Fig ure 3 shows an increased expression of TNF R1 in NOD acini in resting conditions with a negligible expression of TNF R2 in both cases. With the aim of investigating the ability of TNF to induce apoptosis in acinar cells from NOD and control mice sub mandibular glands, we then determined the effect of TNF at 5 and 10 ngml. Figures 4a and 4b show that TNF induced TP53INP1 expression at 5 ngml only in NOD acini and this effect was paralleled by an increased condensation of nuclear chromatin. At a higher concentration, the cytokine could reproduce these effects on normal acinar cells. At the concentration that TNF induced chromatin condensa tion and TP53INP1 expression only in NOD acini, it also increased caspase 3 activity, and increased expression of Bax, TP53INP1 and its own receptor TNF R1, but it did not modify the expression of BclxL.
To analyse whether VIP could prevent or modulate this effect of TNF , we performed the same experiments in the presence or absence of the neu ropeptide. Inhibitory effect of VIP on TNF induced apoptosis We selleck inhibitor first studied the effect of VIP on apoptotic mediators induced by TNF. The preincubation of acinar cells with 100 nM VIP prevented the inducing effect of TNF on all of the apoptotic events shown. As it is shown in Figures 5a and 5b TNF was unable to induce these factors in control acinar cells although it did increase their expressionactivity in the NOD suspen sion. Moreover, VIP only reduced their expression in NOD acini suggesting that VIP modulation mainly affects TNF upregulated factors.
To identify and functionally characterise VIP receptors involved in this acinar cell preparation, we inves tigated VIP receptor expression, cAMP accumulation and amy lase secretion. Figure 6a shows that acinar cells express VPAC1 receptors in both strains of mice. In contrast VPAC 2 knowing it expression could not be detected at any condition tested. Figure 6b shows that VIP receptors on acinar cells are func tional because 100 nM VIP stimulated cAMP accumulation and amylase secretion at the same concentrations used for inhibiting apoptotic signals. Note that in basal and VIP stimu lated conditions acinar cells present a lower amylase secretion confirming in this acinar cell preparation the results shown pre viously in studies of in vivo salivary flow stimulation. Finally, as expected for a VPAC1 cAMP PKA mediated effect, VIP failed to reverse apoptotic signals induced by TNF in acinar cells in the presence of PKA inhibitor H89.