Natriuretic Peptide Receptors Regulate Endothelin Synthesis and Release

Home , Endothelin, Endothelin 1

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 endothelin 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. 3A). A sharp increase in cGMP production microtiter plate reader (Titertek Fluoroscan 11; Flow Labo- was observed with 1 nM rANP, hANP, or pBNP, with EC50 ratories) as described (20). values of 15.7 ± 7.8, 33.5 ± 8.4, and 28 ± 6.5 nM (mean ± Northern Blot Analysis. PTr cells were grown in 100-mm SD), respectively (Fig. 3B). cGMP content markedly in- plates. Subconfluent cultures were incubated with medium containing 0.1 AM ANP or 0.1 ,uM pBNP for 15, 30, 60, 120, A 0 0 or 360 min. Cells were lysed and total RNA was extracted at 0 the end of incubation as described (7). Samples of total RNA (20 ,ug per lane) were fractionated by electrophoresis in a 1% agarose/formaldehyde gel and transferred to a nylon membrane (Nytran; Schleicher & Schuell). 32P-labeled antisense RNA probe for preproendothelin 1 (preproET-1) was pre- ll 40- pared as described (7). Rat cyclophilin cDNA (21) was radiolabeled with 32P by nick-translation and used as a '820. control probe. Hybridization with RNA or cDNA probes and autoradiography were carried out as described (7). Densito- 0 30 60 90 metric scans of the autoradiograms were performed on an LKB 2202 Ultroscan laser densitometer. B Peptide Secretion by PTr Cells. The effects of hANP and pBNP on ET-1 release by PTr cells were tested in cells 80- incubated at 37°C for 0.5, 1, 2, 6, or 24 hr with the natriuretic hormones. The medium (3 ml) was extracted with Sep-Pak DuZA. C18 cartridges (Waters) and ET-1 concentrations were determined by RIA (22). The sensitivity ofthe assay was 0.5 pg per 40 - tube (7.2% coefficient of variation for intraassay error). PTr cells were also analyzed for ANP- and BNP-like immunore- 20- activity by RIA on concentrated extracts ofPTr medium (23); 20-~~~~~~ BNP RIA was performed with a commercial kit (Peninsula n Laboratories). The sensitivity of the assays was 0.7 pg per 10 20 30 100 Al and 0.9 pg per 100 ,ul for ANP and BNP, respectively Time, min (intraassay coefficient of variation, 7.5 and 9%, respectively). Analysis of Experimental Results. Families of competitive FIG. 1. Rate of association (A) and dissociation (B) of 1251-rANP from binding sites on PTr cells. Cells were incubated for the indicated curves for rANP or were binding pBNP receptor interactions times at 220C with 0.1 nM 125I-rANP with or without 1 AuM rANP. analyzed simultaneously with a nonlinear least-squares fit- Results are expressed as percentage of maximum specific binding ting procedure (LIGAND program; ref. 24). Dissociation data (calculated as the difference between total and specific binding). Data were analyzed by the computer model EXPFIT (25), using a represent the mean of two (A) or three (B) experiments each using biexponential decay model. The computer program ALLFIT samples from triplicate wells. Downloaded by guest on September 28, 2021 6498 Medical Sciences: De Feo et al. Proc. Natl. Acad Sci. USA 88 (1991)

U.UU-. 0

0.010 - 0 0 10 X

0~~~ ~ ~ ~ ~ ~~~~0

0.005 -9 -8 -7 -9 -8 -7 M M -12 -10 -8 -6 0ghANP, PBNP, T. log M FIG. 4. Effects of hANP (A) and pBNP (B) on cGMP production by primary cultures of cells derived from human parathyroid ade- FIG. 2. Binding of rANP and pBNP to PTr cells. Analyses of nomas (n), secondary hyperparathyroidism (A), or multiple endo- tracer (125I-rANP, 1-100 pM) binding and homologous (rANP, *) or crine neoplasia, type I (A, o). Cells were treated as described for PI r heterologous (pBNP, A) competitive inhibition experiments with 0.1 cells. Results shown are the averages of triplicate analyses and are nM tracer were derived from the simultaneous fit of 14 curves expressed as percent of control. wherein each point represented triplicate analyses. B, bound; T, total. 3.7 fluorescence units in controls, respectively; mean + SD, n = 5). creased in response to natriuretic hormones (1-100 nM) in hANP and pBNPSyntheslsIncrease ET-1 and Release. primary cultures of human parathyroid tissue (Fig. 4). Northern blot hybridization withyihesmP-labeled antisense cAMP production by PTr cells was not affected by hANP RNA probe for preproET-1 showed a considerable increase or pBNP up to micromolar concentrations under basal con- in message for ET-1 with 0.1a,uM ANP or BNP (Fig. 5). The ditions or after incubation with forskolin or with pertussis response was evident within 15 min and became maximal at toxin. Neither was there an effect on cultured human cells 2 hr. (data not shown). A significant increase in ET-1 content in the culture hANP and pBNP Decrease Na+-K+-CI- Cotransport But medium of PTr cells was detectable after 24-hr incubations Not Na+/K+ Pump Activity of PTr Cells. Na'-K-Cl- with ANP or BNP (control, 23.7 ± 4.2 pg per 106 cells; 0.1 ,uM cotransport was assessed in PTr cells as furosemide-sensitive hANP and pBNP, 33.4 ± 5.7 and 32.5 + 3.2 pg per 106 cells, 'Rb+ influx with ouabain. A Na'-K-Cl- cotransport sys- respectively, P < 0.05 vs. control; 1 ,uM hANP and pBNP, tem was found in PTr cells (Table 1). Treatment of cells with 39.0 ± 6.8 and 40.2 ± 4.8 pg per 106 cells, respectively, P < hANP or pBNP (100 nM) significantly reduced the cotrans- 0.001 vs. control) (Fig. 6). No effect was detectable at shorter port (down 30% with respect to baseline; P < 0.001 and P < incubation times. 0.002, respectively); no effect was evident on Na+/K+ pump ANP- and BNP-Like Material in Medium from PTr Cells. activity. There was no detectable immunoreactivity for ANP or BNP hANP and pBNP Do Not Affect PTr Cell Growth. Prolifer- in culture medium derived from PTr cells (3-5 x 106 cells per dish) kept at 37°C for 1, 2, 6, 24, or 48 hr (n = 5; data not ation of PTr cells was not affected by the addition of hANP shown). or pBNP over a wide range of concentrations (1 pM to 1 ,uM) for 24 hr (10.2 0.8 and 9.8 ± 2.7 fluorescence units after stimulus with 1 ,M hANP or pBNP vs. 11.7 ± 3.6 and 11.8 DISCUSSION The potential influence of the parathyroids on the cardiovascular system was first recognized in 1925 (28). Much later it was found that PTH causes vasodilatation and increases 0 on 3 ' Table 1. Effects of ANP and BNP Na'-K-CPl cotransport and Na+/K+ pump activity in PTr cells 86Rb+ influx, itmol/min per mg of protein 2 'o E Na+-K+-Cl Agent cotransport pump K+ leak 12. Na+/K+ 1 CO None 0.202 ± 0.010 0.319 ± 0.047 0.043 ± 0.004 hANP 0.137 ± 0.006* 0.334 ± 0.136 0.039 ± 0.009 None 0.179 ± 0.012 0.563 ± 0.049 0.101 ± 0.005 20 40 C -12 -8 -4 pBNP 0.122 ± 0.005t 0.484 ± 0.092 0.094 ± 0.036 Time, min log M Cells were keptfor 10 min in 10 mM Hepes/DMEM (Na+, 150 mM; K+, 6 mM; Cl-, 150 mM) at 37rC, then incubated in the same medium FIG. 3. (A) Rate of cGMP production by PTr cells with ANP or containing 1 mM ouabain plus 1 mM furosemide (Sigma) and/or 0.1 BNP. Cells were incubated at 37°C with no additions (o) or with 10 A&M hANP or pBNP. After aspiration of the medium, the cells were nM rANP (-) or pBNP (-). cGMP concentrations was determined as incubated for 5 min in identical fresh medium containing I*RbCl (1 indicated in Materials and Methods. Data represent the mean of two ,uCi/ml). 86Rb uptake was terminated after aspiration ofthe medium experiments carried out with triplicate samples. (B) Dose-response and extensive washing. Intracellular 86Rb was determined by liquid analyses for ANP- or BNP-stimulated cGMP production with PTr scintillation spectrometry. Data are the mean ± SD ofquadruplicate cells in 20-min incubations: A, rANP; e, hANP; *, pBNP. C, control experimental points from one of three similar experiments. (zero concentration). Data represent the mean of two experiments *P < 0.001 with respect to basal. each carried out with quadruplicate samples. tp < 0.002 with respect to basal. Downloaded by guest on September 28, 2021 Medical Sciences: De Feo et al. Proc. Natl. Acad. Sci. USA 88 (1991) 6499 These apparently contradictory influences of the parathyroid glands in hypertensive states remain to be resolved. The increased circulating PTH levels in animal models of hypertension and in some human hypertensive states have been interpreted as secondary to high blood pressure on the one hand or as a contributing factor to hypertension on the other (32-35). The frequent association ofhypertension with hyperparathyroidism without overt renal damage has led to the interpretation that PTH may be a secondary contributing factor or, indeed, may itself cause hypertension (36, 37). The vasoactive peptides newly identified in the parathyroids may represent clues that will lead to better understanding ofthese 0 2 4 6 glands as possible regulators of cardiovascular homeostasis. Time, hr Specific receptors for ANP have been identified in diverse tissues (38, 39). The peptide elicits multiple effects, predom- ANP BNP inantly actions that modulate intravascular volume and sys- 1 2 3 4 5 6 1 2 3 4 D 6 temic hemodynamics (14) through vasorelaxation, natriure- sis, and diuresis (40, 41). A functional interaction between PTH receptors and ANP has been observed in chondropro- es v to"n#af 0 * a n04 t < ET-1 genitor cells of the avian epiphyseal growth plate (12) and in fetal rat bone cultures (13), where ANP inhibits PTH- stimulated cAMP accumulation and [3H]thymidine incorpo- AN UPdAL Cvclo ration. Moreover, endothelin, in a manner unrelated to inotropic action, enhances release of ANP from atria of FIG. 5. Effects of ANP and BNP on expression of mRNA for hypertensive rats (42), from cultured cardiac myocytes (43), ET-1. PTr cells were incubated in medium containing 0.1 t±M ANP and in vivo upon intravenous infusion in dogs (44). (e) or 0.1 tuM BNP (o) for 0 min (lanes 1), 15 min (lanes 2), 30 min The above information led us to test for ANP binding sites (lanes 3), 1 hr (lanes 4), 2 hr (lanes 5), or 6 hr (lanes 6). Total RNA on parathyroid cells; activation of such receptors could was extracted and 20 ,ug of total RNA from each sample was modulate secretion of endothelin with a consequent effect on fractionated in a 1% agarose/formaldehyde gel, transferred to a cardiovascular homeostasis. We also evaluated the effect of nylon membrane, hybridized with 32P-labeled antisense RNA probe BNP, a peptide first identified in porcine brain (15) and later for ET-1, and submitted to autoradiography at -80'C for 12 hr. at 100-fold greater concentrations in porcine heart (45), on Subsequently, RNA on the same membrane was hybridized with a 32P-labeled cDNA probe for cyclophilin and submitted to autorad- PTr cell function and growth. BNP elicits a spectrum of iography for 48 hr. The autoradiograms were scanned by a densit- actions very similar to that ofANP (15); it has therefore been ometer, and the ratio between ET-1 and cyclophilin (Cyclo) messages assumed that BNP may act in concert with ANP to modulate was taken for the ordinate in the graph. body fluids and blood pressure homeostasis. Our studies identify binding sites for ANP on a parathyroid cardiac function, effects that precede significant changes in cell line derived from hyperplastic rat glands (16). Computer circulating calcium (see ref. 2 for review). PTH also exerts analysis of the data showed that a two-site model significantly direct effects on the juxtaglomerular apparatus, adrenal improved the goodness of fit (P < 0.001) for ligand binding to glands, and nerve terminals by stimulating renin (29) and these sites. This suggests the existence of two distinct aldosterone (30) secretion and by enhancing norepinephrine populations of ANP binding sites, a high-affinity, low- release (31). The latter effects on peripheral hormone secre- capacity receptor and a low-affinity, high-capacity binding tory systems, together with the hypercalcemic effects of site. Competitive inhibition experiments using unlabeled PTH, would contribute to an elevation of blood pressure. BNP suggest that, in PTr cells, ANP and BNP bind to the same receptor, as found in other tissues (46). The binding of ANP or BNP to parathyroid cells causes hAN-P pB\P stimulation of cGMP production in either rat (PTr) or human parathyroid cells (Figs. 3 and 4). In PTr cells, a significant 10 increase in basal cGMP production was observed at nanomo- lar concentrations of either peptide. Neither ANP nor BNP affected cAMP production in the rat or human cell systems. ANP and BNP evoked a significant decrease in Na+-K+- Cl- cotransport in PTr cells (Table 1). To our knowledge, this is the first characterization of a prominent Na+-K+-Cl- cotransport system in parathyroid cells. A similar (30o decrease) effect of several vasoactive agents as well as ANP is found in vascular endothelial cells and is mediated through increased intracellular cGMP (18). Conversely, ANP- mediated vasorelaxation is associated with cGMP-dependent stimulation of Na+-K+-CI- cotransport in smooth muscle cells (47). The physiological relevance ofthe effect ofANP and C -7 -6 -6 BNP on Na+-K+-Cl- cotransport in parathyroid cells is not log M known. Modulation of Na'-K-Cl- cotransport in PTr cells may be involved in the response to vasoactive agents or FIG. 6. Modulation of ET-1 release from PTr cells by hANP and neurotransmitters. pBNP. Cell medium was collected after 24-hr incubations at 370C and ET-1 was measured by RIA. Data represent the mean + SD from The effects of ANP on synthetic and secretory processes three experiments each with quadruplicate samples. Open bar shows in various tissues are well known (40, 41). Actions such as the baseline (control, C) value. P < 0.05 for 0.1 jxM hANP or pBNP vs. inhibition of aldosterone synthesis (48) and of renin (49) and baseline, and P < 0.01 for 1 uM hANP or pBNP vs. baseline. vasopressin (50) release may lead to a reduction in blood Downloaded by guest on September 28, 2021 6500 Medical Sciences: De Feo et al. Proc. Natl. Acad. Sci. USA 88 (1991) pressure. We found that both ANP and BNP stimulate the bach, G. D. & Brandi, M. L. (1987) Proc. Natl. Acad. Sci. USA synthesis of ET-1 in PTr cells. Characteristically ET-1 is 84, 3269-3273. recognized as a product of endothelial cells (8). It exhibits 17. Brooker, G., Harper, J. F., Terasaki, W. L. & Moylon, R. D. (1979) Adv. Cyclic Nucleotide Res. 10, 1-33. vasoconstricting activity in vitro and pressor action in vivo 18. O'Donnell, M. E. (1989) Am. J. Physiol. 257, C36-C44. (51); hence it could be a factor in the pathophysiology of 19. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254. hypertension. On the other hand it has yet to be established 20. Richards, W. L., Song, M. K., Krutzch, H., Evarts, R. P., whether endothelin acts as a circulating hormone or merely Maiden, E. & Thorgeirsson, S. S. (1985) Exp. Cell Res. 159, as a local factor (52). 235-246. As shown here, the parathyroid glands are a newly recog- 21. Danielson, P. E., Forss-Petter, S., Brow, M. A., Calavetta, L., Douglass, J., Milner, R. J. & Sutcliffe, J. G. (1988) DNA 7, nized target tissue for actions of the natriuretic and hypoten- 261-267. sive peptides ANP and BNP. The parathyroids represent one 22. Orlando, C., Brandi, M. L., Peri, A., Giannini, S., Fantoni, G., of the tissues most replete with mRNA for ET-1, which may Calabresi, E., Serio, M. & Maggi, M. (1990) Endocrinology 126, act as an autocrine mediator of the effects of calcium on 1780-1782. parathyroid function (7). Natriuretic hormones could influ- 23. Laffi, G., Marra, F., Pinzani, M., Meacci, E., Tosti-Guerra, C., De Feo, M. L. & Gentilini, P. (1989) Liver 9, 315-321. ence secretory processes in parathyroid cells by increasing 24. Munson, P. J. & Rodbard, D. (1980) Anal. Biochem. 107, ET-1 synthesis. One might speculate that beyond local ef- 220-239. fects, stimulation of ET-1 synthesis and release from para- 25. Guardabasso, V., Munson, P. J. & Rodbard, D. (1988) Comp. thyroid cells by natriuretic hormones could be a regulatory Methods Progr. Biomed. 27, 55-63. influence on intravascular volume and vascular smooth mus- 26. De Lean, A., Munson, P. J. & Rodbard, D. (1978) Am. J. cle tone. On the other hand, however, in our in vitro Physiol. 235, E97-E102. experiments the ANP-related peptides have produced rather 27. Duncan, D. B. (1955) Biometrics 11, 1-42. 28. Collip, J. B. & Clark, E. P. (1925) J. Biol. Chem. 64, 485-507. modest increases in ET-1 release. It is uncertain whether 29. Davis, J. 0. & Freeman, R. H. (1976) Physiol. Rev. 56, 1-56. comparable effects in vivo would be sufficient to affect 30. 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