INTRODUCTION

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THE ENDOCRINE HEART produces two hormones with similar spectra of activity, atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP) (5, 22), which are crucial for the maintenance of cardiovascular homeostasis through modulating actions on systems that tend to augment blood volume and pressure. Chronic hemodynamic overload in vivo results in increased atrial synthesis and release of both natriuretic peptides (NP) (6). However, in decompensated heart failure, the actions of the NP cannot overcome the detrimental effects of the renin-angiotensin-aldosterone system, despite the fact that NP receptors can still respond to exogenously administered ANF or BNP (4, 32). Therefore, the study of the factors that modulate NP production is important to understand the development of syndromes such as chronic congestive heart failure.

Mechanical and neuroendocrine factors are involved in the modulation of ANF and BNP gene expression and secretion, but the detailed roles of specific stimuli are not clear. Atrial stretch, both in vivo and in vitro, is considered a major factor in the modulation of the endocrine heart (6). However, in clinical or experimental situations in which atrial overload is experienced, it is not known whether it is stretch per se that results in the maintenance of ANF or BNP secretion and synthesis found in these chronic or subacute overload situations (6). For example, in aortic-banded animals, both the load on the heart and the hypertrophic process independently contribute to increases in NP secretion and synthesis (23). Among the proposed neuroendocrine factors that are involved in the modulation of the endocrine heart, endothelin-1 (ET-1) is the most potent stimulus for ANF and BNP secretion and has been shown to be required for a full response of the endocrine heart to overload (9, 19, 27). Therefore, ET-1 of local or circulating origin may be an important primary stimulus for increased secretion and synthesis of ANF and BNP in response to hemodynamic imbalances.

Conflicting information exists regarding whether ANF and BNP are individually regulated at the level of secretion or gene expression by mechanical stimuli or by ET-1, and whether such effects are maintained over time (1, 16, 21). In a previous study, Bruneau and de Bold (1) showed that constant linear stretch of perifused whole atria for a 4-h period resulted in increased ANF, but not BNP secretion, and did not affect NP gene expression, whereas ET-1 stimulation resulted in increased secretion of both peptides and modestly increased BNP mRNA levels. Therefore, to address the contribution of stretch and ET-1 on ANF and BNP gene expression and secretion in adult atrial tissue over longer time periods and under more physiological conditions than afforded by previous models, we have exploited the long-term stability of a newly developed model of isolated right atria from a rat with which the anatomic conformation of the atrial chamber is maintained. With this model, pulsatile radial stretch can be applied [versus the constant linear stretch previously afforded (1)], and the stability of the preparation allows for a period of stimulation extending to at least 8 h. To ascertain the stability of the tissue over the period of stimulation, we have thoroughly characterized the synthetic and ultrastructural parameters of this model. We show that the secretion of ANF and BNP is not coregulated in response to stretch, and that BNP gene expression is selectively enhanced in a sustained manner by both stimuli. In addition, the expression of the early-response genes Egr-1 and c-myc were also studied as markers of the transcriptional response of the atrial tissue (1).

	MATERIALS AND METHODS

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Isolated right atria. Hearts were removed from male Sprague-Dawley rats (300-350 g, Charles River Laboratories) and placed in a Krebs-Ringer bicarbonate buffer (KRBB) solution. The KRBB contained 78 mM NaCl, 4.7 mM KCl, 2.54 mM CaCl₂ · H₂O, 1.36 mM NaH₂PO₄ · H₂O, 1.16 mM MgCl₂ · H₂O, 25.0 mM NaHCO₃, 20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 2.0 mM sodium glutamate, 4.0 mM sodium fumarate, 2.0 mM sodium lactate, 11.6 mM glucose, 2.46 mU/ml (Humulin R, Eli Lilly) zinc insulin, amino acids and vitamins as in Eagle's Medium (Life Technologies), 0.001% (wt/vol) bovine serum albumin (BSA), 0.01% (wt/vol) gelatin, 100 U/ml penicillin G (Sigma Chemicals), and 0.25 µg/ml fungizone (Life Technologies). The pH was adjusted to 7.40 at 30°C. The osmolarity of the buffer was 300 mosM. The KRBB was filtered through a 0.45-µm filter before use. The dissection procedure was as described previously (2), and the final setup is diagrammed in Fig. 1. Thus the flow of KRBB was from the superior vena cava, into the right atrium, and out the tricuspid valve. The organ chamber contained 50 ml of KRBB, which was replenished at a rate of 2.5 ml/min, and which was gassed with 95% O₂-5% CO₂ throughout the experiment. The level of the bath medium was set at 20 mm above the atrium. To accurately measure intra-atrial pressure, the tips of the pressure transducer and outflow cannulas were positioned at the same height. To adjust the basal intra-atrial pressure to ~0.5 mmHg, the atrium was raised or lowered in relationship to the tips of the outflow and pressure transducer cannulas. For the imposition of a volume overload on the atrium, a solenoid driving a piston was placed midway along the outflow cannula, acting as a valve. The solenoid was under the control of a wave-function generator (Wavetek) with which solenoid closing rate and duration were altered. The solenoid rate was set at 84 cycles/min, which was the fastest rate at which the intra-atrial pressure could be precisely controlled. Closing duration was adjusted to increase intra-atrial pressure.

View larger version (27K): [in this window] [in a new window]   Fig. 1.   Isolated perfused right atrium. See text for detailed description. KRBB, Krebs-Ringer bicarbonate buffer; A. A., amino acids.

For the analysis of ANF and BNP secretion and atrial gene expression, the atria were allowed to equilibrate for 1 h, after which secretion medium was collected for a basal period of 15 min. Perfusion medium was collected at 5-min periods in siliconized glass tubes using a fraction collector (ISCO Retriever II). A 50-µl aliquot was quickly transferred to another tube for use in the ANF radioimmunoassay (RIA); this aliquot and the remainder of the perfusate were immediately placed at 20°C and were thawed only once for use in RIA or for BNP extraction. After the basal collection period, atria were left at the basal intra-atrial pressure or stimulated by stretch or ET-1 (Peninsula Laboratories) for periods of time extending up to 8 h; perfusate was collected initially for two 15-min periods and every 30 min thereafter. Stretch was imposed by increasing the intra-atrial pressure from 0.5 to 2, 4, 8, 12, or 16 mmHg. ET-1 at nine times its final concentration (10⁹-10⁷ M) was infused via the inflow cannula, using a syringe pump (Sage Instruments) set at 0.34 ml/min; the flow from the delivery module was set such that flow into the atrium was maintained at 3 ml/min. At the end of the perfusion period, the tissue was rapidly removed and placed in a dissecting dish containing KRBB. The cannulas were removed, and the remaining ventricular tissue and connective tissue were cut away. The right atrium was flash-frozen in liquid nitrogen for RNA extraction or fixed overnight in 3% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) at 4°C and processed for transmission electron microscopy.

To determine pressure-volume relationships, the flow into the bath was stopped, and the bath inflow cannula was connected to the pressure transducer by a sidearm cannula; the pressure-volume relationships were previously calibrated by adding predetermined volumes of buffer in the organ chamber by means of a micropipette. After increased intra-atrial pressure, the transducer was switched to accept the bath inflow cannula measurements; changes in pressure from increases in fluid column height were calculated as changes in atrial volume.

Measurement of rates of RNA and protein synthesis. Rates of RNA and protein synthesis were measured using a modification of the method of Kent et al. (13). Atria were perfused as described above, except that the KRBB was supplemented with 0.44 mM phenylalanine (Life Technologies). After a 30-min equilibration period, the perfusion medium was switched to KRBB supplemented with 0.1 µCi/ml each of [³H]uridine and [¹⁴C]phenylalanine (NEN/DuPont). The first 20 ml of radioactive KRBB were discarded, and the perfusion medium was recirculated for 1, 3, or 6 h. After the labeling period, the tissue was perfused with unlabeled KRBB for 5 min. The atria were removed and cleaned, and the tissues were homogenized twice for 15 min at 4°C in 1 ml 0.23 N HClO₄. The homogenate was centrifuged at 16,000 g for 10 min at 4°C. The pellet was washed twice with 1 ml of 0.23 N HClO₄ and centrifuged again at 16,000 g for 5 min at 4°C. The final pellet was dissolved in 1.5 ml of 0.3 N NaOH at 37°C for 60 min, followed by cooling to 4°C. To measure protein concentrations, the solution was diluted 1:10 with saline and read by spectrophotometry at 280 nm. RNA concentration was measured by adding 0.2 ml of 2.3 N HClO₄ to 0.5 ml of the sample, centrifuging for 10 min at 16,000 g at 4°C, and measuring RNA content by spectrophotometry with a 1:3 dilution of the supernatant in diethyl pyrocarbonate-treated H₂O. The remainder of the NaOH solubility product was diluted in 10 ml of scintillation fluid and counted in a scintillation counter (Beckman) using a dual-label program.

Reverse-phase high-performance liquid chromatography. Reverse-phase high-performance liquid chromatography chromatography (RP-HPLC) analysis was performed as previously described (8). Perfusate (90 ml) was loaded on a C₁₈ Vydac column (0.78 × 30 cm); RP-HPLC was performed using a linear gradient of solvent B (80% acetonitrile in 0.1% trifluoroacetic acid) from 15 to 55% vs. solvent A (0.1% trifluoroacetic acid) at a flow rate of 1.5 ml/min; 3-ml fractions were collected, freeze-dried with 100 µg heat-treated BSA, and reconstituted with 125 µl BSA-free RIA buffer (26). One hundred microliters of the fractions (for BNP RIA) or a 1:62.5 dilution (for ANF RIA) was assayed for immunoreactive (ir) ANF and irBNP content.

Radioimmunoassays. RIA was performed using the double-antibody technique as previously described (2, 26). A 1:2 dilution of perfusate was used for the ANF RIA. For measurement of BNP release, 10 ml of perfusate were extracted using C₁₈ Sep-Pak cartridges (Waters) as previously described (2). The sensitivity of the BNP RIA was 1.5 pg/tube.

RNA extraction and Northern analysis. Total RNA extraction and Northern blot analysis were performed as previously described (1, 2) with the following ³²P-labeled probes: rat ANF cDNA, rat BNP cDNA, human c-myc cDNA, and mouse Egr-1 cDNA. Mouse 28S rRNA cDNA (for ANF mRNA) or mouse phosphoglycerate kinase cDNA (pgk-1) were used as housekeeping controls to correct for loading and transfer variation. There were no significant differences when mRNA signals were normalized to either 28S rRNA or pgk-1 signals. Autoradiographs exposed in the linear range of the film were quantitated by one-dimensional scanning laser densitometry, using an LKB Ultroscan XL (Pharmacia LKB).

Statistical analysis. All data are reported as means ± SE. Unpaired Student's t-test was used to determine statistical significance between pairs. Analysis of variance (ANOVA) was performed to determine statistical differences among multiple groups. When significance was obtained by ANOVA, Fisher's least squares difference post hoc analysis was used to determine pairwise differences. Significance was accepted at P < 0.05.

	RESULTS

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Characterization of model. The rates of RNA and protein synthesis in the isolated right atria remained constant over time (Fig. 2), indicating that the synthetic function of the tissue remains intact during the perfusion period. Microscopic examination of perfused tissues after 7.5 h showed a well-preserved structure not distinguishable from unperfused atrial tissue (Fig. 3). The molecular form of irANF and irBNP secreted by the atria were the processed forms of each hormone (Fig. 4). The secretion by the isolated atria of processed forms of ANF and BNP indicates that secretion during the experiment was not due to cell leakage. Processing did not change throughout 8 h of perfusion under basal or stimulated conditions (data not shown). Further indication of the state of the atria can be obtained from the observation that nonstimulated atria beat spontaneously without arrythmias (Fig. 5). RNA degradation is a sensitive indicator of cell damage or death; RNA isolated from atria that had been perfused for 9.25 h (1- h equilibration, 15-min basal period, 8-h experimental period) showed no signs of degradation, whether the tissue had been stimulated or not (data not shown). Therefore, biochemical, morphological, and mechanical indexes demonstrate that this atrial preparation retains normal characteristics for the studied time period and is therefore well-suited for studying cardiac endocrine function for time periods extending up to 8 h of stimulation.

View larger version (14K): [in this window] [in a new window]   Fig. 2.   RNA () and protein synthesis () in isolated right atria perfused at an intra-atrial pressure of 0.5 mmHg. Incorporation of [3H]uridine and [14C]leucine was measured in perfused atrial tissue as described in MATERIALS AND METHODS. dpm, Disintegrations/min.

View larger version (166K): [in this window] [in a new window]   Fig. 3.   Transmission electron micrograph of atrial tissue perfused at 30°C for 7.5 h. G, specific atrial granules. Magnification: ×5,700.

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Reverse-phase high-performance liquid chromatography profiles of immunoreactive atrial natriuretic factor (irANF, A) and immunoreactive brain natriuretic peptide (irBNP, B) secreted from isolated right atria in the first 30 min following the equilibration period. Elution positions for rat ANF-(99126), ANF-(1126) (proANF), BNP-(5195), and BNP-(195) (proBNP) are indicated. Gradient conditions were 15-55% B (solvent B: 80% acetonitrile in 0.1% trifluoroacetic acid).

View larger version (26K): [in this window] [in a new window]   Fig. 5.   Effect of stretch (A, C) or endothelin-1 (ET-1, B, D) on atrial contraction rate (A, B) and intra-atrial pressure (C, D). In A and C, atria were perfused with KRBB alone at 0.5 mmHg (, n = 9) or were distended by increasing intra-atrial pressure to 2 mmHg (, n = 3), 4 mmHg (, n = 3), 8 mmHg (, n = 8), 12 mmHg (, n = 3), or 16 mmHg (, n = 3). In B and D, atria were perfused with KRBB alone (, n = 9) or KRBB containing ET-1 at 109 M (, n = 3), 108 M (, n = 8), or 107 M (, n = 2) at time 0.

Effects of stretch or ET-1 on mechanical parameters and irANF secretion. Stretch resulted in an increase in the rate of contraction, which was sustained throughout the stimulation period (Fig. 5A). Pressure-volume relationships for the isolated right atria performed as described in MATERIALS AND METHODS showed that increases in intra-atrial pressure corresponded to increased atrial volume (data not shown). ET-1 dose dependently increased intra-atrial pressure and rate of contraction (Fig. 5, B and D), which is due to the expected inotropic and chronotropic effects of this peptide. Stretch-stimulated secretion of irANF was load dependent (Fig. 6A). irANF secretion was significantly increased after 15 min of stretch and thereafter declined, reaching basal levels after 3 h of stretch. irANF release was dose dependently stimulated by ET-1 after 15 min (Fig. 6B); the stimulated ANF secretion slowly declined, reaching basal levels after 5 h of stimulation. Calculation of total ANF secreted during 6 h showed that maximal ANF secretion was obtained with a load of 8 mmHg or 10⁸ M ET-1 (not shown).

View larger version (22K): [in this window] [in a new window]   Fig. 6.   Effects of stretch (A) or ET-1 (B) on irANF secretion from isolated right atria. In A, atria were perfused with KRBB alone at 0.5 mmHg (, n = 9; control) or were distended by increasing intra-atrial pressure to 2 mmHg (, n = 3), 4 mmHg (, n = 3), 8 mmHg (, n = 8), 12 mmHg (, n = 3), or 16 mmHg ( n = 3). In B, atria were perfused with KRBB alone (, n = 9; control) or KRBB containing ET-1 at 109 M (, n = 3), 108 M (, n = 8), or 107 M (, n = 2) at time 0, as indicated by arrow. * P < 0.05 vs. time 0 and controls (in A refers to 8, 12, or 16 mmHg only); § P < 0.05 vs. controls (by t-test).

Effects of stretch or ET-1 on irBNP secretion. irBNP secretion was stimulated by stretch (8 mmHg) and ET-1 (10⁸ M) in this model (Fig. 7). The load and ET-1 dose were chosen because they maximally stimulate ANF secretion, and in preliminary experiments they were shown to be the minimum effective load or dose for a detectable change in irBNP release. As with ANF, stretch-stimulated irBNP secretion was temporary, returning to basal levels after 2 h of stretch. irBNP secretion peaked after 1.5 h of ET-1 stimulation and thereafter decreased toward basal levels. There was a proportionally larger increase in ANF secreted in response to stretch in the first 15 min of stimulation [+104 ± 19% for irBNP vs. +184 ± 21% for irANF (P < 0.05, n = 5)]; the proportional increases in ANF and BNP secretion were similar for the remainder of the experimental period. This observation indicates that there was an initial preferential increase in ANF secretion in response to stretch, and that thereafter both ANF and BNP were cosecreted. There also was a larger proportional increase in ANF secretion after 60 min of ET-1 stimulation [+99 ± 23% for irBNP vs. +223 ± 46% for irANF (P < 0.05)].

View larger version (17K): [in this window] [in a new window]   Fig. 7.   Secretory response of irBNP to stretch (A) or ET-1 (B). Isolated right atria were either perfused at an intra-atrial pressure of 0.5 mmHg with KRBB alone (, n = 8; control) or were stimulated by increasing intra-atrial pressure to 8 mmHg (; A) or adding ET-1 at 109 M (, n = 5; B) at time 0, as indicated by arrow. * P < 0.05;  P < 0.01;  <0.001 vs. time 0 (by analysis of variance); § P < 0.05 vs. controls (by t-test).

Effects of stretch or ET-1 on atrial gene expression. Densitometric quantitation of Northern blots performed using total RNA isolated from the experimental atrial tissues is shown in Fig. 8. ANF mRNA levels were not affected by stretch or by ET-1 over 8 h of stimulation. Stretch caused a significant increase in BNP mRNA levels after 1.5 h of stimulation; these levels increased further, peaked after 6 h, and remained significantly elevated after 8 h. Egr-1 mRNA levels were significantly increased after 4 h of stretch; the increase was no longer significant after 6 h of stretch. Stretch also resulted in a significant increase in c-myc mRNA levels after 1.5 h, which peaked after 4 h and thereafter declined to control levels. BNP mRNA levels were increased by 10⁸ M ET-1 after 4 h of stimulation; the stimulated increase peaked after 6 h and thereafter decreased slightly, although they were still significantly elevated. ET-1 also stimulated Egr-1 mRNA levels after 4 h; this increase was temporary. c-myc mRNA levels were stimulated by ET-1, gradually increasing over the 8-h period and reaching significance after 8 h.

View larger version (18K): [in this window] [in a new window]   Fig. 8.   Densitometric analysis of Northern blots performed on atrial RNA extracted from right atria that were perfused at 0.5 mmHg intra-atrial pressure (IAP), 8 mmHg IAP (stretch), or 0.5 mmHg IAP with ET-1 (108 M) for 1.5 h (n = 5), 4 h (n = 5), 6 h (n = 3-5), or 8 h (n = 5). Relative signals quantitated are indicated by closed bars for stretch and hatched bars for ET-1. mRNA levels studied are ANF (A), BNP (B), Egr-1 (C), and c-myc (D). Signals were normalized to 28S rRNA signals (for ANF mRNA) or to pgk-1 mRNA signals (for all other mRNAs). Data are expressed as means ± SE of percent change from unstimulated controls. * P < 0.05;  P < 0.01;  P < 0.001 vs. vehicle (by analysis of variance).

	DISCUSSION

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The present studies were carried out with a newly developed model to study atrial secretory function that more accurately retains the functional conformation of the right atrium in vivo: atrial distension is radial, the flow through the atrium is similar to that observed in situ, and the intra-atrial pressures can be set within physiological values. Also, unlike the Langendorff preparation, the potentially confounding presence of ventricular flow-through is avoided. The analysis of biochemical function and ultrastructure shows that the tissue remains intact and fully functional during the period of study and is therefore ideally suited for the study of atrial endocrine function for time periods extending up to 8 h, a considerably longer time frame than that afforded by previous models (1, 21).

Stretch-induced BNP release from the isolated right atria followed a time course that was similar to that of ANF; however, there was initially a proportionally larger release of ANF. The colocalization of ANF and BNP in rat atrial granules (15, 29) implies that both peptides should be released simultaneously. However, the amount of BNP associated with the granules is only a small portion of total cellular BNP and is proportionally very small compared with granule-associated ANF (15, 30). It is likely that the population of granules destined for rapid stretch-induced release includes bihormonal granules as well as a larger proportion of ANF-only granules (15). Not all studies report a concomitant increase in ANF and BNP secretion following atrial overload. Aortocaval fistula in the rat increases ANF and BNP plasma levels simultaneously (29). However, after acute volume loading in humans and in rats, circulating levels of ANF are rapidly increased while plasma BNP levels remain initially at basal levels, increasing only later (17, 19, 31). Similarly, in previous studies by Bruneau and de Bold (1) and Laine et al. (16) that used isolated whole atria, no significant increase in BNP secretion was observed following stretch. This result can be explained by the observation that a more pronounced stretch is required to effect significant changes in BNP secretion than in ANF secretion. Indeed, ANF secretion was stimulated by stretch as low as 2 mmHg, whereas BNP secretion was only significantly increased by a load of 8 mmHg, and this increase was proportionally smaller than that of ANF at the same load. Therefore, because there was a proportionally smaller increase in ANF secretion in our previous study, and hence a smaller degree of stretch, a small change in BNP secretion might not have been detected (1).

Whereas BNP has actions similar to those of ANF, it has features that functionally distinguish it from ANF, such as a lower metabolic clearance rate and an ability to enhance the effects of ANF (12, 25). With the requirement for a larger degree of stretch on in vitro and in vivo data (17, 19, 30, 31), we conclude that BNP secretion may be acutely increased only after a severe load. In such cases the increase in BNP would supplement the actions of ANF. The dual natriuretic peptide system is therefore well adapted to deal with varying degrees of cardiac overload.

ANF and BNP secretions from atrial tissue were rapidly and potently stimulated by ET-1, as previously reported (1). The time course of ET-1-stimulated BNP secretion was similar to that for ANF, suggesting that the population of granules destined for ET-1-stimulated release contains both hormones, or, alternatively, similar signal transduction processes are responsible for the secretion of both peptides from both monohormonal and bihormonal granules. This finding contrasts with our recent observation that stimulation of isolated atria with phenylephrine, which shares signaling pathways in cardiocytes with ET-1, results in the discoordinate secretion of ANF and BNP (2).

Stretch-stimulated ANF release is associated with the rapid depletion of an immediately releasable pool of newly synthesized hormone (20), and acute stimulation of ANF secretion by both stretch and ET-1 is largely independent of protein synthesis (7, 24), suggesting that acutely increased ANF secretion is not immediately accompanied by increased ANF synthesis. Therefore, it is not surprising that ANF mRNA levels remained constant after 8 h of continuous stimulation by stretch or ET-1.

Stretch and ET-1 enhanced the expression of the BNP gene in the isolated right atria, indicating that BNP mRNA levels are more acutely sensitive to these stimuli than ANF. The changes in BNP mRNA levels occurred asynchronously with the changes in BNP secretion, which is reminiscent of volume overload in rats, in which BNP mRNA levels are increased in the atria without a concomitant change in plasma or tissue levels (31). A similar response to stretch has been previously reported in the atria of Langendorff-perfused hearts, although stimulation was only carried out for 2 h (21). Our previous report (1) showed that a constant linear stretch of perifused whole atria did not result in increased BNP mRNA levels. As with BNP secretion, the differences in BNP gene expression between the two models most likely reflect the extent and geometry of the stretch imposed on the atrial cardiocytes. The present work also shows that ET-1 stimulation robustly increases BNP mRNA levels in a sustained manner over 8 h of stimulation, an observation not appreciated by the shorter time course used in our previous study, which showed only a modest and temporary increase in BNP mRNA levels (1). The rapid response of the BNP gene to ET-1 contrasts with observations in cultured neonatal atrial cardiocytes, in which BNP mRNA levels are modestly increased by ET-1 after 16 h of continuous stimulation, after a previous increase in ANF gene expression (28). This emphasizes the intrinsic differences in endocrine function and phenotype between neonatal cardiocytes in culture and adult tissue. Stretch induced a more rapid and sustained increase in BNP mRNA levels than did ET-1, and the pattern of increased BNP mRNA levels for both was more rapid than that which follows stimulation by phenylephrine (2), which implies that different signaling pathways are used by each stimulus. This extends to the expression of the ANF gene, which is enhanced by phenylephrine (2), but not by stretch or ET-1.

It is not immediately obvious why two hormones that are both secreted in response to a stimulus respond differently at the level of transcript abundance. The differences in the response of ANF and BNP to stretch and ET-1 at the levels of gene expression may be related to the relative proportion of peptide stores in the atrial cardiocytes (15, 30), suggesting that the recruitment of BNP synthesis by acute stretch and by ET-1 stimulation is a requirement for the maintenance of BNP secretion in conditions of volume overload, as is found in chronic overload situations (6). The mechanism of regulation of ANF and BNP mRNA levels may underlie the different response of the two genes: BNP mRNA levels are regulated by changes in mRNA stability, whereas ANF gene expression is mostly transcriptionally regulated (11, 18).

Both stretch and ET-1 caused increases in beating rate. However, it is unlikely that increased rate of contraction is responsible for the observed changes in ANF and BNP secretion and gene expression. For both stimuli, the time course of induced secretion did not parallel that of increased contractility, indicating that the two responses are unrelated. Dissociation of contractile activity from stretch- or ET-1-induced ANF and BNP secretion and gene expression has been shown (6, 10, 14, 28). Therefore, the increase in BNP mRNA levels observed in the present study also likely reflects a direct effect by stretch and ET-1, although a contribution of beating rate cannot be entirely discounted.

The expression of Egr-1 and c-myc was stimulated by stretch and ET-1, indicating that the transcriptional response of the tissue is intact. These genes have been characterized in ventricular cardiocytes as genes that respond quickly and transiently to stimuli (3). The Egr-1 gene responds in the atrium with characteristics of an early-response gene to stretch, ET-1, and ₁-adrenergic stimulation (present study and Refs. 1 and 2), albeit exhibiting a delayed and extended response. The response of c-myc to stretch and ₁-adrenergic stimulation (2) also follows a similar type of pattern; however, the greatly delayed effects of ET-1 on c-myc mRNA levels indicate that this gene responds uniquely to this stimulus.

In conclusion, we have shown, using a novel model of isolated right atria from rat, that stretch and ET-1 induce distinct changes in ANF and BNP secretion and gene expression. The expression of Egr-1 and c-myc in response to stretch and ET-1 emphasizes the differences between mechanical and receptor-mediated stimuli and further supports the possibility that these transcription factors are important in the response of the atrium to stretch and ET-1. These results reveal the importance of mechanical- and ET-1-mediated regulation of ANF and BNP synthesis and secretion and suggest that in conditions of atrial overload, these factors help maintain cardiovascular homeostasis via their actions on the endocrine heart. The distinct responses of ANF and BNP secretion and the specific modulation of BNP mRNA levels by stretch and ET-1 show that the endocrine heart has the ability to distinctly and specifically regulate the synthesis and secretion of its secretory products.