Claudette Klein

Adenylyl Cyclase, a Coincidence Detector for Nitric Oxide

Nitric oxide (NO) donors inhibit hormone- and forskolin-stimulated adenylyl cyclase activity in purified plasma membrane preparations from N18TG2 neuroblastoma cells. Northern blot analyses indicate that the predominant isoform of adenylyl cyclase in N18TG2 cells is the type VI. Our experiments eliminate all the known regulatory proteins for this isoform as possible targets of NO. NO decreases the V max of the enzyme without altering the K m for ATP. Occupancy of the substrate-binding site protects the enzyme from the inhibitory effects of NO, suggesting that the conformation of the enzyme determines its sensitivity. The inhibition is reversed by reducing agents, implicating a Cys residue(s) as the target for nitric oxide and anS-nitrosylation as the underlying modification. These findings implicate NO as a novel cellular regulator of the type VI isoform of adenylyl cyclase.

Zinc Inhibition of cAMP Signaling

Zn2+ is required as either a catalytic or structural component for a large number of enzymes and thus contributes to a variety of important biological processes. We report here that low micromolar concentrations of Zn2+ inhibited hormone- or forskolin-stimulated cAMP production in N18TG2 neuroblastoma cells. Similarly, low concentrations inhibited hormone- and forskolin-stimulated adenylyl cyclase (AC) activity in membrane preparations and did so primarily by altering theV max of the enzyme. Zn2+ also inhibited recombinant isoforms, indicating that this reflects a direct interaction with the enzyme. The IC50 for Zn2+inhibition was ∼1–2 μm with a Hill coefficient of 1.33. The dose-response curve for Zn2+ inhibition was identical for AC1, AC5, and AC6 as well as for the C441R mutant of AC5 whose defect appears to be in one of the catalytic metal binding sites. However, AC2 displayed a distinct dose-response curve. These data in combination with the findings that Zn2+ inhibition was not competitive with Mg2+ or Mg2+/ATP suggest that the inhibitory Zn2+ binding site is distinct from the metal binding sites involved in catalysis. The prestimulated enzyme was found to be less susceptible to Zn2+ inhibition, suggesting that the ability of Zn2+ to inhibit AC could be significantly influenced by the coincidence timing of the input signals to the enzyme.

Effects of cyclic AMP pulses on adenylate cyclase and the phosphodiesterase inhibitor of D. discoideum

STARVATION triggers the differentiation of Dictyostelium discoideum amoebae to aggregation competence. The differentiated cells orientate and migrate towards attracting centres, presumably consisting of amoebae which autonomously and rhythmically emit cyclic AMP as a chemotactic signal. Signals, emitted with a periodicity of 5–8 min (refs 1–3, 5), are presumably generated by rhythmic changes in adenylate cyclase activity6, levels of which dramatically increase during the first few hours before the expression of aggregation competence7,8. Cellular responses to external cyclic AMP are probably mediated by plasma membrane receptors9. When stimulated by a pulse of cyclic AMP, amoebae amplify10 and relay1,5,10,11 the chemotactic signal, which results in a coordinated aggregation. The decay of the chemotactic signal is determined by the activity of the extracellular cyclic AMP phosphodiesterase12,13 whose activity is modulated by an inhibitor excreted by cells during the early hours of starvation14. Cyclic AMP pulses have recently also been shown to regulate the differentiation of cells to aggregation competence15. To determine their mechanism of action, we examined the influence of applied cyclic AMP pulses on the two earliest biochemical events implicated in the control of cyclic AMP levels, the rise in adenylate cyclase activity and the appearance of the extracellular phosphodiesterase inhibitor. The results show that adenylate cyclase may exist in active or inactive forms. Applied cyclic AMP pulses seem to induce cell differentiation by activating the enzyme. They also are shown to repress the production of the phosphodiesterase inhibitor.

Nitric oxide and the other cyclic nucleotide

Nitric oxide (NO) participates in the regulation of the daily activities of cells as well as in cytotoxic events. Elucidating the mechanism(s) by which NO carries out its diverse functions has been the goal of numerous laboratories. In the cardiovascular system, evidence indicates that NO mediates its effects via an activation of soluble guanylyl cyclase (sGC). In other tissues, it is not clear if sGC is an exclusive target for NO or what the functions of cGMP might be. It is also unlikely that the diversity of NO actions is explained solely by changes in cGMP. This review focuses on the evidence that NO modulates cAMP signalling, with specific attention to the effects of NO on adenylyl cyclase (AC) as the target of NO regulation.

Direct intra-tumoral injection of zinc-acetate halts tumor growth in a xenograft model of prostate cancer

Intracellular levels of zinc have shown a strong inverse correlation to growth and malignancy of prostate cancer. To date, studies of zinc supplementation in prostate cancer have been equivocal and have not accounted for bioavailability of zinc. Therefore, we hypothesized that direct intra-tumoral injection of zinc could impact prostate cancer growth. In this study, we evaluated the cytotoxic properties of the pH neutral salt zinc acetate on the prostate cancer cell lines PC3, DU145 and LNCaP. Zinc acetate killed prostate cancer cell lines in vitro, independent of androgen sensitivity, in a dose-dependent manner in a range between 200 and 600 μM. Cell death occurred rapidly with 50% cell death by six hours and maximal cell death by 18 hours. We next established a xenograft model of prostate cancer and tested an experimental treatment protocol of direct intra-tumoral injection of zinc acetate. We found that zinc treatments halted the growth of the prostate cancer tumors and substantially extended the survival of the animals, whilst causing no detectable cytoxicity to other tissues. Thus, our studies form a solid proof-of-concept that direct intra-tumoral injection of zinc acetate could be a safe and effective treatment strategy for prostate cancer.

Nitric oxide selectively inhibits adenylyl cyclase isoforms 5 and 6.

We have previously shown that N18TG2 neuroblastoma cells express the type 6 adenylyl cyclase and that preincubation with nitric oxide (NO) attenuates Gs- and forskolin-stimulated activity. Here we show that this inhibition reflects a direct action of NO on the adenylyl cyclase. Preincubation of N18TG2 cell membranes and insect cell membranes expressing recombinant type 5 and type 6 isoforms with NO donors leads to an inhibition of forskolin-stimulated adenylyl cyclase activity. NO donors do not alter the type 1 (representative of the type 1,3,8 family) or type 2 (representative of the type 2,4, 7 family) isoforms expressed in insect cells, even under conditions of compromised assay conditions or a range of temperatures. Thus, the ability of NO to inhibit adenylyl cyclase stimulation is dependent upon the nature of the isoform present, and appears to represent a unique regulation of the type 5,6 isoform family.

Zinc induces ERK-dependent cell death through a specific Ras isoform.

The effect of Zn on p53-independent cell death was examined in IIC9 embryonic fibroblasts. Despite the fact that these cells are p53-minus, Zn-mediated death occurs via an apoptotic mechanism. Death is facilitated by the presence of the Zn ionophore, pyrithione, indicating that intracellular Zn initiates the death response. Our investigations of the mechanism of Zn action demonstrate that Zn induces the death of IIC9 cells in a manner that is ERK-dependent. Expression of dn-(dominant negative)Ras attenuates ERK1/2 activation by Zn, and correspondingly reduces its cytotoxic effects. Raf-RBD pull-down experiments confirm that Zn treatment activates Ras and identified H-Ras as the specific isoform activated. This contrasts the activation of N-Ras that occurs when IIC9 cells are stimulated with thrombin. Thus, although the prolonged activation of the Ras/ERK pathway by Zn is similar to that seen when induced by mitogen, the distinguishing feature appears to be the isoform specificity of Ras activation.

Monoclonal antibodies: Use to detect developmentally regulated antigens on D. discoideum amebae

We have used monoclonal antibodies to detect developmentally regulated cell surface antigens on D. discoideum amebae. A study of an antigen detected using an antibody produced by a hybridoma line implicates a previously undescribed component in the process of cell aggregation. This antigen (consisting of a doublet of 69,000 and 73,000 molecular weight) is first detected during the early hours of cell starvation and is present until cells begin slug formation. The developmental appearance of the antigen is not controlled by cAMP pulses and is distinct from that of Contact A sites. Fab fragments directed against the antigen are potent inhibitors of aggregation but do not inhibit the differentiation of cells to aggregation competence.

Nitric oxide-releasing compounds inhibit Dictyostelium discoideum aggregation without altering cGMP production

The effects of nitric oxide‐releasing compounds on Dictyostelium discoideum cell development and guanylyl cyclase activity were studied. The addition of SNP (sodium nitroprusside) or SIN‐1 (3‐morpholino‐syndnonimine) to starved cells inhibited their differentiation and aggregation in a concentration‐dependent manner. In contrast to mammalian systems, SNP did not significantly affect guanylyl cyclase activity in cell lysates of D. discoideum nor did it stimulate cGMP production in intact cells. The results suggest that the inhibitory effects of NO on D. discoideum cell aggregation are through a mechanism independent of an effect on guanylyl cyclase activity.

cAMP regulation of cell differentiation in Dictyostelium discoideum and the role of the cAMP receptor

The ability of N 6 -(aminohexyl)-adenosine-3′:5′-monophosphate to mimic the actions of cAMP on cell differentiation and chemotaxis was investigated. This cAMP derivative, referred to as “hexyl-cAMP”, could effectively stimulate the differentiation to aggregation competence of wild-type cells and induce this state in several aggregation-minus mutants when administered in the form of pulses. The single addition of high concentrations of this analog to starved cells could, as does cAMP, induce cAMP phosphodiesterase and delay the appearance of the cAMP phosphodiesterase inhibitor. Unlike cAMP, however, hexyl-cAMP does not appear to bind to the cell surface cAMP receptor, as determined by binding studies with [ 3 H]hexyl-cAMP or the ability of the nonradioactive product to compete for [ 3 H]cAMP binding to cells. Correspondingly, hexyl-cAMP did not induced the down regulation of the cAMP receptor, nor did it elicit a chemotactic response from starved cells. The data are discussed in terms of the role of the cAMP receptor in mediating the cells responses to external cAMP.