Cortical adenylyl cyclase 1 is required for thalamocortical synapse maturation and aspects of layer IV barrel development.

Experimental evidence from mutant or genetically altered mice indicates that the formation of barrels and the proper maturation of thalamocortical (TC) synapses in the primary somatosensory (barrel) cortex depend on mechanisms mediated by neural activity. Type 1 adenylyl cyclase (AC1), which catalyzes the formation of cAMP, is stimulated by increases in intracellular Ca(2+) levels in an activity-dependent manner. The AC1 mutant mouse, barrelless (brl), lacks typical barrel cytoarchitecture, and displays presynaptic and postsynaptic functional defects at TC synapses. However, because AC1 is expressed throughout the trigeminal pathway, the barrel cortex phenotype of brl mice may be a consequence of AC1 disruption in cortical or subcortical regions. To examine the role of cortical AC1 in the development of morphological barrels and TC synapses, we generated cortex-specific AC1 knock-out (CxAC1KO) mice. We found that neurons in layer IV form grossly normal barrels and TC axons fill barrel hollows in CxAC1KO mice. In addition, whisker lesion-induced critical period plasticity was not impaired in these mice. However, we found quantitative reductions in the quality of cortical barrel cytoarchitecture and dendritic asymmetry of layer IV barrel neurons in CxAC1KO mice. Electrophysiologically, CxAC1KO mice have deficits in the postsynaptic but not in the presynaptic maturation of TC synapses. These results suggest that activity-dependent postsynaptic AC1-cAMP signaling is required for functional maturation of TC synapses and the development of normal barrel cortex cytoarchitecture. They also suggest that the formation of the gross morphological features of barrels is independent of postsynaptic AC1 in the barrel cortex.

Expression of adenylyl cyclase type I in cochlear inner hair cells.

Expression of calcium/calmodulin-activated adenylyl cyclase type I (ACI) mRNA has been determined in the cochlea and in an organ-of-Corti subdissected tissue fraction by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Amplification products of predicted size were obtained from the mouse cochlea and rat organ of Corti with nucleotide sequences corresponding to respective ACI brain transcripts. In addition, ACI template was detected in a rat inner hair cell cDNA library by PCR. Immunoreactivity to ACI has been localized within the organ of Corti to the inner hair cell, with diaminobenzidine staining found in both the cell body and in the stereocilia. Evidence, thus, has been obtained that both ACI transcript and protein are expressed in the inner hair cell, the primary mechanosensory receptor cell of the cochlea. We hypothesize that ACI is activated by calcium influx through a calcium/calmodulin interaction and that this adenytyl cyclase isoform may have a role in modulation of receptoneural afferent transmission and/or mechanosensory transduction in the cochlea.

Molecular cloning and expression of an adenylyl cyclase from Xenopus laevis oocytes.

We have cloned a cDNA that encodes a novel Xenopus laevis oocyte adenylyl cyclase (xlAC) using oligonucleotides against conserved mammalian adenylyl cyclase regions. The isolated cDNA is 4372 bp long with an open reading frame of 4065 nucleotides which encodes a protein of 1355 amino acids. Comparison of the deduced amino acid sequence with previously cloned mammalian adenylyl cyclases shows a low identity, 19.7% with type 2 rat adenylyl cyclase and 24.2% with type 4 rat adenylyl cyclase, indicating that this Xenopus isoform represents a new member of this protein family. Gene expression studies of the xlAC by reverse PCR showed that this gene is expressed in all oogenesis stages but not during early embryogenesis. Expression of the xlAC in COS-7 cells resulted in increased basal AC activity, that was stimulated by forskolin, Gpp(NH)p and aluminium fluoride, and was insensitive to calcium and calcium-calmodulin (Ca2(+)-CaM).

Calcineurin feedback inhibition of agonist-evoked cAMP formation.

The effects of immunosuppressant blockers of calcineurin (protein phosphatase 2B) on cAMP formation and hormone release were investigated in mouse pituitary tumor (AtT20) cells. Immunosuppressants enhanced corticotropin-releasing factor- and isoproterenol-evoked cAMP production in proportion with their potency to block calcineurin. Further analysis of cAMP production revealed that intracellular Ca2+ derived through voltage-regulated calcium channels reduces cAMP formation induced by corticotropin releasing-factor or beta 2-adrenergic stimulation and that this effect of Ca2+ is inhibited by blockers of calcineurin. AtT20 cells were found to express at least three species of adenylyl cyclase mRNA-encoding types 1 and 6 as well as a novel isotype, which appeared to be the predominant species. In two cell lines expressing very low or undetectable levels of the novel cyclase mRNA (NCB20 and HEK293 cells respectively), corticotropin-releasing factor-induced cAMP formation was not altered upon blockage of calcineurin activity. These data identify calcineurin as a Ca2+ sensor that mediates the negative feedback effect of intracellular Ca2+ on receptor-stimulated cAMP production. Furthermore, the effect of calcineurin on cAMP synthesis appears to be associated with the expression of a novel adenylyl cyclase isotype, which is highly abundant in AtT20 cells.

The characterization of a novel human adenylyl cyclase which is present in brain and other tissues.

We characterized a human cDNA clone which encodes a novel adenylyl cyclase. Data from Southern and Northern blot analysis, and analysis of sequence similarity with a recently cloned mouse adenylyl cyclase (10), indicated that the human adenylyl cyclase was a species variant of type VII adenylyl cyclase. The sequence of the novel human adenylyl cyclase indicated it was a member of the type II adenylyl cyclase family, and we compared the regulatory characteristics of the novel human enzyme with those of type II adenylyl cyclase. The human type VII and rat type II adenylyl cyclases, expressed in human embryonic kidney 293 cells, were activated by prostaglandin E1 (PGE1), but only type VII was activated by isoproterenol. The stimulation of type VII adenylyl cyclase by PGE1 and isoproterenol was attenuated by pretreatment of the cells with staurosporine. Phorbol 12,13-dibutyrate synergistically enhanced the stimulation of both type VII and type II enzyme activity by PGE1 and by the constitutively active Gs mutant Gs (Q227L). The human type VII adenylyl cyclase activity was unresponsive to capacitatively induced changes in intracellular Ca2+. The functional characteristics of human type VII adenylyl cyclase resemble those of the rat type II enzyme, but the enzymes may respond differently to in vivo phosphorylation conditions. While the mRNA for adenylyl cyclase type II was found in several brain areas, the message for type VII adenylyl cyclase was localized primarily to the cerebellar granule cell layer.

Ca(2+)-inhibitable adenylyl cyclase modulates pulmonary artery endothelial cell cAMP content and barrier function.

Maintenance by the endothelium of a semi-permeable barrier is critically important in the exchange of oxygen and carbon dioxide in the lung. Intracellular free Ca2+ ([Ca2+]i) and cAMP are principal determinants of endothelial cell barrier function through their mutually opposing actions on endothelial retraction. However, details of the mechanisms of this antagonism are lacking. The recent discovery that certain adenylyl cyclases (EC 4.6.1.1) could be acutely inhibited by Ca2+ in the intracellular concentration range provided one possible mechanism whereby elevated [Ca2+]i could decrease cAMP content. This possibility was explored in pulmonary artery endothelial cells. The results indicate that a type VI Ca(2+)-inhibitable adenylyl cyclase exists in pulmonary artery endothelial cells and is modulated by physiological changes in [Ca2+]i. Furthermore, the results suggest the inverse relationship between [Ca2+]i and cAMP that is established by Ca(2+)-inhibitable adenylyl cyclase plays a critical role in modulating pulmonary artery endothelial cell permeability. These data provide evidence that susceptibility to inhibition of adenylyl cyclase by Ca2+ can be exploited in modulating a central physiological process.

Molecular cloning and characterization of the type VII isoform of mammalian adenylyl cyclase expressed widely in mouse tissues and in S49 mouse lymphoma cells.

We have isolated a 5199-nucleotide cDNA from a mouse library containing an open reading frame encoding the 1099-amino acid type VII adenylyl cyclase protein. The type VII protein is most closely related in primary structure to an unpublished human adenylyl cyclase clone (GenBank accession no. D25538) and type II adenylyl cyclase. Northern blot analysis demonstrates that the type VII mRNA is most abundant in mouse heart, spleen, and lung. cAMP content rises rapidly in HEK 293 cells overexpressing type VII adenylyl cyclase following treatment with phorbol ester, peaking by 4 min, while cells expressing the type II adenylyl cyclase reach peak accumulation only after 20 min. Increases in intracellular calcium through treatment of type VII-293 cells with either ATP or A23187 alone failed to increase intracellular cAMP content. Phorbol ester treatment acted synergistically with beta-adrenergic stimulation to increase cAMP content in type VII-transformed cells. Pretreatment of type VII-transformed cells with pertussis toxin fails to prevent phorbol ester potentiation of isoproterenol stimulation. Thus the ability of phorbol ester to increase basal and isoproterenol-stimulated type VII activity appears to be a direct effect on this adenylyl cyclase isoform and not the result of modification of the inhibitory G protein, Gi.

Effect of ovarian steroids on hypothalamic norepinephrine-induced adenylate cyclase activity.

The adenylate cyclase activity induced by norepinephrine (10(-8)-10(-9) M) was studied in hypothalamic particulate fractions from female rats. The effect of estradiol on this activity was studied in rats that were ovariectomized in diestrus 1, injected with estradiol benzoate (50 micrograms/kg body weight), and killed 48 h later. The effect of progesterone was studied in fractions from female cycling rats injected in the morning of diestrus 2 with progesterone (2 mg/rat); these animals were killed 30 or 48 h after the steroid injection. The blockade of norepinephrine-induced adenylate cyclase activity by alpha- and beta-blocking agents (10(-8)-10(-9) M) was also evaluated. The enzymatic activity was determined by monitoring the capacity to produce cAMP from ATP at saturated levels; cAMP was assayed by radioimmunoassay. At 48 h after administration, estradiol benzoate increased the norepinephrine-induced adenylate cyclase activity in the hypothalamus. This effect was not changed by the presence of phenoxybenzamine, an alpha-adrenoceptor blocker, but was greatly reduced by propranolol, the beta-adrenoreceptor blocker. In contrast, the progesterone, at 30 and 48 h after injection, decreased the hypothalamic adenylate cyclase activity, and this effect was preferentially antagonized in the presence of phenoxybenzamine rather than propranolol. These results suggest that estrogen and progesterone act selectively on one or another type of adrenergic receptor at the hypothalamic level.

Type VIII adenylyl cyclase. A Ca2+/calmodulin-stimulated enzyme expressed in discrete regions of rat brain.

A cDNA that encodes type VIII adenylyl cyclase has been isolated from two rat brain libraries. The open reading frame encodes a 1248-amino acid protein predicted to have two sets of six transmembrane spans and two putative nucleotide binding domains as is characteristic of other mammalian adenylyl cyclases. Two type VIII messages are detected in rat brain with estimated sizes of 5.5 and 4.4 kilobases. In situ hybridization indicates that the type VIII messages are most abundantly expressed in the granule cells of the dentate gyrus, the pyramidal cells of hippocampal fields CA1-CA3, the entorhinal cortex, and the piriform cortex. Hybridization is also detected in the neocortex, the amygdaloid complex, and regions of the thalamus and hypothalamus. Stable expression of the type VIII cDNA in human embryonal kidney cells leads to the appearance of a novel 165-kDa glycoprotein in the membrane fraction. Stimulation of these cells with agents that increase intracellular Ca2+ results in up to 43-fold increases in cAMP accumulation over that of control cells transfected with the expression vector. Addition of isoproterenol alone does not lead to type VIII-specific effects in intact cells. Adenylyl cyclase activity in membranes prepared from type VIII-transformed cells is stimulated up to 40-fold by the addition of Ca2+/calmodulin (EC50 = 53 nM calmodulin). The addition of activated recombinant alpha subunit of Gs synergistically increases the Ca2+/calmodulin-stimulated activity. A possible role for type VIII adenylyl cyclase in long-term potentiation is discussed.

Expression and characterization of calmodulin-activated (type I) adenylylcyclase.

A complementary DNA that encodes a bovine brain, calmodulin-sensitive (type I) adenylylcyclase has been inserted into the baculovirus genome under the control of the strong polyhedron promoter. Expression of the recombinant adenylylcyclase in Sf9 cells using recombinant baculovirus increases adenylylcyclase activity in cell membranes to 10-20 nmol.min-1.mg-1 (approximately 0.1% of membrane protein). The catalytic activity of the recombinant adenylylcyclase can be stimulated by Gs alpha, calmodulin, or forskolin, and it can be inhibited by adenosine analogs and by G protein beta gamma subunit. The specific activity of the purified recombinant protein approximates 5 mumol.min-1.mg-1. This is similar to that of the enzyme purified from bovine brain. Type I adenylylcyclase has a quasiduplicated structure. There are two membrane-spanning domains, each with six putative transmembrane helices, and there are two presumed nucleotide-binding domains that are about 55% similar to each other. No catalytic activity is detectable when each half of the adenylylcyclase molecule is expressed by itself. However, coexpression of the two halves results in considerable enzymatic activity. Interaction between the two halves of adenylylcyclase may be necessary for catalysis.