The expression of carbonic anhydrases II and IV in the human pancreatic cancer cell line (Capan 1) is associated with bicarbonate ion channels

Abstract

Human pancreatic ductal cells of the Capan 1 cell line differentiate progressively during growth. After the exponential growth phase, the cells elongate and become polarized with their apical poles covered by microvilli and separated from the basolateral pole by tight junctions. In this stationary phase, they form domes, which are thought to result from the exchange of water and electrolytes. In this study, we demonstrated, using patch-clamp techniques, that HCO3- ions exit via the g350 high conductance anionic channel we observed recently at the Capan 1 cell surface. This g350 channel was thought to be either a Cl-/HCO3- antiport or a simple HCO3- channel. The stilbene derivatives 4-acetamido-4 isothiocyano-2-2'-disulfonic acid (SITS) and 4,4' diisothiocyano stilbene-2,2' disulfonic acid (DIDS) reduced both the number of domes and the Cl- and HCO3- flux through the g350 channel. Moreover, using histochemical, immunocytochemical and biochemical methods we showed that Capan 1 cells express a specific pattern of carbonic anhydrases (CA). Two types of CA were detected: the CA II isozyme mainly localized in the cytoplasm, but also found beneath the inner leaflet of the apical plasma membrane, and the CA IV isozyme localized on the outer leaflet of the apical plasma membrane and microvilli. Their molecular masses were 30 (CA II) and 55 kDa (CA IV), respectively. They were expressed continuously during the exponential growth phase, although their activity increased greatly during the stationary phase. Inhibition of dome formation by acetazolamide indicated the existence of a direct relationship between dome formation and CA. Characteristic structures with a central electron-dense core surrounded by a light halo were observed on the surface of cell membrane using histochemical and immunocytochemical methods. These structures were thought to represent a channel, corresponding possibly to CA IV. Our observations suggest that Capan 1 cells, despite their neoplasic transformation, produce HCO3- ions in the same way as normal human pancreatic ductal cells. Capan 1 cells in culture may therefore represent a suitable model for studying pancreatic duct HCO3- secretion at the cellular and molecular levels.

Human Mitochondrial Carbonic Anhydrase VB

“…Recently, we reported that mRNA for CA IV and, to a lesser extent, CA VI are expressed in human pancreas as detected by Northern blotting and reverse transcription-polymerase chain reaction (RT-PCR) (7). CA IV has been found on the cell surface of cultured tumor cells from human pancreatic ductal carcinoma (8). We suspected there may be additional novel CA isozymes expressed in human pancreas and sought to clone them by RT-PCR with human pancreas poly(A) ϩ RNA using primers based on conserved amino acid sequences of two short regions of CA known isozymes (homology probing) (9 -11).…”

Carbonic Anhydrase II Associated with Plasma Membrane in a Human Pancreatic Duct Cell Line (CAPAN-1)

“…This increase was concomitant with the spontaneous and progressive acquisition of a differentiated cell stage. The existence of a relationship between the expression of CA II and differentiated cell status has also been demonstrated in other human pancreatic duct cell lines (Frazier et al 1990) and for enzyme systems such as alkaline phosphatases (Fanjul et al 1991) or CA IV (Mahieu et al 1994;Mairal et al 1996) in this same cell model. In the same way, CA II in vivo is considered to be a marker for the tissue differentiation of excretory ducts in the developing rat parotid gland (Peagler et al 1998).…”

“…On electron microscopic analysis, this immunoreactivity was localized essentially in apical plasma membranes. CA IV has also been found to be confined to these membrane domains (Mahieu et al 1994;Mairal et al 1996). Western blotting confirmed the specificity of the immunocytochemical reactions and provided further support for the presence of CA II associated with apical plasma membranes.…”

“…This secretin-dependent secretion is believed to be regulated by the CFTR (cystic fibrosis transmembrane conductance regulator) (Gray et al 1993). It has been suggested that CA II is responsible for the production of HCO 3 Ϫ ions secreted at the apical poles of duct cells via a Cl Ϫ /HCO 3 Ϫ exchanger (Novak and Greger 1988) and in some species likely by an additional HCO 3 Ϫ channel (Mahieu et al 1994;Ishiguro et al 1996). Various subcellular distributions of CA II have been described.…”

Serial in vivo loss and in vitro gain of Fas expression and function in human cancerous pancreatic duct cells

“…Capan-1 cells represent an excellent model for the in vitro study of human cancerous pancreatic duct cells, as despite their neoplastic transformation, they retain a polarized morphology and the ability to secrete HCO 3 À ions 55,56 and M1 mucins (MUC5AC). 57-59 These cells express Fas, but depending to authors, are sensitive [47][48][49] or resistant 50,51 to Fas-mediated apoptosis.…”