Comparative Biochemistry and Physiology Part B 124 (1999) 397 – 403 www.elsevier.com/locate/cbpb
Comparison of phosphodiesterase isozymes in rodent parotid glands A. Imai *, T. Nashida, H. Shimomura Department of Oral Biochemistry, The Nippon Dental Uni6ersity, School of Dentistry at Niigata, 1 -8 Hamaura-cho, Niigata 951 -8580, Japan Received 4 February 1999; received in revised form 9 August 1999; accepted 16 August 1999
Abstract We investigated phosphodiesterase (PDE) isozymes, which hydrolyze cAMP, in rodent parotid glands (mouse, hamster and guinea pig) in order to clarify the effects of cGMP and Ca/calmodulin on the regulation of cellular cAMP and compared them with those of the rat. More than 80% of the activities were in the supernatant fractions except for the hamster. The isozymes were fractionated using Mono Q ion-exchange column. The mouse parotid PDEs consisted of PDE1 (Ca/calmodulin-dependent), PDE2 (cGMP-stimulated), PDE3 (cGMP-inhibited) and PDE4 (cAMP-specific) similar to those of the rat. PDE3 was not detected in the hamster, and PDE4 was not detected in the guinea pig. PDE activities in the supernatant of the mouse and the hamster were stimulated by cGMP, and that of the guinea pig was stimulated by Ca/calmodulin. These results suggest that various PDE isozymes are present in the parotid gland of several species of order Rodentia. There seems to be differences among the species with regard to the PDE isozymes. © 1999 Elsevier Science Inc. All rights reserved. Keywords: cAMP; Guinea pig; Hamster; Identification; Isozyme; Mouse; Parotid gland; Phosphodiesterase; Rat; Rodent
1. Introduction Cyclic AMP (cAMP), as a second messenger, plays a variety of important roles in cellular systems. In salivary glands, it induces exocytosis through the activation of cAMP-dependent protein kinase (PKA) [1,4,26] and gene expression of secretory proteins such as proline-rich protein [31]. Both synthesis and hydrolysis of cAMP regulate its intracellular levels. It has been considered that cGMP and Ca2 + have some effects on the accumulation and the attenuation of cAMP [6,10,17,28,29]. Atrial natriuretic peptide and carbachol, which both enhance cGMP accumulation, attenuate cAMP accumulation evoked by isoproterenol (IPR) in the rat parotid gland [6,10,17]. These attenuations of cAMP are mediated through the inhibiting GTP-binding (Gi) protein [19,23]. Carbachol enhances forskolin- or IPR-stimulated cAMP accumulation at a low concentration (B 1 mM), and attenuates IPR-stimulated cAMP accumula* Corresponding author. Tel.: +81-25-267-1500; fax: + 81-25-2671134. E-mail address:
[email protected] (A. Imai)
tion at a high concentration (\ 10 mM) in the mouse parotid [27–29]. On the other hand, in IPR-stimulated parotid acini, 3-isobuthyl-1-methylxanthine (IBMX), a non-selective phosphodiesterase (PDE) inhibitor, increases cAMP levels approximately sixfold [30]. PDEs, enzymes that catalyze the hydrolysis of cyclic nucleotides, play a role in signal transduction by regulating the cellular cyclic nucleotides. Nine families have been based on their substrate affinity and specificity, on their selective sensitivity to cofactors and inhibitors, and on their amino acid sequences. These families are PDE1, Ca/calmodulin-dependent; PDE2, cGMP-stimulated; PDE3, cGMP-inhibited; PDE4, cAMP-specific; PDE5, cGMP-specific; PDE6, photoreceptor cGMPspecific; PDE7, cAMP-specific rolipram insensitive; PDE8, cAMP-specific IBMX insensitive; PDE9, cGMP-specific IBMX insensitive [2,8,9,22,24,25]. There are multiple isozymes, multiple splice variants, and different tissue distributions of those isozymes within families [2,18]. We have identified previously a range of PDE families hydrolyzing cAMP in the rat parotid gland: PDE1, PDE2, PDE3 and PDE4 (PDE4A, PDE4B, PDE4C, PDE4D1, PDE4D2 and PDE4D3) [11–13]. It
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has been suggested that Ca/calmodulin and cGMP are regulators of parotid PDEs and that the intracellular cAMP is also regulated by activation of PDE through phosphorylation and expression of PDE [12,13]. In the present study, we investigated the effects of cGMP and Ca/calmodulin on PDE activities in the rodent parotid glands (the mouse, the hamster and the guinea pig) and further identified PDE isozymes hydrolyzing cAMP, to compare the regulatory mechanisms of the cAMP level with those of the rat.
2. Materials and methods
2.1. Materials cAMP, cGMP, calmodulin, milrinone, snake venom (Crotalus atrox) and catalytic subunit of PKA were obtained from Sigma (St Louis, MO). [2,8-3H]cAMP (28.1 Ci/mmol) and [8-14C]adenosine (59.8 Ci/mmol) were obtained from DuPont-New England Nuclear. Cilostamide (Otsuka, Japan), rolipram (Schering) and Ro20-1724 (Roche) were kindly donated. All other chemicals were commercially obtained and were of analytical grade. Mice (ddy), hamsters (Syrian), guinea pigs (Hartley) and rats (Wistar) were mature males and were obtained from SLC (Shizuoka, Japan). Oligonucleotides for PCR primers; 5%-TGGTGTTAGCAACTGAT-3%, 5%-TGGGAACTGGCTTCTAC-3% were purchased from Takara (Kyoto, Japan). Rat glyceraldehyde 3-phosphate dehydrogenase (G3PDH) amplimer set was obtained from Clontech (CA).
2.2. Separation of cAMP PDE acti6ity The animals were anaesthetized with Nembutal (50 mg/kg i.p.) and then killed by cardiac puncture and parotid glands were removed quickly. These procedures were approved by the animal care committee of our university. The parotid glands were sliced and homogenized in 10 vol. of ice-cold buffer containing 20 mM Bis – Tris–HCl (pH 6.5), 2 mM EDTA, 2 mM benzamidine, 1 mM dithiothreitol and 100 mM phenylmethanesulfonyl fluoride with Polytron PT-10 (10 s, four times, at speed 5). The homogenate was centrifuged at 1000× g for 15 min at 4°C, and the supernatant fraction was further centrifuged at 100 000× g for 20 min at 4°C. The 100 000×g supernatant was filtered through a Millipore filter (0.45 mm) and applied to a Mono Q HR5/5 (Pharmacia) ion-exchange column equilibrated with the homogenization buffer. The column was washed with 10 ml of the same buffer and eluted with a linear gradient of 0 – 1.0 M NaCl in the same buffer, and 1-ml fractions were collected at a flow rate of 0.5 ml/min.
2.3. Assay of PDE acti6ity The reaction was initiated by the addition of 30 ml of the fraction to the incubation mixture (100 ml) containing 50 mM Tris–HCl (pH 7.5), 6 mM MgCl2, 2.5 mM dithiothreitol, 0.23 mg/ml bovine serum albumin (BSA) and 1 mM [2,8-3H]cAMP (60 000–120 000 dpm) in the presence or absence of 1 mM cGMP or 100 mM CaCl2 with 1 U calmodulin. The tube was incubated at 30°C for 10 min and heated at 90°C for 3 min for the termination of the reaction. After cooling, 20 ml of [8-14C]adenosine (12 000 dpm) to estimate the recovery and 5 ml of snake venom (5 mg/ml) were added, and the reaction mixtures were incubated at 30°C for 10 min. The reaction was terminated by the addition of 1 ml of anion-exchange resin slurry (Bio-Rad; AG 1× 8). The radioactivity of the supernatant (0.4 ml) was measured by a liquid scintillation counter.
2.4. Determination of Km for the PDE acti6ities and Ki 6alues for the selecti6e inhibitor For determination of Km, 0.25–4.0 mM cAMP was used. Ki values were obtained graphically from reciprocal plots made with a series of different inhibitor concentrations.
2.5. Protein assay The protein concentration was measured using a protein assay kit (Bio-Rad).
2.6. Phosphorylation of PDE by PKA Separated PDE isozymes were preincubated for 5 min at 30°C in the reaction buffer consisting of 50 mM Tris–HCl (pH 7.5), 10 mM magnesium acetate, 0.1 mM ATP, 2.5 mM dithiothreitol and 0.23 mg/ml BSA. After the addition of 5 U of the catalytic subunit of PKA, the reaction solutions were further incubated at 30°C for 10 min.
2.7. Re6erse transcriptase-polymerase chain reaction (RT-PCR) Messenger RNA from the parotid gland was prepared using a messenger RNA isolation kit (Stratagene, CA). First strand cDNA was synthesized from 0.1 mg mRNA with oligo(dT)20-M4 adaptor primer using a RNA PCR kit with AMV RTase (Takara). One hundredth of the first strand cDNA reaction mixture or 1 ng of cDNA (rat brain QUICK-Clone™ cDNA, Clontech) was used for the PCR reactions. PCR reactions were performed in 30 cycles using 2 U of Taq DNA Polymerase (Takara) under the following conditions: denaturing at 94°C for 30 s, annealing at 48°C for 30 s, and primer extension at 72°C for 1 min.
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3. Results The subcellular distributions of PDE activities of the mouse, the hamster, the guinea pig and the rat are shown in Table 1. More than 80% of the activities was in the 100 000×g supernatant fractions of three species, except for the hamster, when the assay was performed without cGMP or Ca/calmodulin. In the hamster, only 69% of PDE activity was in the supernatant fraction. The activities of the supernatant fractions in the presence of 1 mM cGMP increased approximately 120% in the mouse and the hamster, but decreased in the guinea pig and the rat. The PDE activities of all animals tested were augmented in the presence of Ca/ calmodulin, and those increased approximately 130% in the guinea pig and the hamster. The supernatant fractions were loaded onto the Mono Q ion-exchange column to separate the PDE isozymes, because PDE activities were mainly present in the supernatant fractions. The elution profiles showed five, four and three peaks in the parotid of the mouse, the hamster and the guinea pig, respectively (Fig. 1). In a previous study [12], we identified PDE1 (peak 1), PDE2 (peak 2), PDE3 (peak 5) and PDE4 (peak 3) in the rat parotid gland (Fig. 1D). Peak 1 of all animals tested was activated in the presence of Ca/calmodulin, and therefore it was identified as PDE1 (Ca/calmodulin-dependent) activity. Peak 2 was identified as PDE2 (cGMP-stimulated) activity, because it was activated by cGMP and had a high Km for cAMP similar to the rat
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PDE2. Other peaks were characterized by the estimations of Ki values for the selective inhibitors and of Km for cAMP, and by the effect of cGMP (Table 2). Both the mouse peak 5 and the guinea pig peak 3 were identified as PDE3 (cGMP-inhibited) activity, because both peaks were inhibited by cGMP and had low Km values, similar to that of the rat PDE3 and they had lower Ki values for both cilostamide and milrinone (selective inhibitors of PDE3) than those for both rolipram and Ro20-1724 (selective inhibitors of PDE4). The mouse peak 3 was identified as PDE4 activity from the elution position (Fig. 1A,D), Km and Ki values (Table 2). Although the elution positions of peaks 3 and 4 in the hamster were distant from those of the mouse and the rat PDE4 (peak 3 in Fig. 1A,D), the two peaks 3 and 4 had lower Ki values for both rolipram and milrinone than that for cilostamide and were not affected by cGMP. Therefore, they were supposed to be PDE4 (cAMP-specific) subtypes. In the guinea pig, PDE4 was not detected by the conventional method. We further examined the expression of PDE4 by RT-PCR with the primers designed from the PDE4B. In general, PDE4 is detected in most tissues, and PDE4B particularly expresses in the majority of cells [7,16]. Therefore, the expression of PDE4 can be considered by the detection of PDE4B. The bands of RT-PCR products were observed in the mouse, the hamster and the rat, but not in the guinea pig (Fig. 2).
Table 1 Subcellular distribution of PDE activity in parotid gland Animal
Total protein recovery (mg)
PDE activity (pmol of cAMP hydrolyzed/min per sample) Without cGMP and Ca/calmodulin
With cGMP
With Ca/calmodulin
Mouse (n = 8) Homogenate 53.5 1000×g pellet 13.6 100 000×g pellet 7.6 100 000×g supernatant 26.9
13 085 1077 62 9288
16 402 1114 756 11 313
16 402 1030 708 9430
Hamster (n= 3) Homogenate 65.3 1000×g pellet 18.0 100 000×g pellet 2.4 100 000×g supernatant 28.1
3832 903 903 2046
3685 910 910 2428
4450 1104 1104 2675
Guinea pig (n =2) Homogenate 43.9 1000×g pellet 19.5 100 000×g pellet 4.5 100 000×g supernatant 17.1
14 507 1966 659 11 660
11 827 1532 536 9647
22 614 3401 867 15 159
Rat (n = 2) Homogenate 42.6 1000×g pellet 16.4 100 000×g pellet 4.0 100 000×g supernatant 16.8
5038 411 239 4798
3952 335 201 3950
5501 468 246 5111
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Fig. 1. Elution profiles for PDE activities with Mono Q ion-exchange chromatography in the parotid gland of the mouse (A), hamster (B), guinea pig (C) and rat (D) [12]. Peak numbers and fractions collected are indicated. PDE activity was assayed with 1 mM cAMP (), with 1 mM cAMP and 1 mM cGMP (), and with 1 mM cAMP and 100 mM CaCl2 and 1 U calmodulin ( ). The dotted line represents protein concentration, and the solid line represents NaCl concentration.
It was reported that PDE isozymes were stimulated by phosphorylation with PKA [2,3]. We had also reported that PDE3 and PDE4 of the rat parotid were stimulated by treatment with catalytic subunit of PKA [12]. Thus, the PDEs separated were anticipated to be activated by PKA, but this was not observed in the mouse, hamster and guinea pig (Table 3).
4. Discussion The parotid glands are constructed by serous cells in human, mouse and rat. In the rat parotid gland, the dispersed cells are over 95% acinar cells; the remaining 5% are ductal cells [14]. The gland synthesizes and secretes a watery proteinaceous solution [20]. The mouse, the hamster, the guinea pig and the rat belong to the same order, Rodentia, and they are classified in different families from the standpoint of taxology. The mouse and the rat belong to the same family, Muridae; on the other hand, the hamster and the guinea pig belong to Cricetidae and Cavidae, respectively. The peculiarity of the elution profiles of the
parotid PDE activity was demonstrated depending on the animals, though the major PDE activities of all animals were in the supernatant fraction (Table 1). Chiu et al. [5] reported six peaks of PDE activity in the soluble fraction of the mouse parotid gland by isoelectric focusing, but they were not identified. In the present study, we characterized at least five PDE isozymes, including PDE1, PDE2, PDE3 and PDE4, which hydrolyze cAMP, in both the mouse and the rat parotid glands. The elution profile of the rat parotid showed PDE3 (cGMP-inhibited) as the highest peak (peak 5 in Fig. 1D). On the other hand, PDE2 (cGMPstimulated) was the highest peak in the mouse parotid (peak 2 in Fig. 1A), supporting the increase in the supernatant activity by the addition of cGMP. The results may indicate that augmentation of cellular cGMP accumulation increases the PDE activity of the mouse. The regulatory effects of cGMP on PDEs in the rat and the mouse were opposite, that is, cGMP depressed the activity in the rat, but enhanced it in the mouse. Watson et al. reported that carbachol induces the augmentation of cellular cGMP accumulation [29] and decreases cAMP accumulation at a high concentra-
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Table 2 Michaelis constants and inhibition constants of PDEs in parotid gland Animal and PDE fraction
Mouse Peak 1 Peak 2 Peak 3 Peak 4 Peak 5
Km (mM cAMP)
2.86 100 5.95 2.67 1.07
Hamster Peak 1 Peak 2 Peak 3 Peak 4
5.55 25.0 3.22 2.17
Guinea pig Peak 1 Peak 2 Peak 3
1.44 40.0 0.81\
Rat a Peak Peak Peak Peak Peak
5.56 40.0 4.00 1.33 0.78
a
1 2 3 4 5
Ki (mM)
PDE family
Cilostamide
Milrinone
– – 100\ 8.30 3.50
– – 37.0 8.46 7.40
– – 0.228 2.19 62.0
– – 4.40 13.20 95.0
PDE1 PDE2 PDE4 PDE3 and/or PDE4 PDE3
– – 32.0 40.0
– – 1.90 0.90
– – 8.50 13.6
PDE1 PDE2 PDE4 PDE4
– – 57.0
PDE1 PDE2 PDE3
– – 82.0 90.0
Rolipram
– – 4.67
– – 1.60
– – 48.0
– – 73.0 12.5 0.037
– – 22.0 5.00 0.368
– – 0.575 8.40 \100
Ro20-1724
– – 6.45 15.5 \200
PDE1 PDE2 PDE4 PDE3 and/or PDE4 PDE3
Imai et al. [12].
tion ( \ 10 mM) stimulated by forskolin in the mouse [27]. Furthermore, they reported that IBMX (a non-selective inhibitor of PDE) augments IPR-stimulated cAMP accumulation approximately ninefold in the presence of carbachol and that milrinone (selective inhibitor of PDE3) does not alter the cAMP accumulation [30]. Milrinone is about 700 times more potent for PDE3 than for PDE2 in inhibiting the PDE activity [21]. Our results, in which PDE2 showed the predominant activity in the mouse parotid, supported their reports. The elution profile of the hamster supernatant fraction exhibited four peaks consisting of PDE1, PDE2 and two subtypes of PDE4, but no peak of PDE3. The high peak of PDE2 and no peak of PDE3 supported the results, in which the supernatant PDE activities increased approximately 120% in the presence of 1 mM cGMP (Table 1). the ratio of the supernatant PDE activity was 69%, the lowest among the animals tested. PDE activities, which were not affected by cGMP and not inhibited by rolipram, but inhibited by IBMX, were also detected in the pellet fraction. These properties were associated with those of PDE7 (data not shown). In the guinea pig parotid gland, three PDE isozymes, PDE1, PDE2 and PDE3, but not PDE4, were identified. PDE4B, which is detected in the other majority of cells [7,16], was not detected in RT-PCR using the guinea pig parotid mRNA, though detected in the
mouse, the hamster and the rat (Fig. 2). This result supported that the guinea pig has no PDE4 activity (Fig. 1C). Methven et al. reported PDEs modulated by Ca2 + in the guinea pig pancreas, but they have not been identified [15]. In the guinea pig parotid, PDE1 exhibited a remarkably high peak among the four animals tested. It suggested that there was a similar regulation on cAMP level by Ca/calmodulin-dependent PDE between the parotid and the pancreas in the guinea pig.
Fig. 2. RT-PCR from the parotid gland extracts products were separated by electrophoresis on a 1.8% agarose gel and stained with SYBR Green I (Takara). Arrows indicate the positions of PCR products; PDE4B (901 bp) and G3PDH (983 bp). Lanes 1, mouse; 2, hamster; 3, guinea pig; 4, rat; 5, rat brain.
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Table 3 Relative PDE activities after the treatment with PKA catalytic subunita Animal
Peak 1
Peak 2
Peak 3
Peak 4
Mouse
88.69 17.2 (55.19 2.3)
75.49 1.1 (290.19 3.6)
80.3 92.2 (185.4 99.7)
98.0 9 2.1 (129.1 93.1)
Hamster
129.09 31.5 (9.29 3.7)
105.19 3.1 (71.39 1.4)
96.4 912.5 (25.3 9 2.2)
104.6 9 25.4 (43.9 91.3)
Guinea pig
101.6 9 6.7 (64.49 2.4)
73.69 2.1 (174.19 5.6)
76.8 9 1.4 (97.6 9 5.6)
Ratb
142.39 10.0 (57.29 14.7)
101.292.1 (79.79 18.9)
196.4 9 39.8* (128.0 9 38.5)
134.6 914.6 (203.0 9 30.0)
Peak 5 91.4 92.4 (42.7 9 2.8)
150.2 915.1* (280.7 9 32.4)
a Data are means9 S.E. (n=3) and are expressed as percentage of PDE activity at the control value. Values in parentheses are control PDE activity (pmol of cAMP hydrolyzed/min per ml). b Imai et al. [12]. * PB0.05 compared with the control values (as 100 9 S.E.%) using Student’s t-test.
Our results suggested that within Rodentia, cAMP levels in the parotid gland might be regulated by the various PDE isozymes depending on the animals. That is, PDE activities of the mouse and the hamster were stimulated by cGMP, and the activities of the guinea pig were stimulated by Ca/calmodulin. In the rat, PDE3 and PDE4 are activated by phosphorylation [12], but no PDE isozymes of the other three species were significantly activated by the treatment with PKA (Table 3). In the present study, we clarified that PDE isozymes in the parotid glands have a peculiarity depending on the respective families (Cricetidae, Muridae and Cavidae), although the families belong to the same order, Rodentia.
Acknowledgements This work was supported by a grant-in-aid from the Ministry of Education, Science and Culture of Japan (No. 09771553).
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