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The effect of pituitary adenylate cyclase activating polypeptide on cultured rat cardiocytes as a cardioprotective factor
Regulatory Peptides 109 (2002) 107 – 113 www.elsevier.com/locate/regpep
The effect of pituitary adenylate cyclase activating polypeptide on cultured rat cardiocytes as a cardioprotective factor Hirofumi Sano a, Atsuro Miyata a,b,*, Takeshi Horio a, Toshio Nishikimi a, Hisayuki Matsuo a, Kenji Kangawa a b
a National Cardiovascular Center Research Institute, 5-7-1 Fujishirodai, Suita, Osaka 565-8565, Japan Department of Pharmacology, Kagoshima University School of Medicine, 8-35-1 Sakuragaoka, Kagoshima 890-8520, Japan
Abstract In the cardiovascular system, pituitary adenylate cyclase activating polypeptide (PACAP) exhibits not only vasodilation but also positive inotropic action by increasing cardiac output. Then the effect of PACAP in cultured cardiovascular cells was examined. In neonatal rat myocytes, PACAP evoked concentration-dependent increase in intracellular cyclic AMP content more potently than vasoactive intestinal polypeptide (VIP). However, in neonatal rat nonmyocytes, PACAP and VIP showed equal potency. The characterization of the subtype of PACAP/VIP receptors by RT-PCR analysis revealed that PAC1 receptor mRNA is dominantly present in the myocytes, but VPAC2 receptor mRNA is abundant in the nonmyocytes. In the myocytes, PACAP did not change the protein synthesis stimulated by endothelin or by itself. However, PACAP moderately stimulated the secretion of atrial natriuretic polypeptide (ANP). On the other hand, PACAP inhibited the protein synthesis and DNA synthesis of the nonmyocytes. These indicate that PACAP might be involved in the regulation of cardiac hypertrophy and fibrosis as a cardioprotective factor. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Cyclic AMP; Cardiac myocyte; Nonmyocyte; Atrial natriuretic polypeptide
1. Introduction Pituitary adenylate cyclase activating polypeptide (PACAP), a neuropeptide first isolated from ovine hypothalamus, has two forms, one with 38 amino acid residues (PACAP38) and the other with 27 residues (PACAP27) [1,2]. Both PACAPs belong to the secretin/glucagon family and show highest homology (68%) with vasoactive intestinal polypeptide (VIP). PACAPs and VIP share three types of specific receptors, i.e., PAC1 receptor, VPAC1 receptor, and VPAC2 receptor [3 –5]. PAC1 receptor is a PACAPpreferring receptor, exhibiting high affinity for PACAP38 and PACAP27, and much lower affinity for VIP. VPAC1 receptor, and VPAC2 receptor possess similar affinity for PACAPs and VIP. These are members of a rhodopsin superfamily of G protein-coupled receptor with seven transmembrane domains and coupled with G proteins [4]. * Corresponding author. Department of Pharmacology, Kagoshima University School of Medicine, 8-35-1 Sakuragaoka, Kagoshima 890-8520, Japan. Tel.: +81-99-275-5256; fax: +81-99-265-8567. E-mail address: [email protected] (A. Miyata).
In the cardiovascular system, PACAP has been characterized as a potent vasodepressor or vasodilator as well as VIP [6]. In the heart, PACAP-immunoreactive neuronal fibers were identified within cardiac ganglia and interganglionic fiber tracts of guinea pig [7]. Messenger RNA encoding PACAP-selective PAC1 receptor isoforms were also present in guinea pig cardiac ganglia. Intravenous injection of PACAP in cat and sheep provokes an increase in heart rate and enhances the contractile ventricular force [8 –10]. In isolated neonatal pig hearts, PACAP produces positive inotropic and luisitropic effects more potently than VIP [11]. In dog, PACAP causes transient positive followed by negative chronotropic and inotropic responses [12]. The positive inotropic and chronotropic effects of PACAP are attributable to direct stimulation of cardiac myocytes [13,14], whereas the negative chronotropic response can be ascribed to presynaptic regulation of acetylcholine release from intracardiac parasympathetic nerves [15]. On the other hand, in rat, it was demonstrated that PACAP directly stimulates norepinephrine release from cardiac sympathetic nerve terminals [16]. These accumulated evidences indicate that PACAP could be a neurohumoral factor
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to be involved in hemodynamic regulation of the cardiovascular system. With regards to the action of PACAP on cultured cardiocytes (myocytes and nonmyocytes), only stimulatory effects on cyclic AMP accumulation and atrial natriuretic peptide (ANP) release were reported [17]. However, little is known about the effects of PACAP on hypertrophic reaction in rat cardiac myocytes and nonmyocytes individually. Incidentally, most of the cultured nonmyocytes in this study consists of cardiac fibroblasts. Therefore, we conducted this study to examine the direct effects of PACAP on myocyte hypertrophy and ANP release, and on nonmyocyte proliferation and collagen production, using the two types of ventricular cells purified and cultured from neonatal rats.
2. Materials and methods 2.1. Cell cultures Primary cultures of neonatal ventricular myocytes and nonmyocytes (cardiac fibroblasts) were prepared as described previously [18,19]. In brief, apical halves of cardiac ventricles from 1- to 2-day-old Wistar rats were separated and minced in a chilled balanced salt solution (116 mM NaCl, 20 mM HEPES, 12.5 mM NaH2PO4, 5.6 mM glucose, 5.4 mM KCl, and 0.8 mM MgSO4, pH 7.35). Ventricular cardiocytes were dispersed with 0.1% collagenase type II (Worthington Biochemical, Freehold, NJ) by agitation at 37 jC until complete digestion. The dispersed cells were collected and subjected to centrifugation with a discontinuous gradient of 40.5% and 58.5% Percoll (Sigma, St. Louis, MO) in order to separate myocytes from nonmyocytes. After centrifugation at 3000 rpm for 30 min, the upper layer consisted of a mixed population of nonmyocyte cell types, and the lower layer consisted almost exclusively of cardiac myocytes. After incubation on uncoated dishes to remove remaining nonmyocytes, the nonattached viable cells (purified myocytes) were plated at a density of 1.2 105 cells/well on gelatin-coated 24-well culture plates and were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Life Technologies) supplemented with 10% fetal calf serum and antibiotics (50 U/ml penicillin and 50 Ag/ml streptomycin, ICN Biomedicals, Aurora, OH) at 37 jC in humidified air with 5% CO2. After 24 –48 h of incubation in DMEM with fetal calf serum, the culture medium was changed to serumfree DMEM, and the experiments were performed 24 h later. Since this purification procedure has been well established, more than 95% of the cells, which we obtained thus, were cardiac myocytes. Nonmyocyte cells were resuspended in DMEM with 10% fetal calf serum and plated on uncoated 10 cm culture dishes for 30 min. After the plating period, nonadherent cells and debris were washed away and fresh medium was added. The cells were allowed to grow to confluence,
trypsinized, and passaged 1:3. Since this procedure yielded cultures of cells that were almost exclusively fibroblasts by the first passage, fibroblasts at the second or third passage were plated as nonmyocytes at a density of 2 104 cells/ well on 24-well plates and allowed to grow to confluence. After incubation in DMEM with fetal calf serum, the culture medium was changed to serum-free DMEM, and the experiments were performed 48 h later. 2.2. Polymerase chain reaction (PCR) for PACAP/VIP receptor mRNA The procedure of RNA preparation and reverse transcription (RT) is performed in a similar manner to that described previously [20]. In brief, total RNA was extracted with TRIzol reagent (BRL) and poly (A) + RNA was prepared with oligo (dT) 30 latex (Nippon Roche). The first-strand cDNA syntheses were performed with random hexamer and RNase H reverse transcriptase (Superscript II, BRL). The cDNA produced from RT reactions using total RNA from the myocytes or nonmyocytes was amplified using PCR with pairs of primers specific for PACAP, PAC1 receptor, VPAC1 receptor and VPAC2 receptor. For PACAP, the primers used were rPCF (5V-CCGTCCTATTTAGTCAACTCTTTC-3V) and rPCR (5V-TTAACCCTCTGTTTATACCTTTTC-3V), which should give a PCR product of 531 base pairs (bp). For PAC1 receptor, the primers used were PACF (5V-GTGGTGTCCAACTACTTCTG-3V), PACR (5V-TGGAGAGAAGGCGAATAC-3V), which would be expected to produce PCR product for the basic receptor, a single cassette insert (hip, hop1 or hop2) and a double insert (hiphop1 or hiphop2) as bands of 411, 495 and 579 bp, respectively [5]. For VPAC1 receptor, the primers used were PV1F (5V-CCAACTTCTTCTGGCTGC3V), PV1R (5V-CACGAAACCCTGGAAAGA-3V), which should give a PCR product of 470 bp. For VPAC2 receptor, the primers used were PV2F (5V-TGGCGACACTTCTACTGGC-3V), PV2R (5V-GGAAGGAACCAACACATAAC-3V), yielding a predicted PCR product 460 bp in length. Thermal cycle profile: annealing at 54– 57 jC for 1 min, extension at 72 jC for 1.5 min, denaturation at 94 jC for 50 s for a total of 35 cycles. The PCR reaction mixture was electrophoresed on a 1.5% agarose gel and photographed. 2.3. Cyclic AMP assay After preincubation, cardiac myocytes or nonmyocytes grown in 24-well plates were treated for 10 min with various concentrations of PACAP38, PACAP27 or VIP in the presence of 0.5 mM 3-isobutyl-1-methylxanthine (IBMX) as described previously [19]. The reaction was terminated by rapid aspiration of the medium and the addition of icecold 70% ethanol. After each ethanol sample was evaporated by a centrifugal evaporator, the dry residue was dissolved in an assay buffer. The cAMP levels were deter-
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mined by a radioimmunoassay performed with a cAMP assay kit (Yamasa Shoyu, Chiba, Japan), as described previously [19]. 2.4. Analyses of protein, DNA, and collagen syntheses The effects of various agents on protein synthesis in cardiac myocytes were evaluated by the incorporation of [14C] phenylalanine into cells . The effects on DNA and collagen syntheses in nonmyocytes were evaluated by the incorporation of [3H] thymidine and [3H] proline, respectively, since collagen is rich in proline as a collagen hydroxyproline. After the preconditioning period, the culture medium was replaced with fresh serum-free DMEM. Then, endothelin-1, angiotensin II, PACAP38 and VIP (Peptide Institute, Osaka, Japan), were added. For protein synthesis in myocytes or collagen synthesis in nonmyocytes, either 0.2 ACi of [14C] phenylalanine or 0.5 ACi of [3H] proline was added, or then the plates were incubated for 24 h. For DNA synthesis in nonmyocytes, 0.5 ACi of [3H] thymidine was added 12 h after pharmacological treatments, and the cells were further incubated for 12 h. After completion of labeling, the cells were rinsed twice with cold phosphate-buffered saline and incubated with 10% trichloroacetic acid at 4 jC for 30 min. The precipitates were washed twice with cold 95% ethanol and solubilized in 1 M NaOH. The radioactivity of aliquots of the trichloroacetic acid-insoluble material was determined using a liquid scintillation counter.
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2.5. ANP production After cardiac myocytes were treated with PACAP38 or VIP for 24 h, the culture medium was aspirated and stored at 80 jC. The medium (100 Al) was acidified with acetic acid, boiled to inactivate intrinsic proteases, and lyophilized. ANP concentrations in the medium were determined by the sandwich-enzyme immunoassay method (Peninsula Lab., San Carlos, CA), according to the manufacturer’s protocol. 2.6. Calculations and statistical analysis The statistical significance of differences in the results was evaluated using an unpaired analysis of variance, and P values were calculated by Fisher’s method. P < 0.05 was accepted as statistically significant.
3. Results 3.1. Effect of PACAP on cellular cAMP levels on cardiac myocytes and nonmyocytes In cultured cardiac myocytes, PACAP38 and PACAP27 increased the cellular levels of cAMP in a concentrationdependent manner with an EC50 (50% effective concentration) of 3.1 10 10 and 2.0 10 9 M, respectively (Fig. 1A). Meanwhile VIP was much less potent (EC50>1 10 6 M). In nonmyocytes, PACAP38, PACAP27 and VIP aug-
Fig. 1. Effects of PACAP27, PACAP38 and VIP on the production of cellular cAMP in cultured cardiac myocytes (A) and nonmyocytes (B). Values are the mean of four measurements.
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Fig. 2. Expression of three subtypes of PACAP/VIP receptor and PACAP detected by RT-PCR in cultured cardiac myocytes (MC) and nonmyocytes (NMC). M, PAC1, VP1 and VP2 indicate DNA size marker, PAC1 receptor, VPAC1 receptor, and VPAC2 receptor, respectively.
ment intracellular cAMP level in a dose-dependent manner with an EC50 of 8.9 10 10, 3.0 10 9, and 2.4 10 9 M, respectively (Fig. 1B). The maximum cAMP formation by PACAP38 was about 10-fold greater in nonmyocytes than in myocytes. 3.2. Expression of PACAP/VIP receptors in cardiac myocytes and nonmyocytes To examine how three distinct PACAP/VIP receptor subtypes (PAC1 receptor, VPAC1 receptor, and VPAC2 receptor) are expressed in rat cardiac cells, we performed RT-PCR using the specific primers on the mRNAs extracted from rat myocytes and nonmyocytes. As shown in Fig. 2, PAC1 mRNA is expressed dominantly in cultured cardiac myocytes, and faintly in nonmyocytes. Whereas VACP2 mRNA is expressed abundantly in nonmyocytes and less in
cardiac myocytes. Incidentally, VPAC1 receptor was not detected, but PACAP was detected in both cells. The sizes of the amplified bands are in agreement with the predicted fragment size produced with the primers specific for the respective receptor. As regards the size of the bands for PAC1, the mRNA from myocytes showed two bands, whose sizes indicate the expression of the short (no insert) form and the single cassette containing form (hip or hop) of the PAC1 receptor. 3.3. Effects of PACAP and VIP on DNA and collagen syntheses in nonmyocytes The effect of PACAP on collagen synthesis in cultured nonmyocytes under non-stimulated and angiotensin IIstimulated conditions is shown in Fig. 3A. The [3H] proline uptakes into both non-stimulated and stimulated cells were inhibited by PACAP as well as VIP. Then as shown in Fig. 3C and D, PACAP38 and VIP inhibit collagen production in a concentration-dependent manner with similar potency. Furthermore, as shown in Fig. 3B, PACAP38 clearly inhibited the [3H] thymidine uptake in cultured nonmyocytes under the basal condition as well as VIP. 3.4. Effects of PACAP and VIP on protein synthesis and ANP secretion in cardiac myocytes The effect of PACAP and VIP on protein synthesis in cultured cardiac myocytes is shown in Fig. 4A. 10 7 M
Fig. 3. (A) Effects of PACAP38 (P, 10 7 M) and VIP (V, 10 7 M) on collagen synthesis in nonmyocytes under non-stimulated (C: control) and angiotensin II (A, 10 6 M)-stimulated conditions. Values are the mean F S.E. (n = 8) as percentage of 3H-proline uptake as compared with the control, * p < 0.05 vs. control, #p < 0.01 vs. angiotensin II. (C, D) Dose-dependent effect of PACAP38 and VIP on collagen synthesis in nonmyocytes. Values are the same in (A), * p < 0.05 vs. control. (B) Effects of PACAP (P, 10 7 M), VIP (V, 10 7 M) and angiotensin II (A, 10 6 M) on DNA synthesis in nonmyocytes. Values are the mean F S.E. (n = 6) as the percentage of 3H-thymidine uptake as compared with the control, * p < 0.05 vs. control.
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Fig. 4. (A) Effects of PACAP38 (P, 10 7 M) and VIP (V, 10 7 M) on protein synthesis in cultured cardiac myocytes under non-stimulated (C: control) and endothelin-I (E, 10 7 M)-stimulated conditions. Values are the mean F S.E. (n = 8) as the percentage of 14C-phenylalanine uptake as compared with the control. * p < 0.05 vs. control. (B) Effect of PACAP38 (P, 10 7 M), VIP (V, 10 7 M) and endothelin-I (E, 10 7 M) on ANP production. Values are the mean F S.E. (n = 3 f 4) as the percentage change of ANP for 24 h as compared with the control (C).
PACAP38 did not affect the incorporation level of [14C] phenylalanine in the cultured cardiac myocytes under nonstimulated and endothelin-1-stimulated conditions. However, The secretion of ANP was increased by stimulation with 10 7 M PACAP38 but less potently than with 10 7 M endothelin-1 as shown in Fig. 4B. On the other hand, VIP did not affect either protein synthesis or the secretion of ANP at all.
4. Discussion The present study has clearly demonstrated that PACAP38 and PACAP27 evokes concentration-dependent increases in intracellular cyclic AMP content much more potently than VIP in cultured cardiac myocytes. This result is consistent with the previous report [17]. On the other hand, three peptides exhibited equal potency in cultured nonmyocytes. These observations agree well with the expression pattern of three subtypes of PACAP/VIP receptor mRNA, in which PAC1 receptor is expressed dominantly in myocytes, whereas VPAC2 receptor abundantly in nonmyocytes. The weak band also found in the myocyte lane of the VPAC2 receptor is probably due to the contamination of nonmyocytes in the preparation of cardiac myocytes, since it is difficult to completely avoid the contamination of nonmyocytes even if the discontinuous Percoll gradient method were used. A similar explanation could be applied to the faint presence of PAC1 in nonmyocytes. It should be interesting that PAC1 receptor and VPAC2 receptor seem to be expressed separately in two different cell types, since the presence of PAC1, VPAC1 and VPAC2 mRNA were demonstrated in rat neonatal heart (atrial appendages and ventricles) [21]. Further, these expression patterns are very
similar to those seen in the vascular wall, in which PAC1 receptor is expressed dominantly in endothelial cells, whereas VPAC2 exclusively in smooth muscle cells as reported previously [22]. These indicate that those two subtypes might be crucial and share the physiological function of PACAP in the cardiovascular system. In addition, the detection of PACAP mRNA in cultured cells indicates that PACAP might function as a paracrine or autocrine. The present study has, for the first time, demonstrated that PACAP inhibits DNA and collagen syntheses in cultured nonmyocytes under non-stimulated and angiotensin IIstimulated conditions. It is well-known that angiotensin II attenuates the increment of interstitial tissue in hypertrophic heart [23]. It was suggested that the observed inhibitory effects of PACAP on nonmyocytes are probably mediated through a cAMP-dependent process, since two cAMPrelated compounds; that is, 8-bromo cAMP, a cAMP analogue, and forskolin, an activator of adenylate cyclase were previously reported to inhibit DNA and collagen syntheses in nonmyocytes [18]. Thus, The possible function of PACAP may be attenuation of the mitogenesis and collagen production in nonmyocytes. As for the protein synthesis in cardiac myocytes, PACAP did not stimulate the [14C] phenylalanine, suggesting the benefit of avoiding hypertrophy of the cardiac myocytes. In addition, PACAP stimulates the release of atrial natriuretic polypeptide (ANP), a marker for hypertrophic response, even though VIP had no effect on its release [17]. Incidentally potassium ATP channels play a fundamental role in cardiac excitability and potently modulate the stimulated ANP secretion. As well as in the vascular smooth muscle cells, PACAP activates the atrial KATP channels through both PKA and PKC pathways [21]. ANP is well known to
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increase with heart failure and reduce the volume overload by diuresis and natriuresis [24]. Since 8-bromo cAMP and forskolin inhibit the ANP secretion from cardiac myocytes, as in previous observations [25], PACAP may stimulate the ANP release via cAMP-independent mechanism through the PACAP-preferring receptor PAC1, even though the exact mechanism has not been elucidated in the present study. Cardiac hypertrophy occurs in various pathophysiological states, such as hypertension, valvular heart disease and so on. In hypertrophic heart, cardiac myocytes increase in size, and nonmyocytes, which are almost cardiac fibroblasts, increase in number and production of intercellular matrix. These reactions, so-called as cardiac remodeling, make worse in cardiac function [26]. Although the pathophysiological roles of PACAP in such cardiac disorders remain unclear, our present findings may suggest some roles. PACAP in heart has been found to act on nonmyocytes as an inhibitor of cardiac fibrosis through VPAC2 receptor, and on myocytes as a stimulator of ANP secretion through PAC1 receptor, indicating that PACAP could play the role of a neurohumoral factor to inhibit the remodeling at cardiac hypertrophy. In addition, cAMP production is a benefit to increase cardiac output by increment of contractile force. These multiple effects of PACAP on the heart indicate that PACAP functions as a cardioprotective factor in some pathological states, even though the biosynthesis and secretion of PACAP under pathological conditions remains unclear. Anyway, further investigations are necessary to clarify the physiological and pathophysiological significance of PACAP in the heart.
Acknowledgements The authors thank Ms. Kimie Godoh for her secretarial assistance, and Ms. Michiyo Tanaka for her technical assistance. This work was supported in part by Special Coordination Funds for Promoting Science and Technology (Encouragement System of COE) from the Science and Technology Agency of Japan, and Scientific Research Grant-in-Aid 11670161 from the Ministry of Education, Science and Culture of Japan.
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[4] Harmar AJ, Arimura A, Gozes I, et al. Nomenclature of receptors for vasoactive intestinal peptide (VIP) and pituitary adenylate cyclaseactivating polypeptide (PACAP). Pharmacol Rev 1998;50:265 – 70. [5] Spengler D, Waeber C, Pantaloni C, Holsboer F, Bockaert J, Seeburg PH, et al. Differential signal transduction by five splices variants of the PACAP receptor. Nature 1993;365:170 – 5. [6] Vaudry D, Gonzalez BJ, Basille M, Yon L, Fournier A, Vaudry H. Pituitary adenylate cyclase-activating polypeptide and its receptors: from structure and functions. Pharmacol Rev 2000;52:269 – 324. [7] Braas KM, May V, Harakall SA, Hardwick JC, Parsons RL. Pituitary adenylate cyclase-activating polypeptide expression and modulation of neuronal excitability in guinea pig cardiac ganglia. J Neurosci 1998;18:9766 – 79. [8] Minkes RK, McMahon TJ, Higuera TR, Murphy WA, Coy DH, Kadowitz PJ. Analysis of systemic and pulmonary vascular responses to PACAP and VIP: role of adrenal catecholamines. Am J Physiol 1992;263:H1659 – 69. [9] Sawangjaroen K, Dallemagen CR, Cross RB, Curlewis JD. Effects of pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) on the cardiovascular system in sheep. Peptides 1992;13:1029 – 32. [10] Sawangjaroen K, Curlewis JD. Effects of pituitary adenylate cyclaseactivating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) on prolactin, luteinizing hormone and growth hormone secretion in the ewe. J Neuroendocrinol 1994;6:549 – 55. [11] Ross-Ascuitto NT, Ascuitto RJ, Ramage D, Kydon DW, Coy DH, Kadowitz PJ. Pituitary adenylate cyclase-activating polypeptide: a neuropeptide with potent inotropic and coronary vasodilatory effects in neonatal pig hearts. Pediatr Res 1993;34:323 – 8. [12] Hirose M, Furukawa Y, Lakhe M, Chiba S. Regional differences in cardiac effects of pituitary adenylate cyclase-activating polypeptide27 in the isolated dog heart. Eur J Pharmacol 1998;349:269 – 76. [13] Suzuki N, Kasai K, Iino I, Takekoshi K, Oka M, Shimoda SI. Antishock effect of pituitary adenylate cyclase-activating polypeptide (PACAP) on experimental endotoxin shock in dogs. Life Sci 1994; 54:389 – 94. [14] Runcie MJ, Ulman LG, Potter EK. Effects of pituitary adenylate cyclase-activating polypeptide on cardiovascular and respiratory responses in anaesthetized dogs. Regul Pept 1995;60:193 – 200. [15] Seebeck J, Schmidt WE, Kilbinger H, Neumann J, Zimmermann N, Herzig S. PACAP induces bradycardia in guinea-pig heart by stimulation of atrial cholinergic neurons. Naunyn Schmiedeberg’s Arch Pharmacol 1996;354:424 – 30. [16] Whalen EJ, Johnson AK, Lewis SJ. Hemodynamic actions of systemically injected pituitary adenylate cyclase-activating polypeptide-27 in the rat. Eur J Pharmacol 1999;365:205 – 21. [17] Ba¨sler I, Kuhn M, Mu¨ller W, Forssmann W-G. Pituitary adenylate cyclase-activating polypeptide stimulates cardiodilatin/atrial natriuretic peptide (CDD/ANP)- (99 – 126) secretion from cultured neonatal rat myocardiocytes. Eur J Pharmacol 1995;291:335 – 42. [18] Horio T, Nishikimi T, Yoshihara F, Matsuo H, Takishita S, Kangawa K. Effects of adrenomedullin on cultured rat cardiac myocytes and fibroblast. Eur J Pharmacol 1999;382:1 – 9. [19] Nishikimi T, Horio T, Yoshihara F, Nagaya N, Matsuo H, Kangawa K. Effect of adrenomedullin on cAMP and cGMP levels in rat cardiac myocytes and nonmyocytes. Eur J Pharmacol 1998;353:337 – 44. [20] Tamakawa H, Miyata A, Satoh K, Miyake Y, Matsuo H, Arimura A, et al. The augmentation of pituitary adenylate cyclase activating polypeptide (PACAP) in streptozotocin-induced diabetic rats. Peptides 1998;19:1497 – 502. [21] Baron A, Monnier D, Roatti A, Baertschi AJ. Pituitary adenylate cyclase-activating polypeptide activates KATP current in rat atrial myocytes. Am J Physiol, Heart Circ Physiol 2001;280:H1058 – 65. [22] Miyata A, Satou K, Hino J, Tamakawa H, Matsuo H, Kangawa K. Rat aortic smooth muscle cells are bidirectionally regulated in a cell cycledependent manner via PACAP/VIP type 2 receptor. Ann NY Acad Sci 1998;865:73 – 81.
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The effect of pituitary adenylate cyclase activating polypeptide on cultured rat cardiocytes as a cardioprotective factor Hirofumi Sano a, Atsuro Miyata a,b,*, Takeshi Horio a, Toshio Nishikimi a, Hisayuki Matsuo a, Kenji Kangawa a b
a National Cardiovascular Center Research Institute, 5-7-1 Fujishirodai, Suita, Osaka 565-8565, Japan Department of Pharmacology, Kagoshima University School of Medicine, 8-35-1 Sakuragaoka, Kagoshima 890-8520, Japan
Abstract In the cardiovascular system, pituitary adenylate cyclase activating polypeptide (PACAP) exhibits not only vasodilation but also positive inotropic action by increasing cardiac output. Then the effect of PACAP in cultured cardiovascular cells was examined. In neonatal rat myocytes, PACAP evoked concentration-dependent increase in intracellular cyclic AMP content more potently than vasoactive intestinal polypeptide (VIP). However, in neonatal rat nonmyocytes, PACAP and VIP showed equal potency. The characterization of the subtype of PACAP/VIP receptors by RT-PCR analysis revealed that PAC1 receptor mRNA is dominantly present in the myocytes, but VPAC2 receptor mRNA is abundant in the nonmyocytes. In the myocytes, PACAP did not change the protein synthesis stimulated by endothelin or by itself. However, PACAP moderately stimulated the secretion of atrial natriuretic polypeptide (ANP). On the other hand, PACAP inhibited the protein synthesis and DNA synthesis of the nonmyocytes. These indicate that PACAP might be involved in the regulation of cardiac hypertrophy and fibrosis as a cardioprotective factor. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Cyclic AMP; Cardiac myocyte; Nonmyocyte; Atrial natriuretic polypeptide
1. Introduction Pituitary adenylate cyclase activating polypeptide (PACAP), a neuropeptide first isolated from ovine hypothalamus, has two forms, one with 38 amino acid residues (PACAP38) and the other with 27 residues (PACAP27) [1,2]. Both PACAPs belong to the secretin/glucagon family and show highest homology (68%) with vasoactive intestinal polypeptide (VIP). PACAPs and VIP share three types of specific receptors, i.e., PAC1 receptor, VPAC1 receptor, and VPAC2 receptor [3 –5]. PAC1 receptor is a PACAPpreferring receptor, exhibiting high affinity for PACAP38 and PACAP27, and much lower affinity for VIP. VPAC1 receptor, and VPAC2 receptor possess similar affinity for PACAPs and VIP. These are members of a rhodopsin superfamily of G protein-coupled receptor with seven transmembrane domains and coupled with G proteins [4]. * Corresponding author. Department of Pharmacology, Kagoshima University School of Medicine, 8-35-1 Sakuragaoka, Kagoshima 890-8520, Japan. Tel.: +81-99-275-5256; fax: +81-99-265-8567. E-mail address: [email protected] (A. Miyata).
In the cardiovascular system, PACAP has been characterized as a potent vasodepressor or vasodilator as well as VIP [6]. In the heart, PACAP-immunoreactive neuronal fibers were identified within cardiac ganglia and interganglionic fiber tracts of guinea pig [7]. Messenger RNA encoding PACAP-selective PAC1 receptor isoforms were also present in guinea pig cardiac ganglia. Intravenous injection of PACAP in cat and sheep provokes an increase in heart rate and enhances the contractile ventricular force [8 –10]. In isolated neonatal pig hearts, PACAP produces positive inotropic and luisitropic effects more potently than VIP [11]. In dog, PACAP causes transient positive followed by negative chronotropic and inotropic responses [12]. The positive inotropic and chronotropic effects of PACAP are attributable to direct stimulation of cardiac myocytes [13,14], whereas the negative chronotropic response can be ascribed to presynaptic regulation of acetylcholine release from intracardiac parasympathetic nerves [15]. On the other hand, in rat, it was demonstrated that PACAP directly stimulates norepinephrine release from cardiac sympathetic nerve terminals [16]. These accumulated evidences indicate that PACAP could be a neurohumoral factor
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to be involved in hemodynamic regulation of the cardiovascular system. With regards to the action of PACAP on cultured cardiocytes (myocytes and nonmyocytes), only stimulatory effects on cyclic AMP accumulation and atrial natriuretic peptide (ANP) release were reported [17]. However, little is known about the effects of PACAP on hypertrophic reaction in rat cardiac myocytes and nonmyocytes individually. Incidentally, most of the cultured nonmyocytes in this study consists of cardiac fibroblasts. Therefore, we conducted this study to examine the direct effects of PACAP on myocyte hypertrophy and ANP release, and on nonmyocyte proliferation and collagen production, using the two types of ventricular cells purified and cultured from neonatal rats.
2. Materials and methods 2.1. Cell cultures Primary cultures of neonatal ventricular myocytes and nonmyocytes (cardiac fibroblasts) were prepared as described previously [18,19]. In brief, apical halves of cardiac ventricles from 1- to 2-day-old Wistar rats were separated and minced in a chilled balanced salt solution (116 mM NaCl, 20 mM HEPES, 12.5 mM NaH2PO4, 5.6 mM glucose, 5.4 mM KCl, and 0.8 mM MgSO4, pH 7.35). Ventricular cardiocytes were dispersed with 0.1% collagenase type II (Worthington Biochemical, Freehold, NJ) by agitation at 37 jC until complete digestion. The dispersed cells were collected and subjected to centrifugation with a discontinuous gradient of 40.5% and 58.5% Percoll (Sigma, St. Louis, MO) in order to separate myocytes from nonmyocytes. After centrifugation at 3000 rpm for 30 min, the upper layer consisted of a mixed population of nonmyocyte cell types, and the lower layer consisted almost exclusively of cardiac myocytes. After incubation on uncoated dishes to remove remaining nonmyocytes, the nonattached viable cells (purified myocytes) were plated at a density of 1.2 105 cells/well on gelatin-coated 24-well culture plates and were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Life Technologies) supplemented with 10% fetal calf serum and antibiotics (50 U/ml penicillin and 50 Ag/ml streptomycin, ICN Biomedicals, Aurora, OH) at 37 jC in humidified air with 5% CO2. After 24 –48 h of incubation in DMEM with fetal calf serum, the culture medium was changed to serumfree DMEM, and the experiments were performed 24 h later. Since this purification procedure has been well established, more than 95% of the cells, which we obtained thus, were cardiac myocytes. Nonmyocyte cells were resuspended in DMEM with 10% fetal calf serum and plated on uncoated 10 cm culture dishes for 30 min. After the plating period, nonadherent cells and debris were washed away and fresh medium was added. The cells were allowed to grow to confluence,
trypsinized, and passaged 1:3. Since this procedure yielded cultures of cells that were almost exclusively fibroblasts by the first passage, fibroblasts at the second or third passage were plated as nonmyocytes at a density of 2 104 cells/ well on 24-well plates and allowed to grow to confluence. After incubation in DMEM with fetal calf serum, the culture medium was changed to serum-free DMEM, and the experiments were performed 48 h later. 2.2. Polymerase chain reaction (PCR) for PACAP/VIP receptor mRNA The procedure of RNA preparation and reverse transcription (RT) is performed in a similar manner to that described previously [20]. In brief, total RNA was extracted with TRIzol reagent (BRL) and poly (A) + RNA was prepared with oligo (dT) 30 latex (Nippon Roche). The first-strand cDNA syntheses were performed with random hexamer and RNase H reverse transcriptase (Superscript II, BRL). The cDNA produced from RT reactions using total RNA from the myocytes or nonmyocytes was amplified using PCR with pairs of primers specific for PACAP, PAC1 receptor, VPAC1 receptor and VPAC2 receptor. For PACAP, the primers used were rPCF (5V-CCGTCCTATTTAGTCAACTCTTTC-3V) and rPCR (5V-TTAACCCTCTGTTTATACCTTTTC-3V), which should give a PCR product of 531 base pairs (bp). For PAC1 receptor, the primers used were PACF (5V-GTGGTGTCCAACTACTTCTG-3V), PACR (5V-TGGAGAGAAGGCGAATAC-3V), which would be expected to produce PCR product for the basic receptor, a single cassette insert (hip, hop1 or hop2) and a double insert (hiphop1 or hiphop2) as bands of 411, 495 and 579 bp, respectively [5]. For VPAC1 receptor, the primers used were PV1F (5V-CCAACTTCTTCTGGCTGC3V), PV1R (5V-CACGAAACCCTGGAAAGA-3V), which should give a PCR product of 470 bp. For VPAC2 receptor, the primers used were PV2F (5V-TGGCGACACTTCTACTGGC-3V), PV2R (5V-GGAAGGAACCAACACATAAC-3V), yielding a predicted PCR product 460 bp in length. Thermal cycle profile: annealing at 54– 57 jC for 1 min, extension at 72 jC for 1.5 min, denaturation at 94 jC for 50 s for a total of 35 cycles. The PCR reaction mixture was electrophoresed on a 1.5% agarose gel and photographed. 2.3. Cyclic AMP assay After preincubation, cardiac myocytes or nonmyocytes grown in 24-well plates were treated for 10 min with various concentrations of PACAP38, PACAP27 or VIP in the presence of 0.5 mM 3-isobutyl-1-methylxanthine (IBMX) as described previously [19]. The reaction was terminated by rapid aspiration of the medium and the addition of icecold 70% ethanol. After each ethanol sample was evaporated by a centrifugal evaporator, the dry residue was dissolved in an assay buffer. The cAMP levels were deter-
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mined by a radioimmunoassay performed with a cAMP assay kit (Yamasa Shoyu, Chiba, Japan), as described previously [19]. 2.4. Analyses of protein, DNA, and collagen syntheses The effects of various agents on protein synthesis in cardiac myocytes were evaluated by the incorporation of [14C] phenylalanine into cells . The effects on DNA and collagen syntheses in nonmyocytes were evaluated by the incorporation of [3H] thymidine and [3H] proline, respectively, since collagen is rich in proline as a collagen hydroxyproline. After the preconditioning period, the culture medium was replaced with fresh serum-free DMEM. Then, endothelin-1, angiotensin II, PACAP38 and VIP (Peptide Institute, Osaka, Japan), were added. For protein synthesis in myocytes or collagen synthesis in nonmyocytes, either 0.2 ACi of [14C] phenylalanine or 0.5 ACi of [3H] proline was added, or then the plates were incubated for 24 h. For DNA synthesis in nonmyocytes, 0.5 ACi of [3H] thymidine was added 12 h after pharmacological treatments, and the cells were further incubated for 12 h. After completion of labeling, the cells were rinsed twice with cold phosphate-buffered saline and incubated with 10% trichloroacetic acid at 4 jC for 30 min. The precipitates were washed twice with cold 95% ethanol and solubilized in 1 M NaOH. The radioactivity of aliquots of the trichloroacetic acid-insoluble material was determined using a liquid scintillation counter.
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2.5. ANP production After cardiac myocytes were treated with PACAP38 or VIP for 24 h, the culture medium was aspirated and stored at 80 jC. The medium (100 Al) was acidified with acetic acid, boiled to inactivate intrinsic proteases, and lyophilized. ANP concentrations in the medium were determined by the sandwich-enzyme immunoassay method (Peninsula Lab., San Carlos, CA), according to the manufacturer’s protocol. 2.6. Calculations and statistical analysis The statistical significance of differences in the results was evaluated using an unpaired analysis of variance, and P values were calculated by Fisher’s method. P < 0.05 was accepted as statistically significant.
3. Results 3.1. Effect of PACAP on cellular cAMP levels on cardiac myocytes and nonmyocytes In cultured cardiac myocytes, PACAP38 and PACAP27 increased the cellular levels of cAMP in a concentrationdependent manner with an EC50 (50% effective concentration) of 3.1 10 10 and 2.0 10 9 M, respectively (Fig. 1A). Meanwhile VIP was much less potent (EC50>1 10 6 M). In nonmyocytes, PACAP38, PACAP27 and VIP aug-
Fig. 1. Effects of PACAP27, PACAP38 and VIP on the production of cellular cAMP in cultured cardiac myocytes (A) and nonmyocytes (B). Values are the mean of four measurements.
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Fig. 2. Expression of three subtypes of PACAP/VIP receptor and PACAP detected by RT-PCR in cultured cardiac myocytes (MC) and nonmyocytes (NMC). M, PAC1, VP1 and VP2 indicate DNA size marker, PAC1 receptor, VPAC1 receptor, and VPAC2 receptor, respectively.
ment intracellular cAMP level in a dose-dependent manner with an EC50 of 8.9 10 10, 3.0 10 9, and 2.4 10 9 M, respectively (Fig. 1B). The maximum cAMP formation by PACAP38 was about 10-fold greater in nonmyocytes than in myocytes. 3.2. Expression of PACAP/VIP receptors in cardiac myocytes and nonmyocytes To examine how three distinct PACAP/VIP receptor subtypes (PAC1 receptor, VPAC1 receptor, and VPAC2 receptor) are expressed in rat cardiac cells, we performed RT-PCR using the specific primers on the mRNAs extracted from rat myocytes and nonmyocytes. As shown in Fig. 2, PAC1 mRNA is expressed dominantly in cultured cardiac myocytes, and faintly in nonmyocytes. Whereas VACP2 mRNA is expressed abundantly in nonmyocytes and less in
cardiac myocytes. Incidentally, VPAC1 receptor was not detected, but PACAP was detected in both cells. The sizes of the amplified bands are in agreement with the predicted fragment size produced with the primers specific for the respective receptor. As regards the size of the bands for PAC1, the mRNA from myocytes showed two bands, whose sizes indicate the expression of the short (no insert) form and the single cassette containing form (hip or hop) of the PAC1 receptor. 3.3. Effects of PACAP and VIP on DNA and collagen syntheses in nonmyocytes The effect of PACAP on collagen synthesis in cultured nonmyocytes under non-stimulated and angiotensin IIstimulated conditions is shown in Fig. 3A. The [3H] proline uptakes into both non-stimulated and stimulated cells were inhibited by PACAP as well as VIP. Then as shown in Fig. 3C and D, PACAP38 and VIP inhibit collagen production in a concentration-dependent manner with similar potency. Furthermore, as shown in Fig. 3B, PACAP38 clearly inhibited the [3H] thymidine uptake in cultured nonmyocytes under the basal condition as well as VIP. 3.4. Effects of PACAP and VIP on protein synthesis and ANP secretion in cardiac myocytes The effect of PACAP and VIP on protein synthesis in cultured cardiac myocytes is shown in Fig. 4A. 10 7 M
Fig. 3. (A) Effects of PACAP38 (P, 10 7 M) and VIP (V, 10 7 M) on collagen synthesis in nonmyocytes under non-stimulated (C: control) and angiotensin II (A, 10 6 M)-stimulated conditions. Values are the mean F S.E. (n = 8) as percentage of 3H-proline uptake as compared with the control, * p < 0.05 vs. control, #p < 0.01 vs. angiotensin II. (C, D) Dose-dependent effect of PACAP38 and VIP on collagen synthesis in nonmyocytes. Values are the same in (A), * p < 0.05 vs. control. (B) Effects of PACAP (P, 10 7 M), VIP (V, 10 7 M) and angiotensin II (A, 10 6 M) on DNA synthesis in nonmyocytes. Values are the mean F S.E. (n = 6) as the percentage of 3H-thymidine uptake as compared with the control, * p < 0.05 vs. control.
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Fig. 4. (A) Effects of PACAP38 (P, 10 7 M) and VIP (V, 10 7 M) on protein synthesis in cultured cardiac myocytes under non-stimulated (C: control) and endothelin-I (E, 10 7 M)-stimulated conditions. Values are the mean F S.E. (n = 8) as the percentage of 14C-phenylalanine uptake as compared with the control. * p < 0.05 vs. control. (B) Effect of PACAP38 (P, 10 7 M), VIP (V, 10 7 M) and endothelin-I (E, 10 7 M) on ANP production. Values are the mean F S.E. (n = 3 f 4) as the percentage change of ANP for 24 h as compared with the control (C).
PACAP38 did not affect the incorporation level of [14C] phenylalanine in the cultured cardiac myocytes under nonstimulated and endothelin-1-stimulated conditions. However, The secretion of ANP was increased by stimulation with 10 7 M PACAP38 but less potently than with 10 7 M endothelin-1 as shown in Fig. 4B. On the other hand, VIP did not affect either protein synthesis or the secretion of ANP at all.
4. Discussion The present study has clearly demonstrated that PACAP38 and PACAP27 evokes concentration-dependent increases in intracellular cyclic AMP content much more potently than VIP in cultured cardiac myocytes. This result is consistent with the previous report [17]. On the other hand, three peptides exhibited equal potency in cultured nonmyocytes. These observations agree well with the expression pattern of three subtypes of PACAP/VIP receptor mRNA, in which PAC1 receptor is expressed dominantly in myocytes, whereas VPAC2 receptor abundantly in nonmyocytes. The weak band also found in the myocyte lane of the VPAC2 receptor is probably due to the contamination of nonmyocytes in the preparation of cardiac myocytes, since it is difficult to completely avoid the contamination of nonmyocytes even if the discontinuous Percoll gradient method were used. A similar explanation could be applied to the faint presence of PAC1 in nonmyocytes. It should be interesting that PAC1 receptor and VPAC2 receptor seem to be expressed separately in two different cell types, since the presence of PAC1, VPAC1 and VPAC2 mRNA were demonstrated in rat neonatal heart (atrial appendages and ventricles) [21]. Further, these expression patterns are very
similar to those seen in the vascular wall, in which PAC1 receptor is expressed dominantly in endothelial cells, whereas VPAC2 exclusively in smooth muscle cells as reported previously [22]. These indicate that those two subtypes might be crucial and share the physiological function of PACAP in the cardiovascular system. In addition, the detection of PACAP mRNA in cultured cells indicates that PACAP might function as a paracrine or autocrine. The present study has, for the first time, demonstrated that PACAP inhibits DNA and collagen syntheses in cultured nonmyocytes under non-stimulated and angiotensin IIstimulated conditions. It is well-known that angiotensin II attenuates the increment of interstitial tissue in hypertrophic heart [23]. It was suggested that the observed inhibitory effects of PACAP on nonmyocytes are probably mediated through a cAMP-dependent process, since two cAMPrelated compounds; that is, 8-bromo cAMP, a cAMP analogue, and forskolin, an activator of adenylate cyclase were previously reported to inhibit DNA and collagen syntheses in nonmyocytes [18]. Thus, The possible function of PACAP may be attenuation of the mitogenesis and collagen production in nonmyocytes. As for the protein synthesis in cardiac myocytes, PACAP did not stimulate the [14C] phenylalanine, suggesting the benefit of avoiding hypertrophy of the cardiac myocytes. In addition, PACAP stimulates the release of atrial natriuretic polypeptide (ANP), a marker for hypertrophic response, even though VIP had no effect on its release [17]. Incidentally potassium ATP channels play a fundamental role in cardiac excitability and potently modulate the stimulated ANP secretion. As well as in the vascular smooth muscle cells, PACAP activates the atrial KATP channels through both PKA and PKC pathways [21]. ANP is well known to
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increase with heart failure and reduce the volume overload by diuresis and natriuresis [24]. Since 8-bromo cAMP and forskolin inhibit the ANP secretion from cardiac myocytes, as in previous observations [25], PACAP may stimulate the ANP release via cAMP-independent mechanism through the PACAP-preferring receptor PAC1, even though the exact mechanism has not been elucidated in the present study. Cardiac hypertrophy occurs in various pathophysiological states, such as hypertension, valvular heart disease and so on. In hypertrophic heart, cardiac myocytes increase in size, and nonmyocytes, which are almost cardiac fibroblasts, increase in number and production of intercellular matrix. These reactions, so-called as cardiac remodeling, make worse in cardiac function [26]. Although the pathophysiological roles of PACAP in such cardiac disorders remain unclear, our present findings may suggest some roles. PACAP in heart has been found to act on nonmyocytes as an inhibitor of cardiac fibrosis through VPAC2 receptor, and on myocytes as a stimulator of ANP secretion through PAC1 receptor, indicating that PACAP could play the role of a neurohumoral factor to inhibit the remodeling at cardiac hypertrophy. In addition, cAMP production is a benefit to increase cardiac output by increment of contractile force. These multiple effects of PACAP on the heart indicate that PACAP functions as a cardioprotective factor in some pathological states, even though the biosynthesis and secretion of PACAP under pathological conditions remains unclear. Anyway, further investigations are necessary to clarify the physiological and pathophysiological significance of PACAP in the heart.
Acknowledgements The authors thank Ms. Kimie Godoh for her secretarial assistance, and Ms. Michiyo Tanaka for her technical assistance. This work was supported in part by Special Coordination Funds for Promoting Science and Technology (Encouragement System of COE) from the Science and Technology Agency of Japan, and Scientific Research Grant-in-Aid 11670161 from the Ministry of Education, Science and Culture of Japan.
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