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Am J Physiol Heart Circ Physiol 278: H117-H125, 2000;
0363-6135/00 $5.00
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Vol. 278, Issue 1, H117-H125, January 2000

Role of cell contractions in cAMP-induced cardiomyocyte atrial natriuretic peptide release

D. J. Church1, M. C. Rebsamen2, D. Morabito2, V. van der Bent2, M. B. Vallotton2, and U. Lang2

1 Serono, Geneva Pharmaceutical Research Institute, and 2 Division of Endocrinology, University Hospital, CH-1211 Geneva 14, Switzerland


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Incubation of spontaneously beating ventricular cardiomyocytes from neonatal rats with prostaglandin E2 (0.1 µM) or forskolin (0.1 µM) simultaneously increased the rate of cellular contraction and atrial natriuretic peptide (ANP) secretion. Both responses were maximal within 10-20 min of application and were accompanied by three- to fourfold increases in cAMP formation. By contrast, a higher regimen of forskolin (10 µM) promoted a 20- to 30-fold increase in basal cAMP production, which was accompanied by the abolition of contractile activity and ANP release. Low regimens of forskolin (0.1 µM) doubled the occurrence of cytosolic Ca2+ transients associated with monolayer contraction, whereas higher regimens of forskolin (10 µM) completely suppressed Ca2+ transients. Moreover, in quiescent cultures that were pretreated with ryanodine, tetrodotoxin, nifedipine, or butanedione monoxime, prostaglandin E2 (0.1 µM) and forskolin (0.1 µM) failed to elicit significant ANP secretion, suggesting that cAMP-elevating agents promote ANP secretion to a great extent via an increase in cellular contraction frequency in ventricular cardiomyocytes.

ventricular cardiomyocytes; cell contractions; prostaglandin E2; forskolin; cytosolic calcium concentration


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

A LARGE NUMBER of cAMP-stimulating agents, such as beta 2-adrenergic agonists, have been reported to induce atrial natriuretic peptide (ANP) release in cardiomyocytes (for review see Ref. 18), whereas phospholipase C-activating hormones, such as ANG II and vasopressin, stimulate ANP secretion via an increased production of prostaglandin E2 (PGE2) and prostacyclin (5, 30), potent auto- and paracrine modulators of biological functions noted for their Ca2+-mobilizing or/and adenylyl cyclase-stimulating effects (31, 32). These eicosanoids have been shown to stimulate the formation of cardiomyocyte cAMP (6), which activates voltage-gated Ca2+ channels (29), a response leading to myocardial ANP release (23).

However, the effect of cAMP formation on myocardial ANP release remains to be fully elucidated, inasmuch as cAMP production has been shown to promote and inhibit ANP secretion depending on the experimental model under consideration. Several reports demonstrate that increases in cAMP or Ca2+ influx augment myocardial ANP secretion (6, 21, 23, 26), whereas other studies show that cAMP and/or Ca2+ have little effect on ANP secretion per se (20) or even inhibit ANP release altogether (1, 10, 28). In this context, it is likely that the frequency and force at which cardiomyocytes contract play a major role in determining the pathways involved in ANP secretion, inasmuch as positive chronotropic responses are known to stimulate ANP secretion in beating heart tissue and atrial myocytes (3, 16, 25), and the cAMP-elevating agent isoproterenol induces ANP release in electrically paced isolated atria yet has no effect or actually inhibits ANP secretion in the nonbeating atria (23). Thus variations of the cardiomyocyte's overall contractile state could explain the discrepancy in the literature concerning the influence of cAMP in cardiac ANP secretion.

We investigated this hypothesis by studying the time- and concentration-dependent effects of the cAMP-elevating agents forskolin and prostaglandin E2 (PGE2) on cell contraction frequency, cAMP production, cytosolic free Ca2+ concentration ([Ca2+]c), and ANP release in spontaneously beating neonatal rat ventricular cardiomyocytes, an experimental model previously shown to release ANP on stimulation with cAMP-elevating agents.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials

Rat [3-125I-Tyr125]ANP-(99-126) was purchased from Novabiochem (Basel, Switzerland) and 125I-cAMP from Amersham International (Amersham, UK). Ryanodine (Rya), tetrodotoxin (TTX), butanedione monoxime (BDM), and 3-isobutyl-1-methylxanthine (IBMX) were obtained from Sigma Chemical (St. Louis, MO). Forskolin, fura 2-AM, and 1,2-bis-5-methylaminophenoxyl-ethane-N,N,N'-tetraacetoxymethyl acetate (MAPT-AM) were obtained from Calbiochem (San Diego, CA). McCoy's modified 5A medium, Ca2+- and Mg2+-free Hanks' balanced salt solution (HBSS), FCS, trypsin, DNase I (type IV), and insulin (5 µg/ml)-transferrin (5 µg/ml)-selenium (5 ng/ml) medium supplement (ITS) were acquired from GIBCO (Basel, Switzerland). Rabbit anti-alpha -ANP antiserum and alpha -ANP were obtained from Peninsula Labs (Belmont, CA), and nifedipine (Nif) was a generous donation from Bayer (Wuppertal, Germany). PGE2 was kindly donated by Upjohn (Kalamazoo, MI), and anti-cAMP antiserum was a generous gift from Dr. Baukal (National Institutes of Health, Bethesda, MD).

Cell Culture

Spontaneously beating neonatal rat ventricular cardiomyocyte cultures were obtained from 1- to 2-day-old Wistar rats by the trypsin-DNase sequential-digestion method previously described (30). Briefly, ventricular heart tissue was washed with 40 ml of HBSS, cut into small pieces, further washed with 10 ml of HBSS, and enzymatically digested for 8 min with 10 ml of trypsin-DNase (2.5 and 0.3 mg/ml, respectively) at 37°C in a 50-ml sterile conical tube subjected to constant stirring. The supernatant from the first incubation was discarded, 10 ml of fresh enzyme solution were added, and the incubation procedure was repeated. Subsequent supernatants were collected and centrifuged at 1,200 rpm for 4 min, and the resulting cell pellets were resuspended in McCoy's medium containing 10% FCS at 37°C. Once the sequential digestions were terminated, the cells were pooled in McCoy's modified 5A medium containing 10% FCS, 1% ITS, 100 IU/ml penicillin, and 10 mg/ml streptomycin and seeded in 90-mm petri dishes. After 3 h of incubation, the petri dishes were shaken, and the supernatants containing the cardiomyocytes were pooled and seeded in 90-mm petri dishes or six-well culture plates (Costar, Cambridge, MA). Most of the cultured cells (i.e., >90%) began to contract spontaneously within 24-48 h after they were plated (30-60 beats/min) and exhibited positive staining for pro-ANP (30), a precursor form of alpha -ANP and a specific cardiomyocyte marker. Confluent, spontaneously beating cells were used on day 3 of culture for all experiments.

Determination of ANP Secretion

For assessment of ANP release, six-well tissue culture plates containing confluent, spontaneously contracting cardiomyocyte monolayers were washed with 2 ml of Krebs-Ringer buffer containing 0.2% BSA and 0.2% glucose, as previously described (6). After the supernatant was replaced with 1 ml of fresh buffer, cells were incubated at 37°C for various times in the presence of various pharmacological agents, and aliquots of the supernatants were collected and assayed for ANP content according to Shenker et al. (27). Relative affinity of the anti-ANP antiserum for rat atriopeptides was as follows: 100% for human alpha -ANP and rat ANP (i.e., human alpha -[Ile18]ANP), 100% for rat atriopeptin III, 60% for rat ANP-(18-28), 5% for rat atriopeptin II, <0.001% for rat ANP-(13-28), and 0% for rat brain natriuretic peptide, rat C-type natriuretic peptide, ANP-(1-11), and rat atriopeptin I. The detection limit for ANP determinations was 5 pg/ml of incubation medium. The intra- and interassay coefficients of variation were estimated at 5 and 7%, respectively (n = 8). The ED50 for ANP tracer displacement occurred at 48 ± 5 pg (n = 5). Serial dilutions of the incubation media yielded results that paralleled those of synthetic standard.

Determination of cAMP Formation

For cAMP determinations, contracting cells were grown to confluency in six-well tissue culture plates, washed twice with 2 ml of Krebs-Ringer buffer, and incubated with various pharmacological agents at 37°C for 20 min in the presence or absence of 0.5 mM IBMX. cAMP was measured according to Harper and Brooker (9), with acetylation of the samples. Dioxan (70% vol/vol) was used for separation, and bound radioactivity was counted with an LKB multigamma counter. The limit of detection was 30 fmol. Specific binding ranged from 15.4 to 28.9%, and nonspecific binding ranged from 2.1 to 3.3%. Fifty percent tracer displacement occurred at 477.7 ± 53.3 (SE) fmol (n = 18). Intra- and interassay coefficients of variation were 9.5 and 12%, respectively.

Measurement of [Ca2+]c

Conventional fluorometry. [Ca2+]c was determined in confluent monolayers of spontaneously contracting cardiomyocytes grown on glass slides with use of the fluorescent Ca2+ probe fura 2-AM by an adaptation of the method described elsewhere (6). Briefly, the monolayers were washed twice with a modified Krebs-Ringer buffer (136 mM NaCl, 1.8 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 5 mM NaHCO3, 1.2 mM CaCl2, 0.21 mM EGTA, 20 mM HEPES, 5.5 mM glucose, and 1% BSA at pH 7.4), covered with 400 µl of modified Krebs-Ringer buffer containing 2 µM fura 2 and 0.5% BSA, and incubated for 20 min at 37°C. At the end of the loading period, the slides were washed twice with modified Krebs-Ringer buffer, inserted into glass cuvettes containing 3 ml of the same buffer, and placed in the thermostated holder of a Perkin-Elmer LS3 spectrophotometer. Continuous stirring was achieved by means of a magnetic stirrer. Fluorescence of fura 2-loaded monolayers was measured using an excitation wavelength of 340 nm and an emission wavelength of 505 nm. The signal was calibrated with ionomycin and MnCl2 in the presence of excess Ca2+, as described previously (6). Mean diastolic [Ca2+]c was estimated by averaging basal (resting) [Ca2+]c values for each trace over a 20-s interval before stimulation with the various pharmacological agents.

Dual-wavelength microfluorometry. Dual-excitation-wavelength microfluorometry was performed as previously described (30). Briefly, glass coverslips containing fura 2-loaded cells were mounted in a small chamber under an inverted Nikon Diaphot microscope and continuously superfused with BSA-free Krebs-Ringer buffer at 37°C. A micropipette was used to superfuse selected cells with forskolin. A Spex fluorometer was connected to the microscope (epifluorescence mode configuration) with the use of a dichroic mirror (model DM400) and an immersion lens (model F100, Nikon). Excitation at 340 and 380 nm from two separate sources was alternated using a rotating chopper mirror, and cell fluorescence was measured by means of a photomultiplier at 505 nm. Measurements of emitted fluorescence at each excitation wavelength were collected at 50-ms intervals and stored in separate files to permit ratio calculation, whereas calibration of [Ca2+]c was performed as described by Kem et al. (13). According to Kem et al., [Ca2+]c values should not be interpreted too strictly in terms of absolute concentrations because of various potential drawbacks, such as variable hydrolysis within the cell, dye sequestration, photobleaching, and intracellular Ca2+ buffering. However, in the present study we took care to establish a maximal fluorescent ratio (Rmax) under conditions that ensured maximal access of an elevated Ca2+ concentration to the intracellular content. To determine the minimum ratio (Rmin), cells were loaded with the intracellular nonfluorescent Ca2+ chelator MAPT by incubation in a Ca2+-free solution (<10 nM Ca2+) containing 25 µM MAPT-AM and 2 mM EGTA. Rmax was determined by the addition of 3 µM ionomycin and/or 0.2 µM digitonin to cells in a phosphate-free medium containing 10 mM Ca2+. To determine the proportionality constant (beta ), cells were prepared for Rmin measurements and then perfused with the solution used to obtain Rmax, but with excitation wavelengths at 360 nm (the isobestic point of fura 2) and 380 nm to allow correction for fura 2 loss from the cell during manipulations. The values for Rmin, Rmax, and beta  were 0.36 ± 0.17 (n = 6), 3.98 ± 0.61 (n = 5), and 2.13 ± 0.24 (n = 3), respectively.

Statistical Analysis

Student-Fisher unpaired bilateral t-tests and/or analysis of variance by Scheffé's F test criterion for unbalanced groups were used where applicable. P < 0.05 was accepted as statistically significant. Values are means ± SE of at least three separate experiments performed in duplicate or triplicate determinations.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effects of Forskolin and PGE2 on ANP Release and Cellular Contraction Frequency

As illustrated in Fig. 1, incubation of spontaneously contracting neonatal rat ventricular cardiomyocytes with 0.1 µM forskolin led to a rapid significant increase in ANP release from 0.49 ± 0.06 to 0.96 ± 0.12 ng/mg cell protein at 10 min and from 0.92 ± 0.09 to 1.73 ± 0.17 ng/mg cell protein at 20 min (P < 0.05, n = 4-5). Thereafter, forskolin-induced ANP release reached a plateau, whereas basal constitutive ANP production continued to increase linearly. Exposure of cardiomyocyte monolayers to 0.1 µM PGE2 led to a very similar response, increasing ANP secretion to 1.02 ± 0.09 ng/mg cell protein at 10 min and to 1.74 ± 0.12 ng/mg cell protein at 20 min (Fig. 1; P < 0.05, n = 4-5). As shown for forskolin, the stimulatory effect of PGE2 on ANP release reached a plateau within 30 min. Likewise, 0.5 µM forskolin induced a rapid transient increase in ANP secretion, although this response was markedly smaller than that promoted by 0.1 µM forskolin or 0.1 µM PGE2 (Fig. 1). Thus, in ventricular cardiomyocytes, we observed a basal constitutive and a regulated ANP secretion in response to forskolin and PGE2. These results are in agreement with those found previously in endothelin-stimulated cells (12).


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  		Fig. 1.   Forskolin (Fors)- and prostaglandin E2 (PGE2)-induced increases in cardiomyocyte atrial natriuretic peptide (ANP) secretion. Spontaneously beating cultured cardiomyocytes were incubated at 37°C with 0.1-10 µM forskolin or 0.1 µM PGE2 for indicated times, and ANP release was determined. Values are means ± SE of 4-7 experiments performed in triplicate determinations. * P < 0.05 vs. corresponding control.

Incubation of spontaneously contracting cardiomyocyte monolayers with a higher, 1 µM regimen of forskolin slightly increased ANP secretion at 10 min, whereas it had a small inhibitory effect at 30 min. Exposure of cells to a very high concentration of 10 µM forskolin led to the lack of a significant effect on ANP release at 10 min, with the absence of response being followed by a massive inhibition of basal ANP secretion at 30 min (48 ± 6% inhibition, P < 0.05, n = 4).

Interestingly, the stimulatory as well as the inhibitory effects of forskolin on ANP secretion were accompanied by nearly identical effects on cellular contraction frequency. As shown in Fig. 2, determination of cellular contraction frequency by direct microscopic observation of the cell monolayers indicated that forskolin- and PGE2-induced ANP secretion were accompanied by marked increases in monolayer contraction frequency. Incubation of cells for 10 min with 0.1 µM forskolin led to a near doubling of the number of monolayer contractions per unit time (from 42 ± 5 to 77 ± 5 beats/min, P < 0.05, n = 4-5), a response that was followed by a decrease in contraction frequency thereafter. Exposure of cardiomyocytes to 0.5 µM forskolin led to a similar, albeit slightly smaller, increase in contraction frequency at 10 min. Incubation of cells with 0.1 µM PGE2 led to a similar response, with PGE2 promoting a maximal increase in contraction frequency at 20 min (from 43 ± 5 to 74 ± 6 beats/min, P < 0.05, n = 4-5), which dropped to values comparable to basal levels at 30 min (54 ± 6 beats/min, n = 5).


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  		Fig. 2.   Forskolin- and PGE2-induced increases in cardiomyocyte contraction frequency. Spontaneously beating cultured cardiomyocytes were incubated at 37°C with 0.1-10 µM forskolin or 0.1 µM PGE2 for indicated times, and cell contraction frequency was determined by direct microscopic observation. Values are means ± SE of 4-7 experiments performed in triplicate determinations. * P < 0.05 vs. corresponding control.

As illustrated in Fig. 2, exposure of spontaneously contracting cardiomyocytes to a higher concentration of forskolin, i.e., 1 µM, slightly increased cell contraction frequency at 10 min (38 ± 4% increase), whereas it significantly inhibited cell contractions at 30 min (52 ± 6% inhibition, P < 0.05, n = 4). Incubation of spontaneously contracting monolayers with a very high regimen of 10 µM forskolin promoted a decrease in cellular contraction frequency that was apparent within 20 min of application (62 ± 4% inhibition, P < 0.05, n = 4), and cell contractions were fully abolished within 10 min thereafter. Microscopic observation of the monolayers indicated that the cellular arrest induced by 10 µM forskolin was preceded by the cardiomyocytes taking on stellate appearances and beating asynchronously in a fibrillatory manner (data not shown).

Effects of Forskolin and PGE2 on cAMP Production

Forskolin and PGE2 have previously been shown to stimulate cAMP formation in spontaneously beating neonatal rat cardiomyocytes (6). In view of the marked correlation between ANP secretion and the increase in contraction frequency promoted by both of these agents, we further investigated the time course of forskolin- and PGE2-induced cAMP formation in these cells. As shown in Fig. 3A, incubation of cardiomyocytes with 0.1 µM forskolin or 0.1 µM PGE2 in the presence of the phosphodiesterase inhibitor IBMX at 0.5 mM led to rapid six- to sevenfold increases in cellular cAMP formation. The effects were maximal within 20-30 min, with cAMP production increasing at 30 min from 0.92 ± 0.16 to 5.45 ± 0.54 and 5.92 ± 0.49 nmol/mg protein for 0.1 µM forskolin and 0.1 µM PGE2, respectively (Fig. 3A; P < 0.05, n = 3-4). Similar stimulatory effects could also be observed in the absence of IBMX, although they were reduced to three- to fourfold increases in this instance (Fig. 3B; from 0.26 ± 0.04 to 1.09 ± 0.10 and 1.08 ± 0.09 nmol/mg protein for 0.1 µM forskolin and 0.1 µM PGE2, respectively, at 30 min, P < 0.05, n = 3-4). A higher regimen of 0.5 µM forskolin, while leading to submaximal stimulation of ANP release and cell contraction frequency (Figs. 1 and 2), promoted a markedly higher cAMP production, leading within 30 min to an 18- and 12-fold increase in the presence and absence of IBMX, respectively.


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  		Fig. 3.   Effect of forskolin and PGE2 on cAMP formation in rat cardiomyocytes. Spontaneously beating cultured cardiomyocytes were incubated at 37°C for indicated times with 0.1-10 µM forskolin or 0.1 µM PGE2 in presence (A) or absence (B) of 0.5 mM 3-isobutyl-1-methylxanthine and assayed for cAMP formation. Values are means ± SE of 3-5 experiments performed in duplicate. * P < 0.05 vs. corresponding control.

Forskolin- and PGE2-induced increases in ANP release could not be simultaneously determined with cAMP formation, inasmuch as 0.5 mM IBMX stimulated basal ANP secretion after 10-20 min of incubation but inhibited agonist-induced ANP release as well as basal secretion after 30 and 60 min of incubation (results varied considerably, data not shown). Surprisingly, although forskolin (0.1 and 0.5 µM)- and PGE2 (0.1 µM)-induced cAMP formation was positively correlated with increases in cellular contraction frequency and ANP release during the first 10 min of incubation in both cases (Fig. 1), the cAMP responses diverged from the latter during incubation periods exceeding 20 min.

The divergence between the time-dependent inhibitory effect of 0.1 µM forskolin on ANP release and the sustained effect of this agent on cellular cAMP production suggests that relatively small increases in cAMP formation are able to promote ANP secretion in this system, whereas higher levels of cAMP formation are likely to inhibit this process. Consistent with this hypothesis, incubation of spontaneously contracting cardiomyocyte monolayers with higher 1 and 10 µM regimens of forskolin prevented the ANP release observed with lower concentrations of forskolin (Fig. 1). Exposing contracting cardiomyocytes to a very high regimen of 10 µM forskolin not only led to the lack of a significant effect on ANP release at 10 min (Fig. 1) but also resulted in a marked inhibition of basal, constitutive ANP secretion at 30 min (49 ± 6% inhibition, P < 0.05, n = 4).

As shown in Fig. 3, the forskolin-induced inhibition of cellular contraction and ANP secretion was accompanied by a massive increase in cellular cAMP production. Incubation of spontaneously contracting cardiomyocytes with 1 and 10 µM forskolin in the presence of 0.5 mM IBMX led to a 26- and 43-fold increase in cellular cAMP formation, respectively, a response that was maximal within 20-30 min, increasing at 30 min from 0.92 ± 0.16 to 23.12 ± 1.23 nmol/mg protein for 1 µM forskolin and to 39.43 ± 3.34 nmol/mg protein for 10 µM forskolin (P < 0.05, n = 3-4). As shown for the lower regimen of forskolin, the cAMP response was also evident in the absence of IBMX, although values were reduced to 20-25% of those in the presence of IBMX (Fig. 3B; increasing at 30 min for 1 and 10 µM forskolin from 0.26 ± 0.04 to 3.79 and 7.83 ± 0.42 nmol/mg protein, respectively, P < 0.05, n = 3-4). In contrast to the experiments performed with the lower regimen of forskolin, the massive cAMP production induced by 10 µM forskolin appeared to be directly correlated with the inhibition of ANP secretion and cellular contraction frequency (Figs. 1 and 2).

Effect of Forskolin on Cardiomyocyte [Ca2+]c

The effects of cAMP on cardiomyocyte ANP secretion have been attributed to a cAMP-dependent, Ca2+ influx-related event that is at the basis of forskolin-induced ANP release (6, 10). We further investigated the effect of low (0.1 µM) and high (10 µM) regimens of forskolin on cardiomyocyte [Ca2+]c. As shown by the traces in Fig. 4, which are representative of results obtained in four to five separate cultures, Ca2+ fluorometry of cell populations with the Ca2+-sensitive fluorescent probe fura 2 revealed that cultured, spontaneously beating neonatal rat ventricular cardiomyocytes behaved much like a syncytium, such that each contraction of the monolayer was accompagnied by a single Ca2+ transient or "spike" displaying a mean basal (resting) diastolic value of 127 ± 18 nM, a mean systolic (contracted) value of 246 ± 35 nM, and a mean amplitude of 117 ± 16 nM (Fig. 4, left traces; n = 4).


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  		Fig. 4.   Effect of 0.1 and 10 µM forskolin on cardiomyocyte free cytosolic Ca2+ concentration ([Ca2+]c). Spontaneously beating cardiomyocyte monolayers were grown on glass slides, and [Ca2+]c was determined using fluorescent probe fura 2. Traces represent spontaneous Ca2+ transients accompanying cellular contractions (left) and effects of 0.1 and 10 µM forskolin on cardiomyocyte [Ca2+]c (right). Traces are representative of results obtained in 4-5 separate cell cultures.

As illustrated in Fig. 4, top, a 0.1 µM regimen of forskolin increased the frequency of spontaneous Ca2+ transient occurrence from 31 ± 5 to 56 ± 7 transients/min within 5 min of application (P < 0.05, n = 4). Although no effect was observed on Ca2+ transient amplitude, the 0.1 µM regimen of forskolin also promoted a slight sustained but not significant increase in basal diastolic [Ca2+]c (26 ± 8% increase, n = 4). As shown in a previous report from our laboratory (6), very similar results were obtained with 0.1 µM PGE2, although with 0.1 µM PGE2 promoting no changes in basal diastolic [Ca2+]c. In contrast, when the effects of a higher regimen of forskolin were studied, 10 µM completely abolished all Ca2+ transients associated with spontaneous monolayer contraction within 5 min of application (Fig. 4, bottom; n = 5). Except for slight differences in the time at which these stimulatory and inhibitory effects were observed, Ca2+ fluorometry data supported what was previously seen when cell contraction was assessed by visual determination. Similar to our observations with 0.1 µM forskolin, a high regimen of forskolin (10 µM) also induced a small sustained but not significant increase in diastolic [Ca2+]c, as measured in confluent monolayers by fura 2 fluorometry (28 ± 9% increase, n = 4). An intermediate regimen of 1 µM forskolin disrupted the synchronous contractions of cardiomyocyte monolayers, resulting in very variable contraction frequency before completely suppressing cell contractions (data not shown). Thus we measured the effect of 1 µM forskolin on [Ca2+]c in a single beating, fura 2-loaded cardiomyocyte (Fig. 5). In this case, we observed that cardiomyocytes, after showing a rapid and transient increase in cell contraction frequency, ceased beating when exposed to 1 µM forskolin for >10-15 min (n = 3), a situation accompanied by a small sustained but not significant increase in [Ca2+]c, as illustrated by the trace in Fig. 5, which is representative of results obtained in three experiments (31 ± 11% increase, n = 3).


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  		Fig. 5.   Effect of 1 µM forskolin on [Ca2+]c in a single spontaneously beating cardiomyocyte. [Ca2+]c was measured by dual-wavelength fura 2 microfluorometry. Horizontal bar, superfusion with 1 µM forskolin. Traces represent results obtained in 3 separate cell cultures.

Effect of Forskolin and PGE2 on ANP Release in Quiescent Cardiomyocytes

The preceding data indicate that forskolin and PGE2 promote significant ANP secretion in instances where these agents induce modest three- to fourfold increases in cellular cAMP formation that are accompanied by marked increases in cellular contraction frequency. The fact that a high regimen of forskolin simultaneously abolishes monolayer contraction and markedly inhibits basal ANP secretion suggests that the agonist-induced increase in cellular contraction frequency is an important determinant for the cAMP-dependent ANP release in this system. We further tested the effects of 0.1 µM forskolin and 0.1 µM PGE2 on ANP secretion in noncontracting cells treated with 1 µM Rya, 1 µM TTX, 1 µM Nif, or 10 mM BDM, as shown in Fig. 6. Preincubation of cardiomyocytes with 1 µM Rya, 1 µM TTX, 1 µM Nif, or 10 mM BDM for 15 min inhibited cell contractions by 90-100% yet had no effect on basal ANP secretion in these cultures (P < 0.05, n = 8-9). However, pretreatment with Rya or TTX reduced forskolin-induced ANP secretion by 79 ± 9 and 81 ± 7% and PGE2-stimulated ANP release by 67 ± 7 and 69 ± 8%, respectively (P < 0.05, n = 8-9). Abolition of cell contraction with 1 µM Nif or 10 mM BDM led to similar, although more powerful, inhibitory effects, inasmuch as forskolin- and PGE2-induced ANP secretion were fully abolished by the Ca2+ channel blocker Nif or by BDM, known to uncouple membrane depolarization and Ca2+ release from mechanical contraction (Fig. 6). Consequently, it appears that substantial portions (60-80%) of forskolin- and PGE2-induced ANP secretion are indeed mediated by a cAMP-dependent modification of myocyte contraction in this system, a conclusion in agreement with the fact that forskolin promotes ANP release in contracting atrial tissue, but not in quiescent cardiomyocytes (18).


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  		Fig. 6.   Inhibition of forskolin (FSK)- and PGE2-induced ANP release by ryanodine (Rya), tetrodotoxin (TTX), nifedipine (Nif), and butanedione monoxime (BDM). Spontaneously beating cultured cardiomyocytes were pretreated for 15 min with 1 µM Rya, 1 µM TTX, 1 µM Nif, or 10 mM BDM added to cell culture medium. Thereafter, cells were washed and then stimulated with 0.1 µM forskolin or 0.1 µM PGE2 in presence of same cell-arresting agents (Rya, TTX, Nif, or BDM) at 37°C for 15 min, and ANP release was determined. Rya, TTX, Nif, or BDM treatment of these preparations inhibited cellular contraction by 90-100%. Values are means ± SE of 6-11 experiments performed in triplicate determinations. * P < 0.05 vs. corresponding control. + P < 0.05 vs. corresponding maximal stimulation.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Taken together, our results indicate that forskolin and PGE2 promote ANP secretion in spontaneously beating neonatal rat ventricular cardiomyocytes via a cAMP-dependent modification of cellular contraction frequency. In support of this hypothesis, the ANP release-stimulating effects of 0.1 µM forskolin and PGE2 were accompanied by three- to fourfold increases in basal cAMP production and a near doubling of the rate of spontaneous monolayer contraction and Ca2+ transient occurrence. Conversely, forskolin- and PGE2-induced ANP secretion were strongly reduced or failed to occur in quiescent cardiomyocytes preincubated with Rya, TTX, Nif, or BDM, indicating that cellular contraction plays a crucial role in cAMP-induced myocardial ANP release. Interestingly, a 10 µM regimen of forskolin, promoting an ~30-fold increase in cellular cAMP formation, led to the suppression of cell contraction, Ca2+ transient occurrence, and ANP release. In this view, it appears that cAMP-mediated ANP secretion has a low threshold of activation in cultured neonatal rat ventricular cardiomyocytes, inasmuch as relatively minor increases in cAMP formation clearly promote ANP release, whereas major increases abolish spontaneous contraction and ANP secretion altogether.

Overall, these findings are consistent with previous observations indicating that the cAMP-elevating agents forskolin and PGE2 promote ANP secretion in spontaneously contracting, cultured neonatal rat ventricular cardiomyocytes (6) and further appear to resolve a number of questions related to the effects of cAMP on myocardial ANP release. Indeed, it has previously been demonstrated that cAMP-elevating agents and mimetics such as norepinephrine, calcitonin gene-related peptide, and dibutyryl cAMP promote significant ANP secretion in the paced adult rat heart, paced atria, atrial slices, and/or dispersed rat atrial cells (2, 8, 19, 22, 23, 26, 33), suggesting that cAMP is a positive stimulus for myocardial ANP secretion. Nevertheless, studies investigating the effect of forskolin, phosphodiesterase inhibitor, or beta -adrenergic receptor stimulation in cardiomyocyte cultures have clearly shown that increases in cellular cAMP formation promote a decrease in ANP release (1, 10, 11, 28), suggesting that cAMP is actually an inhibitor of ANP secretion in isolated cardiomyocyte systems. These contradictory results obtained in various experimental models have been attributed to numerous causes, including cAMP-dependent Ca2+ influx (10), electrical membrane activity (24), and differences in contraction frequency, contractile resting tension, and/or contractile developed tension in each setting (18, 22).

The present study clearly demonstrates that the stimulatory and inhibitory effects of cAMP on myocardial ANP secretion can be observed in spontaneously beating rat cardiomyocyte preparations and further indicates that the observation of one or the other of these conflicting responses is primarily due to the concentration-dependent effect(s) of cAMP on cellular contractile function. Indeed, when cardiomyocytes were incubated with relatively low (0.1 µM) concentrations of forskolin and PGE2, ambient cAMP levels rose to three to six times basal levels within 10 min in the absence or presence of IBMX, responses that were associated with increases in contraction frequency and "burstlike" releases of ANP. A similar acute ANP release characteristic of regulated secretion has previously been shown for endothelin-induced ANP response in neonatal rat ventricular cardiomyocytes (12).

When a higher regimen of forskolin was administered (10 µM), ambient cAMP levels rose to 17-21 times basal levels within 10 min in the absence or presence of IBMX, and the cardiomyocytes underwent dramatic changes in cell morphology, stopped contracting, and ceased secreting ANP altogether. Confirming the positive and the negative effects of increased cAMP formation, 1 µM forskolin, which induced a ninefold increase in cAMP formation, led to a rapid transient increase in cellular contraction frequency and ANP release followed by a complete suppression of cell contractions that was accompanied by abolition of the ANP response. Interestingly, prolonged exposure of cardiomyocytes to high concentrations of forskolin not only prevented the burstlike regulated ANP release but also inhibited constitutive basal ANP secretion. However, further experiments are required to elucidate the mechanism of this inhibition.

The time courses of ANP release and cell contractions, associated with application of the lower regimen of forskolin (0.1 µM), suggest that it is not the sustained elevation in contraction frequency itself that is at the basis of ANP secretion in this system but, rather, the transition from the slowly beating basal state to the rapidly beating stimulated one. Consistent with this concept, cardiomyocytes failed to release additional ANP after the first 10 min of incubation with 0.1 µM forskolin and 0.1 µM PGE2, despite the fact that both agents promoted increases in contraction frequency for up to 20 min. Conversely, the time course of inhibition of ANP release by 10 µM forskolin was remarkably similar to the effect of 10 µM forskolin on cellular contraction frequency. In agreement with these observations, cAMP-induced ANP secretion did not occur in cells arrested with Rya, TTX, Nif, or BDM, whereas basal ANP secretion was the same as in contracting cardiomyocytes, suggesting that increased cellular contraction frequency plays an important role in the agonist-stimulated, regulated ANP release (i.e., release of ANP from pro-ANP-containing cytoplasmic granules) but not in the constitutive ANP secretion (i.e., secretion occurring soon after ANP formation). In summary, it appears that agonist-induced ANP secretion invariably parallels agonist-induced modifications of basal contraction frequency in cultured spontaneously beating ventricular cardiomyocytes, a conclusion consistent with previous observations indicating that ANP secretion is associated with increases in contraction frequency in other experimental models (1, 3, 16, 24, 25). In cultured ventricular cardiomyocytes, prolonged electrical stimulation of contraction was shown to induce increases in the expression of the cardiac genes ANP and myosin light chain-2 (15).

Although our results shed light on the reasons for which cAMP-elevating agents have been shown to stimulate (2, 6, 8, 23, 26, 30, 33) and inhibit myocardial ANP secretion (1, 10, 11, 28), the mechanism(s) by which cAMP promote(s) these opposite effects on ANP release remains to be fully elucidated. In this context, the effect of cAMP on cardiomyocyte contraction frequency and resulting ANP secretion may be closely related to the effect of this cyclic nucleotide on Ca2+ influx, inasmuch as it has previously been shown that the inhibition of cAMP-induced Ca2+ influx abolishes cAMP-induced ANP release in spontaneously beating cardiomyocytes (6), whereas Ca2+ channel blockade inhibits ANP secretion elicited by increases in contraction frequency in electrically paced perfused rat atria (24). Overall, this suggests that Ca2+ influx is a major factor underlying the effects of cAMP on ANP release, and consistent with this suggestion, it has previously been shown that Ca2+ channel blockade actually counteracts the cAMP-mediated inhibition of ANP release in nonbeating systems, where this is the prevalent response (10). Reinforcing the hypothesis that Ca2+ influx could also be involved in the inhibitory effects of cAMP on cell contraction and ANP secretion, spontaneously beating cardiomyocytes exposed to 1 µM forskolin for 10 min assumed noncontracting stellate conformations associated with a sustained increase in [Ca2+]c, as measured by single-cell fura 2 Ca2+ microfluorometry. In this case, the cells appear to be in a hypercontracted state unfavorable for the liberation of secretory granules.

Although our experiments using BDM further confirm the importance of cell contractions for regulated ANP release, the role of [Ca2+]c remains less clear. We exposed spontaneously beating cardiomyocytes to 10 mM BDM, which is known to greatly depress contractility but appears to affect only slightly [Ca2+]c (4, 17). We observed that cell contractions were inhibited by 90-100% and that the ANP release induced by 0.1 µM forskolin and 0.1 µM PGE2 was completely suppressed, whereas the inhibitory effect of 10 µM forskolin was not significantly affected. Thus our observations suggest that, in ventricular cardiomyocytes, contractile activity is more important for ANP release than [Ca2+]c. However, in this context, observations concerning the effects of BDM on [Ca2+]c are controversial. Indeed, although BDM was found to reduce [Ca2+]c in isolated cardiomyocytes and heart muscle (4, 17), it was also shown to strongly increase Ca2+ transients in isolated papillary muscles from guinea pig heart (14). Clearly, further investigations are necessary to determine the exact mechanisms underlying the cAMP-mediated stimulation and inhibition of ANP release.

In summary, the present study indicates that cAMP is a positive and a negative modulator of ANP secretion in cultured neonatal rat ventricular cardiomyocytes, a dual effect that appears to be mediated by the susceptibility of the cell's secretory machinery to the cytosolic concentration of this cyclic nucleotide. In the normal setting where cells are subject to rhythmically occurring spontaneous contraction, relatively minor (3- to 4-fold) increases in cAMP levels promote significant ANP release, a response that is associated with a marked increase in cellular contraction frequency and fails to occur in arrested preparations. However, when cytosolic cAMP is increased to substantially higher (15- to 20-fold) levels, cellular contraction and ANP secretion are arrested altogether. Although these results do not allow one to draw firm conclusions as to the physiological relevance of a direct action of cAMP-elevating agents on myocardial ANP secretion, they do suggest that PGE2 has direct myocardium-stimulating properties that should be considered in cases of heart failure with elevated levels of circulating prostaglandin. Furthermore, the dual and opposing concentration-dependent effects of cAMP on ANP release observed in this study serve to explain a number of previously conflicting reports on the effect of cAMP on myocardial ANP secretion.


    ACKNOWLEDGEMENTS

We thank C. Gerber-Wicht, M. Rey, and M. Klein for excellent technical assistance.


    FOOTNOTES

This study was supported by Swiss National Science Foundation Grants 31-42295.94 and 31-52935.97 and a grant from the Swiss Foundation of Cardiology.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: U. Lang, Div. of Endocrinology, University Hospital, CH-1211 Geneva 14, Switzerland (E-mail: langu{at}cmu.unige.ch).

Received 24 August 1998; accepted in final form 12 August 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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