Research in
Res Exp Med (1992) 192 : 295-303
Experimental Medicine 9 Springer-Verlag 1992
Phorbol ester attenuates inositol 1,4,5-trisphosphate-induced Ca 2+ release in electropermeabilized rat pancreatic acini Y. Arita, 17. Kimura, Y. Ogami, and H. Nawata The Third Department of Internal Medicine, Faculty of Medicine, Kyushu University, Fukuoka 812, Japan Received October 24, 1991 / accepted April 30, 1992
Summary. To investigate the mechanism of inositol trisphosphate (IP3)-induced Ca 2+ release from the internal Ca 2+ store, we examined the effects of heparin, phorbol ester and cyclic nucleotides on Ca 2+ release induced by carbachol or inositol 1,4,5-trisphosphate (1,4,5-IP3). For monitoring changes of Ca 2+ we used the fluorescent indicator, fura-2, in electropermeabilized rat pancreatic acini. An amount of 100 btg/ml heparin inhibited the Ca 2+ release induced by 1 btM 1,4,5-IP3 in permeabilized acini. Pretreatment with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) for 10 min reduced the release of Ca > induced by 10 laM carbachol and 1 ~tM 1,4,5-IP3 in permeabilized acini. Staurosporine, a protein kinase C inhibitor, blocked the inhibitory effect of TPA. Cytosolic calcium concentration was restored by staurosporine in TPA-treated acini. Although cyclic A M P exaggerated the amylase release induced by carbachol, cyclic A M P and cyclic G M P had no effect on the carbachol-induced release of Ca 2+ in permeabilized acini. These findings suggest that protein kinase C may act at the level of the IP3 receptors or the IP3-0perated Ca 2+ channels of the internal Ca 2+ store and indicate that cyclic nucleotides do not affect the IP3-induced release of Ca 2+ in rat pancreatic acini.
Key words: Exocrine pancreas - Phorbol ester - Calcium mobilization - Cyclic nucleotide - Permeabilized cell Introduction Such agents as carbachol, cholecystokinin (CCK), and bombesin stimulate enzyme secretion in the exocrine pancreas by increasing the cytosolic free calcium ([Ca2+]i) [6]. The increase in [Ca2+]i is mainly derived from intracellular Ca 2+ stores and is related to the initial-burst enzyme secretion in the pancreatic acinar cells [15]. The receptor-triggered hydrolysis of phosphatidylinositol 4,5-bisCorrespondence to: Y. Arita
296 p h o s p h a t e by phospholipase C results in the formation of inositol 1.4.5-trisphosphate (1,4,5-IP3) and diacylglycerol ( D G ) [4]. IP3 releases calcium from an endoplasmic reticulum store by directly o p e n i n g the channels. D G is the physiological activator of protein kinase C (PKC). P h o r b o l esters activate P K C bv substituting for e n d o g e n o u s D G in a variety of tissues [4]. Previous studies have indicated that p h o r b o l esters stimulate e n z y m e secretion and that p r e t r e a t m e n t with p h o r b o l ester attenuates the mobilization of calcium and release of amylase stimulated by C C K and carbachol in pancreatic acini [2, 8, 24]. R e c e n t studies have revealed that P K C is involved in desensitizing muscarinic receptors induced by p h o r b o l esters [16]. H o w e v e r , o t h e r investigations have f o u n d that p h o r b o l ester decreases the release of Ca > induced by IP3 [25]. In this study, we e x a m i n e d the effects of heparin, 1 2 - O - t e t r a d e c a n o y l p h o r bol-13-acetate ( T P A ) , and cyclic nucleotides on the release of Ca 2+ induced by carbachol and IP3 in monitoring the o u t c o m e in electropermeabilized rat pancreatic acini with the fluorescent indicator, fura-2 [1-(2-(5-carboxyoxazol-2'-yl)6 - a m i n o - 5 ' - m e t h y l p h e n o x y l ) - e t h a n e - N , N, N', N'-tetraacetic acid, K salt].
Materials and methods
Chemicals' Fura-2, fura-2/AM (The acetoxymethyl derivative of fura-2), Hepes, collagenase, and EGTA (ethyleneglycol-bis(beta-aminoethyl ester)-tetraacetic acid) were obtained from Wako Pure Chemical Industries (Osaka, Japan). Bovine serum albumin (fraction V), soybean trypsin inhibitor (type I-S), MgATP, creatine phosphate, CPK, IP3, TPA, and carbachol were obtained from Sigma Chemical, St. Louis, Mo., USA. Eagle MEM minimum-essential amino acids and vitamins were obtained from the Nissui Pharmaceutical, Tokyo, Japan; TRIS and EDTA from Nakarai Chemicals, Kyoto, Japan; Triton X from Yoneyama Yakuhin Kogyo, Osaka, Japan; and the Phadebas amylase test from Shionogi, Osaka, Japan. The incubation solution used was Krebs-Ringer-Hepes buffer containing 10mM Hepes, 120mM NaC1, 4.7mM KCI, l m M KH2PO4, 1 mM MgCI:, 2 mM CaCI2, 15 mM glucose. 0.1% (w/v) bovine serum albumin, 0.01% (w/v) soybean trypsin inhibitor, and Eagle MEM essential amino acids and vitamins adjusted with NaOH to pH 7.4 equilibrated with 100% 02.
Preparation of isolated acini Rat pancreatic acini were prepared by the modified method of Amsterdam and Jamieson [1] as reported previously [3]. Male Wistar King albino rats (300-360g) were decapitated and their pancreata rapidly excised. The method is based on two successive incubations (with collagenase in a Krebs-Ringer-Hepes medium, see above) having a divalent cation chelating step in between. After digestion, the tissue was dispersed by pipetting, filtered through nylon gauze, and purified by centrifugation through 4% albumin layer. The viability of the acini obtained by this procedure exceeded 95%, as measured by the trypan blue exclusion method.
Permeabilization of acini Pancreatic acini were washed once in KRH buffer without Ca 2- and bovine serum, but containing 2 mM EDTA, then washed twice, resuspended in a cold medium high in potassium (500 gl) containing 140 mM KC1, 10 mM NaC1, 10 mM Hepes, 1 mM MgCI2, 0.01% trypsin inhibitor, and 5 mM MgATP (pH 6.6). The acini were electroporated with four discharges at 2.7 kV with a time constant of 200 ~_s in the high potassium medium. After permeabilization, the acini were washed twice and resuspended in the high-potassium medium with an ATP-regenerating system (4 mM MgATP, 10 mM creatine phosphate, and 7 IU/ml CPK).
297
Measurement of cytosolic calcium The concentration of cytosolic free calcium in intact pancreatic acini was measured with fura-2/ AM, as reported previously [3]. To measure the [Ca 2+] in electropermeabilized acini, changes in buffer [Ca 2+] were monitored with fura-2 by the modified method of Hill et al. [11]. Samples of permeabilized acini (2-3 • 10v cells in 0.5 ml of the potassium medium) were incubated for 10 rain at 37~ after the addition of the ATP-regenerating system and 0.1 laM fura-2 (non-esterized) was added. [Ca 2+] was measured in 0.5 ml aliquots of cell suspensions in a Schimazu RF-5000 fluorescence spectrophotometer at a setting of 340nm and 380nm excitation and 500 nm emission. Calcium concentration, [Ca2+], was calculated as described previously [9], using a Ca2+/dye dissociation constant for fura-2 of 224nM: [Ca 2+] = Kd X (R-Rmin)/(Rmax-R) x F380min/F380....
Amylase release Permeabilized acini were incubated with secretagogues at 37~ for 30 min after 30-min preincubation in the potassium buffer with the ATP-regenerating system. Amylase release into the extracellular medium during incubation was calculated as a percentage of the total content present in the acini at the beginning of incubation. Amylase activity was assayed by the method of Ceska et al. [7] using Phadebas reagent. Ca z+ concentrations were adjusted as described by van Heeswjik et al. [21].
[Ca~+] (nM)
[C~ +]
COh 1
1rnin
(1,4,5) IP3 1
100 L
[Ca~+] Heparin
100 L-
(1,4,5) IP3
1
Fig. 1. Effect of heparin on carbacholand 1,4,5-IPrinduced Ca 2+ release in electropermeabilized acini. To measure [Ca 2+] in the electropermeabilized acini, changes in buffer [Ca 2+] induced by 10 ~tM carbachol or i gM 1,4,5-IP3 were monitored with fura-2. Each recording is of a single experiment representative of the other five
298
Results Effects o f heparin on Ca2+ release When electropermeabilized acini were incubated in a Ca>-deficient m e d i u m containing A T P and ATP-regenerating system for 10 min at 37~ basal [Ca 2§ was 50-160 nM. Ca > release was induced by 10 btM carbachol and 1.0 laM 1,4,5IP 3 release in permeabilized acini (Fig. 1). The peaks of [Ca 2+] induced by carbachol and 1,4,5-IP3 were 184 + 13% and 193 ___23% above the basal levels, respectively. It was reported that heparin inhibited the IP3-induced Ca :+ release in electropermeabilized pancreatic [3-cells [18]. The stimulation of Ca 2+ release with 1.0 gM IP3 was completely inhibited by 100 pg/ml heparin (Fig. 1). The inhibiting effect of heparin was dose-dependent. Stimulation of Ca -~+ release with 1.0 btM IP3 was slightly attenuated by 1.0 btg/ml heparin (data not shown). ICs0 was about 6 I,tg/inl, a value in good agreement with the previous finding [18]. Heparin also completely inhibited the carbachol-induced release of Ca 2. (data not shown).
Effects of TPA on Ca 2+ release We previously reported that p r e t r e a t m e n t with T P A attenuated the release of Ca 2+ in intact pancreatic acini and that H7 and staurosporine, both potent P K C inhibitors, blocked the inhibitory effect of T P A [3]. When the permeabilized
pretreatment of TPA [Ca~+] (nM)
4ool
CCh
L
1rain
I__ 1
200f lOO'-
[Ca*+] (nM)
pretreatment of TPA IP3 lmin
4~176 I 100 L
I
Fig. 2. Effect of 12-O-tetradecanoylphorbol 13-acetate (TPA) on carbachol- and 1,4,5IP3-induced Ca 2- release in electropermeabilized acini. Dispersed acini were incubated at 37~ in the presence of 10-7 M TPA. To measure [Ca2-] in the electropermeabilized acini, changes in buffer [Ca2~] induced by 10 #M carbachol or i #M IP3 were monitored with fura-2. Each recording is of a single experiment representative of the other five
299 IP3-induced Ca 2"release
CCh-induced Ca 2"release
N.S.
N.S. i
I
I
I
I
control
TPA
I
*
i
I
ontrol
TPA
n~6 ~-P
Res Exp Med (1992) 192 : 295-303
Experimental Medicine 9 Springer-Verlag 1992
Phorbol ester attenuates inositol 1,4,5-trisphosphate-induced Ca 2+ release in electropermeabilized rat pancreatic acini Y. Arita, 17. Kimura, Y. Ogami, and H. Nawata The Third Department of Internal Medicine, Faculty of Medicine, Kyushu University, Fukuoka 812, Japan Received October 24, 1991 / accepted April 30, 1992
Summary. To investigate the mechanism of inositol trisphosphate (IP3)-induced Ca 2+ release from the internal Ca 2+ store, we examined the effects of heparin, phorbol ester and cyclic nucleotides on Ca 2+ release induced by carbachol or inositol 1,4,5-trisphosphate (1,4,5-IP3). For monitoring changes of Ca 2+ we used the fluorescent indicator, fura-2, in electropermeabilized rat pancreatic acini. An amount of 100 btg/ml heparin inhibited the Ca 2+ release induced by 1 btM 1,4,5-IP3 in permeabilized acini. Pretreatment with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) for 10 min reduced the release of Ca > induced by 10 laM carbachol and 1 ~tM 1,4,5-IP3 in permeabilized acini. Staurosporine, a protein kinase C inhibitor, blocked the inhibitory effect of TPA. Cytosolic calcium concentration was restored by staurosporine in TPA-treated acini. Although cyclic A M P exaggerated the amylase release induced by carbachol, cyclic A M P and cyclic G M P had no effect on the carbachol-induced release of Ca 2+ in permeabilized acini. These findings suggest that protein kinase C may act at the level of the IP3 receptors or the IP3-0perated Ca 2+ channels of the internal Ca 2+ store and indicate that cyclic nucleotides do not affect the IP3-induced release of Ca 2+ in rat pancreatic acini.
Key words: Exocrine pancreas - Phorbol ester - Calcium mobilization - Cyclic nucleotide - Permeabilized cell Introduction Such agents as carbachol, cholecystokinin (CCK), and bombesin stimulate enzyme secretion in the exocrine pancreas by increasing the cytosolic free calcium ([Ca2+]i) [6]. The increase in [Ca2+]i is mainly derived from intracellular Ca 2+ stores and is related to the initial-burst enzyme secretion in the pancreatic acinar cells [15]. The receptor-triggered hydrolysis of phosphatidylinositol 4,5-bisCorrespondence to: Y. Arita
296 p h o s p h a t e by phospholipase C results in the formation of inositol 1.4.5-trisphosphate (1,4,5-IP3) and diacylglycerol ( D G ) [4]. IP3 releases calcium from an endoplasmic reticulum store by directly o p e n i n g the channels. D G is the physiological activator of protein kinase C (PKC). P h o r b o l esters activate P K C bv substituting for e n d o g e n o u s D G in a variety of tissues [4]. Previous studies have indicated that p h o r b o l esters stimulate e n z y m e secretion and that p r e t r e a t m e n t with p h o r b o l ester attenuates the mobilization of calcium and release of amylase stimulated by C C K and carbachol in pancreatic acini [2, 8, 24]. R e c e n t studies have revealed that P K C is involved in desensitizing muscarinic receptors induced by p h o r b o l esters [16]. H o w e v e r , o t h e r investigations have f o u n d that p h o r b o l ester decreases the release of Ca > induced by IP3 [25]. In this study, we e x a m i n e d the effects of heparin, 1 2 - O - t e t r a d e c a n o y l p h o r bol-13-acetate ( T P A ) , and cyclic nucleotides on the release of Ca 2+ induced by carbachol and IP3 in monitoring the o u t c o m e in electropermeabilized rat pancreatic acini with the fluorescent indicator, fura-2 [1-(2-(5-carboxyoxazol-2'-yl)6 - a m i n o - 5 ' - m e t h y l p h e n o x y l ) - e t h a n e - N , N, N', N'-tetraacetic acid, K salt].
Materials and methods
Chemicals' Fura-2, fura-2/AM (The acetoxymethyl derivative of fura-2), Hepes, collagenase, and EGTA (ethyleneglycol-bis(beta-aminoethyl ester)-tetraacetic acid) were obtained from Wako Pure Chemical Industries (Osaka, Japan). Bovine serum albumin (fraction V), soybean trypsin inhibitor (type I-S), MgATP, creatine phosphate, CPK, IP3, TPA, and carbachol were obtained from Sigma Chemical, St. Louis, Mo., USA. Eagle MEM minimum-essential amino acids and vitamins were obtained from the Nissui Pharmaceutical, Tokyo, Japan; TRIS and EDTA from Nakarai Chemicals, Kyoto, Japan; Triton X from Yoneyama Yakuhin Kogyo, Osaka, Japan; and the Phadebas amylase test from Shionogi, Osaka, Japan. The incubation solution used was Krebs-Ringer-Hepes buffer containing 10mM Hepes, 120mM NaC1, 4.7mM KCI, l m M KH2PO4, 1 mM MgCI:, 2 mM CaCI2, 15 mM glucose. 0.1% (w/v) bovine serum albumin, 0.01% (w/v) soybean trypsin inhibitor, and Eagle MEM essential amino acids and vitamins adjusted with NaOH to pH 7.4 equilibrated with 100% 02.
Preparation of isolated acini Rat pancreatic acini were prepared by the modified method of Amsterdam and Jamieson [1] as reported previously [3]. Male Wistar King albino rats (300-360g) were decapitated and their pancreata rapidly excised. The method is based on two successive incubations (with collagenase in a Krebs-Ringer-Hepes medium, see above) having a divalent cation chelating step in between. After digestion, the tissue was dispersed by pipetting, filtered through nylon gauze, and purified by centrifugation through 4% albumin layer. The viability of the acini obtained by this procedure exceeded 95%, as measured by the trypan blue exclusion method.
Permeabilization of acini Pancreatic acini were washed once in KRH buffer without Ca 2- and bovine serum, but containing 2 mM EDTA, then washed twice, resuspended in a cold medium high in potassium (500 gl) containing 140 mM KC1, 10 mM NaC1, 10 mM Hepes, 1 mM MgCI2, 0.01% trypsin inhibitor, and 5 mM MgATP (pH 6.6). The acini were electroporated with four discharges at 2.7 kV with a time constant of 200 ~_s in the high potassium medium. After permeabilization, the acini were washed twice and resuspended in the high-potassium medium with an ATP-regenerating system (4 mM MgATP, 10 mM creatine phosphate, and 7 IU/ml CPK).
297
Measurement of cytosolic calcium The concentration of cytosolic free calcium in intact pancreatic acini was measured with fura-2/ AM, as reported previously [3]. To measure the [Ca 2+] in electropermeabilized acini, changes in buffer [Ca 2+] were monitored with fura-2 by the modified method of Hill et al. [11]. Samples of permeabilized acini (2-3 • 10v cells in 0.5 ml of the potassium medium) were incubated for 10 rain at 37~ after the addition of the ATP-regenerating system and 0.1 laM fura-2 (non-esterized) was added. [Ca 2+] was measured in 0.5 ml aliquots of cell suspensions in a Schimazu RF-5000 fluorescence spectrophotometer at a setting of 340nm and 380nm excitation and 500 nm emission. Calcium concentration, [Ca2+], was calculated as described previously [9], using a Ca2+/dye dissociation constant for fura-2 of 224nM: [Ca 2+] = Kd X (R-Rmin)/(Rmax-R) x F380min/F380....
Amylase release Permeabilized acini were incubated with secretagogues at 37~ for 30 min after 30-min preincubation in the potassium buffer with the ATP-regenerating system. Amylase release into the extracellular medium during incubation was calculated as a percentage of the total content present in the acini at the beginning of incubation. Amylase activity was assayed by the method of Ceska et al. [7] using Phadebas reagent. Ca z+ concentrations were adjusted as described by van Heeswjik et al. [21].
[Ca~+] (nM)
[C~ +]
COh 1
1rnin
(1,4,5) IP3 1
100 L
[Ca~+] Heparin
100 L-
(1,4,5) IP3
1
Fig. 1. Effect of heparin on carbacholand 1,4,5-IPrinduced Ca 2+ release in electropermeabilized acini. To measure [Ca 2+] in the electropermeabilized acini, changes in buffer [Ca 2+] induced by 10 ~tM carbachol or i gM 1,4,5-IP3 were monitored with fura-2. Each recording is of a single experiment representative of the other five
298
Results Effects o f heparin on Ca2+ release When electropermeabilized acini were incubated in a Ca>-deficient m e d i u m containing A T P and ATP-regenerating system for 10 min at 37~ basal [Ca 2§ was 50-160 nM. Ca > release was induced by 10 btM carbachol and 1.0 laM 1,4,5IP 3 release in permeabilized acini (Fig. 1). The peaks of [Ca 2+] induced by carbachol and 1,4,5-IP3 were 184 + 13% and 193 ___23% above the basal levels, respectively. It was reported that heparin inhibited the IP3-induced Ca :+ release in electropermeabilized pancreatic [3-cells [18]. The stimulation of Ca 2+ release with 1.0 gM IP3 was completely inhibited by 100 pg/ml heparin (Fig. 1). The inhibiting effect of heparin was dose-dependent. Stimulation of Ca -~+ release with 1.0 btM IP3 was slightly attenuated by 1.0 btg/ml heparin (data not shown). ICs0 was about 6 I,tg/inl, a value in good agreement with the previous finding [18]. Heparin also completely inhibited the carbachol-induced release of Ca 2. (data not shown).
Effects of TPA on Ca 2+ release We previously reported that p r e t r e a t m e n t with T P A attenuated the release of Ca 2+ in intact pancreatic acini and that H7 and staurosporine, both potent P K C inhibitors, blocked the inhibitory effect of T P A [3]. When the permeabilized
pretreatment of TPA [Ca~+] (nM)
4ool
CCh
L
1rain
I__ 1
200f lOO'-
[Ca*+] (nM)
pretreatment of TPA IP3 lmin
4~176 I 100 L
I
Fig. 2. Effect of 12-O-tetradecanoylphorbol 13-acetate (TPA) on carbachol- and 1,4,5IP3-induced Ca 2- release in electropermeabilized acini. Dispersed acini were incubated at 37~ in the presence of 10-7 M TPA. To measure [Ca2-] in the electropermeabilized acini, changes in buffer [Ca2~] induced by 10 #M carbachol or i #M IP3 were monitored with fura-2. Each recording is of a single experiment representative of the other five
299 IP3-induced Ca 2"release
CCh-induced Ca 2"release
N.S.
N.S. i
I
I
I
I
control
TPA
I
*
i
I
ontrol
TPA
n~6 ~-P
