Cytosolic calcium regulates epidermal growth factor endocytosis in rat pancreas and cultured fibroblasts.

Abstract

Cholecystokinin octapeptide (CCK8), the COOH-terminal moiety of cholecystokinin (CCK), exerted a rapid inhibitory effect on total cell-associated 125I-labeled epidermal growth factor As in the case of a number of other polypeptide hormones, high concentrations of EGF decrease the number of EGF receptors on the cell surface through a process called "downregulation." EGF binding and internalization are also modulated by a variety of nonphysiological agents. Thus, binding of EGF to its receptor is altered by phorbol esters (4), cyclamates (5), quinone derivatives (6), and teleocidine B (7), whereas EGF internalization is inhibited by dansylcadaverine, amantadine, and rimantidine (8). The physiological relevance of these observations is not easily apparent, inasmuch as these compounds are not normally found in vivo.Recently, platelet-derived growth factor (9), fibroblast-derived growth factor (10), the neurohypophyseal hormone vasopressin (11), and cholecystokinin (CCK) (12) have also been shown to inhibit EGF binding. These observations suggest that compounds other than EGF may modulate EGF binding activity in vivo. We now report that the inhibitory effect of CCK on EGF binding in isolated rat pancreatic acini may be due to Ca2+-mediated inhibition of EGF internalization. In support of this hypothesis, we demonstrate that the Ca2+ ionophore A23187 inhibits the internalization of 1251I-labeled EGF (1251I-EGF) in A-431 human carcinoma cells and other cultured fibroblasts. Inasmuch as a variety of hormones and neurotransmitters are known to act through Ca2 , it is possible that EGF internalization may be regulated through similar mechanisms in other cell types. MATERIALS AND METHODSPreparation of Acini and Cell Culture. Pancreatic acini were prepared from fasted male Sprague-Dawley rats as reported (13). Freshly isolated acini were incubated for 60 min at 370C in Hepes-buffered Ringer solution that was titrated to pH 7.40, equilibrated with 100% oxygen (13), and resuspended in fresh Hepes/Ringer buffer (0.5-0.8 mg of protein per ml) for use in experiments. Derivation of Rat-1 fibroblasts (14), A-431 human vulvar carcinoma cells (12), and Swiss mouse 3T3 cells (15) has been described. All cell lines were propagated at 370C in a 5% C02/95% air atmosphere in Dulbecco's modified Eagle's medium supplemented with antibiotics and the following serum conditions: 10% newborn calf serum (3T3), 10% calf and 2% newborn calf serum (Rat-1), or 5% newborn calf and 2% fetal calf serum (A-431).Binding Studies. EGF was prepared from mouse submaxillary glands by the method of Savage and Cohen (16) and further purified by reverse-phase high-performance liquid chromatography (14). Biologically active 125I-EGF (14) and 125I-labeled insulin (1251-insulin) (17) were prepared with chloramine T. To measure binding in acini, 2-to 18-ml suspensions of cells were routinely incubated at 37°C with 1251_ EGF (23.4-53.0 ,uCi/,ug; 1 Ci = 37 GBq) or 125I-insulin (20.5 ,uCi/,ug). To separate bound and free hormone, 0.5-to 0.7-ml aliquots of...

Pancreatic gastrin stimulates islet differentiation of transforming growth factor alpha-induced ductular precursor cells.

“…Interactions between EGF and CCK, homologues ofTGFa and gastrin, respectively, have been well studied on pancreatic exocrine cells. CCK decreases EGF receptor binding by stimulating intracellular C kinase activity, which inhibits internalization of receptor bound EGF (28,29). Although the metaplastic ductules contain numerous insulin-positive cells, the islet mass of the TGFa transgenic mice was not increased over controls.…”

Phorbol esters induce transient internalization without degradation of unoccupied epidermal growth factor receptors.

“…Ca2l has been suggested to be involved in association of normally soluble protein kinase C with plasma membranes (37,38). In addition, Ca2l has been shown to act synergistically in altering EGF internalization (39,40).…”

Early events elicited by bombesin and structurally related peptides in quiescent Swiss 3T3 cells. I. Activation of protein kinase C and inhibition of epidermal growth factor binding.

“…It has been suggested that a variety of secretagogues, including bombesin, inhibit 125I-EGF association to pancreatic acinar cells by increasing cytosolic Ca 2÷ in the cells (28,31,46). The following paper (37) reports that bombesin and bombesinrelated peptides induce Ca 2÷ mobilization from intracellular stores and increase cytosolic Ca 2÷ concentration in 3T3 cells (37).…”

“…It has also been reported that bombesin reduces the association of ~:5I-EGF to 3T3 cells (4) and to pancreatic acinar cells, an effect apparently mediated by an increase in the cytosolic Ca 2+ concentration (28,31,46). In the course of our studies concerning the early events elicited by peptides of the bombesin family in quiescent Swiss 3T3 cells, we examined the effect of these peptides on the binding and internalization of ~25I-EGF into Swiss 3T3 cells, and the parts played by protein kinase C and an increase in cytosolic Ca 2+ in mediating the inhibition of t2SI-EGF association caused by bombesin.…”