Gonadotropin-releasing hormone (GnRH) activates oscillatory Ca2+ signaling in pituitary gonadotrophs at a frequency (up to 25 min-1) that is dose-dependent and is determined by the degree of receptor-mediated inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) formation. Similar dose-dependent and frequency-modulated Ca2+ oscillations were elicited by intracellular administration of Ins(1,4,5)P3 and its nonhydrolyzable analogs, consistent with models in which Ins(1,4,5)P3 levels determine the frequency of Ca2+ oscillations but do not fluctuate in synchrony with [Ca2+]i. At constant agonist concentrations, Ca2+ spiking varied in amplitude, with a number of progressively larger transients before the onset of maximal oscillations, followed by a gradual decrease in spike amplitude that was accompanied by an increase in spiking frequency. The decline in the amplitude and increase in frequency of Ca2+ transients during stimulation by GnRH were not related to a decrease in the propagation of the Ca2+ signal within the cell but were associated with gradual depletion of the agonist-sensitive Ca2+ pool. Once initiated, the pattern of Ca2+ spiking was not altered by blockade of receptor occupancy, by inhibition of phospholipase C, or by reduction of extracellular [Ca2+]. Also, the endoplasmic reticulum (Ca2+)-ATPase blocker, thapsigargin, could substitute for Ins(1,4,5)P3 in initiating the oscillatory Ca2+ response. These findings indicate that although the Ins(1,4,5)P3 concentration determines the pattern of transients at the initiation of the oscillatory Ca2+ signal, maintenance of the signal does not require a sustained rise in Ins(1,4,5)P3. Since the frequency of Ca2+ oscillations is also influenced by depletion of luminal [Ca2+], it is possible that the Ins(1,4,5)P3-sensitive channels in the endoplasmic reticulum are tonically inhibited by high intraluminal Ca2+ levels and that Ins(1,4,5)P3 surmounts such inhibition by promoting Ca2+ discharge. When a critical level of Ca2+ discharge is attained, repetitive Ca2+ transients are generated by an autocatalytic mechanism in which a sustained rise in Ins(1,4,5)P3 is not an essential requirement.

Formation of neuromuscular synapses requires a series of inductive interactions between growing motor axons and differentiating muscle cells, culminating in the precise juxtaposition of a highly specialized nerve terminal with a complex molecular structure on the postsynaptic muscle surface. The receptors and signaling pathways mediating these inductive interactions are not known. We have generated mice with a targeted disruption of the gene encoding MuSK, a receptor tyrosine kinase selectively localized to the postsynaptic muscle surface. Neuromuscular synapses do not form in these mice, suggesting a failure in the induction of synapse formation. Together with the results of an accompanying manuscript, our findings indicate that MuSK responds to a critical nerve-derived signal (agrin), and in turn activates signaling cascades responsible for all aspects of synapse formation, including organization of the postsynaptic membrane, synapse-specific transcription, and presynaptic differentiation.

DNA is a frequent target of oxidative damage, and DNA damage removal is therefore a crucial process in prevention of or recovery from degenerative diseases. DNA repair is an essential system for maintaining the inherited nucleotide sequence of genomic DNA over time. Cells engage in efficient DNA repair mechanisms, the activity of which can vary depending on the type of lesion and the developmental stage. Base excision repair (BER) and nucleotide excision repair (NER) are the major repair pathways addressed in this study. BER is the principal mechanism for repair of DNA oxidative lesions, while NER is the mechanism for repair of a variety of helix-distorting lesions such as those caused by UV radiation. Recent studies suggest that NER plays a cooperative role in removal of oxidative lesions. Little is known about the roles of DNA damage sensors and repair factors in terminally differentiated, non-proliferating cells such as neurons, which are vulnerable to oxidative damage from reactive oxygen species generated by endogenous or exogenous agents. We used the human neuroblastoma MSN cell model to investigate whether terminally differentiated neuronal cells respond to lesions cause in the DNA helix, such as UV-induced CPD and the major DNA oxidative lesion 8OHdG, and thereby clarify the role of NER capacity. We observed differences in DNA damage removal depending on the challenge insult and the differentiation state. Differentiated MSN cells, compared with undifferentiated cells, showed greater sensitivity to UVC and decreased DNA damage over time. In contrast, undifferentiated cells displayed genotoxicity induced by oxidative insult and tended to accumulate DNA damage and 8OHdG lesions over time. Our findings suggest the participation of GG-NER, TC-NER and BER proteins in the removal of 8-OHG and CPDs indicating a dynamic role in overall response to damage.

Metals are a threat to human health by increasing disease risk. Experimental data have linked altered miRNA expression with exposure to some metals. MiRNAs comprise a large family of non-coding single-stranded molecules that primarily function to negatively regulate gene expression post-transcriptionally. Although several human populations are exposed to low concentrations of As, Cd and Pb as a mixture, most toxicology research focuses on the individual effects that these metals exert. Thus, this study aims to evaluate global miRNA and mRNA expression changes induced by a metal mixture containing NaAsO2, CdCl2, Pb(C2H3O2)2·3H2O and to predict possible metal-associated disease development under these conditions. Our results show that this metal mixture results in a miRNA expression profile that may be responsible for the mRNA expression changes observed under experimental conditions in which coding proteins are involved in cellular processes, including cell death, growth and proliferation related to the metal-associated inflammatory response and cancer.