By tethering intermediate filaments (IFs) to sites of intercellular adhesion, desmosomes facilitate formation of a supercellular scaffold that imparts mechanical strength to a tissue. However, the role IF–membrane attachments play in strengthening adhesion has not been directly examined. To address this question, we generated Tet-On A431 cells inducibly expressing a desmoplakin (DP) mutant lacking the rod and IF-binding domains (DPNTP). DPNTP localized to the plasma membrane and led to dissociation of IFs from the junctional plaque, without altering total or cell surface distribution of adherens junction or desmosomal proteins. However, a specific decrease in the detergent-insoluble pool of desmoglein suggested a reduced association with the IF cytoskeleton. DPNTP-expressing cell aggregates in suspension or substrate-released cell sheets readily dissociated when subjected to mechanical stress whereas controls remained largely intact. Dissociation occurred without lactate dehydrogenase release, suggesting that loss of tissue integrity was due to reduced adhesion rather than increased cytolysis. JD-1 cells from a patient with a DP COOH-terminal truncation were also more weakly adherent compared with normal keratinocytes. When used in combination with DPNTP, latrunculin A, which disassembles actin filaments and disrupts adherens junctions, led to dissociation up to an order of magnitude greater than either treatment alone. These data provide direct in vitro evidence that IF–membrane attachments regulate adhesive strength and suggest furthermore that actin- and IF-based junctions act synergistically to strengthen adhesion.

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 Zn 2؉ 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 the V max of the enzyme.

No abstract

The regulation of adenylyl cyclase activity by nitric oxide (NO) was studied in rat (Sprague±Dawley) striatal membranes. Three chemically distinct NO donors attenuated forskolinstimulated activity but did not alter basal activity. Maximum inhibition resulted in a 50% decrease in forskolin-stimulated activity, consistent with the presence of multiple isoforms of adenylyl cyclase and our previous ®ndings that only the forskolin-stimulated activity of the type-5 and -6 isoform family of enzymes is inhibited by NO. To monitor primarily the type-5 isoform, we examined the ability of NO donors to attenuate D 1 -agonist-stimulated adenylyl cyclase activity. Under those conditions, complete inhibition was observed. The data indicate that NO attenuates neuromodulator-stimulated cAMP signaling in the striatum.