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British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 2000 Apr;129(8):1723–1731. doi: 10.1038/sj.bjp.0703243

FP-receptor mediated trophic effects of prostanoids in rat ventricular cardiomyocytes

K Pönicke 1, C Giessler 1, M Grapow 1, I Heinroth-Hoffmann 1, K Becker 1, B Osten 2, O-E Brodde 1,*
PMCID: PMC1572001  PMID: 10780979

Abstract

  1. The aim of this study was to characterize the receptor subtype involved in cardiac effects of prostanoids. For this purpose we determined in neonatal and adult rat cardiomyocytes effects of prostanoids on inositol phosphate (InsP)-formation (assessed as accumulation of total [3H]-InsP's in myo-[3H]-inositol pre-labelled cells) and on rate of protein synthesis (assessed as [3H]-phenylalanine incorporation), and on contractile force in left ventricular strips of the rat heart. For comparison, effects of prostanoids on InsP-formation and contractile force were determined in rat thoracic aorta, a classical TP-receptor containing tissue.

  2. Prostanoid increased InsP-formation and rate of protein synthesis in neonatal as well as adult rat cardiomyocytes; the order of potency was in neonatal (PGF2α>PGD2⩾PGE2⩾U 46619>PGE1) and adult (PGF2α>PGD2⩾PGE2>U 46619) rat cardiomyocytes well comparable. Moreover, in electrically driven left ventricular strips PGF2α caused positive inotropic effects (pD2 7.5) whereas U 46619 (up to 1 μM) was uneffective.

  3. In contrast, in rat thoracic aorta U 46619 was about 100 times more potent than PGF2α in increasing InsP-formation and contractile force.

  4. The TP-receptor antagonist SQ 29548 only weakly antagonized prostanoid-induced increases in rate of protein synthesis (pKB about 6) in rat cardiomyocytes but was very potent (pKB about 8–9) in antagonizing prostanoid-induced increases in InsP-formation and contractile force in rat aorta.

  5. We conclude that, in cardiomyocytes of neonatal and adult rats, the prostanoid-receptor mediating increases in InsP-formation and rate of protein synthesis is a FP-receptor. Moreover, stimulation of these cardiac FP-receptors can mediate increases in contractile force.

Keywords: Prostanoid-receptors, neonatal rat cardiomyocytes, adult rat cardiomyocytes, protein synthesis, inositol phosphate, hypertrophy, contractile force

Introduction

It is now generally accepted that stimulation of receptors that couple through Gq/11-protein to phospholipase C (PLC) leads to formation of the second messengers inositol trisphosphate (InsP3) and diacylglycerol (DAG). DAG can activate certain isoforms of protein kinase C (PKC) and growing evidence has accumulated that this pathway is involved in induction of cell growth in various cell systems including cardiac myocytes (for recent review see Sugden & Clerk, 1998; Dorn & Brown, 1999). Thus, several groups have convincingly shown that, in neonatal rat ventricular cardiomyocytes, stimulation of α1-adrenoceptors (Simpson, 1983; Lee et al., 1988; Knowlton et al., 1993) and ETA-receptors (Shubeita et al., 1990; Suzuki et al., 1990; Sugden et al., 1993; Ito et al., 1993, for review see Sugden & Bogoyevitch, 1995) causes increases in rate of protein synthesis. We have recently shown, that in these cells also the TXA2-mimetic U 46619 increased InsP-formation and rate of protein synthesis (Pönicke et al., 1999); the effects of U 46619 appeared, however, not to be mediated by a TP-receptor, since the TP-receptor antagonist SQ 29548 (Ogletree et al., 1985) was only a weak antagonist of these effects with a potency (pKB-value around 6) 100 times less than could be expected for a TP-receptor mediated effect (7.5–9.1, Coleman et al., 1994).

The aim of the present study was, therefore, to find out which prostanoid-receptor subtype might be involved in the growth-promoting effects of U 46619 in the rat cardiomyocytes. For this purpose we firstly studied the effects of a series of prostanoids on InsP-formation and rate of protein synthesis (assessed by [3H]-phenylalanine incorporation) in neonatal rat cardiomyocytes. We secondly repeated these studies in ventricular cardiomyocytes isolated from adult rats in order to find out whether or not prostanoid receptors in neonatal cardiomyocytes resemble those in adult cardiomyocytes. And thirdly for reason of comparison we studied the effects of several prostanoids on InsP-formation and vasoconstriction in rat thoracic aorta, a tissue widely used to study TP-receptor-mediated effects (Jones et al., 1989; Tymkewycz et al., 1991; Wagner et al., 1997).

Methods

Preparation of neonatal rat cardiomyocyte culture

Cardiomyocytes of neonatal rats were isolated as described recently (Pönicke et al., 1997). In order to prevent proliferation of non-myocytes (mainly fibroblasts) and to obtain cardiomyocytes that were nearly free of contamination, cells were prepared and cultured in presence of 10 μM cytosine-β-D-arabinofuranoside.

Preparation of cardiomyocyte culture of adult rats

Cardiomyocytes of adult rats were isolated as described by Piper & Volz (1990) in a slightly modified version. Briefly, 12 weeks old male Wistar rats were anaesthetized with pentobarbitone sodium (35 mg kg−1, i.p.) and 500 U heparin sulphate were injected intraperitoneally. The heart was rapidly excised under artificial ventilation. The calcium-tolerant myocytes were isolated by cardiac retrograde aortic perfusion (Langendorff method) as described by Viko et al. (1995). Freshly isolated left ventricular cells were gently diluted in steril culture medium 199, pH 7.4, supplemented with 10% new-born calf serum. For studies of InsP-formation the resultant suspension of ventricular myocytes was transferred into 75 cm2 cell culture flasks (3.6×104 cells cm−2) and immediately used. For studies of [3H]-phenylalanine incorporation the ventricular myocytes suspension was seeded into 12-well-plates (16,000 cells per well) which had been coated with 4% foetal calf serum in medium 199 for 24 h at 37°C (in a humidified incubator at 5% CO2/95% air) and incubated for 16 h at 37°C. Thereafter, the cultures were rinsed with serum-free Hank's balanced salt solution to remove damaged, rounded and nonattached myocytes and the rod-shaped cells were cultured for the following experiments in serum-free medium M199 supplemented with 2 mM L-carnitine, 5 mM taurine, 5 mM creatine and antibiotics (100 U ml−1 penicillin and 100 μg ml−1 streptomycin). To prevent growth of nonmyocytes, the culture medium was supplemented with 10 μM cytosine-β-D-arabinofuranoside.

Incorporation of [3H]-phenylalanine

Protein synthesis by cardiomyocytes was assessed by incorporation of [3H]-phenylalanine into cells as recently described (Pönicke et al., 1997). Briefly, after addition of [3H]-phenylalanine (0.5 μCi ml−1) at 37°C and various concentrations of prostanoids in the presence or absence of antagonists the cultures were incubated for 20 h at 37°C in 5% CO2/95% air. At the end of the experiments cells were washed with ice-cold 0.9% NaCl-solution and incubated for 24 h at 4°C with 10% trichloroacetic acid. Thereafter precipitates were washed again with 10% trichloroacetic acid and twice with 0.9% NaCl solution. The remaining precipitate on the culture dishes was solubilized in 1 N NaOH supplemented with 0.1% sodium dodecyl sulphate by room temperature for 24 h, and radioactivity was determined in aliquots by the use of a liquid scintillation counter (Beckman LS 6000).

Inositol phosphate formation in neonatal rat cardiomyocytes

InsP-formation in rat cardiomyocytes was determined as recently described (Pönicke et al., 1997). Briefly, after the 24 h incubation (see above) cells were washed with culture medium M199 supplemented with 10% new-born calf serum and 100 U ml−1 penicillin and 100 μg ml−1 streptomycin and incubated for 24 h with myo-[3H]-inositol (2.9 μCi ml−1) at 37°C. Thereafter adherent cells were peeled off by trypsin-EDTA treatment and non-incorporated myo-[3H]-inositol was washed out by centrifugation and resuspension in Hanks' buffered saline solution supplemented with 10 mM LiCl and 1% bovine serum albumin. Aliquots (970 μl) of the cardiomyocyte suspension (2.3×105 cells ml−1) were then incubated with the indicated prostanoids in the presence or absence of antagonists for 60 min at 37°C in a final volume of 1 ml. The incubation was stopped by addition of 1 ml ice-cold methanol and 2 ml chloroform. Total inositol phosphates were separated and determined as recently described (Pönicke et al., 1997). Each data point was determined in quadruplicate in each experiment.

Inositol phosphate formation in adult rat cardiomyocytes

The ventricular myocytes suspension (see above) was incubated for 24 h with myo-[3H]-inositol (2.9 μCi ml−1) at 37°C. Thereafter, non-incorporated myo-[3H]-inositol was washed out by centrifugation and resuspension in Hanks buffered saline solution supplemented with 10 mM LiCl and 1% bovine serum albumin. Aliquotes (970 μl) of the cardiomyocyte suspension (5×104 cells ml−1) were incubated with the indicated prostanoids in a final volume of 1 ml for 45 min at 37°C. Total inositol phosphates were separated and determined as described above.

Inositol phosphate formation in rat ventricular slices

InsP-formation in ventricular slices of rat heart was determined as recently described (Pönicke et al., 1998).

Inositol phosphate formation in rat aortic rings

Male Wistar rats (12 weeks old) were killed by cervical dislocation; the thoracic aorta was removed rapidly and placed into oxygenated Krebs-Henseleit buffer of the following composition (mM): NaCl 108, KCl 4.7, KH2PO4 1.2, MgSO4 1.2, NaHCO3 24.9, CaCl2 1.3, D-glucose 11, EDTA 0.001. Fat surrounding the aorta was carefully removed without stretching the tissue. The aorta was cleaned of adhering fat and connective tissue and cut into rings of 1 mm wide with a tissue shopper. The rings were maintained at 37°C and continuously bubbled with carbogen for 45 min. In this time the buffer was changed twice. Thereafter the aortic rings were preincubated with myo-[3H]-inositol (6 μCi ml−1) for 1 h. After washout of the radioactivity, three rings of each aorta were incubated in a total volume of 330 μl in the presence or absence of prostanoids for 45 min at 37°C. The incubation was stopped by addition of 330 μl ice-cold methanol and 660 μl chloroform. The mixture was vigorously vortexed twice, and thereafter the phases were separated by centrifugation at 820×g for 10 min at 4°C. Aliquots (400 μl) of the upper phase were placed on Dowex AG 1-X8 columns (200 mg per column). Free inositol was eluted twice each with 5 ml H2O and 5 ml of 60 mM ammonium formate. Total inositol phosphates were eluted by addition of 2×1 ml 1 M ammonium formate dissolved in 100 mM formic acid. Each data point was determined in quadruplicate in each experiment.

Preparation of rat ventricular strips

The preparation of ventricular strips of the rat heart were performed as described earlier (Kotchi Kotchi et al., 1998). Briefly, male Wistar rats (12 weeks old) were killed by cervical dislocation; the hearts were rapidly removed, placed into oxygenated modified Tyrode solution containing (mM): NaCl 136.9, KCl 5.4, NaH2PO4 0.42, MgCl 1.05, NaHCO3 25.0, CaCl2 2.5, D-glucose 9.7 equilibrated with carbogen, and trabecular strips (1–2 mm wide, 1–1.5 mm thick, 6–8 mm long) were prepared from the left ventricle. The ventricular strips were mounted in a 10 ml organ bath containing Tyrode solution at 37°C. The strips were electrically stimulated by square wave pulse (5 ms) of about 50% above threshold at a frequency of 1 Hz (Stimulator II, Hugo Sachs Elektronik KG, March-Hugstetten, Germany). The developed tension of the preparation (maintained under a resting tension of 10 mN) was recorded via a strain gauge on a Hellige recorder (Hellige GmbH Freiburg, Germany). After an equilibration time of at least 1 h, the cumulative concentration-response curves for prostanoids were determined.

Preparation of rat aortic strips

Male Wistar rats (12 weeks old) were killed by cervical dislocation. The thoracic aorta was rapidly removed and placed into oxygenated modified Krebs-Henseleit solution containing (mM): NaCl 119, KCl 4.75, KH2PO4 1.19, MgSO4 1.19, NaHCO3 25, CaCl2 2.25, D-glucose 10, EDTA 0.0228, ascorbic acid 0.117. The aorta was cleaned of adhering fat and connective tissue, and cut into helical strips 2 mm in width and 10 mm in length. The strips were placed in 10 ml chambers containing Krebs-Henseleit solution with constant oxygenation (carbogen) at 37°C. The contractile force was measured isometrically using force transducers connected to amplifiers and recorders (Föhr Medical Instruments GmbH, Germany). The resting tension in the aorta was adjusted to 9.81 mN. The strips were allowed to equilibrate for 60 min (bath fluid was replaced at 20 min intervals). Following equilibration, the aortic strips were contracted by treatment with 50 mM KCl and 1 μM phenyl-ephrine to confirm the viability of the smooth muscle. When tension had reached a plateau, 10 μM carbachol was added to verify the functional state of the endothelium. The bath was then washed repeatedly with Krebs-Henseleit solution until the preparations reached their initial tension. After this equilibration period, cumulative concentration-response curves for the prostanoids were determined. For SQ 29548 experiments, aortic strips were incubated for 30 min with SQ 29548 in the indicated concentrations; thereafter cumulative concentration-response curves for prostanoids were determined in presence of SQ 29548.

Drugs

L-[2,3,4,5,6a[3H]-phenylalanine (spec. Activity: 5.03 TBq mmol−1) and myo-[3H]-inositol (spec. activity: 4.25 TBq mmol−1) was purchased from Amersham Buchler (Braunschweig, Germany). PGF2α, PGD2, PGE1, PGE2 were purchased from Saxon Biochemicals (Hannover, Germany), carbocyclin from Calbiochem (Bad Soden, Germany) and L-phenylalanine, cytosine-β-D-arabinofuranoside, sodium dodecyl sulphate, trypsin (crude), L-carnitine, taurine, creatine from Sigma-Aldrich (Deisenhofen, Germany). Hanks' balanced salt solutions, culture medium M199 and penicillin-streptomycin were obtained from Life Technologies (Eggenstein, Germany). U 46619 (9,11-dideoxy-11α,9α-epoxy-methanoprostaglandin F2α) and SQ 29548 [{1S-(1α,2α(Z),3α,4α)}-7-{3-[[2-[(phenylamino)carbonyl]hydrazino]methyl] - 7 - oxabicyclo[2.2.1]hept -2-yl}-5-heptanoic acid] were purchased from Reatec GmbH (Weiterstadt, Germany). All other chemicals were of the highest purity grade commercially available.

Statistical analysis

Data given are means±s.e.mean of n experiments. Agonist-concentration-effect curves were fitted to sigmoid function with fixed slopes at 1.0, and EC50-values were calculated by non-linear regression analysis using the GraphPad Prism 2.01 program (GraphPad Software, San Diego, U.S.A.). In order to find out whether or not, in rat aorta, concentration-effect curves for PGF2α effects on InsP-formation and contraction fitted better to a two-site model or to a one-site model the F-test was used (GraphPad Prism 2.01 program, GraphPad Software, San Diego, U.S.A.).

The apparent SQ 29548 affinity was either calculated using the formula

graphic file with name 129-0703243e1.jpg

(concentration-experiments on helically cut strips of aorta) with A is the concentration of SQ 29548, and CR is the ratio of EC50-values of agonist measured in the presence and absence of SQ 29548 or using the Cheng & Prusoff-equation (Cheng & Prusoff, 1973)

graphic file with name 129-0703243e2.jpg

(InsP-formation experiments on aortic rings) with IC50 concentration of SQ 29548 to inhibit agonist-induced InsP-formation by 50%, [S]=agonist concentration in the assay and EC50=concentration of agonist inducing 50% of maximal InsP-formation (determined as described above). Statistical significance of differences were analysed by paired, two-tailed Student's t-test; a P-value <0.05 was considered significant. All statistical calculations were performed with the GraphPad Prism 2.01 programme.

Results

Effects of prostanoids on InsP-formation and rate of protein synthesis in neonatal rat cardiomyocytes

As discussed above, we had recently shown that in neonatal rat cardiomyocytes, the TXA2-mimetic U 46619 weakly increased InsP-formation and rate of protein synthesis (assessed by [3H]-phenylalanine incorporation), this was antagonized by the TP-receptor antagonist SQ 29548 with a potency (pKB-value about 6) 10–100 time less than could be expected from the affinity of SQ 29548 for a TP-receptor (Coleman et al., 1994). We, therefore, studied the effects of several prostanoids on InsP-formation and rate of protein synthesis to find out whether another prostanoid receptor might be involved. All prostanoids studied (PGF2α, PGD2, PGE2, PGE1, carbocyclin and U 46619) increased concentration dependently InsP-formation (Figure 1A) and [3H]-phenylalanine incorporation (as a measure of rate of protein synthesis, Figure 1B). In both settings PGF2α was about 100 times more potent than U 46619 (Figure 1, Table 1). Although prostanoid concentration-effect curves did not always yield clear maximum (Figure 1) and, hence, pEC50-values could partly be only roughly calculated (Table 1) the order of potency for prostanoids induced increases in InsP-formation and rate of protein synthesis is best described as PGF2α>PGD2⩾PGE2⩾ U46619>PGE1.

Figure 1.

Figure 1

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(A) Prostanoid-induced inositol phosphate (InsP) generation in neonatal rat ventricular cardiomyocytes. Ordinate scale: [3H]-InsP-formation as per cent of basal formation. Abscissa scale: molar concentrations of prostanoids. Basal [3H]-InsP-formation was 1–2% of the incorporated radioactivity and amounted to 1139±144 c.p.m. in control cells (n=22). (B) Prostanoid-induced [3H]-phenylalanine incorporation in neonatal rat ventricular cardiomyocytes. Ordinate scale: [3H]-phenylalanine incorporation as per cent of basal incorporation. Abscissa scale: molar concentrations of prostanoids. Basal [3H]-phenylalanine incorporation in control cells was 3445±185 c.p.m. (n=18). In (A) and (B) values are means and vertical lines show s.e.mean.

Table 1.

Effects of prostanoids on protein synthesis or InsP-formation in neonatal rat cardiomyocytes

graphic file with name 129-0703243t1.jpg

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Effects of prostanoids on InsP-formation and rate of protein synthesis in ventricular cardiomyocytes of adult rats

We next studied whether the characteristics of prostanoid-induced increases in InsP-formation and in rate of protein synthesis might be altered when cardiomyocytes were used that had been isolated from adult rats (12 weeks old). As shown in Figure 2, the order of potency for prostanoid-induced increases in InsP-formation and in rate of protein synthesis (PGF2α>PGD2⩾PGE2>U 46619) was well comparable to that obtained in neonatal rat cardiomyocytes, (c.f. Figure 1). In addition, pEC50-values for prostanoid-induced increases in InsP-formation and rate of protein synthesis (Table 2) were in a similar range as those obtained in neonatal rat cardiomyocytes. Moreover, as in the neonatal rat cardiomyocytes (Pönicke et al., 1999) also in the adult rat cardiomyocytes 10 μM SQ 29548 was only a very weak inhibitor of U 46619-induced increase in rate of protein synthesis (Figure 3). These data, therefore, strongly suggest that in adult rat cardiomyocytes the receptor mediating the effects of prostanoids is very similar to that in neonatal rat cardiomyocytes.

Figure 2.

Figure 2

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(A) Prostanoid-induced inositol phosphate (InsP) generation in adult male rat ventricular cardiomyocytes. Ordinate scale: [3H]-InsP-formation as per cent of basal formation. Abscissa scale: molar concentrations of prostanoids. Basal [3H]-InsP-formation was 1–2% of the incorporated radioactivity and amounted to 589±65 c.p.m. in control cells (n=12). (B) Prostanoid-induced [3H]-phenylalanine incorporation in adult rat ventricular cardiomyocytes. Ordinate scale: [3H]-phenylalanine incorporation as per cent of basal incorporation. Abscissa scale: molar concentrations of several prostanoids. Basal [3H]-phenylalanine incorporation in control cells was 1845± 135 c.p.m. (n=20). In (A) and (B) values are means and vertical lines show s.e.mean.

Table 2.

Effects of prostanoids on protein synthesis or InsP-formation in adult rat cardiomyocytes

graphic file with name 129-0703243t2.jpg

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Figure 3.

Figure 3

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Effects of 1 μM SQ 29548 on U 46619-induced [3H]-phenylalanine incorporation in adult male Wistar rat ventricular cardiomyocytes. Ordinate scale: [3H]-phenylalanine incorporation as per cent of basal incorporation. Abscissa scale: molar concentrations of U 46619. Basal [3H]-phenylalanine incorporation in control cells was 1445±111 c.p.m. (n=4); values are means and vertical lines show s.e.mean. *P<0.05 vs U 46619.

We also studied prostanoid-induced InsP-formation in left ventricular slices of rat heart. As shown in Figure 4, PGF2α (10 nM–100 μM) and U 46619 (10 nM–100 μM) concentration-dependently increased InsP-formation; PGF2α (pEC50=6.6±0.2) however, was about 100 times more potent than U 46619 (pEC50=4.4±0.4).

Figure 4.

Figure 4

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PGF2α or U 46619-induced inositol phosphate (InsP) generation in left ventricular slices of 12 week old male Wistar rats. Ordinate scale: [3H]-InsP-formation as per cent of basal formation. Abscissa scale: molar concentrations of U 46619 or PGF2α. Basal [3H]-InsP-formation was about 0.25% of the incorporated radioactivity and amounted to 92±8 c.p.m. in control ventricular slices (n=10); values are means and vertical lines show s.e.mean.

Effects of PGF2α and U 46619 on force of contraction of isolated, electrically driven left ventricular strips of the rat heart

It is well known that in the heart of several species, stimulation of Gq/11-coupled receptors such as α1-adrenergic or ETA-receptors does not only lead to increases in InsP-formation but also to increases in force of contraction (Wagner & Brodde, 1978; Terzic et al., 1993; Rubanyi & Polokoff, 1994). We, therefore, studied whether PGF2α or U 46619 might cause positive inotropic effects in the rat heart. As shown in Figure 5, PGF2α (0.1 nM–1 μM) caused a concentration-dependent increase in force of contraction of the isolated, electrically driven left ventricular strips; the pD2-value (7.4±0.1) was comparable with its pEC50-value for increases in rate of protein synthesis in the adult rat cardiomyocytes (c.f. Table 2). Maximal increase was comparable with that induced by endothelin-1 (ET-1, pD2=8.0±0.1) via ETA-receptor stimulation, but less than that evoked by noradrenaline (in the presence of 1 μM propranolol, pD2=5.0±0.1) via α1-adrenoceptor stimulation. On the other hand, U 46619 (up to 1 μM) did not significantly affect basal force of contraction of the ventricular strips (Figure 5).

Figure 5.

Figure 5

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Effects of PGF2α, U 46619, noradrenaline (NA) or endothelin (ET)-1 on contractile force of isolated electrically driven left ventricular strips of 12 week old male Wistar rats. Ordinate scale: increase of contractile force in mN; abscissa scale: molar concentrations of the agonists. Basal force of contraction was 2.75±0.2 mN (n=5) in the NA-experiments, 3.04±0.2 mN (n=8) in the ET-1-experiments, 3.4±0.3 mN (n=7) in the PGF2α-experiments and 2.56±0.3 mN (n=4) in the U 46619-experiments; values are means and vertical lines show s.e.mean.

Effects of PGF2α and U 46619 on InsP-formation and force of contraction of rat thoracic aorta

In a final set of experiments we assessed the effects of PGF2α and U 46619 on InsP-formation and force of contraction of rat isolated thoracic aorta, a tissue known to contain a TP-receptor (Jones et al., 1989; Tymkewycz et al., 1991; Wagner et al., 1997). In rings of rat thoracic aorta U 46619 (10 nM–10 μM) caused a pronounced increase in InsP-formation; maximal increase at 10 μM was 928±189% of control (Figure 6A). The pEC50-value for U 46619 was 6.9±0.1 (n=4). The TP-receptor antagonist SQ 29548 (1 nM–10 μM) potently inhibited 1 μM U 46619-induced InsP-formation; pKi-value was 8.2±0.3 (n=3), (Figure 6B). On the contrary, the effect of PGF2α on InsP-formation in rat aortic rings was only weak, maximal increase at 100 μM was 254±28% of control (n=5). It should be noted, that PGF2α-curve fitted significantly better to a two-side model than to a one-side model (F-test: P<0.01); at lower concentrations (up to 1 μM) it increased InsP-formation by only 50%, while the second component started at concentration >1 μM. Ten μM PGF2α-evoked InsP-formation was inhibited by SQ 29548 with an IC50-value of 17.1±2 nM (n=5) (Figure 6B).

Figure 6.

Figure 6

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(A) PGF2α or U 46619-induced inositol phosphate (InsP) generation in rings of thoracic aorta of 12 week old Wistar rats. Ordinate scale: [3H]-InsP-formation as per cent of basal formation. Abscissa scale: molar concentrations of PGF2α or U 46619. Basal [3H]-InsP-formation was 0.5–1.0% of the incorporated radioactivity and amounted to 326±38 c.p.m. in control aortic rings (n=9). (B) Effect of the TP-receptor antagonist SQ 29548 on 1 μM U 46619 or 10 μM PGF2α-induced inositol phosphate (InsP) generation in rings of thoracic aorta of 12 week old male Wistar rats. Ordinate scale: per cent inhibition of agonist-induced [3H]-InsP-formation. Abscissa scale: molar concentrations of SQ 29548. [3H]-InsP-formation was after 1 μM U 46619 1256±380 c.p.m. (n=3) and after 10 μM PGF2α 366±62 c.p.m. (n=5). In (A) and (B) values are means and vertical lines show s.e.mean.

Finally, we studied the effects of U 46619, PGF2α and PGD2 on developed tension in isolated helically cut strips of rat thoracic aorta. All three agonists led to a concentration-dependent contraction of rat aorta. However, U 46619 (pD2-value 8.1±0.1, n=17) was about 100 times more potent than PGF2α or PGD2. Again, concentration-response curve for PGF2α fitted significantly better to a two-side model than to a one-side model (F-test: P<0.001) (Figure 7A).

Figure 7.

Figure 7

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(A) Effects of U 46619, PGF2α or PGD2 on contractile force of helical strips of isolated thoracic aorta of 12 weeks old male Wistar rats. Ordinate scale: changes in force of contraction in mN. Abscissa scale: molar concentrations of U 46619, PGF2α or PGD2. (B) Effect of the TP-receptor antagonist SQ 29548 (10 nM) on U 46619-evoked changes of contractile force of helical strips of rat isolated thoracic aorta. Ordinate scale: changes in mN. Abscissa scale: molar concentrations of U 46619. (C) Effect of the TP-receptor antagonist SQ 29548 (10 nM) on PGF2α-evoked changes of contractile force of helical strips of rat isolated thoracic aorta. Ordinate scale: changes in contractile force in mN. Abscissa scale: molar concentrations of PGF2α. In (A), (B) and (C) values are means and vertical lines show s.e.mean.

The TP-receptor antagonist SQ 29548 (10 nM) significantly shifted the concentration-response curve for U 46619 to the right (Figure 7B); the pKB-value was estimated to 8.8±0.2 (n=10). Similarly, SQ 29548 caused an about 10 fold shift of the concentration-response curve for PGF2α to the right (Figure 7C); moreover, in the presence of SQ 29548, PGF2α concentration-response curve was monophasic (Figure 7C).

Discussion

In the present study the effects of several prostanoids on InsP-formation and rate of protein synthesis (assessed as [3H]-phenylalanine incorporation) were assessed in ventricular cardiomyocytes from neonatal as well as adult rats. In both preparations PGF2α was the most potent prostanoid in inducing increases in InsP-formation and in rate of protein synthesis whereas the TP-receptor agonist U 46619 was only a weak agonist causing effects only in concentrations >1 μM. Similarly, in slices of the left ventricle of the adult rat heart PGF2α was about 100 times more potent than U 46619 in increasing InsP-formation. For all prostanoids studied the order of potency for increasing InsP-formation and rate of protein synthesis was in neonatal (PGF2α>PGD2⩾PGE2⩾U 46619>PGE1) well comparable with that in adult rat cardiomyocytes (PGF2α>PGD2⩾PGE2>U 46619).

This order of potency was in marked contrast to that obtained for prostanoid-effects in rat thoracic aorta, a tissue widely used to study TP-receptor-mediated effects (Jones et al., 1989; Tymkewycz et al., 1991; Wagner et al., 1997). Thus, in slices of rat thoracic aorta U 46619 was much more potent than PGF2α in increasing InsP-formation; maximal increases induced by U 46619 were obtained in concentrations between 1–10 μM, i.e. concentrations where U 46619 just started to evoke effects in the cardiomyocytes. The same held true for prostanoid-induced contractions of the isolated helically cut strip of the thoracic aorta. U 46619 evoked contractions with a potency about 100 times greater than that of PGF2α or PGD2. Moreover, U 46619- as well as PGF2α-effects were inhibited in thoracic aorta by the selective TP-receptor antagonist SQ 29548 with a potency (pKB-values of about 8.0–9.0) that was well in the range of its affinity at the TP-receptor (Coleman et al., 1994). On the other hand, SQ 29548, even in the high concentration of 1 μM, did only marginally affect U 46619-evoked increase in protein synthesis in neonatal (Pönicke et al., 1999) and adult cardiomyocytes (c.f. Figure 3). Thus, the order of potency U 46619 ≫ PGF2α=PGD2 and the high antagonistic potency of SQ 29548 confirm that prostanoid-induced effects in rat thoracic aorta are mediated by a TP-receptor. Accordingly, the present results clearly demonstrate that the prostanoid receptor subtype mediating the hypertrophic response in rat cardiomyocytes is not a TP-receptor; the order of potency for prostanoid-induced effects (PGF2α>PGD2⩾PGE2>U 46619) strongly supports the view that increases in InsP-formation and in rate of protein synthesis are mediated predominantly, if not exclusively, by an FP-receptor (Coleman et al., 1994). It should be noted that this holds true not only for neonatal rat cardiomyocytes (in agreement with data from the literature, Adams et al., 1996; Lai et al., 1996; Kunapuli et al., 1998) but also for cardiomyocytes isolated from left ventricle of the adult rat heart. Thus, these results indicate that (at least for prostanoid mediated effects) data obtained in neonatal cardiomyocytes are comparable with those obtained in adult cardiomyocytes. It has been recently shown that, in sarcolemmal membranes of pig heart an E-type prostaglandin-receptor (EP3) exists that is completely different from the FP-receptor in rat cardiomyocytes: this EP3-receptor couples via a pertussis toxin sensitive G-protein (Gi) to adenylyl cyclase in an inhibitory fashion and exerts antiadrenergic effects (Hohlfeld et al., 1997).

Hoffmann et al. (1993) and Dogan et al. (1997) have shown that, in neonatal rat cardiomyocytes, U 46619 induced increases in Ca2+ transients, and this could be inhibited by the TP-receptor antagonists SK&F95585 (2 μM, Hoffmann et al., 1993) and SQ 29548 (10 μM, Dogan et al., 1997). Thus, it had been proposed that these effects of U 46619 are mediated by a TP receptor. However, these data are not necessarily contradictory to the present results. In both reports U 46619 caused significant effects in concentrations ⩾100 nM, a concentration that was also in the present study threshold concentration for U 46619-induced InsP-formation and in rate of protein synthesis (c.f. Figure 1) in the cardiomyocytes. On the other hand, in the classical TP-receptor system, the thoracic aorta, at this concentration (100 nM) U 46619 caused nearly maximal contraction (c.f. Figure 7). Moreover, concentrations for antagonists used (2 μM for SK&F 95587, i.e. 5–10 times KI (Tymkewycz et al., 1991); 10 μM for SQ 29548, i.e. 100–1000 times KI (Ogletree et al., 1985)) were rather high so that nonspecific effects can not be excluded. It is therefore, well possible that the effects of U 46619 on Ca2+-transients are not mediated by a TP-receptor but (according to the present results) by an FP-receptor.

It has been shown that in the heart of several species, including the rat, stimulation of Gq/11-coupled receptors can lead to increases in contractile force. Thus, many studies have demonstrated that in rat heart stimulation of α1-adrenoceptors (Wagner & Brodde, 1978; Terzic et al., 1993) or ETA-receptors (Rubanyi & Polokoff, 1994) evoked increases in force of contraction on left ventricular preparations. The same holds true also for the FP-receptor: as shown in Figure 4 PGF2α (that causes its effects on the cardiomyocytes via the FP-receptor, see above), concentration-dependently increased force of contraction of the isolated electrically driven left ventricular strip of the rat heart. Its pD2-value (7.4) was in good agreement with its pEC50-value (7.8, see Table 2) for increasing rate of protein synthesis in the adult cardiomyocytes. Maximal increases in contractile force evoked by PGF2α were comparable with those induced by ET-1 (via ETA-receptors) but less than those evoked by noradrenaline (via α1-adrenoceptors). On the other hand, U 46619 (up to 1 μM) did not affect basal force of contraction of the left ventricular strips–in contrast to its effect on the isolated helically cut strips of the thoracic aorta where the 1 μM concentration caused nearly maximal contractile effects (c.f. Figure 7). Thus, also for inducing positive inotropic effects the order of potency PGF2α ≫ U 46619 indicates involvement of an FP-receptor.

The mechanism underlying positive inotropic effects evoked by Gq/11-coupled receptors is not completely understood. Activation of these receptors causes formation of InsP3 and DAG with the former mediating the release of Ca2+ from intracellular stores which might be involved in increases of force of contraction. In addition, however, it has been shown that stimulation of α1-adrenoceptors (for review see Terzic et al., 1993) and ETA-receptors (Krämer et al., 1991; Meyer et al., 1996) increases the Ca2+-sensitivity of myofilaments via activation of the Na+/H+-antiporter and it has been suggested that these effects are (at least partly) due to DAG-induced activation of PKC. It is reasonable to assume that the positive inotropic effect of PGF2α (via the Gq/11-coupled FP-receptor) is brought about by a similar mechanism as that for α1- and ETA-receptors. In fact, Yew et al. (1998) have recently shown, that in isolated rat ventricular cardiomyocytes PGF2α increased single myocyte shortening and reduced resting cell-length in a concentration-dependent manner. This positive inotropic action was inhibited by the Na+/H+-antiporter inhibitor HOE 694 and by the PKC-inhibitor chelerythrine. Thus, the positive inotropic effect of PGF2α appears to be mediated via activation of the Na+/H+-antiporter with the possible involvement of PKC. That would also explain why U 46619 up to 1 μM does not affect force of contraction in the ventricular strips: at this concentration U 46619 does neither in adult cardiomyocytes nor in left ventricular slices stimulate PLC (as demonstrated by the lack of effect of U 46619 on InsP-formation).

In conclusion: In neonatal as well as adult rat ventricular cardiomyocytes prostanoids can increase inositol phosphate formation and rate of protein synthesis; these growth-promoting effects of prostanoids are mediated by activation of an FP-receptor. Studies are now in progress to investigate whether or not prostanoids play a pathophysiologic role in development and/or maintenance of cardiac hypertrophy.

Acknowledgments

The skilful technical assistance of I. Adler, A. Dunemann, A. Hauser, P. Schiewe, M. Niebisch and A. Struppert is gratefully acknowledged. This work was supported by a grant of the Deutsche Forschungsgemeinschaft (DFG OS 131/3-2 to B. Osten and O.-E. Brodde).

Abbreviations

DAG

diacylglycerol

ET-1

endothelin-1

InsP

inositol phosphate

PG

prostaglandin

PKC

protein kinase C

PLC

phospholipase C

TXA2

thromboxane A2

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