ion Transport. Rubidium-86 (DuPont/NEN) uptake was cantly increased the goodness of fit (P < 0.005; k0fn = 2.1988 determined as a measure of Na'-K+-Cl- cotransport and ± 1.5292 min-' and kff2 = 0.0364 ± 0.0072 min-', mean ± Na+/K' pump activity in PTr cells as described by O'Don- SD) (Fig. 1B). Mathematical modeling of competition curves nell (18). Na'-K+-Cl- cotransport was determined as oua- among 1251-rANP, the corresponding unlabeled peptide, and bain-insensitive, furosemide-sensitive K+ flux [i.e., (K+ flux BNP strongly indicated heterogeneity ofANP binding sites in in incubation with ouabain) - (K+ flux with ouabain plus PTr cells. Indeed, the introduction of a second independent furosemide)]. Na+/K+-ATPase-mediated K+ influx was cal- class of sites significantly improved the goodness of fit (P < culated as ouabain-sensitive flux [i.e., (total flux) - (flux in 0.001). Homologous heterologous medium with ouabain)]. Protein content was determined and competition curves for using the Pierce protein assay reagent based on the method rANP and pBNP, obtained by use of a two-site model, are of Bradford (19). shown in Fig. 2. Both ligands bind with high affinity (rANP Cell Proliferation. PTr cells were incubated on microplates pK = 10.7 + 0.24; pBNP pK = 11.04 + 1.6, mean ± SD) to for 24 hr at 37°C in serum- and serum substitute-free medium the low-capacity site (R1, 0.308 ± 0.178 fmol per 106 cells, without phenol red. Various concentrations of hANP or mean ± SD), with lower affinity (rANP pK = 6.91 ± 0.13; pBNP (10 pM to 1 ,uM) were added (five wells for each pBNP pK = 6.48 ± 0.16, mean ± SD) for the high-capacity experimental point) and incubations continued for another 24 site (R2, 2182 ± 1255 fmol per 106 cells, mean ± SD). hr at 37°C. Cells were then washed with PBS and incubated ANP and BNP Increase cGMP Production. cGMP accumu- for 45 min in the same steady-state medium containing lation in PTr cells incubated with 10 nM rANP or pBNP Hoechst 33342 dye (10lg/ml; Polysciences). Chromatin- increased significantly within 1 min, with a further increase associated fluorescence was evaluated in a fluorescence up to 60 min (Fig.