Distinct protein kinases regulate SNAP‐25 expression in chromaffin cells

Abstract

The contribution of distinct Ca(2+)-sensitive protein kinases to the regulation of the expression of the synaptosomal-associated protein SNAP-25 was examined in bovine chromaffin cells. Prolonged incubation with high K(+) (38 mM) or 1,1-dimethyl-4-phenyl-piperazinium (DMPP), a nicotinic receptor agonist, significantly increased SNAP-25 protein and mRNA expression, as assessed by immunoblotting and semi-quantitative RT-PCR analysis. Both stimuli preferentially enhanced mRNA coding for the SNAP-25a isoform. Increase of SNAP-25 expression induced by K(+) or DMPP was inhibited over 70% by KN-62 and KN-93, two Ca(2+)/calmodulin-dependent protein kinase (CaMK) inhibitors, whereas the inactive analogue KN-92 only reduced the expression by 34%. The three compounds also inhibited the high K(+)-elicited [Ca(2+)](i) signal by 40%, suggesting that the effect of KN-62 and KN-93 was a combination of CaMK/ Ca(2+) influx inhibitory actions. Incubation of the cells with mitogen-activated protein kinase (MAPK) inhibitors PD98059 and U0126 reduced protein expression elicited by high K(+) by 50%, but did not modify the response to DMPP. Interestingly, although protein kinase A (PKA) inhibition by H-89 did not affect the high K(+) or DMPP-induced SNAP-25 expression, basal protein levels were significantly modified upon activation or inhibition of this pathway. Basal expression of SNAP-25 was also modified by the protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate, but not by Gö6976, a PKC-alpha inhibitor, suggesting that the Ca(2+)-insensitive PKC-epsilon isoform control basal expression of SNAP-25 in these cells. Taken together, these results provide the first evidence that diverse protein kinases might converge in the induction of SNAP-25 expression in chromaffin cells. The preferential contribution of one or another kinase would depend on the physiological or experimental conditions.

An extremely low frequency magnetic field attenuates insulin secretion from the insulinoma cell line, RIN‐m

“…SNAP-25 is a SNARE protein that is expressed in pancreatic islets and is involved in insulin release [Sadoul et al, 1995;Wheeler et al, 1996]. SNAP-25 mRNA expression was significantly increased when chromaffin cells [Garcia-Palomero et al, 2000;Montiel et al, 2003] and rat granulosa cells [Grosse et al, 2000] were stimulated to release neurotransmitters, and these events were closely related to the influx of calcium ions.…”

Dynamin-2 Regulates Fusion Pore Expansion and Quantal Release through a Mechanism that Involves Actin Dynamics in Neuroendocrine Chromaffin Cells

“…Bovine adrenal chromaffin cells were isolated as previously described [73] and incubated at 37°C in a 5% CO 2 . Cells were kept at 37°C at least 24 hours before the experiments.…”

The Association of Dynamin with Synaptophysin Regulates Quantal Size and Duration of Exocytotic Events in Chromaffin Cells

“…Bovine adrenal chromaffin cells were isolated as previously described (Montiel et al, 2003). Briefly, cells were suspended in 1:1 mixture of DMEM and HamЈs F-12 (DMEM-F12) supplemented with 10% fetal calf serum, 50 IU/ml penicillin, and 100 g/ml gentamicin.…”