Ca2+/Calmodulin-Dependent Cyclic Nucleotide Phosphodiesterase in Cgmp Metabolism in Rabbit Parotid Acinar Cells

Biomedical Research 27 (1) 37-44, 2006

Ca2+/calmodulin-dependent cyclic nucleotide phosphodiesterase in cGMP metabolism in rabbit parotid acinar cells

1 2 2, 3 Nakayasu SAIRENJI , Keitaro SATOH and Hiroshi SUGIYA Departments of 1 Anesthesiology and 2 Physiology, and 3 Research Institute of Oral Science, Nihon University School of Dentistry at Matsudo, Matsudo, Chiba 271-8587, Japan (Received 25 November 2005; and accepted 10 January 2006)

ABSTRACT Muscarinic cholinergic receptor activation provokes cGMP formation in parotid acinar cells. We investigated the involvement of Ca2+/calmodulin-dependent cyclic nucleotide phosphodiesterase (PDE1) in cGMP breakdown in rabbit parotid acinar cells. The muscarinic agonist carbachol stimulated cGMP formation in the cells. The carbachol-induced cGMP formation was enhanced in the presence of 8-methoxymethyl-3-isobutyl-1-methylxanthine (MM-IBMX), a PDE1 inhibitor. cGMP- PDE activity in rabbit parotid acinar cells was reduced by about 25% in the absence of Ca2+/ calmodulin or in the presence of MM-IBMX. Ca2+/calmodulin-dependent cGMP-PDE in rabbit parotid acinar cells was puriﬁed using Calmodulin-Sepharose 4B and Mono Q ion-exchange column chromatography. Two dominant fractions with cGMP-PDE activity, referred to as the P-1 and P-2 fractions, were eluted from the Mono Q ion-exchange column. The Km values for cGMP of PDE in the P-1 and P-2 fractions were 0.82 μM and 0.40 μM, respectively, which were much lower 2+ than that for cAMP. The EC50 for Ca and calmodulin of PDEs in the P-1 and P-2 fractions were 458 nM and 426 nM, respectively, and 32 nM and 137 nM, respectively. Protein bands that cross- reacted with anti-PDE1A antibody were detected. These results suggest that Ca2+/calmodulin-dependent PDE, PDE1A, is involved in cGMP breakdown in rabbit parotid acinar cells.

Cyclic guanosine-3’,5’-monophosphate (cGMP) is tor speciﬁcities, allosteric properties and amino acid an intracellular signal that is involved in the action sequences (12). In the PDE families, 8 families have of various hormones, autacoids, neurotransmitters, been reported to hydrolyze cGMP; Ca2+/calmodulin- and vasoactive substances (27). cGMP synthesis is dependent PDE (PDE1), cGMP-stimulated PDE catalyzed by soluble or particulate guanylyl cyclase, (PDE2), cGMP-inhibited PDE (PDE3), cGMP-bind- and the actions of cGMP are carried out via cGMP ing cGMP-specific PDE (PDE5), photoreceptor mediators such as cGMP-gated ion channels and cGMP-PDE (PDE6), high-affinity cGMP specific cGMP-dependent protein kinases (19). To terminate and IBMX-insensitive PDE (PDE9), and the recent- intracellular cGMP signaling, cGMP is converted to ly recognized PDE10 and PDE11 (12). the inactive 5’GMP by cyclic nucleotide phosphodi- In parotid glands of mouse, rat and rabbit, since esterases (PDEs) (1, 22). PDEs have been classiﬁed secretagogues such as muscarinic agonists provoke into 11 families on the basis of substrate and inhibi- cGMP formation, cGMP is considered to be involved in parotid functions as an intracellular mes- Address correspondence to: Dr. H. Sugiya, Department senger (3, 4, 16, 28). We previously demonstrated of Physiology, Nihon University School of Dentistry at that cGMP production is coupled to nitric oxide (NO) Matsudo, Matsudo, Chiba 271-8587, Japan generation in rabbit parotid acinar cells: muscarinic 2+ Tel: +81-47-360-9325, Fax: +81-47-360-9325 receptor activation resulted in Ca mobilization, E-mail: [email protected] which activated Ca2+/calmodulin-dependent NO syn- 38 N. Sairenji et al. thase to generate NO, and subsequently NO activat- with trypsin (0.5 mg/mL) at 37°C for 10 min. After ed soluble guanylyl cyclase to produce cGMP (16, incubation, the trypsin-treated glands were removed 23). However, the pathway of cGMP breakdown has by centrifugation at 200 g for 1 min. The glands not yet been elucidated. Because cGMP synthesis is were subsequently incubated in Ca2+-Mg2+-free KRB coupled to Ca2+ mobilization and the presence of solution containing 1 mM EGTA, trypsin inhibitor Ca2+/calmodulin-dependent PDE activities have been (0.5 mg/mL) at 37°C for 5 min. After the solution reported in rat, mouse, hamster and guinea-pig pa- was removed by centrifugation (200 g for 1 min), rotid glands (10, 31), it is most likely that the in- the glands were incubated in Ca2+-Mg2+-free KRB 2+ 2+ crease in intracellular Ca concentrations ([Ca ]i) solution without trypsin inhibitor at 37°C for 5 min. provoked by muscarinic receptor activation simulta- After the solution was removed by centrifugation neously activates Ca2+/calmodulin-dependent PDE. (200 g for 1 min), the glands were incubated in Here we investigated the involvement of Ca2+/ KRB solution with collagenase (1.5 mg/mL) at 37°C calmodulin-dependent PDE in cGMP metabolism in for 20 min. The suspension was passed through rabbit parotid acinar cells. eight layers of nylon mesh to separate the dispersed cells from undigested connective tissue, and was gently put on KRB solution containing 4% BSA. MATERIALS AND METHODS After centrifugation (50 g for 5 min), the cells were Materials. Collagenase A, bovine serum albumin suspended in the appropriate amounts of KRB solu- (BSA) and Protease Inhibitors Cocktail (Complete™) tion containing 0.5% BSA and 0.02% trypsin inhibi- were purchased from Roche (Basel, Switzerland). tor. Trypsin (type III), trypsin inhibitor (type I-S), 3-isobutyl-1-methylxanthine (IBMX), Crotalus atrox Determination of cellular cGMP. The dispersed aci- snake venom and bovine brain PDE were obtained nar cells from one rabbit were suspended in 8 mL from Sigma (St. Louis, MO, USA). Calmodulin and of KRB solution containing 0.5% BSA and 0.02% 8-methoxymethyl-3-isobutyl-1-methylxanthine (MM- trypsin inhibitor and incubated at 37°C with the in- IBMX) were obtained from Wako (Osaka, Japan) dicated agents. The cell suspension was separated to and Calbiochem (San Diego, CA, USA), respective- 4 parts (each 2 mL), and incubated with vehicle, ly. AG 50W-X4 resin (200–400 mesh, hydrogen MM-IBMX (0.5 mM), carbachol (10 μM), and MM- form) and a Mono Q ion-exchange column were IBMX/carbachol. Carbachol was added after prein- purchased from Bio-Rad (Hercules, CA, USA) and cubation with vehicle or MM-IBMX for 10 min. At Pharmacia (Piscata Way, NJ, USA), respectively. the indicated time, 200 μL of cell suspension was Centricon-30 and Calmodulin-Sepharose 4B were removed, mixed with 30 μL of 35% perchlroric acid purchased from Amersham (Piscata Way, NJ, USA). and put on ice for 30 min. Then, 60 μL of 17.5% [8-3H] cGMP (277.5 GBq/mmol) and [8-3H]cAMP potassium hydroxide was added to the mixture (1187.7 GBq/mmol) were obtained from PerkinEl- for neutralization, the mixture was centrifuged at mer (Wellesley, MA, USA). An [125I]cGMP radioim- 10,000 g for 5 min, and the supernatant was isolat- munoassay kit was purchased from Yamasa (Tokyo, ed. The cGMP concentration in the supernatant was Japan). Anti-PDE1 antibodies (rabbit, polyclonal) measured using a radioimmunoassay kit (Yamasa). were purchased from FabGennix (Frisco, TX, USA). Isolation of cytosolic fraction and purification of Preparation of parotid acinar cells. Acinar cells of PDE. Parotid acinar cells from 4 rabbits were ho- the parotid glands of male Japanese white rabbits mogenized with 10 mL of 20 mM Tris-HCl (pH 7.4)

(2–2.5 kg) were prepared as previously described containing 2 mM MgCl2, 1 mM dithiothreitol (DTT), (23). The parotid glands were removed and placed 1 mM phenylmethylsulfonyl fluoride (PMSF), in a small volume with Krebs-Ringer-bicarbonate 1.3 mM benzamidine, and Protease Inhibitors Cock- (KRB) solution with the following composition (in tail (Complete™) and centrifuged at 105,000 g for mM): NaCl, 116; KCl, 5.4; MgSO4, 0.8; CaCl2, 1.8; 60 min at 4°C. The supernatant was used as a cyto- NaH2PO4, 0.96; NaHCO3, 25; HEPES (pH 7.4), 5; solic fraction. and glucose, 11.1. The KRB solution was equilibrat- For the puriﬁcation of PDE, the cytosolic fraction ed with an atmosphere of 95% O2/5% CO2. After was used. All the puriﬁcation steps were carried out mincing with a razor, the glands were treated with at 4°C. The supernatant was loaded onto a Calmod- KRB buffer with 0.5% BSA in the presence or ab- ulin-Sepharose 4B column (0.46 × 10 cm) equili- sence of enzyme. First, the glands were incubated brated with buffer A (20 mM Tris-HCl (pH 7.4) cGMP-PDE in parotid 39

containing 2 mM MgCl2, 1 mM DTT, 1 mM PMSF transferred to a nitrocellulose membrane (12 V, and 1.3 mM benzamidine). The column was washed overnight). The membrane was blocked at room with 45 mL of buffer A containing 0.25 mM CaCl2, temperature for 50 min in Tris-buffered saline with and the fractions containing cGMP-PDE activity 3% gelatin, washed briefly with Tris-buffered saline, were eluted with buffer A containing 0.5 mM and probed for 2 h with rabbit anti-PDE1 antibodies EGTA. The cGMP-PDE fractions were collected, (0.2 μg/mL). Blots were washed three times with concentrated to about 10 mL by ultrafiltration using Tris-buffered saline containing 0.05% Tween 20, Cenricon-30 (Amersham), and loaded at 1 mL/min and probed for 90 min with anti-rabbit IgG. Immu- onto a Mono Q ion-exchange column (1 × 10 cm) noreactivity was determined with the ECL chemilu- equilibrated with buffer A. The column was washed minescence reaction (Amersham). with 50 mL of buffer A, and cGMP-PDE was eluted with a 50 mL linear gradient of 0–500 mM NaCl in Protein determination. Protein concentrations were buffer A at a flow rate of 1 mL/min. Fractions (1 mL) determined by the method previously described (2) were collected and assayed for cGMP-PDE activity. with BSA as the standard.

Assay of PDE activity. cGMP-PDE activities were Statistical analysis. Statistical differences were de- determined by the two-step method (9). The enzy- termined using Student’s t test. P values below 0.05 matic reaction was performed in a total volume of were regarded as statistically signiﬁcant, and are in- 0.1 mL. The reaction mixture contained 50 mM Tris- dicated by asterisks (*).

HCl (pH 7.4), 5 mM MgCl2 and substrate (0.5 μM [3H]cGMP; 200,000 dpm) and enzyme. The mixture RESULTS was incubated at 30°C for 10 min, and then the reaction was terminated by heating at 90°C for 5 min. Effect of a PDE1 inhibitor on carbachol-induced 5’-[3H]GMP formed by PDE was converted to cGMP formation in parotid acinar cells [3H]guanosine by the action of nucleotidase (25 μL In rabbit parotid acinar cells, muscarinic receptor of 1 mg/mL snake venom at 30°C for 10 min). The activation has been demonstrated to stimulate cGMP reaction was terminated by the addition of 0.5 mL formation (16). Thus, we ﬁrst studied the effect of of water and denatured protein was removed by MM-IBMX, an inhibitor of PDE1, on cGMP forma- centrifugation (10,000 g, 3 min). Five hundred mi- tion induced by muscarinic receptor activation. As croliters of clear supernatant ﬂuid was applied to a Fig. 1A shows, the muscarinic agonist carbachol 0.5-mL column of AG 50W-X4 resin (200–400 (10 μM) induced cGMP formation in a time-depen- mesh; hydrogen form). The reaction product, dent manner in the absence of the inhibitor. When [3H]guanosine, was eluted with 1.5 mL of 3N am- acinar cells were pretreated with MM-IBMX monium hydroxide after the column was washed (0.5 mM) for 10 min and then stimulated, carbachol- with 15 mL of water, and radioactivity was mea- induced cGMP formation was clearly enhanced. sured using a liquid scintillation counter. The PDE Fig. 1B shows the concentration-dependence of activity was assayed in the presence or absence of cGMP formation on MM-IBMX (0.1 to 1 mM) the indicated concentrations of 1 mM CaCl2 with when cells were stimulated by carbachol (10 μM) 0.4 μM calmodulin or 1 mM EGTA. When the con- for 1 min. These observations suggest that PDE1 is centrations of Ca2+ and calmodulin were changed, involved in the breakdown of cGMP formed by car- the Ca2+ concentrations in the reaction mixture were bachol stimulation. adjusted using fura-2 (8). When cAMP-PDE was assayed, 0.1 μM [3H]cAMP instead of [3H]cGMP was PDE1 activity in parotid acinar cells used as substrate, and [3H]adenosine converted from We next investigated the effect of Ca2+/calmodulin 5’-[3H]AMP formed by PDE was determined. and MM-IBMX on the cGMP-PDE activity in the

cytosolic fraction. In the presence of CaCl2 and Immunoblot analysis. The fractions containing calmodulin, MM-IBMX (100 μM) inhibited cGMP- cGMP-PDE activity eluted from a Calmodulin-Sep- PDE activity to 26.1% of the control level (Table 1). harose 4B column were concentrated by ultraﬁltra- In the presence of EGTA (1 mM) and absence of tion using Cenricon-30 (Amersham) and used as a CaCl2 and calmodulin, cGMP-PDE activity in the sample for immunoblot analysis. The sample (100 μg presence of 1 mM CaCl2 and 0.4 μM calmodulin protein) was resolved by 10% SDS-polyacrylamide was markedly reduced to 27.7% of the control level, gel electrophoresis. The proteins on the gel were but MM-IBMX showed no further effect on the ac- 40 N. Sairenji et al.

Fig. 1 Effect of MM-IBMX on carbachol-induced cGMP formation in rabbit parotid acinar cells. A, Time-course of cGMP levels. After preincubation with (squares) or without (circles) MM-IBMX (0.5 mM) for 10 min, rabbit parotid acinar cells were stimulated with 10 µM carbachol (closed circles and closed squares) or vehicle (open circles and open squares) at 0 min; B, Dose-dependent effect of MM-IBMX on carbachol-induced cGMP formation. After preincubation in the presence of various concentrations of MM-IBMX for 10 min, cells were stimulated with carbachol (10 µM) for 1 min. The increase in cGMP formation in the presence of MM-IBMX is shown as fold of the control value (in the absence of MM-IBMX). Results are means ± SE from 4 experiments. *The values in the presence of MM-IBMX were compared to that in the absence of MM-IBMX.

Table 1. Effect of PDE inhibitors on cGMP-PDE activity in in rabbit parotid acinar cells, PDE1 in the cytosolic rabbit parotid acinar cells fraction of rabbit parotid acinar cells was partially Ca2+/calmodulin purified using calmodulin-affinity and Mono Q ion- Inhibitor Concentration exchange column chromatography. Fig. 2 shows the (+) (−) elution profiles of PDE1 activities from the calmod- (μM) (%) ulin-affinity column (Fig. 2A) and the Mono Q ion- Vehicle 100 27.7 ± 0.9 exchange column (Fig. 2B). Two dominant cGMP- MM-IBMX 50 37.6 ± 0.4 28.4 ± 0.5 PDE activity peaks and several minor peaks were 100 26.1 ± 1.3 25.2 ± 0.5 eluted from the Mono Q ion-exchange column. The IBMX 100 4.9 ± 0.5 3.3 ± 1.0 dominant peaks are referred to as the P-1 and P-2 fractions according to their order of elution from the cGMP-PDE activity in the presence of 1 mM CaCl2 and 0.4 μM 2+ column, and the characteristics of the PDEs in the calmodulin [Ca /calmodulin (+)] was taken as 100%. When the fractions were examined. The specific activities of activity was assayed in the absence of CaCl2 and calmodulin [Ca2+/calmodulin (-)], 1 mM EGTA was added to the reaction cGMP-PDEs in the P-1 and P-2 fractions were mixture. Results are means ± SE from 5 experiments. 423 pmol/min/mg protein and 518 pmol/min/mg protein, respectively. The Km value for cGMP was determined by a tivity (25.2%). The non-selective PDE inhibitor Lineweaver-Burk plot analysis (Fig. 3). The Km val- IBMX (100 μM) almost completely inhibited cGMP- ues of the PDEs in the P-1 and P-2 fractions were PDE activity not only in the presence but also in the 0.82 μM and 0.40 μM, respectively. Regarding the absence of Ca2+/calmodulin. These results strongly affinity for cAMP, the Km values of the PDEs in suggest that about 75% of cGMP-PDE in the cyto- the P-1 and P-2 fractions were 13 μM and 25 μM, solic fraction of rabbit parotid acinar cells is PDE1, respectively (data not shown). Ca2+/ calmodulin-dependent PDE. Fig. 4 summarizes the Ca2+- and calmodulin-dependency of PDEs in the P-1 and P-2 fractions, re- Characterization of cGMP-PDE in rabbit parotid spectively. Omission of Ca2+ or calmodulin from the acinar cells reaction mixture essentially abolished the cGMP- To characterize the Ca2+/calmodulin-dependent PDE PDE activities in both fractions. Although the range cGMP-PDE in parotid 41

61, 60 and 59 kDa protein bands that immunoreacted with anti-PDE1A antibody were detected in the fraction eluted from the calmodulin-afﬁnity column; these bands were smaller than the 63 kDa bovine brain PDE (21). These observations suggest that PDE1A isoforms are expressed in rabbit parotid acinar cells.

DISCUSSION Here we demonstrated that a Ca2+/calmodulin-dependent PDE, PDE1, is involved in regulating cGMP concentrations in rabbit parotid acinar cells. The PDE1 subfamily consists of three different gene products, PDE1A, PDE1B and PDE1C, with different properties, such as their regulatory properties, substrate affinities, activation constants for calmodu- Fig. 2 Elution profiles of Ca2+/calmodulin-dependent PDE lin and molecular weights (1, 5, 12). The heteroge- in rabbit parotid acinar cells on Calmodulin-Sepharose 4B neity of PDE1 isoforms is further increased by column and Mono Q ion-exchange column. A, The cytosolic alternative splicing, creating divergent N- and C-ter- fraction of rabbit parotid acinar cells was applied to a Calmodulin-Sepharose 4B column. PDE activity was eluted mini (1, 5, 12). PDE1 family members have been with 0.5 mM EGTA as indicated by the arrow, and fractions reported to have Km values for cGMP in the range of 2.5 mL were collected. B, Fractions containing PDE ac- of 0.33–5.2 μM (12). The Km values for cAMP of tivity were collected, concentrated, and applied to a Mono the PDE1A and PDE1B isoforms are generally high- Q ion-exchange column. The column was eluted with a er than that of PDE1C. For example, PDE1A2 and 50 mL linear gradient from 0 to 500 mM NaCl (a solid line) at a flow rate of 1 mL/min. Fractions of 1 mL were collect- PDE1B1 in bovine brain have Km values of 35 and ed. The two dominant peaks are referred to as the P-1 and 12 μM for cAMP, respectively (11, 20). Bovine P-2 fractions. PDE1A2 and PDE1B1 expressed in COS-7 cells have Km values of 113 and 24 μM for cAMP, respectively (30). On the other hand, rat PDE1C2 and of Ca2+-dependency for the cGMP-PDE activity in mouse PDE1C1 and PDE1C4/5 expressed in COS-7 the P-2 fraction was wider than that in the P-1 frac- cells have Km values of 1.2, 3.5 and 1.1 μM for 2+ tion, the EC50 values for Ca of the cGMP-PDEs in cAMP, respectively (29, 30). We demonstrated that the P-1 and P-2 fractions were the same, 458 nM the PDE1 enzymes in the P-1 and P-2 fractions had and 426 nM, respectively (Fig. 4A). The EC50 values Km values of 13 μM and 25 μM for cAMP, respec- for calmodulin of the enzymes in the P-1 and P-2 tively, and 0.82 and 0.40 μM for cGMP, respective- fractions were 32 nM and 137 nM, respectively ly. Therefore, the enzymes in the P-1 and P-2 (Fig. 4B). fractions are considered likely to be PDE1A or PDE1B. The 61, 60 and 59 kDa protein bands that PDE1 isoform in rabbit parotid acinar cells were immunoreactive with anti-PDE1A antibody, The PDE1 subfamily consists of three different gene but not anti-PDE1B or anti-PDE1C antibodies, were products, PDE1A, PDE1B and PDE1C (1, 5). To detected in the fraction with cGMP-PDE activity. identify the PDE1 isoform in rabbit parotid acinar The molecular masses of PDE1A in various tissues cells, immunoblot analysis was performed using an- are 58–68 kDa (5). Therefore, the results of immu- tibodies against PDE1 isoforms. For this immunob- noblot analysis suggest that PDE1A isoforms are ex- lot analysis, the fraction eluted from a Calmodulin- pressed in rabbit parotid acinar cells. The PDE1 Sepharose 4B column was concentrated and then enzymes in the P-1 and P-2 fractions appear to be analyzed, because we were not able to collect suffi- alternative splicing variants of PDE1A. cient concentrations of protein for this analysis from The PDE1 enzymes in the P-1 and P-2 fractions 2+ the fractions eluted from a Mono Q ion-exchange had EC50 values of 458 nM and 426 nM for Ca , column. No protein band immunoreacted with anti- respectively, and EC50 values of 32 nM and 137 nM PDE1B or anti-PDE1C antibodies was detected (data for calmodulin, respectively. These results strongly not shown), but, as shown in Fig. 5, approximately suggest that Ca2+ and calmodulin are regulators of 42 N. Sairenji et al.

Fig. 3 Determination of Km for cGMP of Ca2+/calmodulin-dependent PDE in the P-1 and P-2 fractions. Double reciprocal plots of the initial reaction velocity versus the cGMP concentration in the presence of 1.2 µM Ca2+ and 0.4 µM calmodulin.

Fig. 5 Immunoblotting of PDE1A in rabbit parotid acinar

2+ cells. The fraction eluted from the Calmodulin-Sepharose Fig. 4 Ca /calmodulin-dependent PDE activities in the 4B column (100 µg) was used for the immunoblot analysis. P-1 and P-2 fractions. The PDE activities in the P-1 (open Bovine brain PDE (1 µg) was used as a positive control. circle) and P-2 (closed circle) fractions were assayed in the presence of various concentrations of Ca2+ and 0.4 µM calmodulin (A) or various concentrations of calmodulin and 1.2 µM Ca2+ (B). cells from extracellular sites (6, 7, 13, 14, 26). In parotid acinar cells, it is well known that muscarinic 2+ receptor activation provokes an increase in [Ca ]i PDE activation. However, because the total amounts (15, 17, 23, 24). This Ca2+ mobilization consists of of calmodulin do not change rapidly in the cells, it Ca2+ release from intracellular Ca2+ pools mediated 2+ is most likely that changes in [Ca ]i primarily con- by inositol 1,4,5-trisphosphate derived from the re- tribute to the regulation of PDE1 activation. In ceptor-activated hydrolysis of inositol phospholipids, 1321N1 human astrocytoma cells, PDE1 is consid- and Ca2+ entry from extracellular sites (25). Deple- ered to be activated by the entry of Ca2+ into the tion of the intracellular Ca2+ pools triggers Ca2+ en- cGMP-PDE in parotid 43 try, which is termed capacitative Ca2+ entry (18). In 2. Bradford MM (1976) A rapid and sensitive method for the our results, carbachol-induced cGMP formation was quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analyt Biochem 72, 248– enhanced in the presence of the PDE1 inhibitor 254. MM-IBMX, but the control levels of cGMP were 3. 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Hidaka H and Asano T (1976) Platelets cyclic 3’:5’-nucleotide phosphodiesterase released by thrombin and calcium ion- PDE. ophore. J Biol Chem 251, 7508–7516. Although the PDE1 enzymes in the P-1 and P-2 10. Imai A, Nashida T and Shimomura H (1999) Comparison of 2+ fractions had the same EC50 for Ca , the activities phosphodiesterase isozymes in rodent parotid glands. Comp in the P-1 and P-2 fractions were regulated in the Biochem Physiol B 124, 397–403. 2+ 11. Kakkar R, Raju RVS and Sharma RK (1999) Calmodulin- range of 75–1100 nM and 150–870 nM Ca , respec- dependent cyclic nucleotide phosphodiesterase (PDE1). Cell tively. In parotid acinar cells, muscarinic receptor Mol Life Sci 55, 1164–1186. activation induces a rapid, transient and a subse- 12. Lugnier C (2005) Cyclic nucleotide phosphodiesterase (PDE) 2+ superfamily: A new target for the development of specific quently sustained increase in [Ca ]i (15, 17, 23, 24). 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