Localization of the platelet-activating factor receptor to rat pancreatic microvascular endothelial cells

Abstract

Platelet-activating factor (PAF) is a potent lipid autocoid involved in numerous inflammatory processes. Although PAF plays a key role as a mediator of inflammation in acute pancreatitis, the site(s) of action of PAF in the pancreas remains unknown. One of the aims of this study was to identify cell types within the pancreas expressing the PAF receptor using immunohistochemical protocols. Additionally, pancreatic microvascular endothelial cells were isolated and examined for the PAF receptor using immunohistochemistry, reverse transcription-polymerase chain reaction, and intracellular calcium responses to PAF exposure. Immunohistochemical analysis of pancreatic slices using an antibody directed toward the N-terminus of the PAF receptor revealed specific localization to the vascular endothelium with no localization to other pancreatic cell types. Reverse transcription-polymerase chain reaction of RNA isolated from cultured pancreatic islet endothelial cells yielded the predicted amplicon for the PAF receptor. Cultured pancreatic islet endothelial cells responded to PAF as measured by a transient increase in intracellular calcium, which was ameliorated in the presence of a PAF receptor antagonist. The results demonstrate the localization of PAF receptors on the pancreatic vascular endothelium. The presence of PAF receptors on the pancreatic vascular endothelium provides a defined, highly localized target for therapeutic intervention.

Figure 1.

Ethidium bromide-stained agarose gel of RT-PCR using primers for PAF receptor. Lane 1: 100-bp ladder; Lane 2: water; Lane 3: PIEC; Lane 4: AR42J; Lane 5: freshly isolated dispersed acini; Lane 6: kidney; Lane 7: pancreas.

Figure 2.

Hematoxylin and eosin stain of rat pancreas. A: 40× magnification showing acinar cell unit (a) and islet (I). B, upper panel: 100× magnification showing pancreatic microvessels (v) with endothelial cell lining (ec); lower panel : 100× magnification showing pancreatic duct (d).

Figure 3.

Immunohistochemistry of rat pancreas using PAF receptor antibody (A), von Willebrand factor antibody (B), and Ox 2 antibody (C). Antibody-associated HRP activity visualized using diaminobenzidine and tissue counterstained with hematoxylin.

Figure 4.

Immunohistochemistry of rat PIEC using PAF receptor antibody (A), von Willebrand factor antibody (B), and Ox 2 antibody (C). Antibody-associated HRP activity visualized using Vector VIP.

Figure 5.

Intracellular calcium measurements in rat PIEC following exposure to PAF (100 nmol/L). Solid line represents intracellular calcium change in PIEC exposed to PAF. Dotted line represents intracellular calcium change in PIEC preincubated with Lexipafant and exposed to PAF, followed by thrombin stimulation.