Biochimica et Biophysica Acta, 1052 (1990) 285-292
285
Elsevier BBAMCR 12686
Glucose- and concentration-dependence of noradrenalin effects on electrical activity in mouse pancreatic fl cells Michel Joffre and Anne Debuyser Laboratoire de Physiologie Animale, CNRS: URA 290 Biomembranes, UFR Sciences, Poitiers, 'France)
(Received 3 October 1989)
Key words: Electrophysiology;Noradrenalin; et2-Adrenoceptors;Glucose; (Mouse pancreatic fl cells)
The effects of a wide range of noradrenalin (NA) concentrations (10-11--10 -4 M) on the membrane potential and on the glucose-induced electrical activity were investigated with microelectrodes in microdissected mouse islets. In the presence of 11.1 mM glucose, the fl cells exhibited a repetitive activity. NA at more than 1 0 - 7 M induced a rapid hyperpolarization followed by a silent depolarization, then by the appearance of a slowed pace of repetitive activity (dose-dependent effects). NA at less than 10 -7 M did not markedly affect the electrical activity; it only induced a dose-dependent increase in the degree of activity with no change of the potential levels. The glucose-dependence of these effects were then investigated. In the absence of glucose, 10 - s and 10 -6 M NA did not affect the resting membrane potential. Non-stimulatory glucose concentrations (2.8-7.3 mM) progressively decreased the membrane potential. 10 - s M NA did not affect it, while 1 0 - 6 M NA induced a dose-dependent and long-lasting hyperpolarization. In the presence of stimulatory glucose concentrations (7.3-30 mM) the degree of activity increased, 10 - s M NA induced a slight leftward shift and 1 0 - 6 M NA a slight rightward shift of the dose-response curve.
Introduction Catecholamines are important physiological modulators of hormone release by the endocrine pancreas [1]. It is widely agreed that the inhibition of insulin secretion results from an activation of the alpha 2 adrenoceptors present in beta cells [2-6], but the cellular mechanisms are complex and still incompletely understood [7-8]. On the other hand, the stimulation of fl adrenoceptors increases insulin release [9-12]. This effect may, however, be species-dependent and indirect. Since the early studies of Dean and Matthews [13], numerous investigations have defined the burst pattern of the glucose-induced electrical activity in mouse pancreatic fl cells [14-17]. In contrast, only few studies have described the modifications of this electrical activity by catecholamines [13,18-19]. They were shown to induce a sustained silent inhibition [13] or a transient silent inhibition followed by a slowest burst pattern [18].
Abbreviation: NA, noradrenalin. Correspondence: M. Joffre, Laboratoire de Physiologie Animale, CNRS URA 290 Biomembranes, 40 Avenue Recteur Pineau, 86022 Poitiers cedex, France.
The present study was therefore undertaken to specify the effects of various concentrations of noradrenaline on the glucose-induced electrical activity in mouse fl cells and to investigate the glucose-dependence of these effects for low and high noradrenalin concentrations.
Materials and Methods All the experiments were performed on pancreatic islets of female N M R I mice (20-30 g) killed by decapitation 2 h after an intraperitoneal injection of pilocarpine (0.2 ml of 2% pilocarpine in 9%o NaC1), to clear the exocrin pancreas, or after an overnight fasting. Neither treatment had an effect on the glucose-induced electrical activity in fl cells. A piece of pancreas was fixed in a small chamber and islets were then partly dissected under a binocular microscope. The tissue was continuously perifused at 3 7 ° C with a bicarbonate-buffered solution (pH 7.4) which contained (mM): 122 NaC1; 4.7 KC1; 1.1 MgC12; 2.5 CaC12; 20 N a H C O 3. Glucose was added at concentrations ranging from 0 to 30 mM. The solutions were continuously gassed with a mixture of O 2 / C O 2 (95/5). Stock solutions of 10 m M N A (Serva) were prepared in 10 m M ascorbic acid (Serva) and kept frozen. Aliquots were added to media before each experiment. The addition of 10 -4 M ascorbic acid to the perifusion solu-
0167-4889/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
286 tion did not affect the burst pattern of the electrical activity induced by 11.1 m M glucose. Microelectrodes made from glass capillary tubes (Frederick Haer, U.S.A., No. 30.33.1) and filled with potassium citrate solution (2 M, p H 7.2) were used for recording the membrane potential. Their resistance ranged from 100 to 200 M~2. The/3 cells were identified by their typical repetitive activity in the presence of 11.1 m M glucose. The results are illustrated by recordings that are representative of the indicated number of experiments, and by several parameters which were measured: the membrane potential during active and silent phases, the duration of active (plateau) and silent phases (Dp and D~), the frequency of slow waves ( F ) and the degree of activity (A) determined as the percentage of active phase ( D p / ( D p + D~) is the plateau fraction). The responses to glucose and to N A were found to vary from one cell to another. The effects are expressed by the differences between the control values obtained before an application of N A and the values obtained, in the same cell, when the effect of N A reached the steady state. Dose response curves were built from the values obtained in different cells only exposed to one NA concentration or in cells exposed to increasing N A concentrations (cumulative doses) with no period of perifusion with a control medium, since no desensitization to NA was recorded. All the data are given as means (_+ S.E.) of absolute or relative values for a certain number of experiments (n). The statistical significance of the effects of the different noradrenalin concentrations was assessed by paired t-test.
0
A
NA 10 - 8 •
M
0[ E
¢J ~J
o ¢J
B
NA 1Q- 6
M
1:1
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°I o
-60[
c
Fig. 1. Effects of N A on the g l u c o s e - i n d u c e d electrical activity in single m o u s e /3 cells. The c o n c e n t r a t i o n of glucose was 11.1 m M t h r o u g h o u t and N A was a d d e d as i n d i c a t e d by the arrow. R e c o r d s A and B were o b t a i n e d in two different cells. They are r e p r e s e n t a t i v e of the results o b t a i n e d in 7 (10 s M) a n d 11 (10 6 M) different experiments. A: Effects of 10 s M N A . Records b and c were o b t a i n e d on steady state, respectively 10 and 15 min after the a d d i t i o n of NA. B: Effects of 10 - 6 M N A . R e c o r d s b a n d c are the direct c o n t i n u a t i o n of a, T i m e c a l i b r a t i o n (bar): 10 s.
Results
Concentration-dependence of noradrenalin effects in the presence of 11.1 m M glucose The effects of a wide range of noradrenaline concentrations ( 1 0 - t t to 1 0 - 4 M) were investigated in islets perifused with media containing 11.1 m M glucose. When mouse /3 cells were exposed to a medium containing glucose alone, they exhibited a typical electrical activity consisting of oscillations of the membrane potential (slow waves) with bursts of rapid spikes superimposed on the plateau, followed by repolarized silent phases (Fig. 1). 10 - s M noradrenalin did not markedly affect the glucose-induced electrical activity (Fig. 1A). The potential levels were never changed. A slight potentiation of the electrical activity was always recorded on steady state (ranged from 10-15 min). That was confirmed by measuring the degree of activity in 7 cells (Fig. 2D). In contrast, the average values of the other parameters were not affected by 10 - s M N A (Fig. 2A, B and C). Nevertheless, all the individual data demonstrated that
the stimulation by this low concentration lengthened the plateaus, shortened the silent phases and increased the frequency of slow waves (7 out of 7 cells). No change of the pattern, the amplitude and the frequency of the spikes was recorded. In contrast, 10 6 M N A markedly affected the pattern of the recordings, with a transient inhibition of the glucose-induced electrical activity (Fig. 1B). The membrane was initially hyperpolarized and the electrical activity suppressed. That was followed by a silent depolarization to the potential at which the electrical activity appeared in the control medium, then by the reappearance of slow waves. In the steady state, the duration of the active and silent phases were increased, this gives rise to the decrease of the degree of activity and of the frequency of slow waves (Fig. 2). The pattern, the amplitude and the frequency of the spikes were not affected. From 1 0 - l l to 10 s M, N A potentiated the glucoseinduced electrical activity in agreement with the record-
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285
Elsevier BBAMCR 12686
Glucose- and concentration-dependence of noradrenalin effects on electrical activity in mouse pancreatic fl cells Michel Joffre and Anne Debuyser Laboratoire de Physiologie Animale, CNRS: URA 290 Biomembranes, UFR Sciences, Poitiers, 'France)
(Received 3 October 1989)
Key words: Electrophysiology;Noradrenalin; et2-Adrenoceptors;Glucose; (Mouse pancreatic fl cells)
The effects of a wide range of noradrenalin (NA) concentrations (10-11--10 -4 M) on the membrane potential and on the glucose-induced electrical activity were investigated with microelectrodes in microdissected mouse islets. In the presence of 11.1 mM glucose, the fl cells exhibited a repetitive activity. NA at more than 1 0 - 7 M induced a rapid hyperpolarization followed by a silent depolarization, then by the appearance of a slowed pace of repetitive activity (dose-dependent effects). NA at less than 10 -7 M did not markedly affect the electrical activity; it only induced a dose-dependent increase in the degree of activity with no change of the potential levels. The glucose-dependence of these effects were then investigated. In the absence of glucose, 10 - s and 10 -6 M NA did not affect the resting membrane potential. Non-stimulatory glucose concentrations (2.8-7.3 mM) progressively decreased the membrane potential. 10 - s M NA did not affect it, while 1 0 - 6 M NA induced a dose-dependent and long-lasting hyperpolarization. In the presence of stimulatory glucose concentrations (7.3-30 mM) the degree of activity increased, 10 - s M NA induced a slight leftward shift and 1 0 - 6 M NA a slight rightward shift of the dose-response curve.
Introduction Catecholamines are important physiological modulators of hormone release by the endocrine pancreas [1]. It is widely agreed that the inhibition of insulin secretion results from an activation of the alpha 2 adrenoceptors present in beta cells [2-6], but the cellular mechanisms are complex and still incompletely understood [7-8]. On the other hand, the stimulation of fl adrenoceptors increases insulin release [9-12]. This effect may, however, be species-dependent and indirect. Since the early studies of Dean and Matthews [13], numerous investigations have defined the burst pattern of the glucose-induced electrical activity in mouse pancreatic fl cells [14-17]. In contrast, only few studies have described the modifications of this electrical activity by catecholamines [13,18-19]. They were shown to induce a sustained silent inhibition [13] or a transient silent inhibition followed by a slowest burst pattern [18].
Abbreviation: NA, noradrenalin. Correspondence: M. Joffre, Laboratoire de Physiologie Animale, CNRS URA 290 Biomembranes, 40 Avenue Recteur Pineau, 86022 Poitiers cedex, France.
The present study was therefore undertaken to specify the effects of various concentrations of noradrenaline on the glucose-induced electrical activity in mouse fl cells and to investigate the glucose-dependence of these effects for low and high noradrenalin concentrations.
Materials and Methods All the experiments were performed on pancreatic islets of female N M R I mice (20-30 g) killed by decapitation 2 h after an intraperitoneal injection of pilocarpine (0.2 ml of 2% pilocarpine in 9%o NaC1), to clear the exocrin pancreas, or after an overnight fasting. Neither treatment had an effect on the glucose-induced electrical activity in fl cells. A piece of pancreas was fixed in a small chamber and islets were then partly dissected under a binocular microscope. The tissue was continuously perifused at 3 7 ° C with a bicarbonate-buffered solution (pH 7.4) which contained (mM): 122 NaC1; 4.7 KC1; 1.1 MgC12; 2.5 CaC12; 20 N a H C O 3. Glucose was added at concentrations ranging from 0 to 30 mM. The solutions were continuously gassed with a mixture of O 2 / C O 2 (95/5). Stock solutions of 10 m M N A (Serva) were prepared in 10 m M ascorbic acid (Serva) and kept frozen. Aliquots were added to media before each experiment. The addition of 10 -4 M ascorbic acid to the perifusion solu-
0167-4889/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
286 tion did not affect the burst pattern of the electrical activity induced by 11.1 m M glucose. Microelectrodes made from glass capillary tubes (Frederick Haer, U.S.A., No. 30.33.1) and filled with potassium citrate solution (2 M, p H 7.2) were used for recording the membrane potential. Their resistance ranged from 100 to 200 M~2. The/3 cells were identified by their typical repetitive activity in the presence of 11.1 m M glucose. The results are illustrated by recordings that are representative of the indicated number of experiments, and by several parameters which were measured: the membrane potential during active and silent phases, the duration of active (plateau) and silent phases (Dp and D~), the frequency of slow waves ( F ) and the degree of activity (A) determined as the percentage of active phase ( D p / ( D p + D~) is the plateau fraction). The responses to glucose and to N A were found to vary from one cell to another. The effects are expressed by the differences between the control values obtained before an application of N A and the values obtained, in the same cell, when the effect of N A reached the steady state. Dose response curves were built from the values obtained in different cells only exposed to one NA concentration or in cells exposed to increasing N A concentrations (cumulative doses) with no period of perifusion with a control medium, since no desensitization to NA was recorded. All the data are given as means (_+ S.E.) of absolute or relative values for a certain number of experiments (n). The statistical significance of the effects of the different noradrenalin concentrations was assessed by paired t-test.
0
A
NA 10 - 8 •
M
0[ E
¢J ~J
o ¢J
B
NA 1Q- 6
M
1:1
E y
°I o
-60[
c
Fig. 1. Effects of N A on the g l u c o s e - i n d u c e d electrical activity in single m o u s e /3 cells. The c o n c e n t r a t i o n of glucose was 11.1 m M t h r o u g h o u t and N A was a d d e d as i n d i c a t e d by the arrow. R e c o r d s A and B were o b t a i n e d in two different cells. They are r e p r e s e n t a t i v e of the results o b t a i n e d in 7 (10 s M) a n d 11 (10 6 M) different experiments. A: Effects of 10 s M N A . Records b and c were o b t a i n e d on steady state, respectively 10 and 15 min after the a d d i t i o n of NA. B: Effects of 10 - 6 M N A . R e c o r d s b a n d c are the direct c o n t i n u a t i o n of a, T i m e c a l i b r a t i o n (bar): 10 s.
Results
Concentration-dependence of noradrenalin effects in the presence of 11.1 m M glucose The effects of a wide range of noradrenaline concentrations ( 1 0 - t t to 1 0 - 4 M) were investigated in islets perifused with media containing 11.1 m M glucose. When mouse /3 cells were exposed to a medium containing glucose alone, they exhibited a typical electrical activity consisting of oscillations of the membrane potential (slow waves) with bursts of rapid spikes superimposed on the plateau, followed by repolarized silent phases (Fig. 1). 10 - s M noradrenalin did not markedly affect the glucose-induced electrical activity (Fig. 1A). The potential levels were never changed. A slight potentiation of the electrical activity was always recorded on steady state (ranged from 10-15 min). That was confirmed by measuring the degree of activity in 7 cells (Fig. 2D). In contrast, the average values of the other parameters were not affected by 10 - s M N A (Fig. 2A, B and C). Nevertheless, all the individual data demonstrated that
the stimulation by this low concentration lengthened the plateaus, shortened the silent phases and increased the frequency of slow waves (7 out of 7 cells). No change of the pattern, the amplitude and the frequency of the spikes was recorded. In contrast, 10 6 M N A markedly affected the pattern of the recordings, with a transient inhibition of the glucose-induced electrical activity (Fig. 1B). The membrane was initially hyperpolarized and the electrical activity suppressed. That was followed by a silent depolarization to the potential at which the electrical activity appeared in the control medium, then by the reappearance of slow waves. In the steady state, the duration of the active and silent phases were increased, this gives rise to the decrease of the degree of activity and of the frequency of slow waves (Fig. 2). The pattern, the amplitude and the frequency of the spikes were not affected. From 1 0 - l l to 10 s M, N A potentiated the glucoseinduced electrical activity in agreement with the record-
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