PubMed · 2476302
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Z Tulassay. 1989. Serum enzyme changes after ERCP.. https://doi.org/10.1055/s-2007-1012944
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To study the effect of microwaves on immunolabeling, ultrathin cryosections or diluted antibodies were irradiated prior to antibody application, and gold labeling was quantified. In addition, affinity purified, polyclonal antibodies and protein A-gold were applied to ultrathin cryosections of aldehyde-fixed material in the presence and absence of microwaves. Amylase, a soluble protein secreted by pancreatic acinar cells, MHC class II, an integral membrane protein, and 3-(2,4-dinitroanilino)-3-amino-N-methyldipropylamine (DAMP), an exogenously added antigen, were localized with monospecific antibodies. Each was chosen for their contrasting subcellular characteristics. Results demonstrated that for some antigens, antibody labeling efficiency was quantitatively improved by microwave irradiation of sections prior to antibody application. Irradiation of diluted antibodies prior to their application also resulted in improved labeling. In contrast, the results obtained using rapid immunolabeling protocols in the presence of microwaves resulted in labeling levels similar to those obtained in the absence of microwaves. We conclude that microwave irradiation can improve the labeling efficiency of some antibodies. However, improvements in labeling density are dependent on the antigen under study and on variable irradiation times, unique to each antibody. This suggests that the routine use of microwaves to reduce incubation times may not be a viable alternative to bench protocols.
We have studied the role of protein tyrosine phosphorylation in amylase secretion from differentiated AR4-2J cells. The secretagogue bombesin, the protein kinase C activator phorbol 12-myristate 13-acetate (PMA), and the protein-tyrosine phosphatase inhibitor pervanadate induced tyrosine phosphorylation of different proteins, including paxillin and p125(FAK), which was reduced or blocked by the tyrosine kinase inhibitors genistein and tyrphostin B56, respectively. Both PMA and pervanadate continuously increased amylase secretion with a similar time course, reaching the level of bombesin-induced amylase release after 60 min. Their effects were not additive and could be inhibited by preincubation of AR4-2J cells with genistein or tyrphostin B56, respectively. Inhibition of protein kinase C with Ro 31-8220 nearly abolished the effects of PMA, but had no effect on either pervanadate-induced protein tyrosine phosphorylation or amylase secretion. An increase in cytosolic free Ca2+ concentration by thapsigargin or A23187 caused a rapid increase in amylase release within the initial 5 min. In the presence of PMA or pervanadate, amylase secretion was further stimulated to levels comparable to those induced by bombesin after 30 min of stimulation. Inhibition of PMA-induced amylase secretion by Ro 31-8220 was less at elevated cytosolic free Ca2+ concentrations than without Ca2+. Furthermore, an increase in cytosolic free Ca2+ concentration had no effect on protein tyrosine phosphorylation in either the absence or presence of PMA or pervanadate. We therefore conclude that in the cascade of events that lead to bombesin-induced protein secretion from AR4-2J cells, protein tyrosine phosphorylation occurs downstream of protein kinase C activation. A further step in secretion that is Ca2+-dependent occurs distal to protein tyrosine phosphorylation.
To determine whether junctional communication between pancreatic acinar cells contributes to their secretory function in vivo, we have compared wild-type mice, which express the gap junctional proteins connexin32 (Cx32) and connexin26, to mice deficient for the Cx32 gene. Pancreatic acinar cells from Cx32 (-/-) mice failed to express Cx32 as evidenced by reverse transcription-PCR and immunolabeling and showed a marked reduction (4.8- and 25-fold, respectively) in the number and size of gap junctions. Dye transfer studies showed that the extent of intercellular communication was inhibited in Cx32 (-/-) acini. However, electrical coupling was detected by dual patch clamp recording in Cx32 (-/-) acinar cell pairs. Although wild-type and Cx32 (-/-) acini were similarly stimulated to release amylase by carbamylcholine, Cx32 (-/-) acini showed a twofold increase of their basal secretion. This effect was caused by an increase in the proportion of secreting acini, as detected with a reverse hemolytic plaque assay. Blood measurements further revealed that Cx32 (-/-) mice had elevated basal levels of circulating amylase. The results, which demonstrate an inverse relationship between the extent of acinar cell coupling and basal amylase secretion in vivo, support the view that the physiological recruitment of secretory acinar cells is regulated by gap junction mediated intercellular communication.