Cosecretion of atrial and brain natriuretic peptides stimulated by endothelin-1 from cultured rat atrial and ventricular cardiocytes

Cosecretion

of Atria1 and Brain Natriuretic Peptides Stimulated by Endothelin-1 From Cultured Rat Atria1 and Ventricular Cardiocytes Takeshi Horio, Masakazu

Kohno, and Tadanao

Takeda

We examined the secretion of immunoreactive (ir) atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) in primary cultures of atrial and ventricular cardiocytes from neonatal rats, and also investigated the stimulatory effect of endothelin-1 (ET-I) on ir-ANP and ir-BNP release from these cells. After 2 days of culture, rat atrial and ventricular cardiocytes secreted both ir-ANP and ir-BNP into serum-free medium in a time-dependent manner. The amount of ir-BNP released per IO5 ventricular cells was approximately 60% to 70% (1.6 f 0.5 pg/h, 2.5 + 0.4 pg/2 h, 2.6 f 0.4 pg/4 h) of that released from atrial cells (2.8 f 0.4 pg/h, 3.5 f 0.6 pg/2 h, 4.6 + 0.6 pg/4 h), although ir-ANP was secreted almost entirely from atrial cells. ET-1 clearly stimulated secretion of both ir-ANP and ir-BNP in atrial and ventricular cardiocytes. These observations indicate that ir-BNP is cosecreted with ir-ANP not only from atrial cardiocytes, but also from ventricular cardiocytes in the rat, and that ET-1 stimulates the secretion of these natriuretic peptides by a direct mechanism, not through a hemodynamic change. Copyright 0 1993 by W.B. S&n&s Company

S

INCE ATRIAL NATRIURETIC peptide (ANP) was isolated from mammalian cardiac atria, many studies have investigated its role in regulating the homeostatic balance of body fluids and blood pressure. Recently, a novel natriuretic peptide, brain natriuretic peptide (BNP), was isolated originally from porcine brain.’ This peptide, which consists of 26 amino acid residues containing an intramolecular disulfide linkage, has a high sequence homology with ANP and pharmacological properties very similar to those of ANP.’ Subsequently, it has been demonstrated that a considerable concentration of BNP is present in porcine,? human,” and rat hearts4 suggesting that BNP may act as a cardiac hormone rather than as a neuropeptide. In vivo, the secretion of ANP may be mediated by changes in atria1 pressure,’ and its release in vivo and in vitro is known to be stimulated by a newly described vasoconstrictive peptide, endothelin.6,7 However, the agents and conditions that affect the release of BNP remain to be elucidated, and it is not clear whether endothelin stimulates the secretion of both BNP and ANP. In the present study, we examined whether immunoreactive (ir) BNP is cosecreted with ir-ANP from cultured rat atria1 and ventricular cardiocytes. We also studied the stimulatory effect of endothelin-1 (ET-l) on the release of ir-ANP and ir-BNP from these cells. MATERIALS

AND METHODS

Cell Culture and Pharmacological Treatment Primary cultures of neonatal were prepared according to the minor modifications. Briefly, apexes were removed from 2Both tissues were minced into collagenase type II (1 mg/mL. 37°C and by repeated pipetting.

atria1 and ventricular cardiocytes method previously described,* with atria1 appendages and ventricular to 5-day-old Sprague-Dawley rats. small pieces and dissociated with Sigma Chemical, St Louis, MO) at The dissociated cells were centri-

From the First Department of Internal Medicine, Osaka City Universi~ Medical School, Osaka, Japan. Submitted September 16, 1991; accepted April 7, 1992. Address reprint requests to Takeshi Horio, MD, First Department of Internal Medicine, Osaka City University Medical School, I-5-7 Asahi-machi, Abeno-ku, Osaka 545, Japan. Copyright 0 1993 by W.B. Saunders Company 0026-049519314201-0016$03.OOlO 94

fuged and resuspended in Dulbecco’s modified Eagle’s medium ([DMEM] Gibco Laboratories, Grand Island, NY) supplemented with 10% fetal calf serum ([FCS] Gibco), penicillin (50 uU/mL, Gibco) and streptomycin (50 pU/mL, Gibco). Cells were then seeded onto multiwell plates (Becton Dickinson, Lincoln Park, NJ) and maintained in an incubator at constant humidity and temperature (37°C) with 95% air and 5% CO?. After 48 hours, medium was replaced with 2 mL fresh DMEM without FCS, and cells were incubated for the indicated intervals. To assess the effect of ET-l on ANP and BNP secretions, various doses of ET-l (10-i” to 10mh mol/L, Peptide Institute, Osaka, Japan) were added and cells were incubated at 37°C for 1 hour. After incubation, medium was aspirated and centrifuged; then the supernatant was collected and stored at -80°C until time of radioimmunoassay. Cell number was determined with an electronic cell counter (Model ZBl. Coulter Electronics, Hialeah, FL) after trypsinization.

Extraction of ANP and BNP From Culture Medium ANP and BNP were extracted as reported previously for ANP.” Briefly, 1.5 mL culture medium was diluted with 4% acetic acid, and after centrifugation the solution was pumped at the rate of 1 mL/min through a Sep-Pak Cts cartridge (Waters Associates, Milford, MA). After the cartridge was washed with distilled water, adsorbed peptides were eluted with 86% ethanol in 4% acetic acid. After evaporation of the eluate by a centrifugal evaporator (Model RD-31, Yamato Scientific, Tokyo, Japan), the dry residue was dissolved in an assay buffer.

Radioimmunoassay for ANP and BNP Radioimmunoassay for ANP was performed using antibody against synthetic n-human ANP(I-28) obtained from Peninsula Laboratories, Belmont, CA, and ‘ZSI-cu-human ANP( l-28) obtained from Amersham Japan, Tokyo, Japan, as previously reported.9 This antibody reacted 100% with o-human ANP(l-28) and cross-reacted 100% with u-rat ANP(l-28). 57% with o-rat ANP( 18. 28) 27% with a-rat ANP(5-37), and 3% with cu-rat ANP(5-25). It did not show any cross-reactivity with porcine BNP-26, human BNP-32, rat BNP-45. rat BNP-32, somatostatin, vasopressin, or ET-l. The concentration of ir-BNP was measured with antibody against synthetic rat BNP-32 and lZ51-rat BNP-32 (Peninsula Laboratories). This antibody reacted 100% with rat BNP-32 and cross-reacted lOO’% with rat BNP-45. and did not cross-react with human BNP-32. porcine BNP-26, a-human ANP(l-28), n-rat ANP(l-28) vasopressin, angiotensin II, or ET-l. The radioimmunoassay was performed in an assay buffer of 0.01 mol/L sodium phosphate (pH 7.4) containing 0.05 mol/L sodium chloride, 0.1% Metabolism,

Vol42, No 1 (January), 1993: pp 94-96

95

ANP AND BNP RELEASE BY ET-1

bovine serum albumin, 0.1% Nonidet P-40, and 0.01% sodium azide, as recently reported.‘” Briefly, rehydrated antiserum (100 FL) was added to 100 FL of the sample or of standard rat BNP-32 dissolved in the assay buffer, and the mixture was incubated for 24 hours at 4°C. Approximately 15,000 cpm lz51-rat BNP-32 was added to each sample, and the mixture was incubated for an additional 24 hours. After the second 24-hour incubation, 100 FL diluted normal rabbit serum and 100 PL diluted sheep anti-rabbit y-globulin serum were added, and The mixture was again incubated for 24 hours. After the third incubation, the precipitate was collected by centrifugation at 1.700 x g for 30 minutes. The supernatant was removed by aspiration, and the radioactivity of the pellet (lz51) was counted with a gamma counter. The effective range of the standard curve was between 0.5 and 100 pg rat BNP-32, and the 50% intercept was 21 pg rat BNP-32. The interassay variations of ANP and BNP were 11.4% and 12.5%, respectively, and the intraassay variations were 6.3% and 7.3%. respectively.

Statistical significance of the results ANOVA, and P values were calculated

was evaluated by unpaired by Scheffe’s method.]’ All

values were expressed as means 2 SEM. RESULTS

Figure 1 shows the time-course of ir-ANP (Fig 1A) and ir-BNP (Fig 1B) release into medium from cultured rat atria1 and ventricular cardiocytes. After 2 days of culture, both atria1 and ventricular cardiocytes secreted ir-ANP and ir-BNP into serum-free medium in a time-dependent manner. The amount of ir-BNP released per lo5 ventricular cells was approximately 60% to 70% (1.8 ? 0.5 pg/h, 2.5 + 0.4 pg/2 h, 2.6 2 0.4 pg/4 h) of that released from atria1 cells (2.8 +- 0.4 pg/h, 3.5 & 0.6 pg/2 h, 4.6 * 0.6 pg/4 h), while the amount of ir-ANP released per lo5 ventricular cells was less than 10% of that released from atria1 cells. Table 1 shows the release of ir-ANP and ir-BNP from cultured atria1 and ventricular cardiocytes 1 hour after stimulation with various doses of ET-l. The secretion of ir-ANP from both atria1 and ventricular cells was stimulated by ET-1 in a dose-dependent manner (10m8 to 10eh mol/L). The secretion of ir-BNP was stimulated in a

(6)

(A)

I 300 -

2

ET-1 in Cultured Atrial and Ventricular Cardiocytes ET-l (mol/L)

Measurements (pg/hl105

cells)

0

10-10

178 k 14

276 2 20

321 k 52*

414 + 19*

20 + 3

29 -+_8

48 + 8*

64 2 3*

3.7 -+ 0.6

4.9 -c 0.6

7.9 + 0.7*

10.2 k 1.2*

2.9 k 0.6

3.6 k 0.4

3.6 + 0.9

6.3 k 0.9*

10-S

10-6

ir-ANP Atrial cells Ventricular

cells

ir-BNP Atrial cells Ventricular

cells

NOTE. Values are means 2 SEM of results from four dishes. *P < .05 v control.

dose-dependent manner by ET-l (lOeK to 10-h mol/L) atria1 cells, and by 10eh mol/L ET-1 in ventricular cells.

in

DISCUSSION

Statistical Analysis

I

Table 1. Release of ir-ANP and ir-BNP 1 Hour After Stimulation by

3

Time (hrs)

4 Time

(hrs)

Fig 1. Time-course of the release of ir-ANP IA) and ir-BNP (6) into medium from cultured rat atrial (0) and ventricular (0) cardiocytes. Values are means f SEM of results from four dishes.

This study demonstrated that ir-BNP is present at low concentrations in serum-free media of cultured rat atria1 and ventricular cardiocytes, and that ir-BNP is cosecreted with ir-ANP from these cells in a time-dependent manner. Ogawa et all? reported that a considerable amount of rat ir-BNP was secreted into perfusate from isolated rat heart as measured by Langendorff’s method, and this secretion was maintained at approximately 60% even after atria1 removal. Moreover, our perfusion studies in hypertensive rat models with left ventricular hypertrophy indicated that a large amount of BNP was secreted from the hypertrophied ventricles.‘” These observations and our data strongly suggest that rat BNP is secreted in significant amounts from both the ventricle and the atrium, while rat ANP is secreted almost entirely from the atrium. In the present study, we also demonstrated that ET-l stimulates secretion of both ir-ANP and ir-BNP from atria1 and ventricular cardiocytes. Fukuda et a17J3 have shown that endothelin stimulates synthesis and secretion of ANP in cultured rat atria1 cardiocytes. Furthermore, they found that ANP secretion induced by endothelin is attenuated by the Ca’+-channel blocker, nicardipine, but no further stimulation is induced when a Ca?+-channel agonist, BAY-K 8644, is added. Hirata et alI5 reported that rat ventricular cardiocytes have specific receptors for endothelin, and that endothelin induces a receptor-mediated increase in cytosolic free Ca’+ in these cells. On the other hand, Shubeita et allh reported that endothelin stimulates accumulation of diacylglycerol in cultured ventricular myocytes. Recently, it has been shown that BNP is secreted with ANP from cultured porcine atria1 cardiocytes and this secretion is enhanced by a Ca?+ ionophore. ionomycin. and by a protein kinase C activator, tetradecanoylphorbol acetate.17 Taken together, these observations suggest that influx of extracellular Cal+ and activation of protein kinase C are closely involved in the secretory mechanism of BNP by endothelin from cardiomyocytes, as is the case with ANP. BNP has been shown to be almost equipotent to ANP in promoting natriuresis and vasorelaxation,’ whereas endothelin is well known to have a novel vasoconstrictive activity.lx Recently, we demonstrated that ANP and BNP inhibit secretion of ET-1 in cultured human endothelial cellstu This suggests

96

HORIO, KOHNO, AND TAKEDA

the presence of a negative-feedback loop whereby ET-1 stimulates ANP-BNP release, which in turn suppresses ET-l release. Our present study and other reports indicate that the amount of BNP secreted from the rat heart is much less than that of ANP,12 and that the plasma concentration of BNP is also much lower than that of ANP.?O However, previous reports have shown that the relative increase of plasma BNP compared with the level in the control group was much greater than the increase of ANP in some

pathophysiological states, eg, heart failure2’ and deoxycorticosterone acetate salt hypertension.22 Therefore, BNP may act as a novel cardiac hormone by differing somewhat from ANP in secretion behaviors. Further studies are required to clarify the physiological role and significance of BNP.

ACKNOWLEDGMENT We thank

Machiko

Johchi

and Ikuko

Kuno for their technical

assistance.

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