Ca²⁺ is an important intracellular messenger that regulates a number of processes including gene expression. The aim of this study was to investigate the role of Ca²⁺ in the regulation of immediate early genes (IEG). Expression of IEG rapidly activated by a variety of extracellular stimuli is an important part of the transcriptional intracellular machinery. IEG protein products effectively modulate the expression of ‘late’ genes.

Experiments were performed on acutely isolated pancreatic acinar cells from mice. Total RNA was reverse transcribed to cDNA and amplified by 30 cycles of PCR with the primers for c-fos, c-myc, c-jun genes and b-actin (internal control). RT-PCR reaction products were subjected to electrophoresis and relative changes in IEG expression were expressed as a percentage.

Elevated extracellular calcium ([Ca²⁺]_o) levels were alone able to activate expression of IEGs: 1 or 10 mM of Ca²⁺ resulted in 23 ± 4.0 and 44 ± 8.1 % increase for c-fos; 21 ± 6.4 and 28 ± 8.4 % for c-myc; and 15 ± 3.4 and 32 ± 5.7 % for c-jun, respectively, compared with those at 0 Ca²⁺ + 200 mM EGTA; data are expressed as means ± S.E.M., n = 6 in all experiments. Stimulation with calcium-mobilising agonists for 30 min induced much higher levels of expression. A supramaximal concentration of CCK (10 nM) evoked a pronounced [Ca²⁺]_o-dependent rise in the expression level: the IEG cDNA content was lowest in Ca²⁺-free (200 mM EGTA) solution, increased at the physiological level of 1 mM [Ca²⁺]_o and maximal at 10 mM [Ca²⁺]_o. Quantitatively it yielded an increase of: 102 ± 22 and 163 ± 15 % for c-fos; c-myc 73 ± 13 and 106 ± 24% c-jun 49 ± 8 and 59 ± 9 % at 1 and 10 mM of extracellular Ca²⁺, respectively. We suggest that extracellular calcium, through a Ca²⁺ influx mechanism, plays an important role in rapid induction of IEG expression.

We have found that the increase in c-fos expression induced by 10 mM acetylcholine (ACh) was also [Ca²⁺]_o dependent although smaller than for CCK. ACh-induced expression of c-fos in 1 and 10 mM [Ca²⁺]_o increased by 36 ± 8.1 and 62 ± 14.5 %, respectively. The addition of atropine to the 10 mM [Ca²⁺]_o containing solution did not cause any detectable changes in the c-fos expression, providing further evidence that the effect of the ACh-induced increase of c-fos expression at [Ca²⁺]_o = 10 mM is mostly due to increased transmembrane Ca²⁺ influx. Finally, we have shown that incubation of cells for 30 min with 1 mM thapsigargin in 0 Ca²⁺ + 200 mM EGTA solution did not significantly change IEG expression. Incubation of acini in solutions with 1 or 10 mM Ca and 1 mM thapsigargin caused elevation of c-fos expression by 59 ± 8 and 70 ± 7 %, respectively. We conclude that extracellular calcium through a Ca²⁺ influx mechanism plays an important role in the rapid induction of IEG expression.

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