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Functional relation between caffeine- and muscarine-sensitive Ca2+ stores and no Ca2+ releasing action of cyclic adenosine diphosphate-ribose in guinea-pig adrenal chromaffin cells
Neuroscience Letters 326 (2002) 167–170 www.elsevier.com/locate/neulet
Functional relation between caffeine- and muscarine-sensitive Ca 21 stores and no Ca 21 releasing action of cyclic adenosine diphosphate-ribose in guinea-pig adrenal chromaffin cells Toshio Ohta a,*, Arun R. Wakade b, Kazuki Yonekubo a, Shigeo Ito a a
Laboratory of Pharmacology, Department of Biomedical Science, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan b Department of Pharmacology, School of Medicine, Wayne State University, Detroit, MI, 48201, USA Received 21 February 2002; received in revised form 14 March 2002; accepted 19 March 2002
Abstract In voltage-clamped guinea-pig chromaffin cells, muscarine (50 mM) or caffeine (30 mM) produced a transient intracellular Ca 21 concentration ([Ca 21]i) increase, catecholamine release and an outward K 1 current mediated through Ca 21 released from internal Ca 21 stores at a holding potential of 240 mV. Caffeine followed by muscarine failed to evoke these responses, while muscarine followed by caffeine was effective in producing about 30% of [Ca 21]i increase and catecholamine secretion. In cells dialyzed with inositol 1,4,5-trisphosphate (IP3), caffeine failed to produce the [Ca 21]i increase. Intracellular application of cyclic adenosine 5 0 -diphosphate-ribose (cADP-ribose) or 8-bromo cADP-ribose exerted no effect on the resting [Ca 21]i and the caffeine-induced [Ca 21]i increase. These results suggest that IP3-sensitive stores are functionally divided into two subpopulations, sensitive and insensitive to caffeine, and it is unlikely that cADP-ribose plays a role as a Ca 21 releaser in guinea-pig adrenal chromaffin cells. q 2002 Published by Elsevier Science Ireland Ltd. Keywords: Amperometry; Cyclic adenosine 5 0 -diphosphate-ribose; Caffeine; Inositol 1,4,5-trisphosphate; Ca 21 stores; Voltage-clamp
It is generally known that there are two types of intracellular Ca 21 stores, one is sensitive to inositol 1,4,5-trisphosphate (IP3) and the other sensitive to caffeine and ryanodine [2]. In adrenal chromaffin cells, these two types of Ca 21 stores are present independently [4], and they are partly [16] or totally [9] consistent with each other. Intracellular Ca 21 concentration ([Ca 21]i) imaging study revealed that IP3-mobilizing agonists increase [Ca 21]i around the nucleus, while caffeine increases [Ca 21]i overall in chromaffin cells [3]. However, the functional relation between these two Ca 21 stores in the regulation of catecholamine secretion in adrenal chromaffin cells has not been examined. We showed that guinea-pig chromaffin cells possess both caffeine- and muscarine-sensitive stores [12]. Under Ca 21-free conditions, ryanodine produces complete abolition of the [Ca 21]i increase induced by caffeine but only partially inhibits that induced by muscarine. Since muscarine was ineffective in producing a [Ca 21]i transient in cells injected with IP3, we hypothesized that muscarine (IP3)-sensitive stores * Corresponding author. Tel.: 181-11-706-5245; fax: 181-11706-5220. E-mail address: [email protected] (T. Ohta).
are partly consistent with caffeine-sensitive ones. However, in the absence of external Ca 21, it is possible that the removal of external Ca 21 may promote the leakage of Ca 21 at different rates between the two stores. Furthermore, since muscarine and caffeine produce changes in membrane potential [12,14], such potential changes may affect [Ca 21]i mobilization through changes in the activities of voltagedependent Ca 21 channels. Recently, we have reported that under voltage-clamp conditions, [Ca 21]i responses to muscarine or caffeine are mainly mediated through Ca 21 released from internal stores even in the presence of extracellular Ca 21 [13,14]. In the present experiment, therefore, to elucidate the functional relation between internal stores sensitive to muscarine (IP3), we compared the [Ca 21]i increases, catecholamine releases and membrane currents induced by sequentially applied muscarine and caffeine in voltage-clamped guinea-pig adrenal chromaffin cells. Cyclic adenosine diphosphate-ribose (cADP-ribose), which is a metabolite of NAD 1 produced by a catalytic enzyme, adenosine diphosphate (ADP)-ribosyl cyclase, has been reported to be an endogenous agonist for ryanodine receptors and promote Ca 21 release from internal stores [7]. In bovine adrenal chromaffin cells, cADP-ribose evokes
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Ca 21 release from internal stores and ADP-ribosyl cyclase is activated by acetylcholine [11]. A clone of neurosecretory PC12 cells (PC12-16A) is also reported to respond to cADPribose [5]. On the other hand, in bovine chromaffin cells that express an endoplasmic reticulum-targeted aequorin, cADP-ribose exerts no Ca 21 releasing action [1]. Therefore, we examined the effect of cADP-ribose and 8-bromo cADPribose, an antagonist of cADP-ribose injected into cells on the resting [Ca 21]i and the caffeine-induced [Ca 21]i increase in guinea-pig chromaffin cells. All studies were performed under the regulations of the Animal Research Committee in the Graduate School of Veterinary Medicine, Hokkaido University. Male guineapigs (Hartley, 250–400 g) were stunned and bled to death. Adrenal chromaffin cells were prepared by the collagenase digestive method [12]. Cells were superfused with an external solution continuously at a flow rate of 2–3 ml/min through a multibarreled puffer pipette placed close to the cells. All experiments were carried out at room temperature (20–25 8C). The [Ca 21]i in single cells was measured with fura-2 as described previously [12]. Adrenal chromaffin cells were incubated with fura-2 acetoxymethyl ester (fura-2-AM, 5 mM) for 1 h at room temperature to load fura-2 into cells. Membrane currents were measured with the nystatin-perforated or standard whole-cell patch-clamp technique using a patch-clamp amplifier (Axopatch 200B, Axon Instruments, USA) [13]. Catecholamine secretion from single cells was detected amperometrically using carbon microelectrodes [14]. The composition of the external solution was: 134 mM NaCl; 6 mM KCl; 2.5 mM CaCl2; 1.2 mM MgCl2; 10 mM glucose; 10 mM N-[2-hydroxyethyl]piperazine-N 0 -[2-ethanesulfonic acid] (HEPES; pH 7.4 with NaOH). The pipette solution had the following composition: 140 mM KCl; 1.2 mM MgCl2; 1 mM ATPNa2; 10 mM HEPES; 0.05 mM ethylenebis(oxononitrilo)tetraacetate (EGTA; pH 7.2 with KOH). Nystatin (0.1–0.2 mg/ml) was supplemented for perforated-patch-clamp measurement. Under standard whole-cell patch-clamp conditions, the EGTA-free pipette solution including fura-2 (0.1 mM) was used. The drugs used were: caffeine (Wako Pure Chem, Japan); fura-2, Fura-2-AM, and IP3 (Dojindo, Japan); cADP-ribose, 8-bromo cADP-ribose (Sigma, USA). Cyclic ADP-ribose from Yamasa (Japan) was also used. Results are expressed as mean values ^ SEM (n ¼ number of observations), and significance was assessed by using the unpaired Student’s t-test. P , 0:05 was considered significant. At a holding potential of 240 mV, fura-2-loaded guineapig chromaffin cells were sequentially stimulated by muscarine (50 mM) followed by caffeine (30 mM) with a 10-s interval or stimulated in the opposite order (Fig. 1). The first application of muscarine or caffeine rapidly increased the [Ca 21]i concomitant with a transient outward current (Iout), being mediated by the activation of Ca 21-dependent K 1 channels, via Ca 21 released from internal stores [13,14]. Muscarine increased [Ca 21]i more but caused an equal or smaller amplitude of Iout compared with caffeine [14].
Caffeine evoked a slight, if any, [Ca 21]i increase after application of muscarine. On the other hand, after caffeine stimulation, muscarine was effective in evoking a [Ca 21]i increase of about one-third of the first response but Iout induced by muscarine was changed to an inward current. Similar to [Ca 21]i responses, both stimuli applied the first time were effective in producing catecholamine secretion. However, caffeine followed by muscarine failed to evoke catecholamine secretion. On the contrary, muscarine followed by caffeine was about 30% effective in evoking catecholamine secretion (Fig. 1B). To estimate the relationship between caffeine- and muscarine (IP3)-sensitive stores, the effects of IP3 injected into cells on caffeine-induced responses were examined (Fig. 2). As a standard, [Ca 21]i and current responses to nicotine (50 mM) and a depolarizing pulse to 110 mV for 1 s were also examined. In these experiments, cells were conventional whole-cell voltage-clamped at 240 mV. In control cells (without IP3), after achieving the whole-cell mode, application of caffeine produced a transient [Ca 21]i increase in parallel with transient Iout. The second application of caffeine produced almost the same amplitude of these responses as the first ones. Nicotine evoked a slight increase of [Ca 21]i and a rapid inward current, and a depolarizing pulse also produced a transient increase of [Ca 21]i. Using a patch pipette containing IP3 (0.1 mM), a rapid [Ca 21]i increase and small Iout appeared just after the breakthrough of the patch membrane, because IP3 diffused into cells and provoked Ca 21 release from IP3-sensitive stores [12,13]. In cells dialyzed with IP3, no responses were elicited by the first and second application of caffeine, while nicotine- and depolarization-induced [Ca 21]i increases were unchanged (Fig. 3A,B). Unlike IP3, an injection of cADP-ribose never elicited changes in [Ca 21]i and membrane currents (Fig. 2C). In addition, it did not affect the caffeine-induced [Ca 21]i increase and Iout. Moreover, this was also the case in the second responses to caffeine a few minutes after dialysis of cADP-ribose, so that the diffusion rate of cADP-ribose did not affect the caffeineinduced responses. The summarized data are shown in Fig. 3C. Also, no effects of cADP-ribose were exerted at different concentrations used (0.05, 0.1 mM). 8-Bromo cADP-ribose (0.2 mM), an antagonist of cADP-ribose, also failed to affect the caffeine-induced responses (Fig. 2D). In the present experiment, we successfully compared the functional relation of [Ca 21]i increases mediated via stores sensitive to muscarine (IP3) and caffeine in the presence of external Ca 21 under voltage-clamped conditions. The transient [Ca 21]i response to caffeine was completely abolished after application of muscarine or in cells dialyzed with IP3, while muscarine was still effective in evoking a [Ca 21]i increase after caffeine stimulation. We have reported that transient elevations of [Ca 21]i in response to muscarine in voltage-clamped cells mainly originate from Ca 21 released from internal stores [13]. Recently, we also reported that
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caffeine elicits a transient [Ca 21]i increase released from internal stores that results in Iout in voltage-clamped guinea-pig chromaffin cells [14]. These were confirmed with the present experiments. Similar to [Ca 21]i responses, it is reasonable that caffeine was ineffective in exocytosis and Iout after application of muscarine. On the contrary, muscarine could evoke catecholamine secretion, but failed to elicit Iout after caffeine stimulation. These results suggest that a caffeine-insensitive component in muscarine (IP3)sensitive stores contributes to catecholamine release more than activation of Ca 21-dependent K 1 channels, indicating that there is functional heterogeneity of IP3-sensitive Ca 21 stores. Therefore, the present results hypothesize that there are at least functionally two subpopulations of stores sensitive to IP3 in guinea-pig adrenal chromaffin cells, one sensitive to IP3 and caffeine, the other only to IP3. This hypothesis supports our previous report that about 25% of the [Ca 21]i increase in response to muscarine is left after treatment with ryanodine, under which condition the [Ca 21]i increase induced by caffeine is completely abolished [12]. In vascular smooth muscle cells, the internal Ca 21 stores have been functionally divided into two based on the sensitivity to IP3 and caffeine, and designated as Sa and Sb. Sa is sensitive to both caffeine and IP3, and Sb to IP3 only [17]. In chromaffin cells from various species, IP3-sensitive stores have been Fig. 2. Representative effects of IP3, cADP-ribose or 8-bromo cADP-ribose applied intracellularly on the resting [Ca 21]i and caffeine-induced [Ca 21]i increase in the voltage-clamped cells. (A) Control, membrane current (Im) and [Ca 21]i responses without IP3, cADP-ribose or 8-bromo cADP-ribose in the pipette. Responses in cells injected with IP3 (B: 0.1 mM), cADP-ribose (C: 0.1 mM) or 8-bromo cADP-ribose (D: 0.2 mM). Repetitive hyperpolarizing pulses (210 mV, 100 ms) were applied at 1 s intervals before and after the breakthrough of the patch membrane. The transition to the whole-cell mode resulted in increase of the capacitative currents. About 0.5 min after the breakthrough of the patch membrane, cells were stimulated with a sequence of caffeine (30 mM, 20 s), nicotine (50 mM, 10 s), depolarizing pulse to 110 mV for 1 s and caffeine again. Outward currents evoked by depolarizing pulses are tunicated by double ramp lines. Fig. 1. Current, [Ca 21]i and secretory responses to sequentially applied muscarine and caffeine in voltage-clamped guinea-pig chromaffin cells. (A) Representative membrane current (Im), [Ca 21]i and catecholamine secretion induced by muscarine (50 mM, 20 s) and caffeine (30 mM, 20 s) with a 10-s interval (left) and by the reversed order (right). Results from the same cell. Cells were voltage-clamped at 240 mV. An arrowhead next to the current trace in this figure and the next figure indicates the zero current level. (B) Increments of [Ca 21]i (upper) and catecholamine secretory responses (lower) to muscarine followed by caffeine (left) and those induced by the opposite order (right). The increment of [Ca 21]i induced by muscarine (hatched columns) or caffeine (open columns) was estimated as the peak value of [Ca 21]i during the stimulation minus the level of the [Ca 21]i prior to the stimulation. The changes in amperometric current were assessed by calculating the area below the curve (the time-integral) during application of the drugs. Data show mean ^ SEM (n ¼ 7).
reported to overlap with caffeine-sensitive stores completely [9] or partly [16] or to be completely different [4] from caffeine-sensitive stores. Although there may be some species difference, most of these experiments were carried out under Ca 21-free conditions. Therefore, there may be some underestimation due to the different capacities of Ca 21 between caffeine- and IP3-sensitive Ca 21 stores. In the present experiment, we could analyze Ca 21 storemediated responses without removal of external Ca 21 in voltage-clamped cells. Therefore, voltage-clamping may be a good manipulation to examine the function of the internal stores. In bovine chromaffin cells, cADP-ribose is synthesized by nicotinic stimulation and produces Ca 21 release from
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[2] [3]
[4]
[5]
Fig. 3. Summarized effects of IP3 (0.1 mM) and cADP-ribose (0.1 mM) on the resting [Ca 21]i and on the [Ca 21]i increase in response to caffeine (30 mM), nicotine (50 mM) and depolarizing pulses (110 mV, 1 s). Columns show the change in [Ca 21]i induced by the rupture of the patch membrane using patch solution without IP3 and cADP-ribose (A: control), with 0.1 mM IP3 (B), and 0.1 mM cADP-ribose (C). The increment of [Ca 21]i induced by each stimulation was estimated as the peak value of [Ca 21]i during the stimulation minus the level of the [Ca 21]i prior to the stimulation. Data show mean ^ SEM (control, n ¼ 6; IP3, n ¼ 6; cADP-ribose, n ¼ 6). *P , 0:05, significant difference vs. control.
[6]
[7] [8]
[9]
digitonin permeabilized cells [11]. However, in an experiment using endoplasmic reticulum-targeted aequorin, cADP-ribose was reported to have no Ca 21 releasing action [1]. In the present experiment, cADP-ribose injected into cells did not change the resting [Ca 21]i and had no effect on the caffeine-induced responses even a few minutes after its injection. We used two commercially available cADPriboses but the same results were obtained. Moreover, 8bromo cADP-ribose, an antagonist of cADP-ribose, did not affect the caffeine-induced responses. Ryanodine receptors are reported to be divided into three subtypes (RR1,2,3) [6] and cADP-ribose is reported to act on the cardiac (RR2) and brain types (RR3) [15], but not on the skeletal type (RR1) [10]. It has been reported that the RR3 type is expressed in the porcine adrenal gland [8]. Therefore, different types of ryanodine receptors or some splicing variants, which are insensitive to cADP-ribose, might be expressed in guinea-pig chromaffin cells. Overall, from the present results, we conclude that cADP-ribose seems not to function as a Ca 21 releaser in guinea-pig chromaffin cells. This study was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture, Japan and the Uehara Foundation.
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[17] [1] Alonso, M.T., Barrero, M.J., Michelena, P., Carnicero, E., Cuchillo, I., Garcia, A.G., Garcia-Sancho, J., Montero, M. and Alverez, J., Ca 21-induced Ca 21 release in chromaffin
cells seen from inside the ER with targeted aequorin, J. Cell Biol., 144 (1999) 241–254. Berridge, M.J., Neuronal calcium signaling, Neuron, 21 (1998) 13–26. Burgoyne, R.D., Cheek, T.R., Morgan, A., O’Sullivan, A.J., Moreton, R.B., Berridge, M.J., Mata, A.M., Colyer, J., Lee, A.G. and East, J.M., Distribution of two distinct Ca 21ATPase-like proteins and their relationships to the agonist-sensitive calcium store in adrenal chromaffin cells, Nature, 342 (1989) 72–74. Cheek, T.R., Barry, V.A., Berridge, M.J. and Missiaen, L., Bovine adrenal chromaffin cells contain an inositol 1,4,5trisphosphate-insensitive but caffeine-sensitive Ca 21 store that can be regulated by intraluminal free Ca 21, Biochem. J., 275 (1991) 697–701. Clementi, E., Riccio, M., Sciorati, C., Nistico, G. and Meldolesi, J., The type 2 ryanodine receptor of neurosecretory PC12 cells is activated by cyclic ADP-ribose. Role of the nitric oxide/cGMP pathway, J. Biol. Chem., 271 (1996) 17739–17745. Giannini, G., Conti, A., Mammarella, S., Scrobogna, M. and Sorrentino, V., The ryanodine receptor/calcium channel genes are widely and differentially expressed in murine brain and peripheral tissues, J. Cell Biol., 128 (1995) 893– 904. Guse, A.H., Cyclic ADP-ribose: a novel Ca 21-mobilising second messenger, Cell. Signal., 11 (1999) 309–316. Ledbetter, M.W., Preiner, J.K., Louis, C.F. and Mickelson, J.R., Tissue distribution of ryanodine receptor isoforms and alleles determined by reverse transcription polymerase chain reaction, J. Biol. Chem., 269 (1994) 31544–31551. Liu, P.S., Lin, Y.J. and Kao, L.S., Caffeine-sensitive calcium stores in bovine adrenal chromaffin cells, J. Neurochem., 56 (1991) 172–177. Meszaros, L.G., Bak, J. and Chu, A., Cyclic ADP-ribose as an endogenous regulator of the non-skeletal type ryanodine receptor Ca 21 channel, Nature, 364 (1993) 76–79. Morita, K., Kitayama, S. and Dohi, T., Stimulation of cyclic ADP-ribose synthesis by acetylcholine and its role in catecholamine release in bovine adrenal chromaffin cells, J. Biol. Chem., 272 (1997) 21002–21009. Ohta, T., Asano, T., Ito, S., Kitamura, N. and Nakazato, Y., Characteristics of cytosolic Ca 21 elevation induced by muscarinic receptor activation in single adrenal chromaffin cells of the guinea pig, Cell Calcium, 20 (1996) 303–314. Ohta, T., Ito, S. and Nakazato, Y., Ca 21-dependent K 1 currents induced by muscarinic receptor activation in guinea pig adrenal chromaffin cells, J. Neurochem., 70 (1998) 1280–1288. Ohta, T., Wakade, A.R., Nakazato, Y. and Ito, S., Ca 21-dependent K 1 current and exocytosis in responses to caffeine and muscarine in voltage-clamped guinea-pig adrenal chromaffin cells, J. Neurochem., 78 (2001) 1–14. Sonnleitner, A., Conti, A., Bertocchini, F., Schindler, H. and Sorrentino, V., Functional properties of the ryanodine receptor type 3 (RyR3) Ca 21 release channel, EMBO J., 17 (1998) 2790–2798. Sorimachi, M., Yamagami, K. and Nishimura, S., A muscarinic receptor agonist mobilizes Ca 21 from caffeine and inositol-1,4,5-trisphosphate-sensitive Ca 21 stores in cat adrenal chromaffin cells, Brain Res., 571 (1992) 154–158. Yamazawa, T., Iino, M. and Endo, M., Presence of functionally different compartments of the Ca 21 store in single intestinal smooth muscle cells, FEBS Lett., 301 (1992) 181– 184.
Functional relation between caffeine- and muscarine-sensitive Ca 21 stores and no Ca 21 releasing action of cyclic adenosine diphosphate-ribose in guinea-pig adrenal chromaffin cells Toshio Ohta a,*, Arun R. Wakade b, Kazuki Yonekubo a, Shigeo Ito a a
Laboratory of Pharmacology, Department of Biomedical Science, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan b Department of Pharmacology, School of Medicine, Wayne State University, Detroit, MI, 48201, USA Received 21 February 2002; received in revised form 14 March 2002; accepted 19 March 2002
Abstract In voltage-clamped guinea-pig chromaffin cells, muscarine (50 mM) or caffeine (30 mM) produced a transient intracellular Ca 21 concentration ([Ca 21]i) increase, catecholamine release and an outward K 1 current mediated through Ca 21 released from internal Ca 21 stores at a holding potential of 240 mV. Caffeine followed by muscarine failed to evoke these responses, while muscarine followed by caffeine was effective in producing about 30% of [Ca 21]i increase and catecholamine secretion. In cells dialyzed with inositol 1,4,5-trisphosphate (IP3), caffeine failed to produce the [Ca 21]i increase. Intracellular application of cyclic adenosine 5 0 -diphosphate-ribose (cADP-ribose) or 8-bromo cADP-ribose exerted no effect on the resting [Ca 21]i and the caffeine-induced [Ca 21]i increase. These results suggest that IP3-sensitive stores are functionally divided into two subpopulations, sensitive and insensitive to caffeine, and it is unlikely that cADP-ribose plays a role as a Ca 21 releaser in guinea-pig adrenal chromaffin cells. q 2002 Published by Elsevier Science Ireland Ltd. Keywords: Amperometry; Cyclic adenosine 5 0 -diphosphate-ribose; Caffeine; Inositol 1,4,5-trisphosphate; Ca 21 stores; Voltage-clamp
It is generally known that there are two types of intracellular Ca 21 stores, one is sensitive to inositol 1,4,5-trisphosphate (IP3) and the other sensitive to caffeine and ryanodine [2]. In adrenal chromaffin cells, these two types of Ca 21 stores are present independently [4], and they are partly [16] or totally [9] consistent with each other. Intracellular Ca 21 concentration ([Ca 21]i) imaging study revealed that IP3-mobilizing agonists increase [Ca 21]i around the nucleus, while caffeine increases [Ca 21]i overall in chromaffin cells [3]. However, the functional relation between these two Ca 21 stores in the regulation of catecholamine secretion in adrenal chromaffin cells has not been examined. We showed that guinea-pig chromaffin cells possess both caffeine- and muscarine-sensitive stores [12]. Under Ca 21-free conditions, ryanodine produces complete abolition of the [Ca 21]i increase induced by caffeine but only partially inhibits that induced by muscarine. Since muscarine was ineffective in producing a [Ca 21]i transient in cells injected with IP3, we hypothesized that muscarine (IP3)-sensitive stores * Corresponding author. Tel.: 181-11-706-5245; fax: 181-11706-5220. E-mail address: [email protected] (T. Ohta).
are partly consistent with caffeine-sensitive ones. However, in the absence of external Ca 21, it is possible that the removal of external Ca 21 may promote the leakage of Ca 21 at different rates between the two stores. Furthermore, since muscarine and caffeine produce changes in membrane potential [12,14], such potential changes may affect [Ca 21]i mobilization through changes in the activities of voltagedependent Ca 21 channels. Recently, we have reported that under voltage-clamp conditions, [Ca 21]i responses to muscarine or caffeine are mainly mediated through Ca 21 released from internal stores even in the presence of extracellular Ca 21 [13,14]. In the present experiment, therefore, to elucidate the functional relation between internal stores sensitive to muscarine (IP3), we compared the [Ca 21]i increases, catecholamine releases and membrane currents induced by sequentially applied muscarine and caffeine in voltage-clamped guinea-pig adrenal chromaffin cells. Cyclic adenosine diphosphate-ribose (cADP-ribose), which is a metabolite of NAD 1 produced by a catalytic enzyme, adenosine diphosphate (ADP)-ribosyl cyclase, has been reported to be an endogenous agonist for ryanodine receptors and promote Ca 21 release from internal stores [7]. In bovine adrenal chromaffin cells, cADP-ribose evokes
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Ca 21 release from internal stores and ADP-ribosyl cyclase is activated by acetylcholine [11]. A clone of neurosecretory PC12 cells (PC12-16A) is also reported to respond to cADPribose [5]. On the other hand, in bovine chromaffin cells that express an endoplasmic reticulum-targeted aequorin, cADP-ribose exerts no Ca 21 releasing action [1]. Therefore, we examined the effect of cADP-ribose and 8-bromo cADPribose, an antagonist of cADP-ribose injected into cells on the resting [Ca 21]i and the caffeine-induced [Ca 21]i increase in guinea-pig chromaffin cells. All studies were performed under the regulations of the Animal Research Committee in the Graduate School of Veterinary Medicine, Hokkaido University. Male guineapigs (Hartley, 250–400 g) were stunned and bled to death. Adrenal chromaffin cells were prepared by the collagenase digestive method [12]. Cells were superfused with an external solution continuously at a flow rate of 2–3 ml/min through a multibarreled puffer pipette placed close to the cells. All experiments were carried out at room temperature (20–25 8C). The [Ca 21]i in single cells was measured with fura-2 as described previously [12]. Adrenal chromaffin cells were incubated with fura-2 acetoxymethyl ester (fura-2-AM, 5 mM) for 1 h at room temperature to load fura-2 into cells. Membrane currents were measured with the nystatin-perforated or standard whole-cell patch-clamp technique using a patch-clamp amplifier (Axopatch 200B, Axon Instruments, USA) [13]. Catecholamine secretion from single cells was detected amperometrically using carbon microelectrodes [14]. The composition of the external solution was: 134 mM NaCl; 6 mM KCl; 2.5 mM CaCl2; 1.2 mM MgCl2; 10 mM glucose; 10 mM N-[2-hydroxyethyl]piperazine-N 0 -[2-ethanesulfonic acid] (HEPES; pH 7.4 with NaOH). The pipette solution had the following composition: 140 mM KCl; 1.2 mM MgCl2; 1 mM ATPNa2; 10 mM HEPES; 0.05 mM ethylenebis(oxononitrilo)tetraacetate (EGTA; pH 7.2 with KOH). Nystatin (0.1–0.2 mg/ml) was supplemented for perforated-patch-clamp measurement. Under standard whole-cell patch-clamp conditions, the EGTA-free pipette solution including fura-2 (0.1 mM) was used. The drugs used were: caffeine (Wako Pure Chem, Japan); fura-2, Fura-2-AM, and IP3 (Dojindo, Japan); cADP-ribose, 8-bromo cADP-ribose (Sigma, USA). Cyclic ADP-ribose from Yamasa (Japan) was also used. Results are expressed as mean values ^ SEM (n ¼ number of observations), and significance was assessed by using the unpaired Student’s t-test. P , 0:05 was considered significant. At a holding potential of 240 mV, fura-2-loaded guineapig chromaffin cells were sequentially stimulated by muscarine (50 mM) followed by caffeine (30 mM) with a 10-s interval or stimulated in the opposite order (Fig. 1). The first application of muscarine or caffeine rapidly increased the [Ca 21]i concomitant with a transient outward current (Iout), being mediated by the activation of Ca 21-dependent K 1 channels, via Ca 21 released from internal stores [13,14]. Muscarine increased [Ca 21]i more but caused an equal or smaller amplitude of Iout compared with caffeine [14].
Caffeine evoked a slight, if any, [Ca 21]i increase after application of muscarine. On the other hand, after caffeine stimulation, muscarine was effective in evoking a [Ca 21]i increase of about one-third of the first response but Iout induced by muscarine was changed to an inward current. Similar to [Ca 21]i responses, both stimuli applied the first time were effective in producing catecholamine secretion. However, caffeine followed by muscarine failed to evoke catecholamine secretion. On the contrary, muscarine followed by caffeine was about 30% effective in evoking catecholamine secretion (Fig. 1B). To estimate the relationship between caffeine- and muscarine (IP3)-sensitive stores, the effects of IP3 injected into cells on caffeine-induced responses were examined (Fig. 2). As a standard, [Ca 21]i and current responses to nicotine (50 mM) and a depolarizing pulse to 110 mV for 1 s were also examined. In these experiments, cells were conventional whole-cell voltage-clamped at 240 mV. In control cells (without IP3), after achieving the whole-cell mode, application of caffeine produced a transient [Ca 21]i increase in parallel with transient Iout. The second application of caffeine produced almost the same amplitude of these responses as the first ones. Nicotine evoked a slight increase of [Ca 21]i and a rapid inward current, and a depolarizing pulse also produced a transient increase of [Ca 21]i. Using a patch pipette containing IP3 (0.1 mM), a rapid [Ca 21]i increase and small Iout appeared just after the breakthrough of the patch membrane, because IP3 diffused into cells and provoked Ca 21 release from IP3-sensitive stores [12,13]. In cells dialyzed with IP3, no responses were elicited by the first and second application of caffeine, while nicotine- and depolarization-induced [Ca 21]i increases were unchanged (Fig. 3A,B). Unlike IP3, an injection of cADP-ribose never elicited changes in [Ca 21]i and membrane currents (Fig. 2C). In addition, it did not affect the caffeine-induced [Ca 21]i increase and Iout. Moreover, this was also the case in the second responses to caffeine a few minutes after dialysis of cADP-ribose, so that the diffusion rate of cADP-ribose did not affect the caffeineinduced responses. The summarized data are shown in Fig. 3C. Also, no effects of cADP-ribose were exerted at different concentrations used (0.05, 0.1 mM). 8-Bromo cADP-ribose (0.2 mM), an antagonist of cADP-ribose, also failed to affect the caffeine-induced responses (Fig. 2D). In the present experiment, we successfully compared the functional relation of [Ca 21]i increases mediated via stores sensitive to muscarine (IP3) and caffeine in the presence of external Ca 21 under voltage-clamped conditions. The transient [Ca 21]i response to caffeine was completely abolished after application of muscarine or in cells dialyzed with IP3, while muscarine was still effective in evoking a [Ca 21]i increase after caffeine stimulation. We have reported that transient elevations of [Ca 21]i in response to muscarine in voltage-clamped cells mainly originate from Ca 21 released from internal stores [13]. Recently, we also reported that
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caffeine elicits a transient [Ca 21]i increase released from internal stores that results in Iout in voltage-clamped guinea-pig chromaffin cells [14]. These were confirmed with the present experiments. Similar to [Ca 21]i responses, it is reasonable that caffeine was ineffective in exocytosis and Iout after application of muscarine. On the contrary, muscarine could evoke catecholamine secretion, but failed to elicit Iout after caffeine stimulation. These results suggest that a caffeine-insensitive component in muscarine (IP3)sensitive stores contributes to catecholamine release more than activation of Ca 21-dependent K 1 channels, indicating that there is functional heterogeneity of IP3-sensitive Ca 21 stores. Therefore, the present results hypothesize that there are at least functionally two subpopulations of stores sensitive to IP3 in guinea-pig adrenal chromaffin cells, one sensitive to IP3 and caffeine, the other only to IP3. This hypothesis supports our previous report that about 25% of the [Ca 21]i increase in response to muscarine is left after treatment with ryanodine, under which condition the [Ca 21]i increase induced by caffeine is completely abolished [12]. In vascular smooth muscle cells, the internal Ca 21 stores have been functionally divided into two based on the sensitivity to IP3 and caffeine, and designated as Sa and Sb. Sa is sensitive to both caffeine and IP3, and Sb to IP3 only [17]. In chromaffin cells from various species, IP3-sensitive stores have been Fig. 2. Representative effects of IP3, cADP-ribose or 8-bromo cADP-ribose applied intracellularly on the resting [Ca 21]i and caffeine-induced [Ca 21]i increase in the voltage-clamped cells. (A) Control, membrane current (Im) and [Ca 21]i responses without IP3, cADP-ribose or 8-bromo cADP-ribose in the pipette. Responses in cells injected with IP3 (B: 0.1 mM), cADP-ribose (C: 0.1 mM) or 8-bromo cADP-ribose (D: 0.2 mM). Repetitive hyperpolarizing pulses (210 mV, 100 ms) were applied at 1 s intervals before and after the breakthrough of the patch membrane. The transition to the whole-cell mode resulted in increase of the capacitative currents. About 0.5 min after the breakthrough of the patch membrane, cells were stimulated with a sequence of caffeine (30 mM, 20 s), nicotine (50 mM, 10 s), depolarizing pulse to 110 mV for 1 s and caffeine again. Outward currents evoked by depolarizing pulses are tunicated by double ramp lines. Fig. 1. Current, [Ca 21]i and secretory responses to sequentially applied muscarine and caffeine in voltage-clamped guinea-pig chromaffin cells. (A) Representative membrane current (Im), [Ca 21]i and catecholamine secretion induced by muscarine (50 mM, 20 s) and caffeine (30 mM, 20 s) with a 10-s interval (left) and by the reversed order (right). Results from the same cell. Cells were voltage-clamped at 240 mV. An arrowhead next to the current trace in this figure and the next figure indicates the zero current level. (B) Increments of [Ca 21]i (upper) and catecholamine secretory responses (lower) to muscarine followed by caffeine (left) and those induced by the opposite order (right). The increment of [Ca 21]i induced by muscarine (hatched columns) or caffeine (open columns) was estimated as the peak value of [Ca 21]i during the stimulation minus the level of the [Ca 21]i prior to the stimulation. The changes in amperometric current were assessed by calculating the area below the curve (the time-integral) during application of the drugs. Data show mean ^ SEM (n ¼ 7).
reported to overlap with caffeine-sensitive stores completely [9] or partly [16] or to be completely different [4] from caffeine-sensitive stores. Although there may be some species difference, most of these experiments were carried out under Ca 21-free conditions. Therefore, there may be some underestimation due to the different capacities of Ca 21 between caffeine- and IP3-sensitive Ca 21 stores. In the present experiment, we could analyze Ca 21 storemediated responses without removal of external Ca 21 in voltage-clamped cells. Therefore, voltage-clamping may be a good manipulation to examine the function of the internal stores. In bovine chromaffin cells, cADP-ribose is synthesized by nicotinic stimulation and produces Ca 21 release from
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[2] [3]
[4]
[5]
Fig. 3. Summarized effects of IP3 (0.1 mM) and cADP-ribose (0.1 mM) on the resting [Ca 21]i and on the [Ca 21]i increase in response to caffeine (30 mM), nicotine (50 mM) and depolarizing pulses (110 mV, 1 s). Columns show the change in [Ca 21]i induced by the rupture of the patch membrane using patch solution without IP3 and cADP-ribose (A: control), with 0.1 mM IP3 (B), and 0.1 mM cADP-ribose (C). The increment of [Ca 21]i induced by each stimulation was estimated as the peak value of [Ca 21]i during the stimulation minus the level of the [Ca 21]i prior to the stimulation. Data show mean ^ SEM (control, n ¼ 6; IP3, n ¼ 6; cADP-ribose, n ¼ 6). *P , 0:05, significant difference vs. control.
[6]
[7] [8]
[9]
digitonin permeabilized cells [11]. However, in an experiment using endoplasmic reticulum-targeted aequorin, cADP-ribose was reported to have no Ca 21 releasing action [1]. In the present experiment, cADP-ribose injected into cells did not change the resting [Ca 21]i and had no effect on the caffeine-induced responses even a few minutes after its injection. We used two commercially available cADPriboses but the same results were obtained. Moreover, 8bromo cADP-ribose, an antagonist of cADP-ribose, did not affect the caffeine-induced responses. Ryanodine receptors are reported to be divided into three subtypes (RR1,2,3) [6] and cADP-ribose is reported to act on the cardiac (RR2) and brain types (RR3) [15], but not on the skeletal type (RR1) [10]. It has been reported that the RR3 type is expressed in the porcine adrenal gland [8]. Therefore, different types of ryanodine receptors or some splicing variants, which are insensitive to cADP-ribose, might be expressed in guinea-pig chromaffin cells. Overall, from the present results, we conclude that cADP-ribose seems not to function as a Ca 21 releaser in guinea-pig chromaffin cells. This study was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture, Japan and the Uehara Foundation.
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