Treatment of pigeon erythrocyte membranes with cholera toxin and NAD+ enhan the GTP stimulation and suppressed the F activation of the adenylate cyclase [ATP pyrophosphatemlyase (cyclizing), EC 4.6.11. In the presence of NAD+ labeled with aP in the AMP moiety the toxin catalyzed the covalent incorporation of radioactivity into membrane proteins with molecular weights (Mrs) of 200,000, 86,000, and 42,000 Extraction of toxin-treated membranes with Lubrol PX followed by affinity chromatography on a GTP-Sepharose column resulted in a 200-fold purification of the 42,000-M, labeled protein and in its complete separation from the other labeled proteins. The fraction containing the purified GTP-binding component from toxin-treated membranes conferred an enhanced GP-stimulated activity on adenylate cyclase solubilized from nontreated membranes. Likewise, the addition of GTPbinding fraction from nontreated membranes to an enzyme solubilized from toxin-treated membranes restored F-stimulation of the adenylate cyclase. The toxin-induced modification of adenylate cyclase and the incorration of radioactivity into the 42,000-M, protein were partially reversed upon incubation with toxin and nicotinamide at pH (1. The results indicate that cholera toxin affects the adenylate cyclase system by catalyzing an ADP-ribosylation of the 42,000-Mr component bearing the guanyl nucleotide regulatory site.

Chronic morphine administration increases levels of adenylyl cyclase and cAMP-dependent protein kinase (PKA) activity in the locus coeruleus (LC), which contributes to the severalfold activation of LC neurons that occurs during opiate withdrawal. A role for the transcription factor cAMP response elementbinding protein (CREB) in mediating the opiate-induced upregulation of the cAMP pathway has been suggested, but direct evidence is lacking. In the present study, we first demonstrated that the morphine-induced increases in adenylyl cyclase and PKA activity in the LC are associated with selective increases in levels of immunoreactivity of types I and VIII adenylyl cyclase and of the catalytic and type II regulatory subunits of PKA. We next used antisense oligonucleotides directed against CREB to study the role of this transcription factor in mediating these effects. Infusion (5 d) of CREB antisense oligonucleotide directly into the LC significantly reduced levels of CREB immunoreactivity. This effect was sequence-specific and not associated with detectable toxicity. CREB antisense oligonucleotide infusions completely blocked the morphine-induced upregulation of type VIII adenylyl cyclase but not of PKA. The infusions also blocked the morphine-induced upregulation of tyrosine hydroxylase but not of Gi␣, two other proteins induced in the LC by chronic morphine treatment. Electrophysiological studies revealed that intra-LC antisense oligonucleotide infusions completely prevented the morphine-induced increase in spontaneous firing rates of LC neurons in brain slices. This blockade was completely reversed by addition of 8-bromo-cAMP (which activates PKA) but not by addition of forskolin (which activates adenylyl cyclase). Intra-LC infusions of CREB antisense oligonucleotide also reduced the development of physical dependence to opiates, based on attenuation of opiate withdrawal. Together, these findings provide the first direct evidence that CREB mediates the morphine-induced upregulation of specific components of the cAMP pathway in the LC that contribute to physical opiate dependence.Key words: morphine; opiate withdrawal; gene expression; cAMP; adenylyl cyclase; protein kinase A; G-proteins; tyrosine hydroxylase; protein phosphorylationThe locus coeruleus (LC) has served as a usef ul model system in which to study the long-term actions of opiates on target neurons. The LC is the major noradrenergic nucleus in brain, located on the floor of the fourth ventricle in the anterior pons (Dahlstrom and Fuxe, 1965;Foote et al., 1983;Aston-Jones et al., 1996). Under normal conditions, the LC is implicated in controlling attention, vigilance, and activity of the autonomic nervous system. The LC also has been implicated in physical opiate dependence. Whereas acute opiate administration inhibits the activity of LC neurons, their firing rates recover toward control levels after chronic exposure and increase more than fourfold above control levels on administration of an opioid receptor antagonist in vivo (Aghajanian, 1978;Rasmuss...

It has been known for some time that chronic treatment of neuronal cells and tissues with opioids, contrary to their acute effect, leads to an increase in cAMP accumulation. This phenomenon, defined as adenylyl cyclase superactivation, has been implicated in opiate addiction, yet the mechanism by which it is induced remains unclear. Here, we show that this phenomenon can be reproduced and studied in COS-7 cells cotransfected with adenylyl cyclase type V and mu-opioid receptor cDNAs. These cells display acute opioid inhibition of adenylyl cyclase activity, whereas prolonged exposure to the mu-agonist morphine or [-Ala2, N-methyl-Phe4, Gly-ol5]enkephalin leads to a time-dependent superactivation of adenylyl cyclase. This superactivated state is reversible, because it is gradually lost following agonist withdrawal. Adenylyl cyclase superactivation can be prevented by pertussis toxin pretreatment, indicating the involvement of Gi/o proteins, or by cotransfection with the carboxyl terminus of beta-adrenergic receptor kinase or with alpha-transducin (scavengers of Gbetagamma dimers), indicating a role for the G protein betagamma dimers in adenylyl cyclase superactivation. However, contrary to several other Gbetagamma-dependent signal transduction mechanisms (e.g. the extracellular signal-regulated kinase 2/MAP kinase pathway), adenylyl cyclase superactivation is not affected by the Ras dominant negative mutant N17-Ras.

Peptides corresponding to regions of the calmodulin-activated NO-synthase and of the calmodulin dependent adenylyl cyclase, which could represent the calmodulin binding domains of the two proteins, have been synthesized and tested for calmodulin binding. The chosen peptides were those in the sequence of the two proteins which most closely corresponded to the accepted general properties of the calmodulin binding domains, i.e., a hydrophobic sequence containing basic amino acids. In the case of the NO-synthase, the putative high-affinity calmodulin binding domain was identified by urea gel electrophoresis and fluorescence spectroscopy with dansylcalmodulin as peptide NO-30 (amino acids 725-754). The highest affinity calmodulin binding site of the calmodulin-dependent adenylyl cyclase was assigned to peptide AC-28 (amino acids 495-522) by titration with dansylcalmodulin and by the ability to inhibit the calmodulin-stimulated activity of purified calmodulin-stimulated adenylyl cyclase. The sequence 495-522 is located in the unit protruding into the cytosol from the sixth putative transmembrane domain of the molecule. It has the typical hydrophobic/basic composition of canonical calmodulin binding domains, and also contains, like most calmodulin binding domains, an aromatic amino acid in its N-terminal portion. It also contains two Cys residues in the central portion, which is an unusual feature of the calmodulin binding domain of this enzyme.

Non-hydrolysable analogues of GTP, such as GTPyS and GMP-PNP, have pr,~viously been shown to inhibit the formation of¢onstitutive secretory vesicles (CSVs) and immature secretory granules (ISGs) from the trans-Golgi network (TGN). Using a cell-free system, we show here that the formation of these vesicles is also inhibited by [A1F+]-. a compound known to act on trimeric G-proteins. Addition of highly purified G-protem J~Y subunits stimulated, in a differential manner, the oell-free formation of both CSVs an~ ISGs. ADP-ribosylation experiments revealed the presence of a pertussis toxin-sensitive G-protein ~ subunit in the TGN. ~0~'e conclude that trimer=c G-proteins regulate the formation at + secretory vesicles from the TGN.