The developmental changes of sympathetic β-adrenergic nervous system in the rat heart

PATHOPttYSIOLOGY ELSEVIER

Pathophysiology 4 (1997) 33 40

The developmental changes of sympathetic fl-adrenergic nervous system in the rat heart Shin-ichi Maie *, Fumitaka Ohsuzu, Shyuichi Katsushika, Haruo Nakamura t:Trst Department of Medicine, National DeJence Medical College, 3-2 Namiki, Tokorozawa City, 359 Saitama, Japan Received 29 November 1995: received in revised form 18 April 1996; accepted 25 July 1996

Abstract

To clarify the changes of sympathetic fl-adrenergic nervous system in the heart with the advancement of age, the fl-adrenergic receptor concentration was determined and the age-associated changes in tissue distribution of tyrosine hydroxylase were analyzed, using 5, 10 and 30-week-old male Wistar-Kyoto (WKY) rats. Further, the steady-state messenger RNAs of fl-adrenergic receptors (fl-ARmRNA) were evaluated in order to clarify the regulation of these receptors on the level of gene transcription. ¢3-adrenergic receptor concentration (Bmax) in the left ventricular myocardium at the age of 5 weeks was significantly higher than that at the age of 10 weeks (42.6 ± 3.7 vs. 29.8 + 5.0 fmol/mg protein, respectively, p < 0.05). Subsequent significant changes of Bmax were not seen from the age of 10-30 weeks. The manifestation of steady-state fl-ARmRNA was clearly observed at the age of 5 weeks. When the results were compared in terms of relative intensity against the messenger RNAs of glyceraldehyde-3-phosphate-dehydrogenase (GAPDHmRNA), a significant increase in steady-state fl-ARmRNA was observed at the age of 5 weeks, compared with that at 10 weeks (30.8 + 6.7 vs. 4.2_+ 1.0%, respectively, p <0,05). As a result of immunohistochemical staining against tyrosine hydroxylase, granular and linear staining patterns were observed in the ventricular myocardium in all three age groups. However, there were no remarkable changes in the staining for tyrosine hydroxylase in the development from 5 to 30 weeks. In conclusion, this study revealed the age-related decrease in the number of fl-adrenergic receptors in the myocardium from 5 to 10 weeks of age. Further these findings may imply that the age-related decrease in fl-adrenergic receptor is regulated, at least in part, at the transcriptional level. © 1997 Elsevier Science Ireland Ltd.

Keywords: Myocardial development; /~-adrenergic nervous system; /J-adrenergic receptor mRNA; Tyrosine hydroxylase

I. Introduction

Sympathetic fl-adrenergic nervous system in the heart exhibits several effects such as inotropic and chronotropic effects. In addition, the excessive exaggerated fl-adrenergic nervous system may transmit harmful cardiotoxic effects [1,2]. Myocardial cell growth, especially hypertrophy, has been reported to be related to e-adrenergic stimulation in tissue culture [3,4], furthermore, the enhanced fl-adrenergic system has been shown to induce hypertrophy [5,6]. With the ad* Corresponding author. Present address: Department of Internal Medicine, Kawasaki Municipal Hospital, 12-1 Shinkawa-dori, Kawasaki-ku, Kawasaki City, 211 Kanagawa, Japan.

vancement of age, it is feasible that the fl-adrenergic nervous system may be influential on the changes in blood pressure, heart rate, and myocardial cell growth. There have been reports of increased sympathetic fl-adrenergic receptors (fl-AR) in the cardiac muscles in newborns and findings of an age-associated decrease [7-9]. In contrast, a developmental increase [10], as well as no developmental change [11] in these receptors have also been reported. In the current study, we determined the number of fl-adrenergic receptors and the tissue contents of their agonists, such as norepinephrine, in order to identify the age-associated changes in the myocardial fl-adrenergic receptors and in the fl-adrenergic nervous system. We also determined the age-associated changes in the tissue distribution of tyrosine

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hydroxylase, a key enzyme of the tissue norepinephrine synthesizing system, by means of i n m m n o histochemical staining. Further, since il is uncleal whether or not changes in the myocardial //-adrenergic receptors are controlled on the gene lexel, x~c determined steady-state messenger RNAs of /7-adrcnergic receptors and compared them to clarify the regulation on the level of gene transcription.

2. Method Five, 10, and 30-week-old male Wistar K}oto (WKY) rats were used in these experiments. Systolic blood pressure (SBP) and heart rate (HR) were measured by the tail cult method. The rats were anesthetized with diethylether and thcn decapitated. After washing out blood with saline, left ventricles x~erc isolated and weighed, and the Iollowing parameters were measured. 2. I. :}leo,s'tl#'{'melll O/ I£~',VIIW /,or(7~im'l#tri##u COtlCUtlIIYlliOII

The left ventricles were homogenized in 5 ml of t).4 N HCIO 4 solution containing I).4'I;, EDTA and I'!., ascorbate with a Polytron homogenizer. As the internal standard, 3,4-dihydroxy-benzylamine t D B H A ) d i s solved in 0.2 N HCI was added to the homogenate and centrifuged at 10000 rpm for 15 rain. The supernatant was adsorbed with 50 mg of activated alumina and then eluted with 0.2 N ttCI. Tissue norepinephrine was assayed by high performance liquid chromatography (HPLC) using electrochemical detection. The working electrode potential was set al 0.8 V against a Ag/AgCI reference electrode. Catecholamines were separated on a C18 Waters ~Waters, Milford. MA) column (6.0 × 150 mm) at constant l]ow rate of 1 mlimin [12,13].

2.2. J/lelnbrane p#'~7~araliol# aIld /7-aUrcm'r pH 7.4) using a "Polytron PT 10 (Kinematical homogenizer for 3 s at setting 9 and then for 9 s at setting 7. The homogenate was centrifuged at 6 0 0 x g for 10 mm. The supernatant was filtered through a double layer Japanese silk screen (size 12). and centrifuged at 39000 × g for 15 rain. The pellet was resuspended in buffer B (100 mM Tris buffer, 5 mM MgCI> 1 mM EGTA, pH 7.4) and served as the membrane preparation. Procedures were done on ice or at 4°C.

fi-adrenergic receptor antagonist binding studies were performed, using seven concentrations of 25 #1 [':~Ilcyanopindolol ([~e~I]CYP) ranging from 0.025 to 0.6 nM, 100/~1 of membrane preparation (50 100 pg), and 25 t~1 of buffer B or 10 tiM ( + ) propranolol, under incubation al 370( ` for 6(I rain. [~>I]CYP binding was ah'eady saturated and was at equilibrium under these conditions, as shown in a preliminary study at our institute. Binding data were analyzed by the least square linear regression method [14]. All samples were run in duplicate. Protein was measured by the method of Lowry et al. [15].

2.3. Muasureme#zl q//~-aeh'cnergic receptor m R N A ¢/]-,4 RmRNA ) RNA was extracted according to a modification of the method of Chomczynski and Sacchi [16]. Total RNA 112 /~g) was denatured with glyoxal at 50°C for f~0 ram, fractionated by, electrophoresis oll a 1% ztgarose gel, and transferred to nylon membranes. The niembranes were baked at 80°C for 2 h, and prehybridized at 42°C for 2 h in a solution containing "; v SSC, 5 × Denhardt's solution, 50% formamide, !l.l'!;, SDS, 50 mM phosphate buffer, and 250 /lg/ml heat-denatured salmon sperm DNA. And hybridization was performed in the same solution using two lypes of probes simultaneously at 42°C for 16 h: one probe was 3-~P-labeled 1878 base pair /)'-adrenergic receptor complementary D N A probe which was isolated from rat heart [17], and the other was 3eP-labeled 1390 base pair glyceraldehyde-3-phosphate-dehydrogenase ( G A P D H ) complementary D N A probe [18] used as the internal standard. Rat cardiac fl-AR complementary DNA (cDNA) and G A P D H c D N A were donated h~ Milsubishi Kasei K.K. (Kanagawa, Japan). Sequencing o f / t - A R c D N A was performed and h o m o f ogy was previously confirmed. Membranes were washed four times at room ten> perature with 2 × SSC and 0.1% SDS, washed twice at 50°( ' with tl.I > SSC and 0. I% SDS, and air dried: then autoradiography was carried out. Exposed X-ray lihn for 24 h at 70°C was scanned with a laser densitometer to quantitate the intensity of the autoradiogram in the linear range of film exposure. The amount of fi-AR steady-state messenger R N A I m R N A ) was expressed as the percentage o f / f - A R m R N A / G A P D H m R N A , that is relative intensity (%), from the autoradiogram of individual samples. Furthermore, dot blot analysis was performed. Total RNA (1.25 5.0 #g) was denatured, spotted on n3hm membranes, baked, prehybridized, and then hybridized with ~'P-labeled 1878 base pair f l - A R c D N A as for Northern blot analysis previously described.

S. Male et al. / Pathophysiology 4 (1997) 33- 40

35

Table 1 Basic characterization of the WKY rat at the age of 5, 10, and 30 weeks and the myocardial norepinephrine concentration

Systolic blood pressure (mmHg) Heart rate (rain) Body weight (g) LV weight LV/body weight 6%) Norepinephrine (ng/mg wet weight)

5-week-old

[0-week-old

30-week-old

112.6 ± 2.3 401.0 ± 8.7 112.9 ± 2.4 0.378 + 0.013 3.346 ± 0.076 2.760 ± 0.172

142.3 ! 4.9* 300.4 + 8.3* 270.7 + 2.9* 0.792 ± 0.013" 2.927 + 0.035* 1.900 ± 0.071'

142.2 _+2.6* 304.9 ± 6.1" 405.6 _+4.2** 1.161 ± 0.018"* 2.862 ± 0.044* 0.333 ± 0.016"*

* P<0.01 as compared with 5-week-old rats: ** P<0.01 as compared with 10-week-oldWKY rats. Data are expressed as mean + S.E. Autoradiography was carried out and exposed X-ray film was scanned with a laser densitometer.

2.4. Immunohistochemistrv against tyrosine hydroxylase Ventricles were mounted on thin cardboard in OCT embedding medium (Tissue-Tek, Miles, IN), and rapidly frozen in propane prechilled with liquid nitrogen. Briefly, sections 10 mm thick were cut using a cryostat at - 2 5 ° C , mounted on slides precoated with gelatin, and air dried for 1 h at room temperature, Then, preparations were fixed by immersion for 1 h at 4°C in modified Bouin's solution (85 ml of 2% (wt./vol.) paraformaldehyde in 0,1 M phosphate buffer (pH 7.2) and 15 ml of saturated picric acid per 100 ml). These preparations were rinsed in several changes of phosphate-buffered saline (PBS, 0.01 M, pH 7.2) and incubated in normal goat serum. Sections were then rinsed in buffer, incubated with diluted primary antiserum (rabbit anti-tyrosine hydroxylase polyclonal antiserum, Chemicon International, CA) for 18 h at 4°C, rinsed again, and then incubated in biotinylated second antibody (goat antirabbit IgG; Vector, CA) for 1 h at room temperature and washed three times in 0.01 M PBS. The sections were incubated in avidin-biotin complexes and developed with the peroxidase reaction using 0.1% diaminobenzidine. Control was done as follows: instead of the anti-tyrosine hydroxylase antiserum, other preparation was incubated with normal rabbit serum, and then incubated in the second antibody. The development of immunohistochemical staining against tyrosine hydroxylase was visually assessed by two pathologists. Right ventricular free wall, ventricular septum, and left ventricular free wall were visually assessed in a semi-quantitative manner and arbitrarily g r a d e d f rom ( - ) to ( + + ) as follows: grade ( - ) no immunoreactivity detected, grade ( + ) scattered individual nerve fibers, grade ( + + ) a large number of fibers. All data were analyzed by one-way ANOVA followed by Duncan's multiple comparison test [19] and expressed as mean + S.E. Differences were considered significant at a value of p < 0.05.

3. Results Table 1 shows the basic characterization in W K Y rats at the ages of 5, 10, and 30 weeks (n = 10, at each age). Systolic blood pressure was significantly elevated as the age advanced from 5 to 10 weeks (p <0.01), while the heart rate decreased significantly (p < 0.01). During the subsequent period up to the age of 30 weeks, however, both the systolic blood pressure and heart rate showed almost no change. The left ventricular (EV) weight and the body weight significantly increased in accordance with growth (p < 0.01). On the other hand, although the LV/body weight ratio (%0) was significantly decreased from 5 to 10 weeks (p < 0.01), it showed no significant change from 10 to 30 weeks. Changes in the fl-adrenergic nervous system in the ventricular muscle of W K Y rats were examined at the ages 5, 10, and 30 weeks; significant changes in the systolic blood pressure and heart rate were observed with increasing age. The results are discussed below.

3.1. Tissue norepinephrine concentration Left ventricular concentration of norepinephrine was decreased significantly during development of age from 5 to 10 weeks old and to 30 weeks old (Table 1).

3,2, fl-adrenergic receptor (binding experiments against ["-SllCYP ) Fig. 1 shows a typical saturation curve obtained from fi-adrenergic receptor, and the results of Scatchard analysis. Scatchard plots showed a linear regression, and a binding site was revealed where fi-adrenergic receptors of the myocardium demonstrated a uniform affinity to antagonists, As shown by the fl-receptor binding experiments against [125I]CYP, fl-adrenergic receptor concentration (Bmax) in the left ventricular myocardium at the age of 5 weeks was significantly higher (p < 0.05) than that at the age of 10 weeks (42.6 + 3.7 vs. 29.8 + 5.0 fmol/mg protein, respectively). Subsequent significant changes of Bma x w e r e not seen from the age of 10-30 weeks (24.2 + 1.2 fmol/mg protein) (Fig. 2). On the other hand, there were no differences in

36

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Typical saturation assay in left ventricular membrane of WKY

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Typical Scatchard plot of specific [ 125 I] CYP binding in left ventricufar membrane of WKY (8)

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Fig. I. Typical saturation curves tAt and Scatchard analysis (B)el /,-adlcnergic receptor r~>l]CYp binding 1o Iclt xcntricular membranes ot Wister Kyoto (WKY) rats, the dissociation constant (Kd) [br [~:51]CYP between the three age groups (71.4 + 8.5, 72.4 _+ 6.7. and 63.2 ± 0.9 pM, respectively) (Fig. 2). 3.3. /3-adrenergic receptor m R N A

When the differences in the /)'-adrenergic receptor m R N A level were evaluated between the age of 5 and 10 weeks, a significant decrease in the number of Fadrenergic receptor was found. Typical autoradiograms, which were obtained by Northern blot analysis using fl-adrenergic receptor c D N A and G A P D H c D N A as probes, are shown in Fig. 2. In a preliminary study, the //-adrenergic probe detected a band of approximately 1.9 kb on a Northern blot of cardiac total

RNA. perlbrmed under the same hybridization and washing conditions as this experiment (results not ,ho~.n). The manifestation of steady-state fi-A RmR NA was clearly observed at the age of 5 weeks, on the othe," hand. it was not very marked at the age of 10 weeks. When the results were compared in terms of relative intensity against G A P D H m R N A as the internal standard, that is / ~ - A R m R N A . G A P D H m R N A (%}, a significant (p < 0.01) increase in steady-state f l - A R m R N A was observed at the age of 5 weeks, with a significantly higher number of fl-adrenergic receptors, compared with that at 10 weeks (30.8 ;+ 6.7 xs. 4.2 ± 1.0%. respectively) (Fig. 3). The results of dot blot analyses showed thal higher levels of /~'-AR m R N A in 5-week-old rats than in 10-week-old rats at each total RNA volume (I.25, 2.5!)

and 5.00 /tg) in Fig. 4. ~.4. hmJtmto/tislochc/Hi.s'lrl' a£,di/lsl llrosiHu ]l.l,~h'o.\~r/asc

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Fig. 2. Typical northern blot using ,fl-adrenergic receptor eDNA and GAPDH cDNA as probes. The total RNA (12 leg) extracted from left ventricles of 5 and 10-week-old WKY rats was hybridized with x2P-labeled #-adrenergic receptor and GAPDH eDNA as probes. I. 5-week-old; 2, 10-week-old.

As a result of immunohistochemical staining against tyrosine hydroxylase (Fig. 5l, granular and linear staining were observed in the ventricular myocardium at the ages of 5. 10, and 30 weeks. Tyrosine hydroxylase s~aining grades o f the myocardium on the basis of visual evaluation are shown in Table 2. We could not lind remarkable changes in staining grades for tyrosine hydroxylase during the development from 5 to 3() weeks old. Further, although the septum showed strong stainmg grades for tyrosine hydroxylase, no remarkable difference in the regional distribution of staining wets seen.

4. Discussion

As the age advanced from 5 to 10 weeks, a significant elewttion of systolic blood pressure and a significant

37

S. Maie et al. / Pathophysiology 4 (1997) 3 3 - 4 0

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Fig. 3. [~2511 CYP binding experiments were performed in seven rats of each age group: 5, 10 and 30-week-old rats. Binding data were analyzed by the least square linear regression method. The expression level of fl-adrenergic receptor m R N A was determined by Northern blots. fl-adrenergic receptor m R N A level at the age of 5 and 10 weeks was expressed as the relative intensity (%) of the amount of G A P D H m R N A in the autoradiography. Data are shown as mean + S,E.

decrease in the heart rate were observed in WKY rats. During this period of youth with marked changes of SBP and HR, the heart weight increased significantly, in association with the increase in body weight. However, the LV/body weight ratio was significantly decreased during the time from 5 to 10 weeks of age. These findings may suggest that the body develops predominantly compared to the left ventricle from 5 to 10 weeks, and that both the body and the left ventricle develop almost equally from 10 to 30 weeks. The increase in the systolic blood pressure from 5 to 10 weeks old may be due to growth-related increasing density of laser densitometer

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Fig. 4. Dot blot analyses of /]-adrenergic receptor m R N A were performed at the age of 5 and 10 weeks. The relation between total RNA (1.25, 2.5 and 5.0 /~g, 5 each) and the densitometric signal density of autoradiogram is shown. The expression level o f tq-adrenergic receptor m R N A at the age of 5 weeks was higher than that at the age of 10 weeks.

systemic vascular resistance. However, there is a possibility that the changes in systolic blood pressure and heart rate are related to the developmental changes in the fl-adrenergic nervous system. In this study, we examined the changes in the myocardial fl-adrenergic nervous system associated with the advancement of age and the changes of steady-state fl-adrenergic receptor mRNA. The tissue norepinephrine concentration was high at the age of 5 weeks, and a developmental decrease in tissue norepinephrine concentration was seen from 5 to 30 weeks of age. It has been reported that the cardiac concentration of norepinephrine in late gestation is quite low, and the levels rise progressively after birth to reach adult levels by approximately 3 weeks of age [20]. The measured content of tissue norepinephrine actually represented the total amount of mobile and storage pools, as it is impossible to virtually distinguish between synthesis, release, and uptake of norepinephrine in the myocardium and we cannot predict the sympathetic activity on the basis of the norepinephrine content alone. It is well known that congestive heart failure is associated with significant impaired alterations in sympathetic nervous system function. The reduced myocardial fl-adrenergic receptor number and the high levels of serum norepinephrine with lower myocardial catecholamine levels indicate the poor prognosis and severity of heart failure. The increase in norepinephrine concentration at the age of 5 weeks may indicate an enhanced activity of the sympathetic fl-adrenergic system. In addition, the number of fl-adrenergic receptors

38

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4 (1997)3_7

40

Fig. 5. Tyrosine hydroxylase po>ltl,,e iler'~c5 I"200l

(B ..... ) was significantly higher in the myocardium tit the age of 5 weeks, and there was no signiticant changes in the B....... from 10 to 3() weeks old. These results alst~ suggest that the possibility of enhancement of lhe fiadrenergic nervous system in the myocardium tit 5 weeks old. Reports of age-related changes in the number of/7-adrenergic receptors are varied [7 11]. Kojima et al. [21] in a report of the developmental changes in fl-adrenergic receptors, stated that the /~...... vahie for [~H]DHA binding started to increase on post-gestaiion day 20, reached almost the maximum level on neonatal day 6. remained at nearly the same level until neonatal day 20, and then decreased slightly to the adult level. It has not been fully determined whether or not these developmental changes in /J-adrenergic receptors are regulated at the transcriptional level: Northern blot analysis was performed to identify the developmental change of/,¢-adrenergic receptor mRNA corresponding to the age-associated decrease in the number of fladrenergic receptors. As a result, a decrease in the

~tcady-state fi-ARmRNA was observed in agreement x~ith the decrease in the number of fl-adrenergic receplots llom 5 to 10 weeks old. Dot blot analysis also clcmonstrated an age-related marked decrease in /Jadrenergic receptor mRNA. The steady-state fl-adrencrgic receptor mRNA levels reflect the equilibrium between the processes of transcription and degradation. Excn though we did not evaluate the post-transcripllonal events, such as mRNA stability, these results, in this study, suggest that the age-related decrease in the number of fl-adrenergic receptors from 5 to 10 weeks old may be controlled at the transcriptional level. Regarding the possible mechanism for the increase in lhc number of cardiac fl-adrenergic receptors m youth. there have been some reports which suggest incomplete mnervation [22,23]. But it is unclear whether or not F-adrenergic receptor distribution is influenced by the incomplete adrenergic innervation in the myocardium a t the age of 5 weeks. Although precise quantitative evahlation was not made in this immunohistochemical

39

S. Male et a l . / Pathophysiology 4 (1997) 33 40

Table 2 Grades of immunohistochemical staining against tyrosine hydroxylase in the developing ventricles

5-week-old 10-week-old 30-week-old

Right ventricular free wall

Septrum

Left ventricular free wall

(+ +) (+ ) (+ t

(+ +) (+ + ) (+ + )

(+ +) (+ ) (+)

Relative number of stained nerves rated arbitrarily: (+) weakly stained; (+ + ) strongly stained.

staining against tyrosine hydroxylase in the myocardium, visual evaluation by pathologists showed that there were no remarkable changes f r o m the age o f 5 to 30 weeks. It has been reported [22] that there is a marked paucity o f sympathetic nerves in the fetal and n e w b o r n heart c o m p a r e d with the adult, and the heart contains essentially the same density o f nerve fibers at the ages o f 3 - 5 weeks, as that seen in the adult; there was a general trend for the n u m b e r o f immunostained nerves to decline with age due to the age-related increase in fibroelastic tissue, These immunohistochemical findings suggest that the increased fl-adrenergic receptor density is n o t explainable by the c o m p e n s a t o r y development o f the fl-adrenergic system due to incomplete innervation in the m y o c a r d i u m at the age o f 5 weeks, since the immunohistochemical staining against tyrosine hydroxylase corresponded with the fl-adrenergic innervation in the m y o c a r d i u m . As for the regional distribution o f tyrosine hydroxylase in the m y o c a r d i u m , it has been reported [24,25] that the atrium has numerous nerves containing tyrosine hydroxylase c o m p a r e d with the ventricle, and the epicardium h a d more nerves than m y o c a r d i u m and e n d o c a r d i u m in the ventricle; the right and left ventricle had almost the same level o f nerves. In this study, the septum had more a b u n d a n t tyrosine hydroxylase staining, although it could not be fully quantitated. M o r e detailed examination is necessary. The myocardial fl-adrenergic stimulated c A M P production was also regulated by adenylate cyclase activity (catalytic unit) and G T P binding proteins (e.g. G T P binding stimulatory protein and G T P binding inhibitory protein), in addition to fl-adrenergic receptors. It m a y be possible that enhancement o f the fl-adrenergic receptor and its m R N A in the m y o c a r d i u m o f the 5-week-old rat is an adaptive response to supplement the depressed mechanisms before and after c A M P production in youth, namely, G T P - b i n d i n g proteins, adenylate cyclase, phosphodiesterase, c A M P - d e p e n d e n t protein kinase (A-kinase), specific p h o s p h o r y l a t i o n o f cytosolic protein, and some effectors with physiological functions. In conclusion, this study revealed an age-related decrease in the n u m b e r o f fl-adrenergic receptors in the m y o c a r d i u m o f rats f r o m 5 to 10 weeks old. It was considered that this age-related decrease is regulated, at

least in part, at the transcriptional level. Further studies are necessary to elucidate the mechanisms which regulate fl-adrenergic receptors and the fl-adrenergic nervous system during development.

References [1] Collins, P., Billings, C.G., Barer, G.R., Daly, J.J. and Jolly, A. (1975) Quantization of isoprenaline induced changes in the ventricular myocardium. Cardiovasc. Res. 9, 797 806. [2] Benjamin, l.J., Jalil, J.E., Tan, L.B., Cho. K., Weber, K.T. and Clark, W.A. (1989) Isoproterenol induced myocardial fibrosis in relation to myocyte necrosis, Circ. Res. 65 (3), 657-670. [3] Simpson, P., McGrath, A. and Savion, S. (1982) Myocyte hypertrophy in neonatal rat heart cultures and its regulation by serum and catecholamines. Circ. Res. 51, 787-801. [4] Simpson, P. and McGrath, A. (1983) Norepinephrine-stimulated hypertrophy of cultured rat myocardial cells is an alpha 1 adrenergic response. J. Clin. Invest. 72, 732 -738. [5] Xenophontos, X.P., Watson, P.A., Chua, B.H.L., Haneda, T. and Morgan, H.E. (1989) Increased cyclic AMP content accelerates protein synthesis in rat heart. Circ. Res. 65 (3), 647-656. [6] Tse, J., Powell, J.R., Baste, C.A., Priest, R.E. and Kuo, J.F. (1979) Isoproterenol-induced cardiac hypertrophy: modifications in characteristics of p-adrenergic receptor, adenylate cyclase, and ventricular contraction. Endocrinology 105, 246-255. [7] Mader, S.L., Dowing, C.L. and Lunteren, E.V. (1991) Effect of age and hypoxia on fl- adrenergic receptors in rat heart. J. Appl. Physiol. 71 (6), 2094- 2098. [8] Bhalla, R.C., Sharma, R.V. and Ramanathan, S. (1980) Ontogenetic development of isoproterenol subsensitivity of myocardial adenylate cyclase and fl-adrenergic receptors in spontaneously hypertensive rats. Biochim. Biophys. Acta 632, 497-506. [9] Mochizuki, M. and Ogawa, K. (1984) Increase of cardiac [Jadrenergic receptors in young spontaneously hypertensive rats. Jpn. Heart J. 25, 411-423, [10] Kusiak, J.W. and Pitha, J. (1983) Decreased response with age of the cardiac catecholamine sensitive adenylate cyclase system. Life Sci. 3, 1679-1686. [11] Urasawa, K., Murakami, T. and Yasuda, H. (1991) Age-related alterations in adenylyl cyclase system of rat hearts. Jpn. Circ. J. 55, 676--684. [12] Honma, T. (1982) Simultaneous determination of biogenic amines and metabolites in small brain tissues by a high-performance liquid chromatograph connected 1:o an electrochemical detector. Ind. Health 20, 247-258. [13] Salzman, S.K. and Sellers, M.S. (1982) Determination of norepinephrine in brain perfusates using high-performance liquid chromatography with eleetochemical detection. J. Chromatogr. 232, 29-37. [14] Scathard, G, (1949) The attractions of protein for small molecules and ions. Ann. N.Y. Acad. Sci. 51, 660 672.

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Palhophvsioloju'

[15] Lowry, O.H., Rosebrough. N.J.. Farr, A.L. and Randall, R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. (_?hem. 193, 265 275. [16] Chomczynski, P. and Sacchi, N. (1987) Single-step method of RNA isolation by acid guanidium thyocyanate-phcnol-chloroform extraction. Anal. Biochem. 162, 156 159. [17] Gocayne, J.. Bobinson. D.A., Fitzgerald, M.G., ( h u n g , F.. Kelavage, A.R., Lentes, K., Lai, J.. Wang, C., Frase, C.M. and Venter, J.C. {1987) Primary structure of rat cardiac fi-adrenergic and muscarinic cholmergic receptors obtained by automated DNA sequence analysis: further evidence for a mulfigene family. Proc. Natl. Acad. Sci. 84, 8296 83{}(}. [18] Dugaiczyk, A., Haron, J.A.. Stone, E.M., Dennison, O.E., Rothblum. K.N. and Schwartz, R.J. {1983) Cloning and sequencing of a deoxyribonucleic acid copy of glycer aldehyde-3-phosphate dehydrogenase messenger ribonucleic acid isolated from chicken muscle. Biochemistry 22, 1605 1613. [19] Duncan. D.B. (195I) A signilicancc test lk)r difl'erences between ranked treatments in an analysis of variance. Vase. ,1. Sci. 2. 171 189, [20] Friedman, W.F., Pool. P.E., ,lacobowitz, D., Seagren, S.C. and Braunwald. E. (1968) Sympathetic mnervation of the developing

[21]

[22]

[23]

[24]

[25]

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