Biochimica et Biophysica Acta, 1052 (1990) 461-466
461
Elsevier BBAMCR 12697
Propranolol inhibits cyclic AMP accumulation and amylase secretion in parotid acinar cells stimulated by isobutylmethylxanthine and forskolin Taishin Takuma Department of Oral Biochemistry, School of Dentistry, Higashi Nippon Gakuen University, Hokkaido (Japan)
(Received 4 October 1989) (Revised manuscript received16 January 1990)
Key words: Propranolol;cAMP; Amylase;Isobutylmethylxanthine;Forskolin;(Parotid gland acinar cell)
Propranoloi inhibited cyclic AMP (cAMP) accumulation stimulated by 3-isobutyi-l-methylxanthine 0BMX) or forskolin in rat parotid acinar cells. The inhibition by propranolol was highly potent; 10 - 7 M propranolol was sufficient for the maximum inhibition (approx. 50% at 5 rain). The inhibitory effect was observed in both intact and saponin-permeabilized parotid cells, but the effect was more prominent in permeabilized ceils than in intact cells. Other fl-blockers, like alprenolol and atenolol, were as effective as propranolol, but butoxamine (fl2-selective) was slightly less effective. The inhibition by propranolol was similarly detected in the ceils prepared from pertussis-toxin-pretreated rats, suggesting that inhibitory guanine nucleotide regulatory protein (Gi) is not involved in the inhibitory mechanism. Propranolol also inhibited the exocytosis of amylase stimulated by IBMX or forskolin. In the presence of propranolol and IBMX, the responsiveness of saponin-permeabilized ceils to exogenous cAMP was markedly increased, indicating that propranoloi neither promotes the degradation of cAMP nor prevents the inhibitory effect of IBMX on cAMP phosphodiesterase.
Introduction Although several lines of evidence indicate that cAMP is a major mediator for the exocytosis of amylase from parotid acinar cells [1,2], the regulatory mechanism by cAMP is totally unknown, including the role of cAMPdependent protein kinase [3]. Basically, it has not yet been established what concentration of cAMP is necessary for amylase release, since, unlike calcium, the cellular level of free cAMP cannot be determined directly. For resolution of this question, the application of permeabilized cells seemed to be useful [4,5]. Even in saponin-permeabilized parotid cells, however, a millimolar concentration of cAMP was necessary for the maximum anylase release, probably because cAMP phosphodiesterase strictly prevents the access of exogenous cAMP to the regulatory site of exocytosis [5]. In
addition, the phosphodiesterase inhibitor IBMX by itself maximally stimulated amylase release, suggesting that basal adenylate cyclase activity in these cells is strong enough to provide sufficient cAMP for maximal amylase secretion when phosphodiesterase activity is inhibited [5]. Propranolol is the prototype of 't-blockers' and is widely used for both clinical and investigative purposes [6]. In the parotid gland, propranolol completely inhibits amylase secretion and cAMP accumulation evoked by fl-agonists [1]. In the course of screening for adenylate cyclase inhibitors, I found that propranolol inhibits amylase release and cAMP accumulation in the parotid cells stimulated not only by isoproterenol (a fl-agonist), but also by IBMX. Furthermore, propranolol also inhibited the effect of forskohn, a direct activator of adenylate cyclase. Results obtained suggest that inhibitory G protein (Gi) is not involved in the inhibitory mechanism.
Abbreviations: IBMX, 3-ibobutyl-l-methylxanthine;R-PIA, ( -)N 6((R)-phenylisopropyl)adenosine; BCA, bicinchoninic acid; BSA, bovine serum albuminl; DTI', dithiothreitol.
Materials and Methods
Correspondence: T. Takuma, Department of Oral Biochemistry, School of Dentistry, Higashi Nippon Gakuen University, Tobetsu, Hokkaido 061-02, Japan.
Materials
The materials used in this study and their sources were as follows: DL-propranolol, L-alprenolol, atenolol,
0167-4889/90/$03.50 © 1990 Elsevier Science Publishers B.V. (BiomedicalDivision)
462 procaine, isoproterenol, IBMX, GDPflS and cAMP from Sigma (St. Louis, MO, U.S.A.); R-PIA from Behringer Manheim Yamanouchi (Tokyo, Japan); 2',5'-dideoxyadenosine from Pharmacia LKB (Uppsala, Sweden); saponin and atropine from Merck (Darmstadt, F.R.G.); phentolamine from Ciba-Geigy (Takarazuka, Japan); cyclic AMP assay kit from Yamasa Shoyu (Chiba, Japan); pertussis toxin (islet-activating protein) from Funakoshi (Tokyo, Japan); BCA protein assay reagent from Pierce (Rockford, IL, U.S.A.); [a-32p]NAD from Du Pont, Daiich Pure Chemicals (Tokyo, Japan); and butoxamine, a gift from Burroughs Wellcome (Research Triangle Park, NC, U.S.A.). Treatment of rats with pertussis toxin Male rats of the Wistar strain were injected intravenously with pertussis toxin (0.5 #g/100 g body weight), 3 days before killing, for preparation of parotid acinar cells [7]. Cycfic A M P assay Cyclic AMP levels were measured according to the method described by Tojyo et al. [8]. Parotid cells, prepared as described previously [3,5], were incubated for 5-30 min at 37°C with various agents or vehicles in 1 ml of either regular Hank's medium or Ca 2+-free KC1 medium composed of 140 mM KC1, 20 mM Na-Hepes (pH 7.2), 1 mM EGTA, 2 mM MgSO4, 10 /~g/ml Phenol red, 1 mg/ml bovine serum albumin (BSA) and 20 #g/ml saponin. After incubation, the cells were immediately boiled for 3 rain in their incubation medium, cooled on ice, and then centrifuged at 900 × g for 5 min. Cyclic AMP concentration in the resulting supernatant was determined using a cAMP assay kit and was expressed as pmol/mg protein. A D P-ribosy lation Parotid cells prepared from normal or pertussistoxin-injected rats were homogenized with 20 mM NaHepes (pH 7.2), 1 mM EGTA, 1 mM EDTA and 1 mM dithiothreitol (DTI') (buffer A) in a Teflon-glass homogenizer. The homogenates were centrifuged at 400 × g for 5 rain and 24000 × g for 15 rain. The resulting pellets (400-24000 x g) were suspended in the same buffer (approx. 8 mg protein/m1) and stored at - 80 o C until use. Pertussis toxin (200 /~g/ml) was freshly activated by incubation with 20 mM D'VF, 4 mM ATP, 1 mg/ml BSA and 40 mM Na-Hepes (pH 7.2) at 30 °C for 20 rain. ADP-ribosylation in vitro was carried out at 30 o C for 60 min in a reaction mixture (200/~1) containing parotid membrane prepared as above (approx. 4 mg/ml), pertussis toxin (20 /~g/ml), (0t-32p)NAD (40 /~Ci, 7-10 #M), 10 mM thymidine, 1 mM ATP, 0.1 mM GTP and 10 mM potassium phosphate (pH 7.2). After incubation the mixture was diluted with ice-cold buffer A (2 ml) and centrifuged at 24000 × g for 15 rain. The
pellets were dissolved in 100 #1 of Laernmli sample buffer [9] and boiled for 5 min. SDS-PAGE was performed on 12.5% gel and autoradiograms were prepared using Fuji X-ray films. Other procedures Amylase release from intact and saponin-permeabilized parotid cells were measured as described previously [3,5]. Protein was determined by the BCA protein assay method using BSA as a standard. Results
Effect on cAMP accumulation Cyclic AMP accumulation stimulated by isoproterenol, forskolin and IBMX was observed both in intact and saponin-permeabilized parotid cells incubated in normal Hanks' medium and Ca2+-free KCI medium containing 20/xg/ml saponin, respectively, although the cAMP level induced by isoproterenol was markedly reduced in the permeabilized cell (Fig. 1). 10/~M propranolol, a nonselective /t-antagonist (1/1 :f12), in" hibited cAMP accumulation evoked not only b y isoproterenol, but also by forskolin or IBMX. The inhibition in isoproterenol-treated cells was complete in both intact and permeabilize£1 cells, whereas that in forskolin- or IBMX-treated ones was incomplete and varied with incubation conditions. Figs. 2 and 3 show the time-courses of cAMP accumulation. Propranolol dearly inhibited cAMP accumulation produced by forskolin in both intact and saponin-permeabilized cells: in intact cells, the inhibition rate was maximal at 5 min (approx. 50%) and gradually decreased (approx. 20% at 30 rain); but in permeabilizexi cells, the rate (approx. 50%) was apparently constant throughout the incubation period(Fig.
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Fig. 1. Effect of propranoiol on cAMP accumulation in intact or saponin-permeabilized parotid cells stimulated by isoprotcrenol, forskolin, or IBMX. Parotid cells were incubated at 37aC for 5 rain with 1/~M isoproterenol, 10 ~tM forskolin, 1 (for intact cells) or 0.1 (for permeabilized cells) mM IBMX, with or without 10 ~M propranolol in either normal Hanks' medium or Ca-free KC1 medium containing 20 p g / m l saponin. Data shown axe means+S.D. (n = 4 - 8 ) . CON, control; ISO, isoproterenol; FRK, forskolin; IMX, IBMX. Open column, without propranolol; solid column, with propranolol.
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461
Elsevier BBAMCR 12697
Propranolol inhibits cyclic AMP accumulation and amylase secretion in parotid acinar cells stimulated by isobutylmethylxanthine and forskolin Taishin Takuma Department of Oral Biochemistry, School of Dentistry, Higashi Nippon Gakuen University, Hokkaido (Japan)
(Received 4 October 1989) (Revised manuscript received16 January 1990)
Key words: Propranolol;cAMP; Amylase;Isobutylmethylxanthine;Forskolin;(Parotid gland acinar cell)
Propranoloi inhibited cyclic AMP (cAMP) accumulation stimulated by 3-isobutyi-l-methylxanthine 0BMX) or forskolin in rat parotid acinar cells. The inhibition by propranolol was highly potent; 10 - 7 M propranolol was sufficient for the maximum inhibition (approx. 50% at 5 rain). The inhibitory effect was observed in both intact and saponin-permeabilized parotid cells, but the effect was more prominent in permeabilized ceils than in intact cells. Other fl-blockers, like alprenolol and atenolol, were as effective as propranolol, but butoxamine (fl2-selective) was slightly less effective. The inhibition by propranolol was similarly detected in the ceils prepared from pertussis-toxin-pretreated rats, suggesting that inhibitory guanine nucleotide regulatory protein (Gi) is not involved in the inhibitory mechanism. Propranolol also inhibited the exocytosis of amylase stimulated by IBMX or forskolin. In the presence of propranolol and IBMX, the responsiveness of saponin-permeabilized ceils to exogenous cAMP was markedly increased, indicating that propranoloi neither promotes the degradation of cAMP nor prevents the inhibitory effect of IBMX on cAMP phosphodiesterase.
Introduction Although several lines of evidence indicate that cAMP is a major mediator for the exocytosis of amylase from parotid acinar cells [1,2], the regulatory mechanism by cAMP is totally unknown, including the role of cAMPdependent protein kinase [3]. Basically, it has not yet been established what concentration of cAMP is necessary for amylase release, since, unlike calcium, the cellular level of free cAMP cannot be determined directly. For resolution of this question, the application of permeabilized cells seemed to be useful [4,5]. Even in saponin-permeabilized parotid cells, however, a millimolar concentration of cAMP was necessary for the maximum anylase release, probably because cAMP phosphodiesterase strictly prevents the access of exogenous cAMP to the regulatory site of exocytosis [5]. In
addition, the phosphodiesterase inhibitor IBMX by itself maximally stimulated amylase release, suggesting that basal adenylate cyclase activity in these cells is strong enough to provide sufficient cAMP for maximal amylase secretion when phosphodiesterase activity is inhibited [5]. Propranolol is the prototype of 't-blockers' and is widely used for both clinical and investigative purposes [6]. In the parotid gland, propranolol completely inhibits amylase secretion and cAMP accumulation evoked by fl-agonists [1]. In the course of screening for adenylate cyclase inhibitors, I found that propranolol inhibits amylase release and cAMP accumulation in the parotid cells stimulated not only by isoproterenol (a fl-agonist), but also by IBMX. Furthermore, propranolol also inhibited the effect of forskohn, a direct activator of adenylate cyclase. Results obtained suggest that inhibitory G protein (Gi) is not involved in the inhibitory mechanism.
Abbreviations: IBMX, 3-ibobutyl-l-methylxanthine;R-PIA, ( -)N 6((R)-phenylisopropyl)adenosine; BCA, bicinchoninic acid; BSA, bovine serum albuminl; DTI', dithiothreitol.
Materials and Methods
Correspondence: T. Takuma, Department of Oral Biochemistry, School of Dentistry, Higashi Nippon Gakuen University, Tobetsu, Hokkaido 061-02, Japan.
Materials
The materials used in this study and their sources were as follows: DL-propranolol, L-alprenolol, atenolol,
0167-4889/90/$03.50 © 1990 Elsevier Science Publishers B.V. (BiomedicalDivision)
462 procaine, isoproterenol, IBMX, GDPflS and cAMP from Sigma (St. Louis, MO, U.S.A.); R-PIA from Behringer Manheim Yamanouchi (Tokyo, Japan); 2',5'-dideoxyadenosine from Pharmacia LKB (Uppsala, Sweden); saponin and atropine from Merck (Darmstadt, F.R.G.); phentolamine from Ciba-Geigy (Takarazuka, Japan); cyclic AMP assay kit from Yamasa Shoyu (Chiba, Japan); pertussis toxin (islet-activating protein) from Funakoshi (Tokyo, Japan); BCA protein assay reagent from Pierce (Rockford, IL, U.S.A.); [a-32p]NAD from Du Pont, Daiich Pure Chemicals (Tokyo, Japan); and butoxamine, a gift from Burroughs Wellcome (Research Triangle Park, NC, U.S.A.). Treatment of rats with pertussis toxin Male rats of the Wistar strain were injected intravenously with pertussis toxin (0.5 #g/100 g body weight), 3 days before killing, for preparation of parotid acinar cells [7]. Cycfic A M P assay Cyclic AMP levels were measured according to the method described by Tojyo et al. [8]. Parotid cells, prepared as described previously [3,5], were incubated for 5-30 min at 37°C with various agents or vehicles in 1 ml of either regular Hank's medium or Ca 2+-free KC1 medium composed of 140 mM KC1, 20 mM Na-Hepes (pH 7.2), 1 mM EGTA, 2 mM MgSO4, 10 /~g/ml Phenol red, 1 mg/ml bovine serum albumin (BSA) and 20 #g/ml saponin. After incubation, the cells were immediately boiled for 3 rain in their incubation medium, cooled on ice, and then centrifuged at 900 × g for 5 min. Cyclic AMP concentration in the resulting supernatant was determined using a cAMP assay kit and was expressed as pmol/mg protein. A D P-ribosy lation Parotid cells prepared from normal or pertussistoxin-injected rats were homogenized with 20 mM NaHepes (pH 7.2), 1 mM EGTA, 1 mM EDTA and 1 mM dithiothreitol (DTI') (buffer A) in a Teflon-glass homogenizer. The homogenates were centrifuged at 400 × g for 5 rain and 24000 × g for 15 rain. The resulting pellets (400-24000 x g) were suspended in the same buffer (approx. 8 mg protein/m1) and stored at - 80 o C until use. Pertussis toxin (200 /~g/ml) was freshly activated by incubation with 20 mM D'VF, 4 mM ATP, 1 mg/ml BSA and 40 mM Na-Hepes (pH 7.2) at 30 °C for 20 rain. ADP-ribosylation in vitro was carried out at 30 o C for 60 min in a reaction mixture (200/~1) containing parotid membrane prepared as above (approx. 4 mg/ml), pertussis toxin (20 /~g/ml), (0t-32p)NAD (40 /~Ci, 7-10 #M), 10 mM thymidine, 1 mM ATP, 0.1 mM GTP and 10 mM potassium phosphate (pH 7.2). After incubation the mixture was diluted with ice-cold buffer A (2 ml) and centrifuged at 24000 × g for 15 rain. The
pellets were dissolved in 100 #1 of Laernmli sample buffer [9] and boiled for 5 min. SDS-PAGE was performed on 12.5% gel and autoradiograms were prepared using Fuji X-ray films. Other procedures Amylase release from intact and saponin-permeabilized parotid cells were measured as described previously [3,5]. Protein was determined by the BCA protein assay method using BSA as a standard. Results
Effect on cAMP accumulation Cyclic AMP accumulation stimulated by isoproterenol, forskolin and IBMX was observed both in intact and saponin-permeabilized parotid cells incubated in normal Hanks' medium and Ca2+-free KCI medium containing 20/xg/ml saponin, respectively, although the cAMP level induced by isoproterenol was markedly reduced in the permeabilized cell (Fig. 1). 10/~M propranolol, a nonselective /t-antagonist (1/1 :f12), in" hibited cAMP accumulation evoked not only b y isoproterenol, but also by forskolin or IBMX. The inhibition in isoproterenol-treated cells was complete in both intact and permeabilize£1 cells, whereas that in forskolin- or IBMX-treated ones was incomplete and varied with incubation conditions. Figs. 2 and 3 show the time-courses of cAMP accumulation. Propranolol dearly inhibited cAMP accumulation produced by forskolin in both intact and saponin-permeabilized cells: in intact cells, the inhibition rate was maximal at 5 min (approx. 50%) and gradually decreased (approx. 20% at 30 rain); but in permeabilizexi cells, the rate (approx. 50%) was apparently constant throughout the incubation period(Fig.
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Fig. 1. Effect of propranoiol on cAMP accumulation in intact or saponin-permeabilized parotid cells stimulated by isoprotcrenol, forskolin, or IBMX. Parotid cells were incubated at 37aC for 5 rain with 1/~M isoproterenol, 10 ~tM forskolin, 1 (for intact cells) or 0.1 (for permeabilized cells) mM IBMX, with or without 10 ~M propranolol in either normal Hanks' medium or Ca-free KC1 medium containing 20 p g / m l saponin. Data shown axe means+S.D. (n = 4 - 8 ) . CON, control; ISO, isoproterenol; FRK, forskolin; IMX, IBMX. Open column, without propranolol; solid column, with propranolol.
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