Abstract. Sympathetic neurons depend on nerve growth factor (NGF) for their survival both in vivo and in vitro. In culture, the neurons die after NGF withdrawal by an autonomous cell death program but whether these neurons die by apoptosis is under debate. Using vital DNA stains and in situ nick translation, we show here that extensive chromatin condensation and DNA fragmentation Occur before plasma membrane breakdown during the death of NGFdeprived rat sympathetic neurons in culture. Furthermore, kinetic analysis of chromatin condensation events within the call population is consistent with a model which postulates that after NGF deprivation nearly all of the neurons die in this manner. Although the dying neurons display membrane blebbing, cell fragmentation into apoptotic bodies does not occur. Apoptotic events proceed rapidly at around the time neurons become committed to die, regardless of neuronal culture age. However the duration of NGF deprivation required to commit neurons to die, and the rate at which apoptosis occurs, increase with culture age. Thus, within the first week of culture, apoptosis is the predominant form of cell death in sympathetic neurons.

We have previously reported that G alpha s is expressed at considerably higher levels in myometrium taken from pregnant than from nonpregnant women. In the present study we have determined adenylyl cyclase activity in myometrial membranes by measuring the conversion of [alpha-32P]ATP to [32P]cAMP and have measured guanosine triphosphate-binding protein expression by immunoblotting with specific antibodies. Here we report that the increase in G alpha s expression in pregnant myometrium is associated with a significant increase in G alpha s-coupled adenylyl cyclase activity, as estimated by incubating myometrial membranes in the presence of 5'-guanylyl-imidodiphosphate with and without prostaglandin E2. Moreover, in myometrium from women in spontaneous labor G alpha s levels and G alpha s-coupled adenylyl cyclase activity are reduced to the levels observed in nonpregnant tissue. There was no apparent change in forskolin-stimulated adenylyl cyclase activity in nonpregnant, pregnant, and laboring tissue. The increase in G alpha s expression in pregnant myometrium may facilitate agonist-induced cAMP formation, resulting in prolonged relaxation of the uterus during gestation. Down-regulation of G alpha s would decrease the relaxing effect exerted by cAMP and may be a triggering mechanism for the initiation of labor.

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We compared the effects of guanine nucleotides and Mg2+ on ADP-ribosylation of rat brain and liver membrane proteins catalysed by Bordetella pertussis toxin (IAP) and cholera toxin (CT). Labelling of proteins in the presence of [alpha-32P]NAD+, ATP and CT required GTP or guanosine 5'-[gamma-thio]triphosphate (GTP [S]). In contrast, labelling of one (liver) or two (brain) polypeptides by IAP was enhanced by guanosine 5'-[beta-thio]diphosphate (GDP[S]) or GTP, but was blocked by GTP[S] or guanosine 5'-[beta, gamma-imido]triphosphate (p[NH]ppG). The order of labelling intensity was GDP[S] greater than GTP greater than no addition greater than GTP[S] = p [NH]ppG. Mg2+ increased labelling by CT, but decreased labelling by IAP. In addition, Mg2+ potentiated the effects of the guanine nucleotides, increasing the inhibitory effects of GTP[S] and the activatory effects of GDP[S] or GTP. Preincubating liver membranes at 30 degrees C in the presence of 10 mm-MgCl2 inhibited labelling by IAP irreversibly. Pretreatment of liver membranes with 4.95 mM-N-ethylmaleimide decreased labelling by CT by approximately 15%, but almost completely blocked labelling by IAP. These results suggest that the undissociated, GDP-bound, conformation of Ni, the inhibitory GTP-binding protein of adenylate cyclase, is the preferred substrate for ADP-ribosylation by IAP. This conformation, which is prevalent in native membranes, is sensitive to temperature, Mg2+ ions and alkylating agents such as N-ethylmaleimide. At 30 degrees C, Mg2+ may cause dissociation and denaturation of Ni in native membranes.