Hydrogen peroxide promotes transformation of rat liver non-neoplastic epithelial cells through activation of epidermal growth factor receptor.

Previous studies demonstrated that hydrogen peroxide (H(2)O(2)) is a tumor promoter in the rat liver epithelial cell line T51B. We investigated the pathway linking H(2)O(2) to tumor promotion. H(2)O(2) can directly induce tyrosine phosphorylation of epidermal growth factor receptor (EGFR). H(2)O(2) and epidermal growth factor exerted similar effects on the induction of early growth response genes, disruption of gap junction communication, triggering of calcium inflow, and promotion of transformation. Furthermore, the effect of H(2)O(2) on tumor promotion was blocked by abrogation of EGFR activation. Our results suggested that tumor promotion by H(2)O(2) is mediated mainly through activation of EGFR in T51B cells.

Anti-GM3 antibodies activate calcium inflow and inhibit platelet-derived growth factor beta receptors (PDGFbetar) in T51B rat liver epithelial cells.

Glycolipids expressed in the plasma membrane regulate a variety of cellular processes including intracellular calcium dynamics. We used flow cytometry to characterize the glycoconjugates on the plasma membrane of T51B liver epithelial cells. Antibodies against glycolipids found to be present were tested for their ability elevate intracellular calcium. An antibody against GM3 (DH2) nearly doubles intracellular calcium while an antibody against type II chains (1B2) increases calcium to nearly four times the baseline level, similar to levels obtained with epidermal growth factor (EGF). The antibodies stimulated calcium inflow but did not trigger calcium release from internal stores. In addition DH2 but not 1B2 inhibited platelet-derived growth factor beta receptor (PDGFbetar) function. This is the first demonstration of activation of calcium inflow by agents that bind GM3 and type II chains. The ganglioside-mediated calcium inflow is likely to stimulate secretion by these liver cells.

Involvement of mitochondria in intracellular calcium sequestration by rat gonadotropes.

Intracellular Ca2+ ([Ca2+]i) dynamics were studied in identified rat gonadotropes using the whole-cell patch-clamp technique in conjunction with Indo-1 photometry. The kinetics of depolarization-induced [Ca2+]i transients vary with Ca2+ load. In addition to a rapid initial decay, large (> 500 nM) [Ca2+]i transients have a slow plateau phase. Application of the mitochondrial inhibitor carbonyl cyanide m-chlorophenylhydrazone (CCCP) significantly slows the decay of [Ca2+]i transients, consistent with stopping uptake of Ca2+ by mitochondria. CCCP causes a small increase of [Ca2+]i at rest. After a large Ca2+ entry the amount is much larger, consistent with release from a mitochondrial Ca2+ pool that fills during cytoplasmic Ca2+ loading. The rate of Ca2+ uptake by mitochondria is dependent upon [Ca2+]i. Consistent with previous studies, gonadotropin releasing hormone (GnRH) induces [Ca2+]i oscillations. The mitochondrial inhibitors CCCP and cyanide (CN-) terminate these oscillations. The mitochondrial ATP-synthase inhibitor oligomycin reduces the frequency and increases the amplitude of the oscillations. In the presence of ruthenium red (a non-specific blocker of the mitochondrial Ca(2+)-uniporter) in the pipette, GnRH does not induce rhythmic [Ca2+]i oscillations. We suggest that mitochondria play a significant role in the rapid clearance of cytosolic Ca2+ loads in gonadotropes and participate in GnRH-induced periodic [Ca2+]i oscillations.

Kinetic basis for the voltage-dependent inhibition of N-type calcium current by somatostatin and norepinephrine in chick sympathetic neurons.

Neurotransmitter inhibition of calcium currents (ICa) can be relieved by large depolarizing prepulses. This effect has been postulated to be due either to the voltage-dependent unbinding of an inhibitory molecule from the channel or to a slow voltage-dependent gating step intrinsic to the modulated channel. According to the first hypothesis, the rate of reinhibition (reblock) following a depolarizing prepulse should depend on the concentration of active inhibitory molecules and thus should increase with the extent of inhibition. To distinguish between these models we examined the actions of norepinephrine (NE) and somatostatin (SS) on high-threshold calcium currents in chick sympathetic ganglia, using whole-cell voltage-clamp methods. As previously described in other systems, both NE and SS inhibit omega-conotoxin-sensitive N-type Ca2+ current in a voltage-dependent manner. Pertussis toxin (PTX) pretreatment prevents the inhibition of the current, while replacing GTP in the patch pipette with GTP-gamma-S results in irreversible inhibition, consistent with the involvement of a PTX-sensitive G-protein. The inhibitory responses to NE and SS are not additive, suggesting that they act at a common locus. The inhibitory response to repeated applications of NE or SS desensitizes, with little evidence for cross desensitization. The inhibition of ICa is relieved by a 15 msec prepulse to +100 mV. Following repolarization to -80 mV, ICa slowly reblocks. During prolonged applications of NE or SS the extent of inhibition decreases due to desensitization and reblock kinetics are significantly slowed (time constant increases from 60 msec to > 100 msec for both NE and SS). These results are well fit by a quantitative model in which the kinetics of reblock reflect the binding of an inhibitory molecule to the channel.