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Intracellular free calcium mobilization in PC12h cells determined by using fura-2
265
I
P.mo.064
Matsumoto,
S., Yamada, K. and Togari, A.
It is weI known that some neurotransmitters and highly concentrated potassium (SOmM KCl,high K+) mobifize intracellular free calcium ([Ca”]i) which affects various cellular functions. Many investigators have tried to classify calcium channels from the physiological point of view using a patch clamp method, but there are few investigations which tried to clarify the participation of calcium channels in [Ca”], mobilization. To investigate the mobilization of [Cat3 Ji, we used PC12h cells as the material, because this cell line has many advantages, for example I) both r~ptor-operate calcium channel and voltag~ependent calcium channel can be activated by carbachol and high K’, 2) this cell line seems to be a suitable material for investigating the cellular differentiation. PC12h cells were maintained as monolayers in Dulbecco’s modified Pagle’s medium supplemented with 5 $5 fetal calf serum and 5 % horse serum. For the experiments, the cells were loaded with 3 FM fura-2/AM for 30 minutes at 37OC and were suspended in Hanks’ balanced salt solution. Carbachol activated the phosphatidy~ositol turnover and caused a rise of [Ca2+lj in PC12h cells. In normal Hanks’ balanced salt solution. 10 or 100 PM carbachol caused a diphasic rise of [Cat+],; first, transient phase and second, plateau phase. The first transient phase disappeared by the pretreatment with 40 mM caffeine. In calcium-deficient Hanks’ balanced salt solution, the second plateau phase was not observed. In normal Hanks’ balanced salt solution, a calcium channel blocker (20 PM verapamil), which is known to block L-type voltage-dependent calcium channel, prevented the second plateau phased partially, but did not the first transient phase at all. High K+ also caused a diphasic rise of [Ca’+]i; first, tr~sient phase and second, plateau phase, in normal Hanks’ balanced salt solution by activating the voltage-dependent calcium channel. Both the first and the second phases were not observed in calcium-deficient Hanks’ balanced salt solution. 20 gM verapamil prevented the second plateau phase intensely, but did not the first transient phase at all. In conclusion, we presume as follows: 1) The rise of [Ca’+]; by 10 or 100 pM carbachol is dependent on two calcium sources, that is, an influx of calcium trout the receptor-operated calcium channel, which was prevented by 20 gM verapamil partially and a release of calcium from the intracellular calcium storage, which was sensitive to caffeine. 2) The rise of [Ca2+]i by high K+ is dependent on the extracellular calcium and is separated into two phases. The first transient phase was not prevented by 20 FM verapamil but the second plateau phase was prevented by 20 PM verapamil intensely. So we assume that the second plateau phase is dependent on L-type voltage-dependent calcium channel, and the first transient phase is dependent on non L-type voltage-dependent calcium channel (No~cky et al., 1985). Now, we are looking for drugs which affect [Ca2’fi mobilization, especially those which prevent the first transient phase induced by high I(” (we have tried to use Ni2+, but Ni2+ did not prevent the first transient phase induced by high K’). Furthermore, we want to understand the reason why there exist various types of calcium channels in a si@e cell in view of cellular functions. Referenca? Nowyeky,M.C..Fox, A.P., and Tsien. R.W., 1985, Three types of neuronal calcium with different calcium agonist sensitivity, Nature 316, 440.
I
P.mo.064
Matsumoto,
S., Yamada, K. and Togari, A.
It is weI known that some neurotransmitters and highly concentrated potassium (SOmM KCl,high K+) mobifize intracellular free calcium ([Ca”]i) which affects various cellular functions. Many investigators have tried to classify calcium channels from the physiological point of view using a patch clamp method, but there are few investigations which tried to clarify the participation of calcium channels in [Ca”], mobilization. To investigate the mobilization of [Cat3 Ji, we used PC12h cells as the material, because this cell line has many advantages, for example I) both r~ptor-operate calcium channel and voltag~ependent calcium channel can be activated by carbachol and high K’, 2) this cell line seems to be a suitable material for investigating the cellular differentiation. PC12h cells were maintained as monolayers in Dulbecco’s modified Pagle’s medium supplemented with 5 $5 fetal calf serum and 5 % horse serum. For the experiments, the cells were loaded with 3 FM fura-2/AM for 30 minutes at 37OC and were suspended in Hanks’ balanced salt solution. Carbachol activated the phosphatidy~ositol turnover and caused a rise of [Ca2+lj in PC12h cells. In normal Hanks’ balanced salt solution. 10 or 100 PM carbachol caused a diphasic rise of [Cat+],; first, transient phase and second, plateau phase. The first transient phase disappeared by the pretreatment with 40 mM caffeine. In calcium-deficient Hanks’ balanced salt solution, the second plateau phase was not observed. In normal Hanks’ balanced salt solution, a calcium channel blocker (20 PM verapamil), which is known to block L-type voltage-dependent calcium channel, prevented the second plateau phased partially, but did not the first transient phase at all. High K+ also caused a diphasic rise of [Ca’+]i; first, tr~sient phase and second, plateau phase, in normal Hanks’ balanced salt solution by activating the voltage-dependent calcium channel. Both the first and the second phases were not observed in calcium-deficient Hanks’ balanced salt solution. 20 gM verapamil prevented the second plateau phase intensely, but did not the first transient phase at all. In conclusion, we presume as follows: 1) The rise of [Ca’+]; by 10 or 100 pM carbachol is dependent on two calcium sources, that is, an influx of calcium trout the receptor-operated calcium channel, which was prevented by 20 gM verapamil partially and a release of calcium from the intracellular calcium storage, which was sensitive to caffeine. 2) The rise of [Ca2+]i by high K+ is dependent on the extracellular calcium and is separated into two phases. The first transient phase was not prevented by 20 FM verapamil but the second plateau phase was prevented by 20 PM verapamil intensely. So we assume that the second plateau phase is dependent on L-type voltage-dependent calcium channel, and the first transient phase is dependent on non L-type voltage-dependent calcium channel (No~cky et al., 1985). Now, we are looking for drugs which affect [Ca2’fi mobilization, especially those which prevent the first transient phase induced by high I(” (we have tried to use Ni2+, but Ni2+ did not prevent the first transient phase induced by high K’). Furthermore, we want to understand the reason why there exist various types of calcium channels in a si@e cell in view of cellular functions. Referenca? Nowyeky,M.C..Fox, A.P., and Tsien. R.W., 1985, Three types of neuronal calcium with different calcium agonist sensitivity, Nature 316, 440.