Impairment of adenylyl cyclase signal transduction in mecobalamin-deficient rats

ELSEVIER

European Journal of Pharmacology Molecular Pharmacology Section 291 ( 1995) 35 l-358

molecular

pharmacology

Impairment of adenylyl cyclase signal transduction in mecobalamin-deficient rats Shinichi Hatta a7*, Masayuki Watanabe b, Hiroshi Ikeda b, Hiroki Kamada b, Toshikazu Saito b, Hideyo Ohshika a a Department of Pharmacology, School of Medicine, Sapporo Medical University, South-l, West-l 7, Chuo-ku, Sapporo 060, Japan b Department of Neuropsychiatry, School of Medicine, Sapporo Medical University, South-l, West-16, Chowku, Sapporo 060, Japan Received 29 May 1995; revised 7 August 1995; accepted 11 August 1995

Abstract This study examined alterations in the /3-adrenoceptor-G,-adenylyl cyclase system in cerebral cortex membranes from vitamin for 15 weeks. Basal, 5’-guanylylimidodiphosphate (GppNHp)-, B,,-deficient rats fed a diet lacking vitamin B,, (mecobalamin) isoproterenol-, and forskolin-stimulated adenylyl cyclase activities were significantly reduced in mecobalamin-deficient rats compared with those in control rats. However, no significant differences were observed in the amount and function of G,, estimated by immunoblotting and guanine nucleotide photoaffinity labeling, respectively, or in the densities and the dissociation constants of /?-adrenoceptors, estimated by [‘25~] pindolol binding, between control and the deficient rats. These results indicate that vitamin B,, deficiency results in the impairment of the coupling among the /?-adrenoceptor, G,, and the catalytic subunit of adenylyl cyclase, and in dysfunction of the catalytic subunit of the enzyme, suggesting that vitamin B ,* participates in the regulation of neuronal adenylyl cyclase signal transduction. Keywords: Vitamin B I2 deficiency; Adenylyl cyclase; PAdrenoceptor;

G,; Signal transduction

1. Introduction It has been suggested that psychiatric and behavioral symptoms such as depression, schizophrenia, and dementia can be attributed to a deficiency of vitamin B r2, which is often associated with those neuropsychiatric illness (Zucker et al., 1981; Lindenbaum et al., 1988; Dommisse, 1991; Healton et al., 1991). Low levels of vitamin B,, have also been reported in dementia of the Alzheimer type and the vitamin deficiency is thought to be related to cognitive impairment in Alzheimer patients (Levitt and Karlinsky, 1992; Kristensen et al., 1993). Furthermore, several studies have indicated that methyl-vitamin B ,* [mecobalamin] has clinical effects in non-24-hour sleep-wake syndrome and delayed sleep phase syndrome (Kamgar-Parsi et al., 1983; Okawa et al., 1990). On the other hand, vitamin B,, is known to be involved in the synthesis of S-adenosyl-Lmethionine (Allen et al., 19931, which has been demonstrated to possess an antidepressive effect (Camey et al.,

* Corresponding author. Tel.: 81-11-611-2111; Fax: 81-11-612-5861. 0922.4106/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDI 0922-4106(95)00127-l

1986; Dommisse, 1991). Thus, there are several lines of evidence to suggest that vitamin B,, participates in the regulation of cellular responsiveness, especially in neuronal systems. However, the molecular mechanisms of vitamin B r2 involvement in the regulation of neuronal cell function remain unclear. Many cellular functions are regulated in response to extracellular signals across plasma membranes. Receptors for these signals are quite often coupled to GTP-binding proteins (G proteins) that mediate those cellular functions by connecting with cellular effecters such as adenylyl cyclase, cyclic GMP phosphodiesterase, and phospholipases, as well as the modulation of activities of various ion channels (Gilman, 1987; Bimbaumer et al., 1990). The adenylyl cyclase [EC 4.6.1. l] system is a membrane-associated complex, and the receptor-mediated stimulation and inhibition of the enzyme are mediated by two distinct G proteins, termed G, and Gi, respectively. These G proteins are heterotrimeric proteins consisting of three subunits, (Y, p, and y, and associated with plasma membranes. The LY subunits exchange GDP to GTP, dissociate from the Pr subunits, and associate with the catalytic subunits of

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adenylyl cyclase, resulting in stimulation or inhibition of adenylyl cyclase. The catalytic subunit of adenylyl cyclase is the enzyme responsible for production of cyclic AMP, which is a critical regulator of cellular function as an intracellular second messenger in signal transduction. The /3-adrenoceptor-adenylyl cyclase complex is one of the best-characterized hormonal signal-transduction systems that mediate intracellular effects through the G s protein. Agonist occupancy of this receptor results in activation of G s, which is in turn coupled to the effector unit, adenylyl cyclase (Gilman, 1987; Birnbaumer et al., 1990). Our recent studies using mecobalamin-deficient rats (Watanabe et al., 1995) have shown that forskolin- and Mn-induced activation of adenylyl cyclase were decreased in the cerebral cortex of mecobalamin-deficient rats compared with those of control rats. In addition, forskolin binding in brain slices, estimated by [3HI forskolin autoradiography, was observed to be reduced in the deficient rats. It seemed that vitamin B12 deficiency resulted in dysfunction of the catalytic subunits of adenylyl cyclase or coupling between G s and the catalytic subunits of the enzyme. This was the first indication that vitamin B12 may participate in the regulation of the function of adenylyl cyclase catalytic subunits. On the other hand, it has been suggested previously that S-adenosyl-L-methionine, acting as a methyl donor, participates in the methylation of phosphatidylethanolamine, which leads to increased membrane fluidity, consequently enhancing the coupling of the /3-adrenoceptor with the adenylyl cyclase system (Hirata and Axelrod, 1978; Hirata et al., 1979). Since vitamin B12 contributes to synthesis of S-adenosyl-L-methionine, vitamin B12 deficiency might exert its influence on the /3adrenoceptor function in the activation of adenylyl cyclase. To further characterize the role of vitamin B12 in the regulation of neuronal signal transduction, the present study examines alterations in the /3-adrenoceptor-Gs protein-adenylyl cyclase system by vitamin B12 deficiency in the cerebral cortex in rats fed with or without mecobalamin for 15 weeks. The results presented here suggest that vitamin B12 deficiency results in impairment of coupling processes among the /3-adrenoceptor, Gs and adenylyl cyclase, as well as in dysfunction of the catalytic subunits of adenylyl cyclase.

2. Materials and methods

2.1. Tissue preparation Two-month-old male Sprague-Dawley rats were housed under standard laboratory conditions of lighting (12 h light/dark cycle) and temperature (24 + 2°C). Rats were fed 6 tzg of mecobalamin per 100 g of the standard laboratory diet (as control) or the standard diet without mecobalamin for 15 weeks and allowed tap water ad libitum. The body weight of rats was 301 + 2 g at the

beginning and 491 + 10 g at the end. There was no significant difference in the rate at which the rats gained weight between control and mecobalamin-deficient rats. The blood concentration of vitamin B~2 in the deficient rats was observed to have declined to about 6% of that in control rats at 14 weeks. After the 15-week treatment, rats were killed and the brain was dissected as described by Heffner et al. (1980). Cortex membranes were prepared as described previously (Hatta et al., 1986) and stored at -80°C until use.

2.2. Adenylyl cyclase assay Cerebral cortex membranes were thawed and resuspended in a buffer containing 20 mM Hepes (pH 7.5), 5 mM MgC12, l mM dithiothreitol, and 0.3 mM phenylmethylsulfonyl fluoride. Adenylyl cyclase activity in cortex membranes was assayed as described previously (Hatta et al., 1986; Hatta et al., 1995). Membranes (30-40 /xg) were incubated with or without the indicated reagents for l0 min at 30°C in 100 /zl of medium containing 15 mM Hepes (pH 7.5), 0.05 mM ATP, [ol-32p]ATP (approximately 2 × l 0 6 cpm/tube), 5 mM MgCI 2, 1 mM EGTA, 1 mM dithiothreitol, 0.05 mM cyclic AMP, 60 mM NaC1, 0.25 m g / m l bovine serum albumin, 0.5 mM 3-isobutyl-1methylxanthine, 1 U / m l adenosine deaminase, and a nucleotide triphosphate-regenerating system consisting of 0.5 mg of creatine phosphate, 0.14 mg of creatine phosphokinase, and 15 U / m l myokinase. The reaction was quenched by the addition of 0.1 ml of a solution containing 2% sodium dodecyl sulfate (SDS), 1.4 mM cyclic AMP, and 40 mM ATP, and the cyclic [32p]AMP formed was isolated by the method of Salomon (1979).

2.3. P3(4-azidoanilido)-P C5'-GTP (AAGTP) photoaffinity labeling [32p]AAGTP was synthesized by the method of Pfeuffer (1977). Photoaffinity labeling of cerebral cortex membranes with [32p]AAGTP was performed as described previously (Hatta et al., 1986; Hatta et al., 1995). Cerebral cortex membranes were washed and resuspended in 2 mM Hepes (pH 7.4)/1 mM MgCI 2. Membrane suspensions (2-3 mg of protein/ml) were incubated with 0.1 /xM [32p]AAGTP for 5 min at 30°C, and the reaction was terminated by dilution with the above ice-cold buffer followed by centrifugation at 1 5 0 0 0 × g for 10 min to remove unbound [32P]AAGTP. Membranes were washed again and resuspended in the same buffer followed by 5 min of UV photolysis on ice with a Spectroline UV lamp (254 nm, 9W) at a distance of 3 cm. The reaction was quenched with ice-cold 2 mM Hepes (pH 7.4)/1 mM MgC12/4 mM dithiothreitol, followed by centrifugation at 15000 × g for 10 min. Membrane pellets were dissolved in 3% SDS Laemmli sample buffer (Laemmli, 1970) with 50 mM dithiothreitol and electophoresed in 10%

S. Hatta et a l . / European Journal of Pharmacology - Molecular Pharmacology Section 291 (1995) 351-358

SDS/polyacrylamide gels by the procedure of Laemmli (Laemmli, 1970). After electrophoresis, gels were stained with Coomassie Blue, dried and autoradiographed with Kodak XAR-5 film. After autoradiography, the radioactivity in each band was determined by excision of the band in the dried gel corresponding to the radiolabeled band on the autoradiograph and counting in a Beckman LS-5801 scintillation counter.

353

ing 150 mM NaCI. Bound and free ligands were separated by rapid vacuum filtration through Whatman G F / B filters using a cell harvester (Brandel). The filters were washed three times with stopping buffer and bound radioactivity was counted in an LKB gamma counter. Specific binding was defined as the difference in the amount of bound radioactive ligand obtained in the absence and presence of 10 p.M DL-propranolol. Data were analyzed using the Scatchard transformation.

2.4. lmmunoblotting Immunoblotting of G proteins was performed by the procedure described previously (Ozawa et al., 1993; Hatta et al., 1994). Briefly, cerebral cortex membranes (5 /zg of membrane protein/lane) were dissolved in 3% Laemmli sample buffer with 50 mM dithiothreitol and electrophoresed in 10% SDS/polyacrylamide gels. Protein was transferred to nitrocellulose filters by electroelution as described by Towbin et al. (1979). After transfer, filters were blocked by a 1-h incubation with 3% bovine serum albumin in a buffer of 10 mM Tris (pH 7.5), 500 mM NaCI, and 0.1% Tween 20 (TBS-T). Nitrocellulose filters were then incubated with TBS-T containing 0.1% bovine serum albumin and anti-G protein a subunit antibodies (Du Pont-NEN) at 1/5000 dilution. After incubation for 14-16 h at room temperature, filters were washed in TBS-T three times and then incubated with TBS-T containing 0.1% bovine serum albumin and horseradish peroxidase-linked anti-rabbit Ig (F(ab') 2) (Amersham) at 1/5000 dilution for 1 h at room temperature. Filters were washed three times with TBS-T and immunoreactivity was detected with an enhanced chemiluminescence Western blot detection system (Amersham) followed by exposure to enhanced chemiluminescence HYPER film (Amersham). The developed autoradiographs were analyzed by laser densitometry (model SLR-2D/1D, Biomed Research Instruments). To standardize integrated optical density against a known amount of G,~ subunit by our immunoblotting methods, purified Go~ protein was used as a standard. The individual integrated optical densities of G,~ subunits were normalized relative to each other by comparison against a pooled standard sample of normal rat cerebral cortex membranes containing 397.89 +_ 4.71 pmol of G o J m g membrane protein, which was run every time.

2.6. Protein determination and statistics Protein was determined by the Commassie Blue binding method (Bradford, 1976) with bovine serum albumin as a standard. Data were analyzed for statistical significance using a two-tailed Student's t test. Values of P < 0.05 were taken to indicate statistical significance.

2.7. Materials [a-32p]ATP (800 Ci/mmol; 1 Ci = GBq) and ['251](-)-pindolol (2200 Ci/mmol) were purchased from Du Pont-New England Nuclear (Tokyo). [ c~-32P]GTP (400 Ci/mmol) were from Amersham (Tokyo). GppNHp was from Boehringer Mannheim (Mannheim). L-Isoproterenol bitartrate and DL-propranolol hydrochloride were from Sigma (St. Louis). Forskolin was from Calbiochem (La Jolla). p-Azidoaniline was from Aldrich (Tokyo). G protein antibodies R M / 1 , A S / 7 , G C / 2 , and QL specific to G s a , G i l a / / G i 2 a , Goa, and G q , / G ) l ~ subunits, respectively, were obtained from Du Pont-New England Nuclear. Horseradish peroxidase-linked anti-rabbit Ig (F(ab') 2) was from Amersham. Purified Go~ from bovine brain was

3o0t 250~

[] Cont

2.5. fl-Adrenoceptor binding Measurement of fl-adrenoceptors in cerebral cortex membranes was performed using [125I](-)-pindolol as described previously (Takemura et al., 1995) with minor modifications. Membranes ( ~ 200 /zg protein) were incubated with [125I]pindolol (25-500 pM) at 30°C for 50 rain in 25 mM Tris-HC1 (pH 7.4) containing 5 mM MgC12, 0.4 mM EGTA, 120 mM NaC1, and l mM sodium ascorbate in a total volume of 500pA. The reaction was terminated by addition of ice-cold 10 mM Tris-HCl (pH 7.4) contain-

BIIsal

Forskolln

Fig. l. Forskolin-stimulated adenylyl cyclase activity in cerebral cortex membranes from control (Cont) and mecobalamin-deficient (Lack) rats. The concentration of forskolin used was 100 p.M. The values are means + S.E.M. of 6 experiments using separate membrane preparations. " P < 0.05 and " " P < 0.01 indicate significant differences compared with the corresponding control values.

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S. Hatta et a l . / European Journal of Pharmacology - Molecular Pharmacology Section 291 (1995) 351-358 220

~200

•

Cont Lack

o

Lack

Cont

~.

/ ~

g~o 160 <

~140 -

O.SH SL

120

~100

•

<

80

• , -// CO

................. 8.0

7.0

8,0

5.0

4.0

- Log [GppNHp] (M)

=~ 7~7¸

Fig. 2. GppNHp-dependent activation of adenylyl cyclase in cerebral cortex membranes from control (Cont) and mecobalamin-deficient (Lack) rats. The basal activities in control and mecobalamin-deficient rats were 98.45 ±4.15 and 82.75-+ 1.40 p m o l / m g of protein/min, respectively. The values are means -+ S,E.M. of 6 experiments using separate membrane preparations. * P < 0.05 indicates a significant difference compared with the corresponding control values.

i

provided by Prof. T. Katada (University of Tokyo). All other reagents used were of analytical grade. i~Ii~ 3.

Results ;5 ~i~?~

3.1. Alterations in adenylyl cyclase activation

•

While forskolin (100/zM) effectively stimulated adenylyl cyclase in control rats, forskolin-induced activation of the enzyme was significantly reduced in mecobalamin-deficient rats compared with that in controls (Fig. 1). In

1'oL

.

17:~ii!..... ii

!~;%~!i!i:!i¸

Fig. 4. Immunoblotting of G protein subunits in cerebral cortex membranes from control (Cont) and mecobalamin-deficient (Lack) rats. The results are representative of 6 similar experiments. Immunoblotting was performed using polyclonal rabbit antisera against G protein subunits ( R M / I , A S / 7 , G C / 2 , and QL specific to G~,~, GH,/Gi2,~, Go~, and Gqc~/GII . subunits, respectively) as described in Materials and methods.

Table 1 Quantitative estimation of the G protein ot subunits and the [~2 P]AAGTP labeling to G proteins in control and mecobalamin-deficient (Lack) rats

.

Control 100 t Io,-.

~7~ %~

Lack

Amount of G, (pmol G o . / m g protein) It . . . . . . . . . . . . . . . . .

0.0

7.o

6.0

s.o

4.o

- Log[Isoproterenol(M) ] Fig. 3. Activation of isoproterenol-sensitive adenylyl cyclase activity in cerebral cortex membranes from control (Cont) and mecobalamin-deficient (Lack) rats. Adenylyl cyclase activity was assayed with the indicated concentrations of isoproterenol in the presence of 10 -6 M GppNHp. The values are expressed as a percentage of the activity obtained with GppNHp alone and are shown as means -+ S.E.M. of 6 experiments using separate membrane preparations. The activities with 10 -6 M GppNHp in control and mecobalamin-deficient rats were 137.79 -+ 8.85 and 114.26 + 2.09 p m o l / m g of protein/rain, respectively. * P < 0.05 indicates a significant difference compared with the corresponding control values.

G~, H G~, L Gil ~ and Gi2 . Go, Gq,~ and G] ~,,

60.87-+ 3.26 24.78+ 4.95 174.43 ± 10.24 325.75 -+ 18.08 259.27-+ 10.06

61.68± 25.49_+ 179.05 ± 314.43 ± 267.30±

2.50 4.64 9.20 21.69 9.17

[ 32P]AAGTP labeling (cpm) GsH G~L Gi/o G~2

444--+ 7 271± 24 2048 ± 181 10615:58

417+ 263_ 2182 ± 978±

15 16 153 70

The values are means-I-S.E.M, of 6 experiments using separate membrane preparations.

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S. Hatta et al. /European Journal of Pharmacology - MolecularPharmacology Section 291 (1995) 351-358

addition, basal activity in the deficient rats was significantly lower than that in controls. These results confirmed our previous findings (Watanabe et al., 1995), suggesting dysfunction of the catalytic subunit of adenylyl cyclase in mecobalamin-deficient rats. GppNHp, a hydrolysis-resistant GTP analog, can activate G~ without activation of receptors. Subsequently, the activated G~ (t~ s) stimulates adenylyl cyclase through direct interaction with the catalytic subunits of adenylyl cyclase. When cerebral cortex membranes were incubated with GppNHp (10 - 8 ~ 10 -4 M), adenylyl cyclase was effectively stimulated by GppNHp with a maximum stimulation of ~ 194% of the basal activity (Fig. 2). In contrast, GppNHp-dependent stimulation of the enzyme was significantly lower ( ~ 167%) in membranes from mecobalamindeficient rats. Reduction of adenylyl cyclase activation in mecobalamin-deficient rats was further demonstrated with respect to /3-adrenoceptor stimulation of the enzyme. As shown in Fig. 3, activation of adenylyl cyclase with isoproterenol (10 - 8 ~ 10 -4 M) in the presence of 10 -6 M GppNHp was significantly reduced in the deficient rats ( ~ 110% of the GppNHp-stimulated activity) compared with that in controls ( ~ 127%). 3.2. Quantitative and qualitative estimation o f G proteins

We next examined the amounts and functions of G proteins in cerebral cortex membranes from control and mecobalamin-deficient rats. Fig. 4 shows representative

Cont

Lack

GsH GsL Gi/o

400' o

i

Control

00'

i' --o 200 E b IIL ~ 100 m 0

0

10

20

30

40

50

60

70

Bound Ilmol/mg protelnl

Fig. 6. Scatchard plot of [125I]pindololbinding to cerebral cortex membranes from control (Cont) and mecobalamin-deficient(Lack) rats. The values are means (+ S.E.M.) of 6 experiments using separate membrane preparations.

immunoblots obtained in the assay of G protein a subunits. Under the same exposure conditions, equivalent amounts of cortex membrane protein were probed with various antisera against G protein oz subunits (Gs~, Gi~, Goa and Gqa). There were no significant differences in the protein levels of G protein ct subunits in cortex membranes from control and mecobalamin-deficient rats (Fig. 4 and Table 1). The functional qualities of G proteins, G s and Gi, were evaluated using the hydrolysis-resistant photoaffinity GTP analog AAGTP. A A G T P has been used as a probe to study the behavior of synaptic membrane G proteins without removing those proteins from the membranes (Hatta et al., 1986; Gordon and Rasenick, 1988; Ozawa et al., 1993). A A G T P binding corresponding to Gs,~ or Gi~ in cerebral cortex membranes was previously identified (Hatta et al., 1986). As shown in Fig. 5 and Table 1, no difference was observed in [32p]AAGTP binding to Gs~ and Gi~ between controls and mecobalamin-deficient rats. 3.3. fl-Adrenoceptor binding

•

Ga2 Fig. 5. Photoaffinity labeling of [~2P]AAGTPin cerebral cortex membranes from control (Cont) and mecobalamin-deficient (Lack) rats. The autoradiographs are representative of 6 similar experiments. GsH and GsL refer to the 52-kDa and 45-kDa cholera toxin substrates, respectively. Gi/o refers to the GTP-binding pertussis toxin substrates clustered around 40 kDa. These include Gi and Go. G32 refers to a 32-kDa AAGTP-binding protein which appears to be particularly abundant in rat brain. It is not yet clear whether this species functions in signal transduction.

To characterize /3-adrenoceptors, [125I]pindolol binding was performed in cerebral cortex membranes from control and mecobalamin-deficient rats. Scatchard analysis of the

Table 2 Binding characteristics of [1251]pindolol in cerebral cortex membranes from control (Cont) and mecobalamin-deficient(Lack) rats Treatment

Cont Lack

Bmax

Kd

(fmol/mg protein)

(pM)

62.64 -t- 1.37 60.63 + 1.63

182.26+ 8.36 181.97+ 9.80

The values are means :t:S.E.M. of 6 experiments using separate membrane preparations.

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saturation isotherm of [125I]pindolol binding is shown in Fig. 6 and the binding characteristics are summarized in Table 2. No significant differences were found either in the density of receptors (Bmax) or in the dissociation constant (K d) between control and mecobalamin-deficient rats.

4. Discussion It is generally accepted that vitamin B12 plays an important role in a number of metabolic pathways in the central nervous system. The vitamin is involved in onecarbon metabolism, which generates methyl groups for the production of such essential substances as monoamine neurotransmitters and phospholipids (Cbanarin et al., 1989). The deficiency of vitamin B12 has been implicated in a demyelinating disease of the brain and spinal cord (Lindenbaum et al., 1988; Healton et al., 1991). Furthermore, it is suggested that vitamin B~2 deficiency causes a wide variety of psychiatric and behavioral symptoms (Zucker et al., 1981; Lindenbaum et al., 1988; Dommisse, 1991). In addition, amelioration of sleep-wake rhythm disorders with vitamin B lz has been documented (Kamgar-Parsi et al., 1983; Okawa et al., 1990). In spite of these findings indicating that vitamin B~2 may contribute to the regulation of neuronal cell function, little is known regarding the mechanism by which it modulates this function. To elucidate the role of vitamin B12 in the regulation of cellular function, we have investigated alterations in the adenylyl cyclase signal transduction in the cerebral cortex in vitamin B lz-deficient rats. The initial efforts from our laboratory to characterize participation of vitamin B j2 in the neuronal signal transduction suggested that vitamin B~2 deficiency causes dysfunction of the catalytic subunits of adenylyl cyclase (Watanabe et al., 1995). In the present study, we found that forskolin-, GppNHp-, and isoproterenol-induced activations of adenylyl cyclase were significantly reduced in cerebral cortex membranes from mecobalamin-deficient rats compared with those in control rats. These results indicate that vitamin B12 deficiency resulted in impairment of the function of the fladrenoceptor-Gs-adenylyl cyclase signal transduction system. Forskolin is known to interact with the catalytic subunit of adenylyl cyclase with high affinity in the presence of activated G protein ( a s) and with low affinity in the absence of ot~ (Seamon and Daly, 1986). Thus, the reduced responsiveness of the enzyme to 100 /zM forskolin observed in mecobalamin-deficient rats (Fig. 1) indicated dysfunction of the catalytic subunit of adenylyl cyclase, which was in consistent with our previous finding (Watanabe et al., 1995) showing a decline in the activation of the enzyme with forskolin (40 ~M) or manganese (20 raM) and in [3H]forskolin binding in the deficient rats. In addition to the dysfunction of the catalytic subunit of adenylyl cyclase, the present result showing a decrease in

the ability of GppNHp to activate the enzyme (Fig. 2) appears to indicate that Gs-mediated regulation of adenylyl cyclase was impaired by vitamin B~2 deficiency. The decrease in GppNHp-stimulated adenylyl cyclase in mecobalamin-deficient rats might simply reflect the dysfunction of the adenylyl cyclase catalytic subunits. However, the impaired function of the catalytic subunit alone seems to be insufficient to account for the reduction in GppNHp-dependent activation of adenylyl cyclase in the deficient rats, since the percentage increments in GppNHp-stimulated activity of the enzyme over the basal activity declined with increasing concentrations of GppNHp (Fig. 2). As was the case with GppNHp, /3-adrenoceptor agonist isoproterenol-dependent activation of the enzyme, i.e. the percentage increments in the activation of adenylyl cyclase with isporoterenol and GppNHp over the activity of GppNHp alone, declined progressively with increasing concentrations of isoproterenol in the deficient rats (Fig. 3). This result suggested that /3-adrenoceptor-mediated activation of adenylyl cyclase was also impaired by vitamin Bl2 deficiency. No alterations in the amount and function of G s protein (Figs. 4 and 5, and Table 1) or in the function of/3-adrenoceptor, i.e., the density (Bma×) and the dissociation constant (Ka) (Fig. 6 and Table 2), were observed between control and mecobalamin-deficient rats. Thus, G S and G i proteins and the fl-adrenoceptor themselves did not seem to be affected by vitamin B~2 deficiency. It is likely, therefore, that alterations in /3-adrenoceptor-mediated and Gs-mediated regulation of adenylyl cyclase in the deficient rats resulted from impairment of both the functional coupling between the /3-adrenoceptor and G~ and between G~ and the catalytic subunit of the enzyme. The reasons for the impaired coupling among the /3adrenoceptor, G s, and the catalytic subunit of adenylyl cyclase and for the dysfunction of the catalytic subunit of the enzyme in vitamin Bj2 deficiency are not clear at present, but there are several possible explanations. It has been demonstrated that adenylyl cyclase functionality depends on the maintenance of a suitable membrane environment (McMurchie, 1988). Changes in lipid components of synaptic membranes, which are associated with the adenylyl cyclase system, can influence many properties of this signaling mechanism (Stubbs and Smith, 1984). Dietary-related alterations in the adenylyl cyclase system have also been reported (Wince and Rutledge, 1981; Neelands and Clandinin, 1983). In addition, alterations in membrane fluidity have been suggested to be involved in the process of adenylyl cyclase activation (Helmreich and Elson, 1984; Houslay, 1985; Needham et al., 1985). Vitamin Bt2 is known to be involved in the synthesis of S-adenosyl-L-methionine (Allen et al., 1993), which participates as a methyl donor in the conversion of phosphatidylethanolamine to phosphatidyl-N-monomethyl-ethanolamine, and then to phosphatidylcholine (Hirata and Axelrod, 1978). Therefore, vitamin Bl2 can be assumed to

s. Hatta et a l . / European Journal of Pharmacology - Molecular Pharmacology Section 291 (1995) 351-358

participate in the methylation of phosphatidylethanolamine through the synthesis of S-adenosyl-L-methionine. It has been suggested that the methylated product phosphatidylN-monomethylethanolamine leads to increased membrane fluidity, consequently enhancing the coupling of the /3adrenoceptor with G s in rat reticulocytes (Hirata and Axelrod, 1978; Hirata et al., 1979). In addition, phosphatidylcholine, which is formed by further methylation of phosphatidyl-N-monomethylethanolamine, has been demonstrated to contribute to the functional coupling between G s and the catalytic subunit of adenylyl cyclase (Ross, 1982; Calorini et al., 1993) and act as a dominant factor regulating the function of the catalytic subunit of the enzyme (Hebdon et al., 1981; Diaz-Laviada et al., 1991). Thus, it is feasible that vitamin B~2 contributes to /3-adrenoceptorGs-adenylyl cyclase signal transduction by providing a favorable environment for coupling among the /3-adrenoceptor, G s, and the catalytic subunit of adenylyl cyclase and for activation of the catalytic subunit of the enzyme. Although levels of S-adenosyl-L-methionine and phosphatidylcholine were not estimated in the present study, a decrease in the concentration of S-adenosyl-L-methionine or phosphatidylcholine has been shown to be associated with vitamin B12 deficiency (Surtees et al., 1991; Kennedy et al., 1992). A deficit of phosphatidylcholine has been shown to cause a substantial decrease in basal and G smediated stimulation of adenylyl cyclase activities in B A L B / c 3T3 cells (Calorini et al., 1993). It may be expected, therefore, that vitamin B ~2 deficiency results in reduction in the methylation of phosphatidylethanolamine, leading to impairment of the functional coupling among the signal transducing components in the adenylyl cyclase system and the dysfunction of the catalytic subunit of adenylyl cyclase. This hypothesis will serve as the basis for further studies of the participation of vitamin B 12 in the neuronal signal transduction cascade. Efforts to understand the mechanism of impairment of the adenylyl cyclase system related to vitamin B~2 deficiency are now under way. In s u m m a r y , using vitamin Blz-deficient rats, we demonstrated that vitamin B12 deficiency results in the impairment of /3-adrenoceptor-G~-adenylyl cyclase signal transduction. Such impairment of the adenylyl cyclase system may be expected to result in an attenuated response in production of cyclic A M P in neuronal cells, which might be related to certain neuropsychiatric illness or symptoms. Thus, the possibility that vitamin B~2 participates in the regulation of the adenylyl cyclase signal transduction in neuronal cells remains intriguing.

Acknowledgements We thank Prof. Toshiaki Katada for the gift of purified Go~ protein. W e also thank Esai Pharmaceutical Co., Ltd. for the generous gift of mecobalamin.

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