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cAMP kinase has been shown to mediate the cAMP pathway for regulation of Cl- channels in lymphocytes, but the mediator of an alternative, Ca2+ pathway has not been identified. We show here that Ca2+ ionophore activates Cl- currents in cell-attached and whole-cell patch-clamp recordings of Jurkat T lymphocytes, but this activation is not direct. The effect of Ca2+ ionophore on whole-cell Cl- currents is inhibited by a specific peptide inhibitor of multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase). Furthermore, Cl- channels are activated in excised patches by purified CaM kinase in a fashion that mimics the effect of Ca2+ ionophore in cell-attached recordings. These results suggest that CaM kinase mediates the Ca2+ pathway of Cl- channel activation.

The insulin-like growth factor-II/mannose 6-phosphate receptor (IGF-II/man6PR) can directly interact with and activate Gi-2, a GTP binding protein (G protein). We found that the segment of residues 2410-2423 in the human IGF-II/man6PR activates Gi-2 in a manner similar to G-coupled receptors. We observed a hierarchy of the segment action when tested on various G proteins, with an order of Gi-2 greater than Gi-1 approximately Gi-3 greater than Go. The segment had no effect on Gs or low molecular weight G proteins. The segment action depended on its primary structure and was potentiated when the segment was connected with a part of the receptor transmembrane region. Finally, the Gi-2-activating function of the human IGF-II/man6PR could be blocked by an antibody against the segment, indicating a critical role for this small region of the receptor.

We previously reported that insulin-like growth factor-II (IGF-II) stimulates both calcium influx and DNA synthesis by acting on the cell surface IGF-II receptor (IGF-IIR) in a manner sensitive to pertussis toxin, and recently demonstrated that IGF-II binding to the IGF-IIR gives rise to functional changes of purified Gi-2, a GTP-binding protein (G protein) in phospholipid vesicles as well as in broken cell membranes. On the other hand, a variety of evidence indicates that the IGF-IIR binds mannose 6-phosphate (man6P) with high affinity probably at a receptor extracellular region different from the IGF-II-binding site. In the present study, we examined whether man6P stimulation of the IGF-IIR evokes the activation of Gi-2 in phospholipid vesicles and in native cell membranes. In vesicles reconstituted with purified rat IGF-IIR and bovine Gi-2, man6P did not stimulate GDP dissociation from Gi-2 even in concentrations up to 10 mM, while IGF-II dose-dependently facilitated GDP release from Gi-2 with an EC50 of 6 nM. The stimulatory effect of IGF-II was not observed in vesicles reconstituted with Gi-2 alone. In addition, also in a native environment of cell membranes, man6P did not affect an endogenous 40-kDa protein or exogenously added purified Gi-2 as assessed with reduction of the pertussis toxin-catalyzed ADP-ribosylation. These results indicate that the IGF-IIR does not activate Gi-like proteins upon man6P binding in phospholipid vesicles and in native cellular membranes, whereas the receptor activates Gi-like proteins upon IGF-II binding in both environments. Thus, we postulate that the IGF-IIR dissimilarly responds to the two structurally unrelated ligands, IGF-II and man6P, in the linkage function with G proteins.

Effect of pertussis toxin treatment on the actions of insulin-like growth factor-I (IGF-I) was examined in Balb/c 3T3 cells. In competent cells primed with epidermal growth factor, IGF-I stimulated both calcium influx and DNA synthesis. When these primed competent cells were treated with various concentrations of pertussis toxin for 2 hrs, IGF-I-induced calcium influx and DNA synthesis were inhibited in a similar dose dependent manner. The inhibitory action of pertussis toxin well coincided with the toxin-induced ADP-ribosylation of a 41 K-Da protein. These results suggest a possible involvement of a pertussis toxin-sensitive GTP-binding protein in the action of IGF-I.

This paper reports an autopsy case of a 78-year-old male with multiple nodules in the liver developed after long-termed administration of phosphate diethylstilbestrol (PDES) for prostatic cancer. Large part of these nodules were suspected to be well differentiated hepatocellular carcinoma with high level of serum alpha-fetoprotein (AFP) up to 3,400 ng/ml, but a part of them was evaluated to be a borderline between hepatocellular carcinoma and adenoma with mild cellular atypism. The liver other than the nodules showed liver fibrosis associated with liver cell dysplasia and peliosis hepatis-like change. This is a unique autopsy case of hepatocellular carcinoma closely related to diethylstilbestrol (DES) therapy for prostatic cancer.

In competent Balb/c 3T3 cells primed with epidermal growth factor (primed competent cells), insulin-like growth factor-II (IGF-II) stimulated calcium influx in a concentration dependent manner with the ED50 of 450 pM. When receptor-bound [125I]IGF-II was cross-linked by use of disuccinimidyl suberate, a 240 K-Da protein was radiolabeled. Excess amount of unlabeled IGF-II inhibited the affinity-labeling of the 240 K-Da protein. To further examine whether IGF-II stimulates calcium influx by acting on the type II IGF receptor, we employed polyclonal antibody raised against rat type II IGF receptor, R-II-PABl. This antibody immunoprecipitated the type II IGF receptor and inhibited IGF-II binding in Balb/c 3T3 cell membrane without affecting IGF-I binding. In primed competent cells, R-II-PABl elicited an agonistic action in stimulating [3H]thymidine incorporation. Under the same condition, R-II-PABl elicited a marked stimulation of calcium influx. These results suggest that, in Balb/c 3T3 cells, 1) relatively low concentrations of IGF-II act mainly on the type II IGF receptor; 2) the type II IGF receptor is coupled to a calcium gating system; and 3) binding of a ligand to the type II IGF receptor leads to the stimulation of DNA synthesis.

To determine the role of calcium in the action of insulin-like growth factor II (IGF-II), we have examined the effect of multiplication stimulating activity, the rat IGF-II, on cytoplasmic-free calcium concentration, [Ca2+]c, in aequorin-loaded Balb/c 3T3 cells. IGF-II does not cause any change in [Ca2+]c in quiescent cells. By contrast, IGF-II induces changes in [Ca2+]c in platelet-derived growth factor(PDGF) - pretreated competent cells: when competent cells are incubated with epidermal growth factor (EGF) for 10 min, subsequent IGF-II induces an immediate increase in [Ca2+]c. Without EGF treatment, IGF-II does not cause any increase in [Ca2+]c. The priming action of EGF is time dependent, requiring approximately 10 min for the maximum effect. The IGF-II-mediated increase in [Ca2+]c is totally dependent on extracellular calcium and is blocked by lanthanum. When DNA synthesis in PDGF-treated competent cells is assessed by measuring [3H]thymidine incorporation, IGF-II by itself has only a small effect. Likewise, a brief treatment with EGF results in only a small increase in [3H]thymidine incorporation. By contrast, in competent cells briefly treated with EGF, IGF-II causes a marked stimulation of [3H]thymidine incorporation. These results indicate that IGF-II increases [Ca2+]c in competent Balb/c 3T3 cells treated with EGF by stimulating calcium influx and that IGF-II-stimulated calcium influx may be related causally to its action on cell proliferation.