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Cardiovascular Research 1998 37(3):718-728; doi:10.1016/S0008-6363(97)00245-9
© 1998 by European Society of Cardiology
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Copyright © 1998, European Society of Cardiology

Stimulation of P2Y receptors activates c-fos gene expression and inhibits DNA synthesis in cultured cardiac fibroblasts

Jing-Sheng Zhenga, Lydia O'Neilla, Xilin Longa, Tania E Webbb, Eric A Barnardb, Edward G Lakattaa and Marvin O Boluyta,*

aLaboratory of Cardiovascular Science, Gerontology Research Center, National Institute on Aging, National Institute of Health, Baltimore, MD 21224, USA
bMolecular Neurobiology Unit, Division of Basic Medical Sciences, Royal Free Hospital School of Medicine, Rowland Hill Street, Hampstead, London, NW3 2PF, UK

* Corresponding author. Present address: The University of Michigan, Kinesiology; Dept. of Movement Science, 401 Washtenaw Avenue, Ann Arbor, MI 48109-2214. Tel. (+1-313) 647 7645; Fax (+1-313) 936 1925; E-mail: boluytm@umich.edu

Received 15 April 1997; accepted 12 September 1997


   Abstract
Top
 Abstract
1 Introduction
 2 Materials and methods
 3 Results
 4 Discussion
 References
 
Objectives: The aims of this study were to determine (1) whether neonatal rat cardiac fibroblasts (CAFB) express P2Y receptors; (2) whether CAFB respond to extracellular ATP by inducing expression of c-fos mRNA; and (3) whether extracellular ATP modulates norepinephrine (NE)-stimulated cell growth in CAFB. Methods: Expression of P2Y1 and P2Y2 receptors and induction of c-fos were examined by Northern blot analysis. CAFB growth was assessed by measuring [3H]thymidine incorporation and DNA content. P2Y receptor pharmacology was studied using various ATP analogues. Results: Northern blot analysis of polyA enriched RNA confirmed that at least 2 subtypes of P2Y receptors (P2Y1 and P2Y2) are expressed in cultured CAFB. Extracellular ATP induced the expression of c-fos mRNA through a pathway that was sensitive to inhibitors of protein kinase C (PKC), but not to inhibitors of intracellular Ca2+ signaling. Extracellular ATP inhibited the NE-stimulated increases in DNA content and in [3H]thymidine incorporation into DNA. Whereas the potency order for stimulation of c-fos expression was ATP = UTP > ADP > adenosine, the potency order to inhibit the NE-induced increase of [3H]thymidine incorporation into DNA was ATP > ADP > UTP > adenosine. Conclusions: These data demonstrate that CAFB express both P2Y1 and P2Y2 receptor mRNA and that CAFB respond to P2Y receptor stimulation by induction of c-fos and inhibition of DNA synthesis. These findings suggest that the effects of ATP on [3H]thymidine incorporation into DNA and on expression of c-fos mRNA are exerted via distinct P2Y receptor subtypes.

KEYWORDS P2Y receptor; Rat cardiac fibroblast; Cell proliferation; c-fos


   1 Introduction
Top
 Abstract
 1 Introduction
2 Materials and methods
 3 Results
 4 Discussion
 References
 
The heart is composed of cardiac myocytes and non-myocytes including fibroblasts, vascular smooth muscle cells, and endothelial cells. Cardiac fibroblasts (CAFB) account for about two thirds of the total cell population in the heart and are the primary source of collagen and fibronectin which are major components of the extracellular matrix [1, 2]. The matrix connects cardiac myocytes to one another and maintains myocyte alignment during the cardiac cycle. CAFB influence myocyte growth and function by elaborating matrix and by secreting growth factors that act in a paracrine manner [2–4]. It is therefore important to identify and understand factors that influence CAFB gene expression and growth.

ATP is commonly conceptualized as the currency of intracellular energy metabolism. Evidence gathered over the last two decades, however, has established a role for ATP outside the cell as an intercellular messenger as well [5, 6]. Extracellular ATP exerts its effects by binding to two classes of P2 receptors (formerly referred to as P2-purinergic), those coupled to G-proteins (P2Y) and those that are intrinsic ion channels (P2X) [7, 8]. At least seven P2X [9]and seven P2Y receptor subtypes have been cloned. In the P2Y family, P2Y1 is a purine-specific receptor first isolated from a chick brain [10], that has a wide tissue distribution. UTP and ATP are equipotent at the P2Y2 receptor [11], which is also widely distributed among mammalian tissues. The P2Y3, P2Y4, and P2Y5 receptors each exhibit distinctive agonist potency orders and are not widely distributed [7, 12–16]. The P2Y6 receptor exhibits an agonist potency order that favors UTP over ATP and has a wide tissue distribution [17, 18]. The P2Y7 receptor has an agonist potency order favoring ATP and ADP over UTP and is widely distributed as well [19]. Thus far, expression of the P2Y1 P2Y2, P2Y4, P2Y6, and P2Y7 has been demonstrated in heart tissue [20].

Upon nerve stimulation ATP is co-released with norepinephrine (NE) from the sympathetic nerve terminals in the heart [5]. Under certain conditions, ATP (and UTP) can be released by platelet degranulation, and from anoxic myocytes or damaged cells [6, 21–23]. In addition to ATP, other related nucleotides capable of acting on P2 receptors may also be produced locally by the above-mentioned mechanisms. By binding to various P2 receptors, extracellular ATP (and its congeners) can act on a number of intracellular effector systems and can thereby elicit diverse responses in the heart, including induction of inward cation currents, activation of the Cl–/HCO3– exchanger, elevation of intracellular Ca2+ concentration, enhancement of phosphotidylinositol turnover, and reduction of the cAMP concentration [24–29]. We have recently shown that extracellular ATP, like NE and many other hypertrophy-stimulating agents, induces expression of the immediate early genes c-fos, and junB in cultured neonatal cardiac myocytes [26]. While NE induces hypertrophic growth of neonatal cardiac myocytes, however, ATP inhibits NE-stimulated hypertrophy of myocytes [30]. Taken together, these findings demonstrate the potential for extracellular ATP to influence heart function and growth by virtue of its effects on cardiac myocytes. Whether or not CAFB are influenced by P2 receptor stimulation is not known.

This study was undertaken to determine whether CAFB possess the necessary requirements to be influenced by extracellular ATP. Initial results indicated that CAFB express at least two P2 receptor subtypes and that extracellular ATP causes a rapid transient induction of c-fos in CAFB. We therefore proceeded to investigate signaling characteristics of the CAFB response to ATP and to determine whether ATP affects fibroblast growth.


   2 Materials and methods
Top
 Abstract
 1 Introduction
 2 Materials and methods
3 Results
 4 Discussion
 References
 
All cells used in this study were derived from Wistar rats obtained from the colony maintained at the Gerontology Research Center. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH Publication No. 85-23, revised 1985).

2.1 Preparation of neonatal CAFB and neonatal cardiac myocytes
Neonatal CAFB were isolated as previously described [26, 30, 31]with some modifications. Hearts from 1–2 day old Wistar rats were removed, the ventricles were separated, then trisected and digested with collagenase type II (0.5 mg/ml, Worthington) and pancreatin (0.6 mg/ml, Sigma) for 20 min at 37°C. The cell supernatant was collected by centrifugation and the pellet resuspended in 100% horse serum. The above steps were repeated 7–10 times until the hearts were completely digested. The cells were combined, washed and centrifuged through a discontinuous Percoll gradient of 1050, 1062 and 1082 g/ml, respectively. The band at the 1062/1082 interface was used for isolating total myocyte RNA directly. The band at the top of the 1050 layer was used for CAFB culture. After three passages with trypsin, fibroblasts were resuspended in culture medium containing 4:1 Dulbecco's modified Eagle's medium (DMEM)/medium 199 (M199; GIBCO Laboratory, NY) supplemented with 10% horse serum, 5% fetal bovine serum and penicillin (100 unit/ml)/streptomycin (100 µg/ml, GIBCO Laboratory) at 37°C for 24 h in humidified air with sufficient CO2 to maintain the pH at 7.3. The medium was then changed to serum-free DMEM/M199 medium. All experiments were initiated 24 h after the change to serum-free culture medium.

2.2 Adult myocyte preparation
Adult rat cardiac myocytes were isolated using the procedure described previously by Janczewski and Lakatta [32]. Briefly, rats were anesthetized with pentobarbitone sodium (50 mg/kg), the heart was removed and perfused retrogradely with a nominally Ca2+-free bicarbonate buffer at 37°C for 5 min. The perfusate was then switched to a Ca2+-free bicarbonate solution containing collagenase (1 mg/ml) and protease (0.04 mg/ml). After perfusion with this medium for 10 min, the ventricles were cut off and disaggregated. Single myocytes were then rinsed in a bicarbonate solution containing 0.25 mM CaCl2, and finally resuspended in HEPES buffer containing 1.0 mM CaCl2.

2.3 Northern blot analysis for c-fos and P2Y1 and P2Y2 studies
Total RNA was isolated from cultured CAFB as described by Chirgwin and coworkers [33]. Poly(A+) RNA was prepared from total RNA using oligo(dT) cellulose chromatography (Pharmacia, Sweden). After denaturation with formamide and formaldehyde, equal amounts of RNA (10 µg/lane) were size fractionated by electrophoresis through a 1% agarose gel containing 3% formaldehyde. RNA was electrophoretically transferred to a nylon (Duralon) membrane at 5 V/cm, crosslinked by ultraviolet irradiation (120 mJ) and then hybridized at 62°C with either a Formula -radiolabeled cDNA probe for rat c-fos [34], or at 60°C with an end-labeled oligonucleotide to ribosomal 18S as described by Church and Gilbert [35]. Membranes were washed twice with low stringency wash at the hybridization temperature for 30 min each, and 3 times with high stringency wash at the hybridization temperature for 20 min each. Hybridization intensity was quantified in disintegrations per min directly from blots using a Betascope 603 (Betagen Corp., Waltham, MA). Signals obtained in this manner are linear and non-saturable. The signal from each sample was normalized to the signal obtained with a synthetic oligonucleotide probe specific for the 18S ribosomal RNA. For P2Y1 or P2Y2 studies, poly(A)+ RNA prepared from CAFB, neonatal myocytes and adult myocytes was separated by formaldehyde/1.0% agarose gel electrophoresis and transferred to a nylon membrane. Membranes containing polyA+ enriched RNA were hybridized with the P2Y1 and P2Y2 cDNA probes at 42°C in 50% formamide, %X Denhardt's solution, 5xSSPE and 0.1% SDS overnight [34]. Membranes were washed with 2xSSC and 0.1% SDS once at 22°C for 30 min and twice at 50°C for 30 min each. Membranes containing total RNA were hybridized at 63.5°C with P2Y1 and P2Y2 cDNA probes overnight and washed at the hybridization temperature as described [35].

2.4 Probes
A 531 bp fragment of the rat P2Y1 receptor encoding cDNA [36]was generated by RT-PCR from rat brain RNA using 27-mer oligonucleotide primers of the following sequence: sense primer 5'-TGCATCAGTGCACAGAGGTACAGTGGC-3', antisense primer 5'-GGTCCACACAGCTGTTGAGACTTGCTA-3'. Similarly, a 514 bp fragment of cDNA encoding the rat P2Y2 receptor [37]was amplified using oligonucleotide primers of the following sequence: sense primer 5'-AGCATCCTCTTCCTCACCTGCATCAGC-3', antisense primer 5'-CGGGTGATCTTATACGCCATGTTGATG-3'. Amplification products were subcloned into the pCRII vector (In Vitrogen) and verified by complete sequencing of the inserts.

The probe used to detect c-fos mRNA, which was generously provided by Michael T. Crow, was an approximately 500 bp Formula -ATP labelled cDNA synthesized by the vector pCRcfos(500) by in vitro RNA transcription [38]. pCRcfos(500) Encodes approximately 500 bp of the conserved coding region of the rat c-fos cDNA and was generated by a polymerase chain reaction using primers complementary to the published sequence [39]and cloned into the vector pCRII according to the manufacture's instructions (In Vitrogen Corp, San Diego, CA). The complete sequence of the cDNA insert derived by PCR was verified by the dideoxy-mediated chain-termination method [40]. The probe for 18S RNA was a synthetic end-labeled oligonucleotide as previously described [41].

2.5 Measurement of intracellular Ca2+ concentration
Neonatal CAFB were cultured on coverslips coated with gelatin. Before loading with indo-1 AM, the incubation solution was changed from DMEM/M199 to an N-2-hydrozyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffer solution containing (in mM) 137 NaCl, 5.4 KCl, 1.5 CaCl2, 1.2 MgSO4, and 20 HEPES at pH 7.4. CAFB were then loaded with indo-1 AM (25 µM) for 20–30 min at 37°C. Indo-1 fluorescence was excited at 350 nm. Paired photomultipliers collected indo-1 emission by simultaneously measuring spectral windows of 391–434 and 457–507 nm selected by bandpass interference filters [42]. Since indo-1 AM may be partially hydrolyzed and the Ca2+ indicator may be compartmentalized in mitochondria or other organelles, calibration of intracellular Ca2+ concentration (Cai) in neonatal CAFB remains uncertain. The 410/490 nm ratio of emitted fluorescence was taken as an index of intracellular Ca2+ concentration, as described previously [42]. Cai measurements were performed at room temperature.

2.6 [3H]-Thymidine incorporation into DNA and measurement of DNA content
Total DNA content and [3H]-thymidine incorporation into DNA were assayed as parameters of cell growth and proliferation. CAFB were grown to 50% confluence in 6-well plates in the plating medium. 24 H before experiments, cells were provided with serum-free maintenance medium. Serum-starved CAFB were then supplemented with 1 µCi [3H]thymidine in the absence (control) or in the presence of ATP, NE, or serum for 24 h. At the end of the labeling periods, radiolabeled medium was aspirated, and washed two times each with ice-cold PBS and 10% TCA. Cells were incubated with 10% TCA on ice for at least 1 h. Cell precipitates were then solubilized in 0.5% NaOH/1% SDS for 1 h. Radioactivity was determined by liquid scintillation counting. DNA content was measured fluorometically using PicoGreen dsDNA Quantitation Reagent (Molecular Probes, Inc.) by a procedure described by Rye et al. [43], with calf thymus DNA as a standard.

2.7 Statistics
Data were expressed as mean±SE. For mRNA experiments, the control group was arbitrarily assigned a value of ‘1’ and deviation of experimental groups from control tested with the t-statistic [44]. P values for multiple comparisons were corrected by the Bonferoni method [44]. For DNA content and [3H]-thymidine incorporation experiments two-way ANOVA was employed. Post-hoc comparisons between groups were made with Tukey's procedure. A value of p<0.05 was considered to be significant.


   3 Results
Top
 Abstract
 1 Introduction
 2 Materials and methods
 3 Results
4 Discussion
 References
 
3.1 Expression of P2Y receptors in neonatal rat CAFB and cardiac myocytes
To determine whether CAFB and myocytes express P2 receptors, Northern blot analysis was performed using P2Y1 and P2Y2 cDNAs to probe membranes containing poly(A)+ enriched RNA isolated from cultured rat CAFB (passage 3), and neonatal and adult rat cardiac myocytes, both of which were not cultured, but frozen immediately after isolation for subsequent RNA extraction. The blots exhibited hybridization of both P2Y1 and P2Y2 receptor probes with each of the 3 cell types studied (Fig. 1). The two bands detected with the P2Y1 probe are most likely due to the presence of two forms of the rat P2Y1 receptor with differing 3'-untranslated region extension, as has been reported previously for the human receptor [45]. When normalized to the levels of GAPDH mRNA, the relative levels of P2Y1 were 1.00, 1.58, 1.05 and relative levels of P2Y2 were 1.00, 0.60, 0.62 in CAFB, adult cardiac myocytes, and neonatal cardiac myocytes, respectively.


Figure 1
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  		Fig. 1 Northern blot analysis of poly(A)+ RNA isolated from CAFB and myocytes with the P2Y1 (A,C) and P2Y2 (B,D) cDNAs as probes. A and B: Poly(A)+ RNA (5 µg each lane) from cultured CAFB, freshly isolated adult cardiac myocytes and from freshly isolated Percoll purified neonatal cardiac myocytes was separated by gel electrophoresis. Transfer to nylon membranes and hybridization were performed as described for each in Materials and Methods followed by autoradiography. Autoradiography was carried out by standard procedures to obtain images. C and D: Total RNA (20 µg/lane) from various rat tissues was size fractionated by gel electrophoresis. Hybridization conditions differed from that used for panels A and B, as described in Materials and Methods. After hybridization with the P2Y1 or P2Y2 probe, each membrane was stripped and hybridized serially with probes for the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and 18S ribosomal RNA. Quantitative measurements in signal for all four panels and images shown in panels C and D were made using a Betascope 603.

 
To verify the specificity of the probes used for P2Y1 and P2Y2 receptors, Northern blots containing 20 µg of total RNA from various tissues were analyzed. Signals for P2Y1 were not distinguishable from background in lanes containing rat liver and rat testis (Fig. 1C). The lane containing rat brain was negative on the P2Y2 blot (Fig. 1D). This pattern is consistent with previously published tissue specificities for these subtypes [11, 36].

3.2 Extracellular ATP stimulates expression of c-fos mRNA in CAFB
The presence of P2 receptor mRNA in CAFB suggested that functional receptors might be present. We therefore sought to determine whether extracellular ATP would induce c-fos gene expression in CAFB. Addition of 100 µM ATP to the cultured CAFB for 30 min increased expression of the immediate early gene, c-fos 8-fold (P<0.05; Fig. 2), while 2 µM NE increased c-fos 2-fold (NS). In response to 100 µM ATP, levels of c-fos mRNA increased by 15 min, peaked at 30 min (8 fold), and decreased to baseline by 1 h (Fig. 3). ATP increased c-fos gene expression in a dose-dependent manner, with the maximal effect observed at 100 µM. 50% of the maximal effect was observed at approximately 25 µM ATP (Fig. 4).


Figure 2
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  		Fig. 2 Effect of ATP on c-fos mRNA levels in cultured CAFB. After culture for 24 h in serum-free medium, CAFB were incubated with vehicle (control), or ATP (100 µM) for 30 min. Total RNA was isolated and levels of c-fos mRNA were quantitated by Northern blot analysis. Blots were subsequently stripped and probed with a synthetic oligonucleotide specific for the 18S ribosomal RNA. A Betascope 603 apparatus (Betagen) was employed to quantitate radioactive signals. The ratio of c-fos/18S mRNA levels is presented to compensate for possible variations in RNA loading. Data are expressed relative to control (arbitrarily assigned a value of 1.0) as mean±S.E. (n=11). *, p<0.05 vs. control (one-sample t-test). Inset: Representative Northern blot showing the effect of ATP on the expression of c-fos mRNA.

 

Figure 3
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  		Fig. 3 Time course of the ATP-induced increase in c-fos gene expression. Serum-starved CAFB were treated with ATP (100 µM) for the length of time indicated on the x-axis. Data are expressed relative to control (arbitrarily assigned a value of 1.0) as mean±S.E. (n=3). Inset shows a representative Northern blot experiment.

 

Figure 4
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  		Fig. 4 Dose response relationship for the ATP-induced increase in c-fos gene expression in CAFB. Serum-starved CAFB were treated with ATP at different concentrations for 30 min. Data were expressed relative to control (arbitrarily assigned a value of 1.0) as mean±S.E. (n=4). Inset shows a representative Northern blot result.

 
3.3 The ATP-stimulated induction of c-fos is mediated by activation of P2 receptors
To determine whether the effect of ATP on c-fos gene expression was mediated by P1 or P2 receptors in CAFB, we used a battery of adenosine nucleotide analogues to differentiate P1 and P2 receptors. The potency order to induce expression of c-fos mRNA was as follows: ATP > ADP > adenosine, while the non-hydrolyzable ATP analogue, ATP{gamma}S mimicked the effect of ATP (Fig. 5A), indicating that the ATP-stimulated induction of c-fos in CAFB is mediated by P2 receptors. A second battery of triphosphate nucleotides used to differentiate P2 receptor subtypes yielded the following potency order: ATP = UTP > ITP = CTP (Fig. 5B). This set of results is most concordant with involvement of the P2Y2 (formerly referred to as P2U) receptor in the induction of c-fos mRNA in CAFB, although the small response to CTP suggests the involvement of other P2 receptor subtypes as well.


Figure 5
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  		Fig. 5 A) Comparison of the effects of ATP and its metabolites on c-fos gene expression. Serum-free CAFB cultures were incubated with ATP (100 µM), ADP (100 µM), or adenosine (100 µM). c-fos mRNA levels were measured as described in Fig. 1. Data are expressed relative to control (arbitrarily assigned a value of 1.0) as mean±S.E. (n=3–7). *, p<0.05 vs. control (one-sample t-test, Bonferroni correction). Also shown are the effects of a non-hydrolyzable analogue of ATP, ATP{gamma}S (100 µM; n=2). Inset is a representative Northern blot result. B) Comparison of the effects of various triphosphate nucleotides on c-fos gene expression. Serum-free cell cultures were incubated with ATP (100 µM), UTP (100 µM), ITP (100 µM) or CTP (100 µM). c-fos mRNA Levels were measured as described in Fig. 1. Data were expressed relative to control (arbitrarily assigned a value of 1.0) as mean±S.E. (n=3–5). *, p<0.05 vs. control (one-sample t-test, Bonferroni correction).

 
3.4 Intracellular signalling mechanisms of the ATP-mediated induction of c-fos mRNA
We next investigated whether or not several candidate second messengers are generated after activation of P2 receptors by ATP using c-fos induction as an endpoint. Whereas NE did not increase intracellular calcium concentration ([Ca2+]i), addition of ATP increased the intracellular Ca2+ concentration (Fig. 6). The latency of the ATP-induced increase in [Ca2+]i suggests that this response is mediated via metabotropic P2 receptors (P2Y) rather than intrinsic ion channels (P2X), although a possible delay in the delivery of ATP to the cells renders this conclusion tentative. Pretreatment with the intracellular Ca2+ chelator, BAPTA-AM significantly decreased, but did not abolish the ATP-induced increase in [Ca2+]i. These findings suggested that the increase in [Ca2+]i may be important in the responses elicited by ATP. Therefore, we determined whether or not the intracellular Ca2+ chelator, BAPTA-AM or an inhibitor of the Ca2+-calmodulin (CaM) dependent kinase II, KN-62 would modulate the ATP-stimulated induction of c-fos mRNA. Pretreatment with either BAPTA-AM or KN-62 had no discernable effect on the ATP-induced increase in c-fos mRNA (Fig. 7), suggesting that the ATP-stimulated increase in [Ca2+]i does not play a crucial role in regulating the ATP-induced increase in expression of c-fos mRNA.


Figure 6
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  		Fig. 6 Effects of NE, ATP, BAPTA+ATP on Cai in neonatal CAFB. Cai was measured by loading CAFB with indo-1 AM (25 µM) for 20–30 min at 37°C. Indo-1 fluorescence was excited at 350 nM and the 410/490 nm ratio of emitted fluorescence was taken as an index of Cai, as described previously (Spurgeon et al. 1990).

 

Figure 7
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  		Fig. 7 Effects of KN-62 and BAPTA on the ATP-induced increase in c-fos gene expression. CAFB were pretreated with KN-62 or BAPTA-AM for 30 min followed by addition of ATP for another 30 min. c-fos mRNA Levels were measured as described in Fig. 1. Data were expressed relative to control (arbitrarily assigned a value of 1.0) as mean±S.E. (n=12–14). Inset shows a representative Northern blot result.

 
To determine whether a PKC-dependent pathway was involved in the ATP-induced c-fos response, down-regulation of PKC by overnight pretreatment with TPA or pretreatment with a general kinase inhibitor, staurosporin, for 30 min prior to addition ATP were employed. Each inhibited the ATP-induced increase in expression of c-fos mRNA (Fig. 8). While the magnitude of the reduction was approximately 50% in each case, the effect of staurosporine was statistically significant (p=0.042) while that of TPA was marginal (p=0.058). Taken together, these data suggest that activation of PKC may play a role in the ATP-induced increase in expression c-fos mRNA.


Figure 8
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  		Fig. 8 Effects of TPA and staurosporine on the ATP-stimulated elevation of c-fos mRNA levels. Serum-starved CAFB were incubated with ATP (100 µM) for 30', or pretreated with either TPA (1 µM) overnight or staurosporine (50 nM) for 30 min, followed by addition of 100 µM ATP for another 30 min. Data are expressed as mean±S.E. (n=5–11). #, p<0.05 vs. ATP alone (independent samples t-tests, Bonferroni correction).

 
3.5 Inhibition of DNA synthesis by extracellular ATP
Having observed evidence for functional P2 receptors on cultured CAFB, we proceeded to determine whether P2 receptor stimulation would impact a physiologically relevant process in CAFB. To this end we used total DNA content and [3H]-thymidine incorporation into DNA as indices of cell proliferation in CAFB. Extracellular ATP inhibited the NE stimulated increase in DNA content in CAFB cultures (Fig. 9A) and also inhibited the increase in incorporation of [3H]-thymidine into DNA increased by NE (Fig. 9B). 2-Way ANOVA yielded an independent effect of ATP on DNA content and incorporation of labeled thymidine, while in the case of label incorporation there was also a significant interactive effect of ATP with NE. Together, these indices provide evidence that extracellular ATP exerts inhibitory effects on both basal and NE stimulated CAFB proliferation.


Figure 9
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  		Fig. 9 Effects of ATP on the NE-stimulated increase in DNA content and NE-induced increase of [3H]-thymidine incorporation into DNA in CAFB. A) DNA content was determined as described in Methods. Data were expressed as mean±S.E (n=9). 2-way ANOVA yielded significant independent effects of both NE (p=0.028) and ATP (p=0.001), but there was no significant interactive effect. *, p<0.05 vs. control; #, p<0.05 vs. NE (Tukey's procedure). B) Serum-starved CAFB cultures were incubated with ATP (100 µM) and/or NE (2 µM) and [3H]-thymidine (0.1 µci/ml) for 24 h. Cells were washed with PBS, and incubated with 0.5% SDS at 37°C for 10 min. After the supernatant was transferred to a 15 ml tube, 20% TCA was added to the cells. Cell lysates were saved and filtered through GFC Whatman 2.4 cm filter. After washing with 10% TCA, 5% TCA, and 95% ethanol, the filter was air-dried. [3H]-thymidine incorporation was determined using a beta counter. Data were expressed relative to control (arbitrarily assigned a value of 1.0) as mean±S.E (n=24–26). Mean value of control was 29 523 cpm/dish. 2-way ANOVA yielded significant main effects for both NE (p=0.007) and ATP (p<0.001), as well as a significant interaction between NE and ATP (p=0.005). *, p<0.05 vs. control; #, p<0.05 vs. NE (Tukey's procedure).

 
To determine whether the continued presence of ATP is required to achieve a maximum inhibitory effect on [3H]-thymidine incorporation into DNA, cultures were first pretreated with ATP for 1 h, and then the medium containing ATP was removed and replaced with fresh serum-free medium containing only NE. [3H]-thymidine incorporation was measured over the next 24 h. Under these conditions, the extent of ATP inhibition was similar to that when ATP was present throughout the entire 24 h period of NE treatment (Fig. 10). To determine whether the inhibitory effect of ATP was mediated by activation of the same P2 receptor subtype as that responsible for the c-fos response, a battery of ATP analogues was employed. The potency order for inhibition of the NE-stimulated increase in incorporation of [3H]-thymidine into DNA was as follows: ATP = ADPβS > ATP{gamma}S > ADP > UTP, while adenosine and {alpha},β-meATP did not inhibit the NE-induced increase in Formula -thymidine incorporation into DNA. These data indicate that at least 2 pharmacologically distinct P2 receptor subtypes are present in cultured neonatal rat CAFB and show that the P2 receptor subtype responsible for the observed inhibitory effect of ATP on [3H]-thymidine incorporation into DNA is distinct from the subtype that stimulates expression of c-fos mRNA in CAFB.


Figure 10
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  		Fig. 10 Effects of ATP analogues or 1 h pretreatment with ATP on the NE-induced increase of [3H]-thymidine incorporation into DNA. Procedures are described in Materials and Methods. Data were expressed relative to control (arbitrarily assigned a value of 1.0) as mean±S.E. (n=9).

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Materials and methods
 3 Results
 4 Discussion
References
 
In the present study, we demonstrate that cultured neonatal rat CAFB express mRNA for both P2Y1 and P2Y2 receptors. Extracellular ATP and other P2 receptor agonists stimulate the transient induction of c-fos gene expression in a dose-dependent manner. The induction of c-fos gene expression by ATP appears to be regulated by protein kinase C, but not by the ATP induced increase in [Ca2+]i. Importantly, extracellular ATP and several ATP analogues inhibit the NE-stimulated incorporation of [3H]-thymidine into DNA. Experiments with a battery of P2 receptor agonists demonstrate that the effects of ATP on the NE-stimulated increase in incorporation of [3H]-thymidine into DNA and on c-fos gene expression are exerted via different P2 receptor subtypes.

Our data show that an ATP-induced increase in [Ca2+]i precedes the ATP-induced c-fos, which suggested that [Ca2+]i might play an important role in the ATP-induced expression of c-fos mRNA. However, pretreatment with the intracellular Ca2+ chelator, BAPTA-AM which markedly reduced the ATP-induced increase in [Ca2+]i did not inhibit the ATP-induced increase in expression c-fos mRNA in CAFB. Likewise, KN-62, a specific inhibitor of Ca2+-calmodulin kinase II failed to modulate the ATP-induced increase in expression of c-fos mRNA suggesting that the CaM kinase II system is not involved in the transmission of signals from the P2 receptor to the nuclear effectors of c-fos gene expression. Both down regulation of PKC with phorbol ester and pretreatment with staurosporine partially inhibited the c-fos response, suggesting that PKC may play a role in regulating the ATP-induced increase in expression of c-fos mRNA. The lack of complete inhibition by either of these agents suggests the involvement of other signal transduction pathways and/or of TPA and staurosporine insensitive isoforms of PKC. The former possibility should be evaluated with specific inhibitors of other pathways such as the mitogen activated protein kinase (MAPK) pathway which is activated by extracellular ATP in cardiac myocytes [30]. The latter possibility could be evaluated using inhibitors of PKC with different modes of action, such as calphostin C and chelerythrine.

A number of studies have shown that mitogenic agents such as NE, angiotensin II, and endothelin induce expression of c-fos mRNA, increase production of collagen, increase DNA synthesis and stimulate proliferation in CAFB [4, 46–49]. In the present study, in contrast to the effect of NE, ATP did not increase incorporation of [3H]-thymidine into DNA in CAFB (Fig. 9). Furthermore, extracellular ATP inhibited the NE-induced increase in [3H]-thymidine incorporation into DNA and the NE stimulated increase in DNA content. These data demonstrate a dissociation between increased expression of c-fos mRNA and cell proliferation in CAFB. These data are consistent with our previous studies in neonatal cardiac myocytes [26, 30]in which we demonstrated that extracellular ATP increased expression of c-fos, but inhibited the NE-induced cardiac hypertrophy. Thus, in contrast to the mitogenic effect of ATP on 3T3 cells [50], ATP inhibits mitogens in both myocytes [26, 30]and fibroblasts of cardiac origin. The reason for the different responses to extracellular ATP among cell types is not clear, but possible explanations include the potential presence of different P2 receptor subtypes, and cell-type specific differences in post-receptor signal transduction. The development of more discriminating pharmacologic agents for P2 receptor subtypes would greatly aid the resolution of these questions.

One explanation for the dissociation of c-fos activation from proliferation by ATP is that ATP may fail to stimulate a sustained elevation in AP-1 complex formation. The effects of c-fos are realized through the formation of AP-1 complexes consisting of either Fos-Jun heterodimers or Jun-Jun homodimers. Transient activation of c-jun is insufficient to mediate AP-1 dependent gene transcription, while sustained elevation of c-jun leads to enhanced AP-1 dependent transcriptional activity [51]. Thus, it is possible that CAFB proliferation requires sustained elevation of AP-1 binding activity and may be dependent on sustained elevations in both Fos and Jun proteins. On the other hand, even sustained AP-1 activity may not be sufficient for CAFB proliferation. Given the multitude, complexity, and redundancy of signal transduction pathways, it seems possible (even likely) that one or more additional inputs are required to initiate and sustain the processes required for cell proliferation. For example, signaling through the rapamycin sensitive 4E binding protein and p70 S6 kinase pathways may be a separate requirement for the protein synthesis necessary for sustained CAFB proliferation [52].

The present study shows that CAFB express both P2Y1 and P2Y2 receptors (Fig. 1), and we have provided evidence elsewhere that P2Y4 and P2Y6 receptors are expressed in CAFB as well [20]. Thus, CAFB express multiple P2 receptor mRNA, but more importantly, they exhibit physiological responses that can be differentiated by their pharmacological profile. The efficacy order of ATP analogues to stimulate expression of c-fos mRNA is most consistent with the potency order of the P2Y2 receptor, while the ATP induced inhibition of the NE-stimulated increase in [3H]-thymidine incorporation into DNA in CAFB was mediated by a distinct P2 receptor(s), possibly including the P2Y1 receptor, as indicated by the different efficacy of UTP (Fig. 5, Fig. 10). Although the pharmacological approach used here cannot rule out other as yet unidentified P2 receptor subtypes in the responses studied, the present study demonstrates that different receptors mediate different physiological responses in CAFB.

While the physiological effects and relevance of extracellular ATP in vivo are not well established, it seems likely that extracellular ATP in the heart may emanate from a variety of sources. Platelet degranulation, and cell lysis may contribute to local accumulation of extracellular ATP [22, 23]. ATP is released into the perfusate of the working rat heart in response to hypoxia [53]and can also be released from hypoxic cardiomyocytes [21]. The primary physiological source of ATP, however, is secretion from sympathic nerve terminals where it is co-stored and co-released with norepinephrine by nerve stimulation [5, 6]. These cotransmitters may interact with each other to exert their ultimate physiological effects. In adult rat cardiac myocytes, for example, NE does not increase intracellular Ca2+ concentration, but greatly potentiates the ATP-induced increase in intracellular Ca2+ concentration [24]. On the other hand, ATP inhibits the NE-induced increase in protein accumulation and cell size in cultured rat neonatal cardiac myocytes [26]. The present findings show the ATP is capable of modifying the effects of NE on CAFB proliferation. Since CAFB not only play an important role in maintaining and remodeling matrix, but also influence myocyte growth and function [54], it seems likely then that extracellular ATP has considerable relevance to heart structure and function, particularly in diseased states that are characterized by ventricular remodeling.

Time for primary review 36 days.


   Acknowledgements
 
The authors are grateful to Bruce Ziman for preparing adult cardiac myocytes, to Michael T. Crow for providing the c-fos probe, and to Sharon Wright for secretarial assistance. T.E.W. and E.A.B. thank the Wellcome Trust for research support.


   References
Top
 Abstract
 1 Introduction
 2 Materials and methods
 3 Results
 4 Discussion
 References
 

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