Localization of adenylyl and guanylyl cyclase in rat brain by in situ hybridization: comparison with calmodulin mRNA distribution (original) (raw)
Related papers
Journal of Neuroendocrinology, 1994
Only three (Types I, II, V) of the six currently-described subtypes of adenylyl cyclase are prominently expressed in the rat brain. These species are differently sensitive to Ca2+, βγ subunits of G-proteins and protein kinase C. A knowledge of the susceptibility of the cAMP-signalling system in particular brain regions to these diverse modes of regulation can shed light on the mechanism of action of the neurotransmitters that modify neuronal activity in such regions. Cyclic AMP is extensively involved in the physiological functions of the hypothalamus. We have used in situ hybridization histochemistry with synthetic oligonucleotides to examine the expression in the rat hypothalamus of the three major brain subtypes of adenylyl cyclase-Ca2+/calmodulin-stimulable (Type I), Ca2+-insensitive (Type II) and Ca2+ -inhibitable (Type V). The hypothalamus expresses high levels only of Type II mRNA, particularly in the supraoptic and paraventricular nuclei. Curiously, the strong expression of the Ca2+-insensitive Type II mRNA and the lack of expression of the major brain specific Type I mRNA does not correlate with the adenylyl cyclase activity, which is largely Ca2+/calmodulin stimulable in plasma membranes prepared from the hypothalamus.
Synapse, 1993
Physiological studies and inferences from invertebrate models implicate Ca2+/calmodulin-sensitive adenylyl cyclase with memory processes. However, Ca2+/calmodulin-insensitive adenylyl cyclase also occurs in brain, and its neuronal functions are less clear. Two oligonucleotide probes, based on rat cDNAs for Types I and II adenylyl cyclase, which appear to correspond to these functional forms, were used to conduct in situ hybridization analysis of the relative abundance and localization of these two species in the rat brain as a first step in evaluating their neuronal role. Quite discrete patterns of expression were encountered; in some areas, both species were co-expressed, but in others, little overlap was observed. The differential expression of the two mRNAs suggests that discrete roles may be fulfilled by the two adenylyl cyclases in neural tissues. © Wiley-Liss, Inc.
Neurochemical Research, 1986
The subcellular distribution of Ca2+/calmodulin-stimulated adenylate cyclase activity was studied in comparison with that of guanine nucleotide-stimulated cyclase activity. The distributions of these activities were similar among the crude fractions but differed among the purified subsynaptosomal fractions. The specific activity of Ca2+/calmodulin-stimulated cyclase was highest in a light synaptic membrane fraction, which has few, if any, postsynaptic densities, whereas that of guanine nucleotide-stimulated cyclase was highest in a heavier synaptic membrane fraction rich in postsynaptic densities. These results suggest thai the CaZ+/ calmodulin-stimulated cyclase has, at least in part, a different cellular or subcellular location than the guanine nucleotide-stimulated cyclase.
Journal of Biological Chemistry, 1994
A cDNA that encodes type VI11 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. T w o type VI11 messages are detected in rat brain with estimated sizes of 5.5 and 4.4 kilobases. In situ hybridization indicates that the type VI11 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 VI11 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 (EC, = 53 n~ calmodulin). The addition of activated recombinant a subunit of G, synergistically increases the Ca2+/calmodulin-stimulated activity. A possible role for type VI11 adenylyl cyclase in long-term potentiation is discussed. The synthesis of cyclic AMP (CAMP) by mammalian, membrane-bound adenylyl cyclases is modulated by hormones and neurotransmitters acting via receptors that couple agonist binding to regulation of the enzyme through direct activation by G proteins and cross-talk with other signaling pathways (1, 2). While adenylyl cyclase activity is ubiquitous in mammalian tissues, it has been demonstrated both biochemically and through molecular cloning that various forms of the enzyme exist that differ widely with regard to tissue distribution, abun-~ ~ ~~ GM46395 (to J. K.) and GM32483 (to D. M. F. C.
Journal of Cellular Biochemistry, 1988
Ca2+, through the mediation of calmodulin, stimulates the activity of brain adenylate cyclase. The growing awareness that fluctuating Ca2+ concentrations play a major role in intracellular signalling prompted the present study, which aimed to investigate the implications for neurotransmitter (receptor) regulation of enzymatic activity of this calmodulin regulation. The role of Ca2+/calmodulin in regulating neurotransmitter-mediated inhibition and stimulation was assessed in a number of rat brain areas. Ca*+/calmodulin stimulated adenylate cyclase activity in EGTA-washed plasma preparations from each region studied-from 1 .%fold (in striatum) to 3.4-fold (in cerebral cortex). The fold-stimulation produced by Ca2+/calmodulin was decreased in the presence of GTP, forskolin, or Mn2'. In EGTA-washed membranes, receptor-mediated inhibition of adenylate cyclase was strictly dependent upon Ca*+/calmodulin stimulation in all regions, except striatum. A requirement for Mg2+ in combination with Ca*+/calmodulin to observe neurotransmitter-mediated inhibition was also observed. In contrast, receptormediated stimulation of activity was much greater in the absence of Ca2+/ calmodulin. The findings demonstrate that ambient Ca*' concentrations, in concert with endogenous calmodulin, may play a central role in dictating whether inhibition or stimulation of adenylate cyclase by neurotransmitters may proceed.
Adenylyl Cyclases: mRNA and Characteristics of Enzyme Activity in Three Areas of Brain
Journal of Neurochemistry, 2002
RNase protection assays were used in a comparative analysis of the quantities of mRNA for five "calcium-sensitive" (types I, Ill, V, VI, and VIII) adenylyl cyclases and one "calcium-insensitive" (type II) adenylyl cyclase in mouse cerebral cortex, cerebellum, and nucleus accumbens. The mRNA levels for type V adenylyl cyclase were dominant in the nucleus accumbens. Type V adenylyl cyclase mRNA was also found in the cerebral cortex and at low levels in the cerebellum. Type I adenylyl cyclase mRNA was the major form in the cerebellum with 15-50-fold higher levels compared with other adenylyl cyclase mRNAs. Type I adenylyl cyclase mRNA was also the most prominent adenylyl cyclase mRNA in the cerebral cortex, although the mRNA levels of other adenylyl cyclase forms were more comparable to those of the type I enzyme in this brain area. The mRNA levels for adenylyl cyclase types II, Ill, VI, and VIII were intermediate to low depending on the brain area. Cell membranes from the nucleus accumbens demonstrated adenylyl cyclase activity that was synergistically activated by concomitant addition of GTP and forskolin to assay mixtures, reflecting a characteristic of type V adenylyl cyclase protein. Calcium/calmodulin stimulated adenylyl cyclase activity in membranes from all three brain areas. However, synergistic activation of adenylyl cyclase activity by GTP and calcium/calmodulin was noted only with cortical membranes, and this characteristic may reflect the presence of type VIII adenylyl cyclase mRNA in the cortex. Although mRNA for type VIII adenylyl cyclase was almost equivalent in the cortex and cerebellum, the lack of a synergistic effect of GTP plus calcium/calmodulin on the cerebellar enzyme activity may be a result of the significant dominance of type I adenylyl cyclase mRNA (and protein) in the cerebellum. In general, the mRNA levels for the various adenylyl cyclases were predictive of the regulatory characteristics of adenylyl cyclase activity in membranes of the brain areas studied.
Neuroscience, 2000
The Ca 2ϩ -calmodulin stimulated AC1 and Ca 2ϩ -insensitive AC2 are major isoforms of adenylyl cyclase, playing an important role in synaptic plasticity in the mammalian brain. We studied the pattern of expression of AC1 and AC2 genes in the hippocampus of C57BL/6 mice. We found that there were differences in their patterns of distribution in the dentate gyrus. AC1 messenger RNA was detected both in the dentate granule cell bodies and the corresponding molecular field whereas AC2 messenger RNA was preferentially distributed in the dentate granule cell layer, suggesting that AC1 and AC2 messenger RNA are differentially regulated in the dentate gyrus. In order to examine the regulation of AC1 and AC2 expression in response to synaptic deafferentation and reinnervation, the distribution patterns of the two AC messenger RNA in the hippocampal fields and the parietal cortex were analysed 2, 5, 9 and 30 days following an unilateral entorhinal cortex lesion. Interestingly, we found significantly reduced levels of AC1 hybridization signal following the lesion whereas the level of AC2 messenger RNA remained unaffected in all lesioned groups. The changes in AC1 messenger RNA were transient, with a maximal reduction at five days postlesion, and were restricted to the granule cell bodies and stratum moleculare of the deafferented dentate gyrus. No significant change in AC1 messenger RNA levels was detected in other hippocampal fields nor for any other postlesion times studied.
Expression of Soluble Guanylyl Cyclase Gene in Adult Rat Brain
European Journal of Neuroscience, 1994
The synthesis of the intracellular messenger, cyclic GMP, is catalysed by particulate or soluble guanylyl cyclase (sGCY). sGCY is activated by nitric oxide, a compound with putative neurotransmitter functions, especially in long-term potentiation. Hybridization histochemistry with a probe complementary to the rat lung large (al) subunit was used to assess the exact localization of sGCY mRNA in the rat brain. Many cells in the olfactory bulb contained sGCY mRNA. In the whole cerebral cortex, sGCY mRNA was found in all layers, with a predominance in layers 11-111. A similar pattern was found in the olfactory tuberculum, in continuation with the piriform cortex and the cortical amygdaloid nucleus. All parts of the striatum expressed sGCY mRNA. sGCY mRNA was also found in the habenula medialis, in the pinealis in some diencephalic nuclei, and in the granule cell layers of the cerebellum. This study provides a description of the normal anatomy of sGCY gene expression in the rat forebrain as a basis for the study of the modulation of expression after physiological and pharmacological manipulations.
Proceedings of The National Academy of Sciences, 1995
Only three isoforms of adenylyl cyclase (EC 4.6.1.1) mRNAs (AC1, -2, and -5) are expressed at high levels in rat brain. ACI occurs predominantly in hippocampus and cerebellum, AC5 is restricted to the basal ganglia, whereas AC2 is more widely expressed, but at much lower levels. The distribution and abundance of adenylyl cyclase protein were examined by immunohistochemistry with an antiserum that recognizes a peptide sequence shared by all known mammalian adenylyl cyclase isoforms. The immunoreactivity in striatum and hippocampus could be readily interpreted within the context of previous in situ hybridization studies. However, extending the information that could be gathered by comparisons with in situ hybridization analysis, it was apparent that staining was confined to the neuropil-corresponding to immunoreactive dendrites and axon terminals. Electron microscopy indicated a remarkably selective subcellular distribution of adenylyl cyclase protein. In the CAl area of the hippocampus, the densest immunoreactivity was seen in postsynaptic densities in dendritic spine heads. Labeled presynaptic axon terminals were also observed, indicating the participation of adenylyl cyclase in the regulation of neurotransmitter release. The selective concentration of adenylyl cyclases at synaptic sites provides morphological data for understanding the pre-and postsynaptic roles of adenylyl cyclase in discrete neuronal circuits in rat brain. The apparent clustering of adenylyl cyclases, coupled with other data that suggest higher-order associations of regulatory elements including G proteins, N-methyl-D-aspartate receptors, and cAMP-dependent protein kinases, suggests not only that the primary structural information has been encoded to render the cAMP system responsive to the Ca2+-signaling system but also that higher-order strictures are in place to ensure that Ca2+ signals are economically delivered and propagated.