Introduction

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Increased plasma levels of neuropeptide Y (NPY), a neurotransmitter in the sympathetic innervation of the heart and vessels, are found in patients with cardiovascular diseases, including hypertension and heart failure (Hulting et al., 1990). Although a causal role for circulating NPY in these diseases has not been established, a consistent observation is the correlation between plasma concentration and severity of left ventricular hypertrophy.

NPY and the related peptide YY (PYY) and pancreatic polypeptide (PP) activate six different receptor populations in a variety of tissues and cells (Balasubramanian, 1997; Michel et al., 1997). Identification of receptor subtypes was based initially on the rank order of affinities or activities of NPY analogs and fragments in radioligand binding studies or bioassay systems (McDermott et al., 1993). [Leu³¹Pro³⁴]-NPY is an agonist with high affinity for NPY Y₁, Y₄, and Y₅ receptors but devoid of activity at NPY Y₂ receptors, whereas peptide YY_3-36 (PYY_3-36) is selective for NPY Y₂ and NPY Y₅ receptors relative to other NPY Y receptor subtypes (Michel et al., 1997). [D-Trp³⁴]-NPY (Parker et al., 2000) and [Ala³¹,Aib³²]-NPY (Cabrele et al., 2000) are potent and selective NPY Y₅ receptor agonists, whereas WX-143-B is a highly selective agonist at NPY Y₄ receptors (Balasubramaniam et al., 2001). More recently, development of nonpeptide NPY Y receptor antagonists has assisted greatly in receptor classification (Cabrele and Beck-Sickinger, 2000). BVD-42 is a selective antagonist at NPY Y₁ receptors (Balasubramaniam et al., 2001), whereas BIIE0246 is a selective antagonist at NPY Y₂ receptors (King et al., 2000). Availability of such compounds provides opportunity for reevaluation of the NPY Y receptor subtypes present in ventricular myocardium.

At the cellular level, myocardial hypertrophy is based on increased mass, not number, of myocardial cells because adult cardiomyocytes do not divide. Increased mass is achieved by increased synthesis of de novo protein, due primarily to increases in ribosomal RNA content, and to reduced degradation of existing protein (Schluter et al., 1995). NPY attenuates protein degradation in healthy cardiomyocytes maintained in short-term, serum-free culture, a model of relevance to initiation of hypertrophy in vivo, but it does not influence protein synthesis (Millar et al., 1994). In contrast, after 1 week in serum-supplemented media, cardiomyocytes lose their rod-shaped appearance and obtain a spread morphology (redifferentiated form) (Schluter et al., 1995), which may parody maintenance of established hypertrophy in vivo; these cells acquire responsiveness to the peptide for protein synthesis (Millar et al., 1994). A role for the cytokine TGF- in acquisition of this responsiveness of the cells to the peptide has been proposed (Goldberg et al., 1998). It is not certain, however, how the in vitro redifferentiation model correlates temporally with disease progression in vivo. Involvement of the various receptor subtype(s) mediating the hypertrophic response to NPY also needs to be addressed. Although a recent report has provided evidence for a role for NPY Y₅ receptors in activating the MAPK hypertrophic signaling cascade in neonatal mouse cardiomyocytes in vitro (Pellieux et al., 2000), the receptor subtype(s) mediating protein turnover has not been investigated.

The spontaneously hypertensive strain of Wistar rat (SHR) is a genetic model of chronic pressure overload displaying many similarities to human essential hypertension (Doggrell and Brown, 1998). Wistar Kyoto rats (WKYs) provide an appropriate normotensive control strain. Hypertension develops in SHRs 6 to 8 weeks after birth, and significant myocardial hypertrophy becomes evident at 14 to 20 weeks. Increased cardiac mass can be attributed to progressive increases in the transverse diameter of individual cardiomyocytes as sarcomeres are added in parallel and to proliferation of nonmyocytes together with enhanced synthesis of extracellular matrix proteins. Because alterations in cardiac performance may reflect the influence of many factors, including intrinsic muscle properties, loading conditions, and altered systemic and/or coronary hemodynamics, investigation of cardiomyocytes obtained ex vivo from SHR is a useful approach to dissect out the contribution of adaptations intrinsic to the myocytes themselves from those of fibrosis and nonmyocyte proliferation (Brooksby et al., 1992). We have found that cardiomyocyte protein mass is not different between strains at 8 and 12 weeks but is elevated significantly in SHR cells at 16 to 24 weeks relative to WKY cells. Parallel changes in cell width have been observed, indicative of concentric cardiomyocyte hypertrophy in response to pressure overload (Bell et al., 2002). As such, a hypertrophic window at 8 to 20 weeks, incorporating baseline and developmental aspects has been identified, which will assist in application of the model in the investigation of pathogenetic mechanisms.

There are few reports of effects of hypertrophic factors generally, and none relating to NPY specifically, at the cellular level in cardiomyocytes isolated from hearts that are already diseased. The aim of the study was to determine whether NPY-related signaling pathways are active in cardiomyocytes isolated from SHRs at the onset, or become associated with the progression of the hypertrophic process in vivo, and if so to characterize NPY Y receptor subtype involvement.

	Materials and Methods

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Solutions. Serum-free creatinine-carnitine-taurine (CCT) medium for the culture of cardiomyocytes consisted of modified glutamine-free medium M199 supplemented with Earle's salts, 15 mM HEPES, 5 mM creatinine, 2 mM l-carnitine, 5 mM taurine, 100 µM ascorbic acid, 100 IU ml¹ penicillin, and 100 µg ml¹ streptomycin. Medium was also supplemented with 10 µM cytosine -D-arabinofuranoside to prevent growth of nonmyocytes. The composition of the Ca²⁺-free Krebs-Ringer solution used in the isolation of cardiomyocytes was as follows: 100 mM NaCl, 2.6 mM KCl, 25 mM NaHCO₃, 1.2 mM MgSO₄, 1.2 mM KH₂PO₄, and 11 mM glucose. This solution was gassed with 95% O₂/5% CO₂ and maintained at pH 7.4 at 37°C. The composition of the phosphate-buffered saline (PBS) was as follows: 137 mM NaCl, 1.5 mM KH₂PO₄, and 1.0 mM Na₂HPO₄, pH 7.4. The composition of DNA assay solution was 1.985 M NaCl and 25 mM Na₂HPO₄, pH 7.4. Bisbenzamide was dissolved in water (0.2 mg ml¹). This stock solution was diluted 1:200 with DNA assay solution to give a working concentration of 1 µg ml¹. Solutions of bisbenzamide are very sensitive to light and were therefore kept in the dark before use. The stock solution was stable for 6 months in the dark at 4°C. Dilute solutions were prepared daily.

Experimental Model. Male spontaneously hypertensive rats and age- and sex-matched WKY normotensive rats were used in the study. These animals were obtained from Harlan (Blackthorn, Oxon, UK) at 4 weeks of age and maintained at the Laboratory Service Unit (The Queen's University of Belfast) before sampling at 8, 12, 16, 20, and 24 weeks of age. Animals were housed four per cage and were given free access to normal rat chow and tap water. Systolic blood pressure was determined by tail cuff sphygmomanometry (Harvard Instruments, Chicago, IL), and the body weight of each animal was determined immediately before use. The study was performed in accordance with UK Government Home Office Guidance on the Operation of the Animals (Scientific Procedures) Act 1986, published by Her Majesty's Stationary Office (London, England).

Isolation and Culture of Cardiomyocytes. Ventricular cardiomyocytes were isolated as described previously (Millar et al., 1994). Briefly, rats were subjected to deep isoflurane anesthesia and their hearts excised. The excised hearts from two rats were immediately immersed in ice-cold saline to remove residual blood and reduce the risk of clotting, slow the metabolic rate, and delay the onset of hypoxia. The hearts were each weighed for subsequent determination of heart weight/body weight ratio, before simultaneous perfusion using a Langendorff apparatus with Ca²⁺-free Krebs-Ringer solution containing collagenase (0.4 mg ml¹) until they became flaccid. The two hearts were chopped finely, and the mince was pooled and agitated gently in the same medium to dissociate individual cells. The resulting cell suspension was filtered to remove undigested material and the cells were sedimented at 750 rpm for 4 min. Ca²⁺ tolerance of the cells was restored gently by resuspending the sediment in Krebs-Ringer solution containing a progressively higher concentration of Ca²⁺ to a final concentration of 1 mM. The cell suspension (3-4 ml) was then layered gently onto a 4% (w/v) albumin solution (12.5 ml), contained in a tube 20 cm in length and 1 cm in internal diameter, to sediment viable cardiomyocytes and effectively remove nonmuscle cells and cell debris. The resultant sediment was resuspended in serum-free CCT medium. Cells derived from the two hearts were pooled, mixed thoroughly, and resuspended at a concentration of 1.5 × 10⁵ viable cardiomyocytes ml¹. Petri dishes were preincubated for 2 h with fetal calf serum (4% v/v) in medium M199. Aliquots of cell suspension (1 ml) were pipetted gently onto 35-mm-diameter Petri dishes. After 1 h, viable cardiomyocytes had attached to the surface of the dishes. The dishes were washed with fresh CCT medium to remove nonattached cells and cell debris, and the attached cells were incubated at 37°C for 24 h in CCT medium (1 ml) containing the appropriate concentrations of the various hypertrophic stimuli and/or antagonists as specified in the experimental protocols. Under all experimental conditions, cardiomyocytes remained mechanically quiescent.

Incorporation of l-[U-¹⁴C]Phenylalanine and Total Mass of Cellular Protein and Total Content of Cellular DNA. The extent of de novo synthesis of protein in the cell cultures was estimated by measuring uptake of radiolabeled amino acid into cellular protein. The cells were exposed for 24 h to l-[U-¹⁴C]phenylalanine (0.1 µC_i ml¹ culture medium). Incorporation of radioactivity into the acid-insoluble cell fraction was determined. At the end of the chosen period of incubation, experiments were terminated by removal of the supernatant medium from the dishes. The attached cells were washed with an aliquot (1 ml) of ice-cold PBS, before the addition of an aliquot (1 ml) of ice-cold trichloroacetic acid (10% w/v). After storage overnight at 4°C, the acid containing the intracellular precursor pool was removed from the dishes, and the attached cells were washed with an aliquot (1 ml) of PBS. The precipitate remaining on the culture dishes was dissolved in an aliquot (1 ml) of 0.1 M NaOH/0.01% (w/v) sodium dodecyl sulfate by overnight incubation at 37°C. In these samples, concentration of protein was determined by the colorimetric method of Lowry et al. (1951), the concentration of DNA in the neutralized sample was determined by a spectrophotometric method in which bisbenzamide dye was incorporated into DNA (Mullan et al., 1997), and the radioactivity was counted. The ratio of protein to DNA per culture served as the parameter of cell mass and the ratio of l-[U-¹⁴C]phenylalanine incorporated to DNA per culture served as a measure of de novo synthesis of protein. The concentration of DNA is a reliable measure of cell number because adult cardiomyocytes do not proliferate in culture (Schluter et al., 1995); also nonmyocyte cells are virtually absent from these fresh cultures, and the very few present cannot proliferate because they would be killed by the presence of cytosine -D-arabinofuranoside (Piper and Volz, 1990).

Semiquantitative Reverse Transcription-Polymerase Chain Reaction (PCR). Total cellular RNA was isolated by a modification of the acid guanidinium thiocyanate-phenol-chloroform method of Chomczynski and Sacchi (1987). First-strand cDNA was synthesized from 2 µg of total RNA in a 20-µl reaction volume using random decamers and Moloney-murine leukemia virus reverse transcriptase (Reverse-iT kit; Abgene, Surrey, UK). The resultant cDNA was amplified by PCR following a standard PCR protocol: each PCR reaction (25 µl) contained 4 µg of cDNA, 1× reaction buffer (1 mM Tris-HCl and 5 mM KCl, pH 8.3), 1.5 mM MgCl₂, 200 µM of each dNTP, 1.25 U of DNA polymerase (Thermus Icelandicus, Red Hot DNA polymerase; Abgene), and 1 µM of each gene-specific forward and backward primer. The gene-specific primers (Table 1) were based upon those reported previously in the literature (Sciore et al., 1990; Goumain et al., 1998; Kobayashi et al., 1999; Lynch et al., 1999). After an initial denaturation at 94°C for 4 min the following cycling profile was used: denaturation at 94°C for 30 s, annealing at each suitable temperature (Table 1) for 40 s, and extension at 72°C for 60 s. Amplification was performed over several cycles (Table 1) and was ended with a final extension at 72°C for 5 min and cooling to 15°C for 5 min. All PCR reactions were performed in duplicate. In preliminary experiments, the amplification cycles for each gene were determined in a range such that the amount of PCR product was in proportion to the amplification cycle. The PCR products were electrophoresed on a 2.0% agarose gel and stained with ethidium bromide. The gels were visualized under ultraviolet illumination and photographed and analyzed using a GeneGenius Gel documentation system with Gene Tools analysis software (Syngene, Cambridge, UK). Band intensity was expressed as the target mRNA to GAPDH mRNA ratio.

Materials. Neuropeptide Y (human and rat), [Leu³¹Pro³⁴]-NPY (porcine), and peptide YY_3-36 (human) were supplied by Bachem UK Ltd. (St. Helen's, Merseyside, England). The neuropeptide Y₂ receptor antagonist BIIE0246 was obtained from Boehringer Ingleheim GmbH (Ingleheim, Germany). The Y₄-selective agonist WX-143B, the Y₅-selective agonist [D-Trp³⁴]-NPY, and the Y₁-selective antagonist BVD-42 were prepared by Professor Ambi Balasubramaniam (University of Cincinnati Medical Center, Cincinnati, OH). Bovine serum albumin (catalog number A7030), l-carnitine, creatine, taurine, cytosine--arabinofuranoside, DNA (sodium salt, from calf thymus), phorbol 12-myristate 13-acetate, and assay kits for the determination of microprotein were obtained from Sigma Chemical (Poole, Dorset, UK). Liquid scintillation fluid was obtained from BDH (Poole, Dorset, UK). Collagenase B was purchased from Roche Applied Science (Mannheim, Germany). Medium M199 (glutamine-free with Earle's salts), fetal bovine serum, and penicillin (5000 IU)/streptomycin (5 mg ml¹) were supplied by Invitrogen (Paisley, Scotland, UK). Bisbenzamide (H 33258) was purchased from Riedel-de-Haen (Seelze, Germany). Plastic Petri dishes were obtained from Falcon (BD Biosciences, Oxford, UK). l-[U-¹⁴C]Phenylalanine was supplied by Amersham Biosciences UK, Ltd. (Little Chalfont, Buckinghamshire, UK). Thermus Icelanicus (Red Hot Taq Polymerase) and Reverse-iT kits were obtained from Abgene and dNTPs were obtained from Bioline (UK) Ltd. (London, UK). Primers were custom synthesized by the Oligonucleotide Synthesis Unit (The Queen's University of Belfast). All other chemicals were of analytical grade and purchased from BDH.

Data Analysis. In each experiment, the total population of cells contained in culture plates was obtained from a pooled suspension prepared from two hearts. Under each condition (in the absence/presence of peptide at various concentrations, with or without antagonist), the average value measured in three culture plates was calculated for each parameter ([¹⁴C]phenylalanine incorporation or protein DNA content¹). Replicate data were obtained for n preparations (4  n  8), and the mean value ± S.E.M. was calculated. Data were analyzed statistically using a one- or two-factor repeated measures analysis of variance (SPSS-PC, version 8.0; SPSS, Inc. Chicago, IL). If P < 0.05 for the overall effect of concentration under a particular condition, differences between the mean values at a particular concentration (x₁) and at baseline (x₀) were tested by calculation of the t statistic as (x₁  x₀)/ residual mean square (2/n). Data from PCR experiments were presented as mean value ± S.E.M. of six hearts and analyzed by two-way analysis of variance.

	Results

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Validation of SHR Model. Systolic blood pressure was not different between strains at 7 weeks of age (151 ± 7 mm Hg, n = 4, SHR versus 143 ± 3 mm Hg, n = 4, WKY). Thereafter, systolic blood pressure increased with age to a sustained value of 229 ± 13 mm Hg (n = 4) in SHR at 14 weeks but was unchanged in WKY (151 ± 11 mm Hg, n = 4). A significant difference between strains was observed from 11 weeks onward (195 ± 11 mm Hg, n = 4, SHR versus 154 ± 10 mm Hg, n = 4, WKY). Heart weight/body weight ratios did not differ between strains at 8 and 12 weeks but were significantly greater in SHR at 16, 20, and 24 weeks than in age-matched WKY. The maximum difference of 14.6% was observed at 24 weeks (0.00464 ± 0.00015, n = 8, SHR versus 0.00405 ± 0.00015, n = 4, WKY; P < 0.05). Cardiomyocyte protein mass increased marginally with age in WKY from 37.8 ± 1.2 µg of protein/µg of DNA (n = 4) at 8 weeks to 41.6 ± 5.4 µg of protein/µg of DNA (n = 5) at 24 weeks. Differences were not statistically significant. In contrast, cardiomyocyte protein mass increased progressively from 38.0 ± 0.5 µg of protein/µg of DNA (n = 4) at 8 weeks of age to 51.6 ± 3.1 µg of protein/µg of DNA (n = 4) at 24 weeks such that cellular protein mass was 24.0% greater than that of WKY cells.

Temporal Dependence of the Hypertrophic Effect of Neuropeptide Y. NPY did not increase cellular protein mass above basal levels in cardiomyocytes from SHRs at 8 and 12 weeks (Figs. 1a and 2a). However, cellular protein mass was increased in SHR cells at 16 and 20 weeks in response to NPY; maximum responses were 9.2 ± 2.1% (n = 8, P < 0.05) and 10.5 ± 5.2% (n = 8, P = N.S.) greater than basal values and occurred at 10⁸ and 10⁹ M, respectively (Figs. 3a and 4a). No increases in cellular protein mass were evident in SHR cells at 24 weeks (Fig. 5a) or in WKY cells at any age (8-24 weeks). Indeed, cellular protein mass was decreased significantly and in a concentration-dependent manner in response to NPY in WKY cells at 12 weeks (Fig. 2a); the maximum decrease, observed in response to NPY (10⁶ M), was 24.6 ± 3.7% (n = 8). NPY did not increase de novo protein synthesis, as evidenced by [¹⁴C]phenylalanine incorporation into cellular protein in cardiomyocytes from SHRs at 8 weeks (Fig. 1b). In contrast, NPY increased de novo protein synthesis significantly and in a concentration-dependent manner in SHR cells at 12 weeks (Fig. 2b); the maximum response, observed at a concentration of 10⁶ M, was 12.6 ± 2.1% (n = 8, P < 0.05). SHR cells were more sensitive to NPY at 16 weeks (Fig. 3b); the concentration dependence of the effect of the peptide was shifted to the left compared with that at 12 weeks, and the maximum response, which was observed at 10⁸ M, was also greater (20.1 ± 4.2%, n = 8). The increase in de novo protein synthesis in response to NPY was smaller in SHR cells at 20 weeks than at 12 and 16 weeks (Fig. 4b); the maximum response obtained at 20 weeks was 9.4 ± 1.8% (n = 8) and was observed at 10⁷ M. NPY did not increase de novo protein synthesis in SHR cells at 24 weeks (Fig. 5b). In contrast to the effect of NPY in cardiomyocytes from SHRs, the peptide was devoid of activity in WKY cells at all ages.

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Concentration dependence of the effect of neuropeptide Y on total mass of cellular protein (a) and incorporation of l-[U-14C]phenylalanine (b) into cellular protein of ventricular cardiomyocytes isolated from the hearts of 8-week-old SHRs () and age-matched WKYs () and maintained in short-term (24 h), serum-free culture. Data are expressed as percent differences from basal values and are the means ± S.E.M. of six experiments. +, significant difference from basal value (+, P < 0.05); *, significant difference between strains (*, P < 0.05).

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Concentration dependence of the effect of neuropeptide Y on total mass of cellular protein (a) and incorporation of l-[U-14C]phenylalanine (b) into cellular protein of ventricular cardiomyocytes isolated from the hearts of 12-week-old SHRs () and age-matched WKYs () and maintained in short-term (24 h), serum-free culture. Data are expressed as percent differences from basal values and are the means ± S.E.M. of eight experiments. +, significant difference from basal value (+, P < 0.05); *, significant difference between strains (*, P < 0.05).

View larger version (13K): [in this window] [in a new window]   Fig. 3.   Concentration dependence of the effect of neuropeptide Y on total mass of cellular protein (a) and incorporation of l-[U-14C]phenylalanine (b) into cellular protein of ventricular cardiomyocytes isolated from the hearts of 16-week-old SHRs () and age-matched WKYs () and maintained in short-term (24 h), serum-free culture. Data are expressed as percent differences from basal values and are the means ± S.E.M. of eight experiments. +, significant difference from basal value (+, P < 0.05); *, significant difference between strains (*, P < 0.05).

View larger version (13K): [in this window] [in a new window]   Fig. 4.   Concentration dependence of the effect of neuropeptide Y on total mass of cellular protein (a) and incorporation of l-[U-14C]phenylalanine (b) into cellular protein of ventricular cardiomyocytes isolated from the hearts of 20-week-old SHRs () and age-matched WKYs () and maintained in short-term (24 h), serum-free culture. Data are expressed as percent differences from basal values and are the means ± S.E.M. of eight experiments. +, significant difference from basal value (+, P < 0.05); *, significant difference between strains (*, P < 0.05).

View larger version (14K): [in this window] [in a new window]   Fig. 5.   Concentration dependence of the effect of neuropeptide Y on total mass of cellular protein (a) and incorporation of l-[U14C]phenylalanine (b) into cellular protein of ventricular cardiomyocytes isolated from the hearts of 24-week-old SHRs () and age-matched WKYs () and maintained in short-term (24 h), serum-free culture. Data are expressed as percent differences from basal values and are the means ± S.E.M. of four experiments. +, significant difference from basal value (+, P < 0.05); *, significant difference between strains (*, P < 0.05).

Receptor Subtype-Selective Agonists. PYY_3-36, which displays some selectivity for NPY Y₂ and NPY Y₅ receptors over other NPY Y receptor subtypes (Table 2), increased de novo protein synthesis in SHR cells at 16 weeks (P < 0.05) maximally by 16.2 ± 5.1% at 10⁶ M (n = 4) (Fig. 6a). PYY_3-36 did not increase de novo protein synthesis significantly in SHR cells at 20 weeks; the maximum response, observed at 10⁷ M, was 4.7 ± 6.7% (n = 4) (data not shown). The peptide did not increase de novo protein synthesis or cellular protein mass in SHR cells at 24 weeks (data not shown) and was devoid of activity in WKY cells (16-24 weeks). The NPY Y₄-selective agonist WX-143B and the NPY Y₅-selective agonist [D-Trp-³⁴]-NPY increased de novo protein synthesis significantly and in a concentration-dependent manner in cardiomyocytes from SHRs at 16 weeks. The maximum response to WX-143B was 14.0 ± 3.2% (n = 7) and was observed at 10⁶ M (Fig. 6b). The maximum response to [D-Trp-³⁴]-NPY was 17.8 ± 5.2% (n = 7) and was observed at 10⁶ M (Fig. 6c). [Leu³¹Pro³⁴]-NPY did not increase de novo protein synthesis (Fig. 6d) significantly in cardiomyocytes from either strain at 16 weeks.

View larger version (23K): [in this window] [in a new window]   Fig. 6.   Concentration dependence of the effect of PYY3-36 (a), WX-143B (b), [D-Trp34]-NPY (c), and [Leu31Pro34]-NPY (d) on the incorporation of l-[U-14C]phenylalanine into cellular protein of ventricular cardiomyocytes isolated from the hearts of 16-week-old SHRs and maintained in short-term (24 h), serum-free culture. Data are expressed as percent differences from basal values and are the means ± S.E.M. of four to seven experiments. *, significant difference from basal value (*, P < 0.05).

Receptor Subtype-Selective Antagonists. The NPY Y₁-selective antagonist BVD-42 (2 × 10⁷ M) (K_i values of 4.6 × 10¹⁰ M, 6.2 × 10⁷ M, 6.5 × 10⁸ M, and 7.9 × 10⁶ M at Y₁, Y₂, Y₄, and Y₅ receptors, respectively; Balasubramaniam et al., 2001) (Fig. 7a), and the NPY Y₂-selective antagonist BIIE0246 (2 × 10⁷ M) (IC₅₀ value of 3.3 × 10⁹ M at Y₂ receptor; IC₅₀ values >1 × 10⁶ M at Y₁, Y₄, and Y₅ receptors; Doods et al., 1999) (Fig. 7b) did not alter de novo protein synthesis per se and did not attenuate responses to the NPY Y receptor agonists NPY (10⁷ M) and PPY_3-36 (10⁷ M) or to the phorbol ester phorbol 12-myristrate 13-acetate, used as a negative control. Indeed, BVD-42 tended to potentiate the responses to each of these stimuli although increases were not significantly greater than control values.

View larger version (23K): [in this window] [in a new window]   Fig. 7.   Effect of BVD-42 (2 × 107 M) (a) and BIIE-0234 (2 × 107 M) (b) on the incorporation of l-[U-14C]phenylalanine into cellular protein of ventricular cardiomyocytes isolated from the hearts of 16-week-old SHRs and maintained in short-term (24 h), serum-free culture in the presence of NPY (107 M), PYY3-36 (107 M), and phorbol 12-myristate 13-acetate (107 M). Data are expressed as percent differences from basal values and are the means ± S.E.M. of five experiments. *, significant difference from value in the absence of antagonist (*, P < 0.05).

Expression of TGF-, NPY, and NPY Y₅ Receptor mRNA. PCR amplification products for TGF-, NPY, and NPY Y₅ receptor were detected at 271, 329, and 524 base pairs, respectively. TGF- mRNA expression was not altered in WKY cardiomyocytes with advancing age (8-24 weeks). However, levels of expression of the cytokine tended to be higher in SHR cardiomyocytes relative to WKY cells at 8 to 20 weeks although differences did not reach statistical significance (Fig. 8a). NPY Y₅ receptor mRNA was expressed abundantly in all SHR and WKY cardiomyocytes; no differences were apparent in the levels of expression detected with respect to age or strain (Fig. 8b). Much lower levels of expression of NPY mRNA were detected in both SHR and WKY cells; no differences were apparent with respect to age or to strain (Fig. 8c).

View larger version (29K): [in this window] [in a new window]   Fig. 8.   Expression of TGF-, pro NPY, and NPY Y5 receptor mRNA in ventricular cardiomyocytes isolated from WKY and SHR between 8 to 24 weeks of age. Reverse transcription-PCR was performed and mRNA levels were normalized to GAPDH internal control. Above graphs: representative photographs of PCR products. Data are expressed as mean ± S.E.M. (n = 6).

	Discussion

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In agreement with previous in vitro studies using normal ventricular cardiomyocytes from healthy adult Wistar rats (Millar et al., 1994; Goldberg et al., 1998), NPY did not increase de novo protein synthesis in nonhypertrophied cells obtained ex vivo from SHRs at 8 weeks, and from WKYs at any age (8-24 weeks), indicating that NPY does not "initiate" hypertrophic growth in vivo. Inability of NPY to increase protein mass in WKY cells and in nonhypertrophied SHR cells is, however, at variance with data obtained previously using in vitro models, which indicated that NPY, in contrast to other "hypertrophic" stimuli, initiated increases in protein mass by attenuating degradation of existing cellular protein. This observation might represent an influence of NPY on cellular metabolism rather than a genuinely hypertrophic response, because it is not accompanied by increased capacity for protein synthesis. NPY reduced cellular protein mass significantly in WKY cells at 12 weeks. This result indicates that the direction of the acute influence of NPY on protein degradation could depend on underlying metabolic status of the cells, or may change over time due to normal developmental growth and ageing, with NPY exerting a transient negative feedback role in preventing further physiological growth in healthy young adult hearts when optimum cardiac enlargement has been achieved.

NPY enhanced de novo protein synthesis, accompanied by increased mass, in hypertrophying cardiomyocytes obtained from SHRs at 12 to 20 weeks, maximally at 16 weeks. These data are in agreement with previous studies in which NPY enhanced protein synthesis in redifferentiated cardiomyocytes (Millar et al., 1994). Taken together, these findings support a role for NPY in progression of preexisting hypertrophic growth and make it possible to relate the in vitro redifferentiated model temporally to changes occurring in vivo during disease progression. Induction of hypertrophic responsiveness to the -adrenoceptor agonist isoprenaline has also been demonstrated in redifferentiated cells in vitro and occurs transiently at 16 weeks of age in SHR cells ex vivo (Bell et al., 2002) subsequent to initiation of hypertrophic growth.

TGF- is an autocrine mediator for induction of hypertrophic responsiveness to isoprenaline and NPY (Goldberg et al., 1998; Taimor et al., 1999) in redifferentiated cardiomyocytes in vitro. TGF- mRNA was expressed in cardiomyocytes and levels of expression tended to be greater in SHR cells than in WKY cells at 8 to 20 weeks, which corresponds to onset of hypertension and initiation and subsequent progression of hypertrophic growth, supporting a role for TGF- as an autocrine factor in acquisition of hypertrophic responsiveness to NPY in vivo. It is possible that levels of expression of this cytokine in left ventricles of SHRs were underestimated because mRNA was extracted from a pooled suspension of left and right ventricular cardiomyocytes and enhanced expression of TGF- in right ventricular cardiomyocytes might not occur because the right chamber is not subjected to pressure overload. Secretion of TGF- as a paracrine mediator from nonmyocytes in vivo should also be considered.

NPY did not stimulate de novo protein synthesis in SHR cardiomyocytes at 24 weeks. Indeed, there was a tendency to reduced protein mass in the presence of NPY. At this time, the maximum extent of cardiomyocyte hypertrophy is achieved; protein mass and cell width are increased maximally relative to WKY cells and basal rates of protein synthesis are markedly reduced (Bell et al., 2002). These data suggest that the association of NPY Y receptors with hypertrophic growth is a transient process, which is inactivated once the full extent of hypertrophic adaptation of the left ventricle in response to pressure overload is realized.

NPY Y₅ receptors have been implicated in potentiation of phenylephrine-induced MAPK signaling in neonatal cardiomyocytes (Pellieux et al., 2000). In contrast, NPY Y₁ and NPY Y₂ receptors mediate MAPK signaling pathways associated with vascular smooth muscle growth (Nie and Selbie, 1998). In a previous investigation in adult ventricular cardiomyocytes, we reported that a positive contractile effect of NPY was mediated by NPY Y₁ receptors, whereas a negative contractile effect was mediated by NPY Y₂ receptors (McDermott et al., 1997). Both receptor subtypes were also implicated in NPY-mediated protein turnover in freshly isolated cardiomyocytes in vitro (S. M. Nicholl and D. Bell, unpublished observation). NPY Y₁ and NPY Y₂ receptor involvement was concluded on the basis of the rank order of potency of agonists, including [Leu³¹Pro³⁴]-NPY and PYY_3-36, and use of peptide analogs, such as bis(31/31')[Cys³¹Trp³²Nva³⁴]-NPY 31-36, a putative NPY Y₁ subtype-selective antagonist, and T₄[NPY33-36]₄, a putative NPY Y₂ subtype-selective antagonist (Grouzmann et al., 1997), both of which had relatively weak antagonistic activity against NPY. Identification of additional NPY Y receptor subtypes and recent evidence questioning the selectivity of these analogs indicate that early reports in which the characterization of NPY Y receptor subtypes present on cardiomyocytes was attempted should be reviewed cautiously (Balasubramaniam, 1997; Michel et al., 1997).

[D-Trp³⁴]-NPY (Parker et al., 2000) potently increased de novo protein synthesis to a similar extent to NPY in SHR cardiomyocytes at 16 weeks. Taken together with lack of antagonism of responses to PYY_3-36 and NPY by BIIE-0246 (Doods et al., 1999; King et al., 2000; Table 2), these data support a role for NPY Y₅ receptors, rather than NPY Y₂ receptors, in enhanced protein synthesis. A definitive answer will require use of more highly selective agonists (Cabrele et al., 2000) and antagonists at NPY Y₅ receptors; such compounds are not commercially available at present. NPY Y₅ mRNA was expressed abundantly in cardiomyocytes, although no differences were observed with respect to age or strain; these data might indicate that NPY Y₅ receptor-effector coupling becomes altered, perhaps under the influence of TGF-, such that cells acquire responsiveness to NPY for de novo protein synthesis during disease progression.

Low activity of [Leu³¹Pro³⁴]-NPY, an agonist with high affinity for NPY Y₁ receptors (Fuhlendorff et al., 1990), together with lack of antagonism of the response to NPY by BVD-42, a selective antagonist at NPY Y₁ receptors (Balasubramaniam et al., 2001), indicates that NPY Y₁ receptors are not involved in increased protein synthesis in SHR cells. The weak action of [Leu³¹Pro³⁴]-NPY might reflect its interaction at NPY Y₄ or NPY Y₅ receptors, at which it has some activity (Gehlert et al., 1997). Although the potent activity of WX-143B, which is incompatible with an action via any other Y receptor subtype (Table 2), indicates that NPY Y₄ receptors could also be linked to a hypertrophic pathway, it is very unlikely that NPY Y₄ receptors would mediate the hypertrophic response to NPY in vivo, because the endogenous peptide has very low affinity for Y₄ receptors (Table 2; Gregor et al., 1996). In addition, BVD-42 (2 × 10⁷ M) did not attenuate the action of NPY, although the majority of Y₄ receptors would have been blocked at this concentration (K_i value Y₄ receptor = 6.5 × 10⁸ M; Balasubramaniam et al., 2001).

In contrast to the declining response to NPY (>10 nM) at 16 weeks (Fig. 3), responses to micromolar concentrations of PYY_3-36, [D-Trp³⁴]-NPY, and WX-143-B were not reduced relative to nanomolar concentrations, indicating that this phenomenon is unlikely to be attributed to receptor desensitization at supramaximal concentrations of NPY but perhaps instead to the unmasking of a negative effect on protein turnover to counteract excessive hypertrophic adaptation. Such an action of NPY might conceivably be mediated by Y₁ receptors. In contrast to [Leu³¹Pro³⁴]-NPY and NPY itself, PYY_3-36, [D-Trp³⁴]-NPY and WX-143-B have relatively low affinity for the Y₁ receptor and would be less likely to exhibit evidence of "self-inhibition". Furthermore, BVD-42 did display a tendency to enhance the actions of NPY and PYY_3-36 (Fig. 7), suggesting blockade of a negative effect.

The activity of the sympathetic nervous system is increased in an important subgroup of patients with essential hypertension (Esler et al., 1988) and in experimental models of hypertension, including SHR (Dechamplain, 1990); this generalized pattern is also present in the heart as evidenced by increased cardiac noradrenaline spill-over to plasma (Castellano and Bohm, 1997). NPY is predominantly found as a cotransmitter with noradrenaline in the sympathetic innervation, although an appreciable amount of NPY is localized separately in intrinsic myocardial neurons (McDermott et al., 1993) and non-neuronal sources, including platelets (Ogawa et al., 1992) and cardiomyocytes (Millar et al., 1991). PYY_3-36 represents a major molecular form of PYY in human plasma (Grandt et al., 1992). Plasma concentration (Zukowska-Grojec et al., 1993) and platelet content (Ogawa et al., 1992) of NPY are increased in the SHR. Although we identified low levels of expression of NPY mRNA in cardiomyocytes from SHR and WKY, indicating a possible autocrine role for NPY, there was no evidence that this expression was altered with respect to age or strain.

In conclusion, it is probable that the temporary recruitment of NPY Y₅ receptors to hypertrophic signaling pathways during the active progression of the initial process of cardiomyocyte hypertrophy would enable the endogenous peptide(s) to make a significant contribution to cardiac remodeling in response to pressure overload. NPY Y₅ receptors may therefore represent a novel therapeutic target for drugs designed to prevent or regress left ventricular hypertrophy.