Abstract
Although methionine sulfoxide reductase (Msr) is known to modulate the activity of multiple functional proteins, the roles of Msr in pancreatic stellate cell physiology have not been reported. In the present work we investigated expression and function of Msr in freshly isolated and cultured rat pancreatic stellate cells. Msr expression was determined by RT-PCR, Western blot and immunocytochemistry. Msr over-expression was achieved by transfection with adenovirus vectors. Pancreatic stellate cells were co-cultured with pancreatic acinar cells AR4-2J in monolayer culture. Pancreatic stellate and acinar cell function was monitored by Fura-2 calcium imaging. Rat pancreatic stellate cells were found to express MsrA, B1, B2, their expressions diminished in culture. Over-expressions of MsrA, B1 or B2 were found to enhance ATP-stimulated calcium increase but decreased reactive oxygen species generation and lipopolysaccharide-elicited IL-1 production. Pancreatic stellate cell-co-culture with AR4-2J blunted cholecystokinin- and acetylcholine-stimulated calcium increases in AR4-2J, depending on acinar/stellate cell ratio, this inhibition was reversed by MsrA, B1 over-expression in stellate cells or by Met supplementation in the co-culture medium. These data suggest that Msr play important roles in pancreatic stellate cell function and the stellate cells may serve as a brake mechanism on pancreatic acinar cell calcium signaling modulated by stellate cell Msr expression.
Highlights
MsrA, B1 over-expression in stellate cells or by Met supplementation in the co-culture medium. These data suggest that methionine sulfoxide reductase (Msr) play important roles in pancreatic stellate cell function and the stellate cells may serve as a brake mechanism on pancreatic acinar cell calcium signaling modulated by stellate cell Msr expression
In view of the strategic location of the stellate cells surrounding the basal plasma membrane of the acinar cells where all the acinar cell surface receptors are located, we suggest that pancreatic stellate cells provide a brake mechanism on pancreatic acinar cell surface receptor activation and calcium signaling
RT-PCR measurements of mRNA contents of Msr revealed that MsrA, B1, B2 were all expressed in the freshly isolated rat pancreatic stellate cells, the expression level gradually decreased with time in culture, which was up to 4 weeks after isolation (Figure 1A)
Summary
Reactive oxygen species (O2 . , H2 O2 , OH. , 1 O2 , HClO) can all oxidize both structural and signaling proteins, at the sulfur-containing cysteine (Cys) and methionine (Met) residues [1,2,3].Met residue oxidation to Met sulfoxide [Met(O)] often results in major changes in protein activity: activation of calcium/calmodulin-dependent protein kinase II and of BK channels, inhibition of Kv channels, inhibition of the fibrillation process of apolipoproteins, β-amyloid peptide, α-synuclein [2]and loss of calcium sensitivity in calcium-sensing protein domains [4].Met oxidation to Met(O) is reversed by the enzyme methionine sulfoxide reductase (Msr): MsrA reduces methionine-(S)-sulfoxide, MsrB reduces methionine-(R)-sulfoxide, back to Met [5,6]. 1 O2 , HClO) can all oxidize both structural and signaling proteins, at the sulfur-containing cysteine (Cys) and methionine (Met) residues [1,2,3]. Met residue oxidation to Met sulfoxide [Met(O)] often results in major changes in protein activity: activation of calcium/calmodulin-dependent protein kinase II and of BK channels, inhibition of Kv channels, inhibition of the fibrillation process of apolipoproteins, β-amyloid peptide, α-synuclein [2]. Loss of calcium sensitivity in calcium-sensing protein domains [4]. Met oxidation to Met(O) is reversed by the enzyme methionine sulfoxide reductase (Msr): MsrA reduces methionine-(S)-sulfoxide, MsrB reduces methionine-(R)-sulfoxide, back to Met [5,6]. The enzyme Msr has been found to play important roles in cellular physiology and pathophysiology. Mammals have at least 4 separate genes (msrA, msrB1-3) [1,2,7] to encode MsrA [8,9], MsrB1 [9,10], MsrB2 [11], MsrB3 [12].
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