Vasoactive intestinal peptide inhibits TNF-alpha-induced apoptotic events in acinar cells from nonobese diabetic mice submandibular glands.
BACKGROUND
The role of apoptotic secretory epithelium as a pro-inflammatory triggering factor of exocrine dysfunction is currently explored in Sjogren's syndrome patients and in the nonobese diabetic (NOD) mouse model. Vasoactive intestinal peptide (VIP) has anti-inflammatory effects in various models of chronic inflammation. Our goal was to analyse the effect of TNF-alpha on apoptotic mediators in isolated acinar cells from NOD submandibular gland and their modulation by VIP.


METHODS
Acinar cells were isolated from submandibular glands of 16-week-old NOD females with salivary flow decline. Age-matched BALB/c females or eight-week-old NOD females were used as controls. Apoptotic mediators and TNF-alpha receptor expression were assessed by immunoblotting and RT-PCR, caspase 3 activity was assessed by optical density at 405 nm with Ac-DEVD-pNA as a substrate and chromatin condensation by Hoechst stain. They were evaluated in resting conditions and after a 3.5 or 6 hours of culture with TNF-alpha. VIP effects in acinar cells were assessed at 100 nM in TNF-alpha-treated cultures and VIP receptor functional assays by radio immunoassay (cAMP) or enzymatic detection (amylase).


RESULTS
NOD acinar cells at 16 weeks present an increased expression of TNF-alpha receptor1 together with increased Bax, tumour protein 53-induced nuclear protein1alpha (TP53INP1alpha), caspase 3 activity and chromatin condensation. Acini from NOD mice were more sensitive to TNF-alpha-induced increases of apoptotic mediators than control cells. VIP inhibited TNF-alpha-induced apoptotic events through functional VPAC1 receptors coupled to the protein kinase A (PKA) signalling pathway.


CONCLUSIONS
Our results indicate that acinar cells isolated from submandibular glands of NOD mice with salivary dysfunction are more sensitive to apoptosis induced by TNF-alpha which could be prevented by VIP through a PKA-mediated pathway.
Calafat
Mario
M
Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Pabellón II, Buenos Aires, Argentina. mcalafat@qb.fcen.uba.ar
Larocca
Luciana
L
Roca
Valeria
V
Hauk
Vanesa
V
Pregi
Nicolás
N
Nesse
Alcira
A
Pérez Leirós
Claudia
C
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D015153:Blotting, Western; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004195:Disease Models, Animal; D005260:Female; D051379:Mice; D008807:Mice, Inbred BALB C; D016688:Mice, Inbred NOD; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000502:physiology; D012859:Sjogren's Syndrome / Q000378:metabolism* / Q000473:pathology; D013363:Submandibular Gland / Q000378:metabolism* / Q000473:pathology; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*; D014660:Vasoactive Intestinal Peptide / Q000378:metabolism*
D014409:Tumor Necrosis Factor-alpha; D014660:Vasoactive Intestinal Peptide; D017868:Cyclic AMP-Dependent Protein Kinases