Complementary DNA's that encode an adenylyl cyclase were isolated from a bovine brain library. Most of the deduced amino acid sequence of 1134 residues is divisible into two alternating sets of hydrophobic and hydrophilic domains. Each of the two large hydrophobic domains appears to contain six transmembrane spans. Each of the two large hydrophilic domains contains a sequence that is homologous to a single cytoplasmic domain of several guanylyl cyclases; these sequences may represent nucleotide binding sites. An unexpected topographical resemblance between adenylyl cyclase and various plasma membrane channels and transporters was observed. This structural complexity suggests possible, unappreciated functions for this important enzyme.

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...

Biochemical, immunological, and molecular cloning studies have suggested the existence of multiple forms of adenylyl cyclase (EC 4.6.1.1). An adenylyl cyclase cDNA clone (type II) was isolated from a rat brain library and found to encode a protein of 1090 amino acids that was homologous to but distinct from the previously described Ca2+/calmodulin-stimulated adenylyl cyclase from bovine brain. Expression of the type II cDNA in an insect cell line resulted in an increased level of adenylyl cyclase activity that was insensitive to Ca2+/calmodulin. Addition of activated Gs alpha protein to type II-containing membranes increased enzyme activity. The mRNA encoding the type II protein was expressed at high levels in brain tissue and at low levels in olfactory epithelium and lung. The existence of multiple adenylyl cyclase enzymes may provide for complex and distinct modes of biochemical regulation of cAMP levels in the brain.

Recombinant adenylyl cyclase isozyme Types I, II, VI, VII, and three splice variants of Type VIII were compared for their sensitivity to P-site-mediated inhibition by several adenine nucleoside derivatives and by the family of recently synthesized adenine nucleoside 3-polyphosphates (Dé saubry, L., Shoshani, I., and Johnson, R. A. (1996) J. Biol. Chem. 271, 14028 -14034). Inhibitory potencies were dependent on isozyme type, the mode of activation of the respective isozymes, and on P-site ligand. For the nucleoside derivatives potency typically followed the order 2,5-dideoxyadenosine (2,5-ddAdo) > ␤-adenosine > 9-(cyclopentyl)-adenine (9-CP-Ade) 9-(tetrahydrofuryl)-adenine (9-THF-Ade; SQ 22,536), with the exception of Type II adenylyl cyclase, which was essentially insensitive to inhibition by 9-CP-Ade. For the adenine nucleoside 3-polyphosphates inhibitory potency followed the order Ado < 2-dAdo < 2,5-ddAdo and 3-mono-< 3-di-< 3-triphosphate. Differences in potency of these ligands were noted between isozymes. The most potent ligand was 2,5-dd-3-ATP with IC 50 values of 40 -300 nM. The data demonstrate isozyme selectivity for some ligands, suggesting the possibility of isozyme-selective inhibitors to take advantage of differences in P-site domains among adenylyl cyclase isozymes. Differential expression of adenylyl cyclase isozymes may dictate the physiological sensitivity and hence importance of this regulatory mechanism in different cells or tissues.Adenylyl cyclase is potently and directly inhibited by analogs of adenosine via a domain referred to as the P-site from its requirement for an intact purine moiety (1-4). Domains for catalysis and inhibition have been distinguished by use of enzyme purification (5, 6), inhibition kinetics (7, 8), site-specific covalent ligands (9), and selective amino acid substitutions (10). These data suggest that the P-site is distinct from, yet homologous to and interacting with, the catalytic domain. The observation that purified native and recombinant Type I adenylyl cyclases are inhibited by P-site ligands, although exhibiting decreased sensitivity to inhibition (4 -6, 11), establishes the locus of the P-site on the enzyme per se and that inhibition is not via cell surface receptors or G-proteins.P-site-mediated inhibition has been characterized pharmacologically (1, 2, 4, 12-16). Inhibition requires an intact adenine moiety, and potency of inhibition is increased substantially for deoxyribose and especially 3Ј-phosphorylribose adenine nucleosides. Inhibitory potency follows the order: 3Ј-mono-Ͻ 3Ј-di Ͻ 3Ј-triphosphate and adenosine (Ado) Ͻ 2Ј-deoxy (d) 1 -Ado Ͻ 2Ј,5Ј-ddAdo, with 2Ј,5Ј-dd-3Ј-ATP being the most potent ligand and exhibiting an IC 50 ϳ40 nM (15). In addition, tolerance for large substitutions at the 3Ј-position and for other ribose modifications has been demonstrated (1, 2, 4).We reported previously that levels of 2Ј-d-3Ј-AMP and 3Ј-AMP varied considerably in different tissues and were dependent on the metabolic state of the animal (17). Moreover, sensitivity o...

is the counterion for ATP. The differences in the structural and enzymatic properties of these three variants are discussed.A variety of hormones, neurotransmitters, and olfactants regulate the synthesis of cAMP by adenylyl cyclases (ACs). 1Many of these agents act through three component, G proteincoupled systems that are capable of modulating the activity of an AC (for review, see Ref. 1). Alternatively, seemingly independent signaling pathways may generate other second messengers or activate kinases that subsequently regulate AC activity by signal cross-talk. The importance of the latter class of regulatory mechanisms has become apparent with the realization that the different AC isoforms are distinguished by their ability to provide a unique integrated response to coincident stimuli.Eight full-length mammalian ACs have been characterized (2-14), and other partial cDNA sequences have been reported (15,16). The existence of additional forms which are derived by alternative splicing of the messages is consistent with the sequence differences in the amino-terminal domains encoded by cDNA clones for both type V (6, 17) and type VI (7-10). A half-molecule variant of the canine type V has been described (18), and the expression of other AC variants is suggested by the detection of multiple type V and type VIII transcripts on RNA blots under high stringency conditions (6, 9, 11, 13). Thus far, unique functional properties have not been ascribed to any specific splice variant.The mammalian ACs share a common topography. The amino-terminal domains are all predicted to be cytoplasmic, but they vary dramatically in both sequence and length. The only well conserved sequences among the ACs are found in the so-called C 1a and C 2a regions, which are two large cytoplasmic domains of over 200 amino acids each (5,19,20). The conserved domains are homologous to each other and to the catalytic domain of the guanylyl cyclases (2, 21). Based on this similarity, they are considered to be nucleotide binding domains and have recently been shown to be sufficient to confer enzymatic activity (22). Each of these domains is preceded by a large hydrophobic region of variable sequence, which includes six transmembrane spans based on hydropathy analysis. Consensus N-linked glycosylation site(s) are always present in at least one putative extracellular domain associated with the second set of six transmembrane spans (2-14). On the carboxyl-terminal side of the first putative nucleotide binding domain is a highly variable region called C 1b (5,19,20), which is a site for type-specific regulation (23-25). The corresponding region following the second nucleotide binding domain, C 2b , is only present in types I, III, and VIII (2, 4, 13). While similarities among these isoforms may underlie their common enzymatic function, their differences at the amino acid level presumably account for variable responsiveness to a variety of regulatory influences.Type VIII AC is a Ca 2ϩ /calmodulin-stimulated isoform abundantly expressed in discrete regions of...