Adenylyl Cyclase mRNA Research Articles

B-adrenergic/cAMP signaling in doxorubicin-induced cardiotoxicity

Doxorubicin (Dox) is one of the most commonly used chemotherapy but also one of the most cardiotoxic. As of today, only the iron chelator Dexrazoxane has been FDA and EMA approved as a cardioprotective agent for Dox but its usage is still controversial. Thus, there is an urgent need to identify new targets to develop an effective protection against Dox-induced cardiotoxicity (DIC) without compromising its anti-tumor action. Well-characterized DIC mechanisms include topoisomerase II inhibition, DNA damages, induction of ROS and bioenergetic distress that lead to cardiomyocyte death. Alternative mechanisms are emerging such as an alteration of the β-adrenergic system. Our objective here was to study DIC mechanisms with a specific focus on how Dox treatment affects the β-adrenergic/cAMP signaling cascade. We used FRET-based sensors for cAMP targeted to two different cellular compartments, cytosol and nucleus, and measured the cAMP response to the β-adrenergic agonist isoprenaline (Iso, 10 nM) in adult rat ventricular myocytes after 24 h treatment with 1 or 10 μM Dox. Basal cAMP concentration was measured by Elisa. Adenylyl cyclase (AC5 and AC6 isoforms) mRNA was measured by RT-qPCR and phosphodiesterase (PDE) isoform expression and activity by western blot and radioenzymatic assay, respectively. cAMP response to Iso was decreased by 25% at 1 μM and > 50% at 10 μM Dox in both cytosol and nucleus. The maximal response to 20 μM L858051 (a forskolin analog) + 200 μM IBMX (a PDE inhibitor) on top of Iso was also decreased by Dox. Dox treatment did not change basal cAMP level nor PDE activity, but decreased AC5 and AC6 mRNA and PDE4 protein expression. Doxorubicin treatment downregulates the β-adrenergic/cAMP cascade in ventricular myocytes by decreasing adenylyl cyclase expression and maximal activity. This may contribute to the Dox-induced cardiotoxicity mechanisms.

Mammalian Taste Cells Express Functional Olfactory Receptors.

The peripheral taste and olfactory systems in mammals are separate and independent sensory systems. In the current model of chemosensation, gustatory, and olfactory receptors are genetically divergent families expressed in anatomically distinct locations that project to disparate downstream targets. Although information from the 2 sensory systems merges to form the perception of flavor, the first cross talk is thought to occur centrally, in the insular cortex. Recent studies have shown that gustatory and olfactory receptors are expressed throughout the body and serve as chemical sensors in multiple tissues. Olfactory receptor cDNA has been detected in the tongue, yet the presence of physiologically functional olfactory receptors in taste cells has not yet been demonstrated. Here we report that olfactory receptors are functionally expressed in taste papillae. We found expression of olfactory receptors in the taste papillae of green fluorescent protein-expressing transgenic mice and, using immunocytochemistry and real-time quantitative polymerase chain reaction experiments, the presence of olfactory signal transduction molecules and olfactory receptors in cultured human fungiform taste papilla (HBO) cells. Both HBO cells and mouse taste papilla cells responded to odorants. Knockdown of adenylyl cyclase mRNA by specific small inhibitory RNA and pharmacological block of adenylyl cyclase eliminated these responses, leading us to hypothesize that the gustatory system may receive olfactory information in the periphery. These results provide the first direct evidence of the presence of functional olfactory receptors in mammalian taste cells. Our results also demonstrate that the initial integration of gustatory and olfactory information may occur as early as the taste receptor cells.

Adenylyl cyclase mRNA localizes to the posterior of polarized DICTYOSTELIUM cells during chemotaxis

BackgroundIn Dictyostelium discoideum, vesicular transport of the adenylyl cyclase A (ACA) to the posterior of polarized cells is essential to relay exogenous 3′,5′-cyclic adenosine monophosphate (cAMP) signals during chemotaxis and for the collective migration of cells in head-to-tail arrangements called streams.ResultsUsing fluorescence in situ hybridization (FISH), we discovered that the ACA mRNA is asymmetrically distributed at the posterior of polarized cells. Using both standard estimators and Monte Carlo simulation methods, we found that the ACA mRNA enrichment depends on the position of the cell within a stream, with the posterior localization of ACA mRNA being strongest for cells at the end of a stream. By monitoring the recovery of ACA-YFP after cycloheximide (CHX) treatment, we observed that ACA mRNA and newly synthesized ACA-YFP first emerge as fluorescent punctae that later accumulate to the posterior of cells. We also found that the ACA mRNA localization requires 3′ ACA cis-acting elements.ConclusionsTogether, our findings suggest that the asymmetric distribution of ACA mRNA allows the local translation and accumulation of ACA protein at the posterior of cells. These data represent a novel functional role for localized translation in the relay of chemotactic signal during chemotaxis.

Enhanced expression of ADCY1 underlies aberrant neuronal signalling and behaviour in a syndromic autism model

Fragile X syndrome (FXS), caused by the loss of functional FMRP, is a leading cause of autism. Neurons lacking FMRP show aberrant mRNA translation and intracellular signalling. Here, we identify that, in Fmr1 knockout neurons, type 1 adenylyl cyclase (Adcy1) mRNA translation is enhanced, leading to excessive production of ADCY1 protein and insensitivity to neuronal stimulation. Genetic reduction of Adcy1 normalizes the aberrant ERK1/2- and PI3K-mediated signalling, attenuates excessive protein synthesis and corrects dendritic spine abnormality in Fmr1 knockout mice. Genetic reduction of Adcy1 also ameliorates autism-related symptoms including repetitive behaviour, defective social interaction and audiogenic seizures. Moreover, peripheral administration of NB001, an experimental compound that preferentially suppresses ADCY1 activity over other ADCY subtypes, attenuates the behavioural abnormalities in Fmr1 knockout mice. These results demonstrate a connection between the elevated Adcy1 translation and abnormal ERK1/2 signalling and behavioural symptoms in FXS.

CAMP-mediated secretion of brain-derived neurotrophic factor in developing airway smooth muscle

Moderate hyperoxic exposure in preterm infants contributes to subsequent airway dysfunction and to risk of developing recurrent wheeze and asthma. The regulatory mechanisms that can contribute to hyperoxia-induced airway dysfunction are still under investigation. Recent studies in mice show that hyperoxia increases brain-derived neurotrophic factor (BDNF), a growth factor that increases airway smooth muscle (ASM) proliferation and contractility. We assessed the mechanisms underlying effects of moderate hyperoxia (50% O2) on BDNF expression and secretion in developing human ASM. Hyperoxia increased BDNF secretion, but did not alter endogenous BDNF mRNA or intracellular protein levels. Exposure to hyperoxia significantly increased [Ca2+]i responses to histamine, an effect blunted by the BDNF chelator TrkB-Fc. Hyperoxia also increased ASM cAMP levels, associated with reduced PDE4 activity, but did not alter protein kinase A (PKA) activity or adenylyl cyclase mRNA levels. However, 50% O2 increased expression of Epac2, which is activated by cAMP and can regulate protein secretion. Silencing RNA studies indicated that Epac2, but not Epac1, is important for hyperoxia-induced BDNF secretion, while PKA inhibition did not influence BDNF secretion. In turn, BDNF had autocrine effects of enhancing ASM cAMP levels, an effect inhibited by TrkB and BDNF siRNAs. Together, these novel studies suggest that hyperoxia can modulate BDNF secretion, via cAMP-mediated Epac2 activation in ASM, resulting in a positive feedback effect of BDNF-mediated elevation in cAMP levels. The potential functional role of this pathway is to sustain BDNF secretion following hyperoxic stimulus, leading to enhanced ASM contractility and proliferation.

Docosahexaenoyl ethanolamide improves glucose uptake and alters endocannabinoid system gene expression in proliferating and differentiating C2C12 myoblasts

Skeletal muscle is a major storage site for glycogen and a focus for understanding insulin resistance and type-2-diabetes. New evidence indicates that overactivation of the peripheral endocannabinoid system (ECS) in skeletal muscle diminishes insulin sensitivity. Specific n-6 and n-3 polyunsaturated fatty acids (PUFA) are precursors for the biosynthesis of ligands that bind to and activate the cannabinoid receptors. The function of the ECS and action of PUFA in skeletal muscle glucose uptake was investigated in proliferating and differentiated C2C12 myoblasts treated with either 25 μM of arachidonate (AA) or docosahexaenoate (DHA), 25 μM of EC [anandamide (AEA), 2-arachidonoylglycerol (2-AG), docosahexaenoylethanolamide (DHEA)], 1 μM of CB1 antagonist NESS0327, and CB2 inverse agonist AM630. Compared to the BSA vehicle control cell cultures in both proliferating and differentiated myoblasts those treated with DHEA, the EC derived from the n-3 PUFA DHA, had higher 24 h glucose uptake, while AEA and 2-AG, the EC derived from the n-6 PUFA AA, had lower basal glucose uptake. Adenylyl cyclase mRNA was higher in myoblasts treated with DHA in both proliferating and differentiated states while those treated with AEA or 2-AG were lower compared to the control cell cultures. Western blot and qPCR analysis showed higher expression of the cannabinoid receptors in differentiated myoblasts treated with DHA while the opposite was observed with AA. These findings indicate a compensatory effect of DHA and DHEA compared to AA-derived ligands on the ECS and associated ECS gene expression and higher glucose uptake in myoblasts.

S129 Decreased cAMP Production in Lung Fibroblasts from Patients with Idiopathic Pulmonary Fibrosis

RationaleIdiopathic pulmonary fibrosis (IPF) is a fatal lung disease with unknown aetiology and no effective therapy. Myofibroblasts are the primary effector cells in the pathogenesis of IPF and differentiation from...

Aberrant cAMP Metabolism in NF1 Malignant Peripheral Nerve Sheath Tumor Cells

Malignant peripheral nerve sheath (MPNST) cell lines derived from patients with neurofibromatosis type 1 (NF!) were found to have basal cAMP levels which are two-fold higher than cAMP levels in normal human adult Schwann cells (nHSC). PCR analysis also revealed that normal adult human Schwann cells express mRNA for types Ill, IV, and IX adenylyl cyclase (AC) while NF1 MPNST cells express AC mRNA of types II, V, and VIII in addition to expressing all the isoforms of normal adult human Schwann cells. Further PCR analysis revealed that NF1 MPNST lines express mRNA for EP2 and EP4 prostaglandin receptors whereas nHSC only express mRNA for the EP2 receptor. Exogenous prostaglandins alone or in combination with PDGF BB induced greater increases in cAMP levels and proliferation of NF1 MPNST cells compared to nHSC. We conclude that aberrant cAMP signaling in NF1 MPNST cells contributes to tumor formation in NF1 patients.

A timely characterization of vasopressin-sensitive adenylyl cyclase isoforms in the mouse inner medullary collecting duct

second messenger signaling in the kidney mediates several of the organ's basic physiological functions including the regulation of blood pressure, control of fluid volume, and maintenance of the body's water homeostasis in times of duress. Tight regulation of the second messenger cAMP is critical

Bicarbonate‐regulated soluble Adenylyl Cyclase (sAC) mRNA expression in peripheral and central chemoreceptors

Bicarbonate‐regulated soluble Adenylyl Cyclase (sAC) mRNA expression in peripheral and central chemoreceptors

Altered collecting duct adenylyl cyclase content in collecting duct endothelin-1 knockout mice

BackgroundEndothelin-1 (ET-1) inhibition of vasopressin (AVP)-stimulated water reabsorption by the inner medullary collecting duct (IMCD) is associated with reduced cAMP accumulation. To determine the effect of ET-1 deficiency, AVP-stimulated cAMP responsiveness was assessed in IMCD from mice with collecting duct-specific deletion of ET-1 (CD ET-1 KO) and from control animals.MethodsCyclic AMP production, adenylyl cyclase (AC) mRNA, and AC protein were measured in acutely isolated IMCD.ResultsCD ET-1 KO IMCD had enhanced AVP-stimulated cAMP accumulation. Inhibition of calcium-stimulated AC using BAPTA did not prevent enhanced AVP responsiveness in CD ET-1 KO IMCD. Factors known to be modified by ET-1, including nitric oxide, cyclooxygenase metabolites, and superoxide did not affect the increased AVP responsiveness of CD ET-1 KO IMCD. Differential V2 receptor or G-protein activity was not involved since CD ET-1 KO IMCD had increased cAMP accumulation in response to forskolin and/or cholera toxin. CD ET-1 KO did not affect mRNA or protein levels of AC3, one of the major known collecting duct AC isoforms. However, the other known major collecting duct AC isoform (AC5/6) did have increased protein levels in CD ET-1 KO IMCD, although AC5 (weak signal) and 6 mRNA levels were unchanged.ConclusionET-1 deficiency increases IMCD AC5/6 content, an effect that may synergize with acute ET-1 inhibition of AVP-stimulated cAMP accumulation.

Lipopolysaccharide attenuates mRNA levels of several adenylyl cyclase isoforms in vivo

Signals that elevate intracellular levels of cyclic adenosine monophosphate (cAMP) are among the factors that control lipopolysaccharide (LPS)-mediated inflammatory mediator production by macrophages. cAMP signaling is also involved in maintaining body functions that are commonly impaired in sepsis, including the endothelial cell barrier function and heart function. Several agents successfully used for sepsis intervention target cAMP signaling, and it was recently shown that liver and lung may be protected from inflammation injury by cAMP-elevating phosphodiesterase inhibitors. Here, we show that LPS attenuates adenylyl cyclase (AC) mRNA levels in liver, lung, heart, spleen and kidney in an animal model of endotoxemia, and in macrophages from liver and lung. In particular, AC5, AC6, AC7 and AC9 mRNA were reduced in most tissues examined and in tissue macrophages. In Kupffer cells, prostaglandin E2-mediated cAMP production was inhibited by LPS treatment. The reduction in AC mRNA by LPS would be expected to lead to a lowered potential for cAMP production in most organs, and in particular, changes in AC6 mRNA may affect endothelial cell barrier function and heart function. In contrast, AC4 mRNA was elevated in heart and lung. The present work indicates a possible mechanism for LPS-mediated alteration of cAMP signaling in vivo.

Cellular Localisation of Adenylyl Cyclase: A Post-genome Perspective

The intracellular messenger cAMP is essential for vital processes ranging from ovulation to cognition. There are 10 genes for adenylyl cyclase (AC), the biosynthetic enzyme of cAMP. Nine of these encode membrane-bound proteins and one gives rise to soluble AC. The understanding of the biological significance of this molecular diversity is incomplete. Membrane-bound ACs conform to the same structural blueprint but have markedly different regulatory characteristics. AC mRNAs are differentially distributed in the body suggesting non-redundant physiological functions. The subcellular localisation of AC isoforms has not been examined in detail. Here we discuss the current knowledge on the intracellular targeting of AC isoforms, and highlight the technical problems of AC detection, some of which appear to be caused by the poor quality-control of commercially supplied antibodies. The principal message is that intracellular targeting of ACs may be isoform-specific and also dependent on the cellular context of expression.

Inactivation of the Mouse Adenylyl Cyclase 3 Gene Disrupts Male Fertility and Spermatozoon Function

Mammalian spermatids and spermatozoa express functional G protein-coupled receptors. However, bicarbonate-regulated soluble adenylyl cyclase (AC), the major AC present in these cells, is not directly coupled to G proteins. To understand how G protein-coupled receptors signal in spermatozoa, we investigated whether a conventional transmembrane cyclase is present and biologically active in these cells. Here, we provide evidence for expression of type 3 AC (AC3) in male germ cells and describe the effects of disruption of the AC3 gene on fertility and function of mouse spermatozoa. As previously reported in rat, AC3 mRNA is expressed in mouse testes and localized, together with soluble AC mRNA, mainly in postmeiotic germ cells. AC3 protein was detected by immunolocalization in round and elongating spermatids in a region corresponding to the developing acrosome and was retained in the mature spermatozoa of the epididymis. Forskolin caused a small increase in cAMP production in mouse spermatozoa, but this increase could not be detected in the AC3(-/-) mice. Inactivation of the AC3 gene did not have overt effects on spermatogenesis; however, AC3(-/-) males were subfertile with only three litters generated by 11 males over a period of 6 months. When used in in vitro fertilization, spermatozoa from these AC3(-/-) mice produced few embryos, but their fertilizing ability was restored after removal of the zona pellucida. Despite an apparently normal structure, these spermatozoa had decreased motility and showed an increase in spontaneous acrosome reactions. These data support the hypothesis that AC3 is required for normal spermatid or spermatozoa function and male fertility.

1,25‐dihydroxyvitamin D3 stimulated protein kinase C phosphorylation of type VI adenylyl cyclase inhibits parathyroid hormone signal transduction in rat osteoblastic UMR 106‐01 cells

1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) treatment of osteoblastic cells was shown previously to attenuate Parathyroid hormone (PTH) response by inhibiting adenylyl cyclase (AC) activity. In this study, we have investigated the mechanism by which 1,25(OH)(2)D(3) inhibits AC in rat osteoblastic UMR 106-01 cells. 1,25(OH)(2)D(3) treatment inhibited both PTH and forskolin-stimulated AC activity by 25%-50% within 12 min in a concentration-dependent manner suggesting a direct inhibition of the AC enzyme. Treatment with 25(OH)D(3) had no effect on basal or stimulated AC activity. We determined the profile of AC subtypes expressed in UMR cells and found AC VI to be the dominant subtype accounting for 50% of AC mRNA. Since AC VI can be inhibited by protein kinase C (PKC) phosphorylation, we examined 1,25(OH)(2)D(3) activation of various PKC isoforms. 1,25(OH)(2)D(3) increased the membrane translocation of PKC-betaI, -delta, and -zeta with a concomitant increase in PKC activity. The translocation of PKC-betaI and -delta was blocked by the PLC inhibitor U73122 whereas that of PKC-zeta was abolished by the PI-3 kinase inhibitor wortmannin. The attenuation of cAMP production by 1,25(OH)(2)D(3) was antagonized by the PKC inhibitors Go6850, calphostin C, and wortmannin, but not by a calmodulin kinase II (CaMKII) inhibitor. Treatment with 1,25(OH)(2)D(3) for 20 min increased AC VI phosphorylation by 10.8-fold and this was blocked partially by Go6850 and partially by wortmannin but was unaffected by CaMKII inhibitor. These results demonstrate that 1,25(OH)(2)D(3) activation of PKC isoforms leads to phosphorylation of AC VI and inhibition of PTH-activation of this pathway in osteoblasts.

Effects of age and spatial learning on adenylyl cyclase mRNA expression in the mouse hippocampus

Adenylyl cyclase (AC) subtypes have been implicated in memory processes and synaptic plasticity. In the present study, the effects of aging and learning on Ca 2+/calmodulin-stimulable AC1, Ca 2+-insensitive AC2 and Ca 2+/calcineurin-inhibited AC9 mRNA level were compared in the dorsal hippocampus of young-adult and aged C57BL/6 mice using in situ hybridization. Both AC1 and AC9 mRNA expression were downregulated in aged hippocampus, whereas AC2 mRNA remained unchanged, suggesting differential sensitivities to the aging process. We next examined AC mRNA expression in the hippocampus after spatial learning in the Morris water maze. Acquisition of the spatial task was associated with an increase of AC1 and AC9 mRNA levels in both young-adult and aged groups, suggesting that Ca 2+-sensitive ACs are oppositely regulated by aging and learning. However, aged-trained mice had reduced AC1 and AC9, but greater AC2, mRNA levels relative to young-trained mice and age-related learning impairments were correlated with reduced AC1 expression in area CA1. We suggest that reduced levels of hippocampal AC1 mRNA may greatly contribute to age-related defects in spatial memory.

Molecular mechanisms of reduced beta-adrenergic signaling in the aged heart as revealed by genomic profiling.

Myocardial aging leads to a reduction of beta-adrenergic receptor-induced metabolic and contractile responsiveness. We hypothesize that a change in the patterns of gene expression is important in these age-related events. To test this, hearts were harvested from young and aged male rats (3-4 and 20-22 mo, respectively). Total mRNA was extracted and prepared for hybridization to Affymetrix U34A GeneChips. Filtering criteria, involving fold change and a statistical significance cutoff were employed, yielding 263 probe pairs exhibiting differential signals. Of the 163 annotated genes, at least 56 (34%) were classified as signaling/cell communication. Of these 56, approximately half were directly involved in G protein-coupled receptor signaling pathways. We next determined which of these changes might be involved in anti-adrenergic activity and identified 19 potentially important gene products. Importantly, we observed a decrease in beta1-adrenergic receptor and adenylyl cyclase mRNAs, whereas the mRNA encoding beta-arrestin increased. Furthermore, the results demonstrate an increase in mRNAs encoding the adenosine A1 receptor and phospholipase D, which could increase anti-adrenergic effects. Moreover, the mRNAs encoding the muscarinic M3 receptor, nicotinic acetylcholine receptor beta3, and nicotinic acetylcholine receptor-related protein were increased as was the mRNA encoding guanylate kinase-associated protein. Interestingly, we also observed eight mRNAs whose abundance changed three- to sixfold with aging that could be considered as being compensatory. Although these results do not prove causality, they demonstrate that cardiac aging is associated with changes in the profiles of gene expression and that many of these changes may contribute to reduced adrenergic signaling.

Differential Expression of Membrane and Soluble Adenylyl Cyclase Isoforms in Cytotrophoblast Cells and Syncytiotrophoblasts of Human Placenta

Adenylyl cyclase (AC) activity is ubiquitous in mammalian cells, and various forms of this enzyme exist that widely differ with regard to tissue distribution, abundance, and modes of regulation. Human placenta is made, among others, of cytotrophoblast cells and syncytiotrophoblasts. This latter is a polynucleate structure that originates from the differentiation of proliferative mononucleated cytotrophoblast cells, the placental stem cell, into syncytiotrophoblasts. In vitro, this differentiation process is associated with a concomitant increase in cellular levels of cAMP and enhanced expression of genes representative of syncytiotrophoblasts endocrine activity. Thus, in this study we evaluated the differential distribution of AC isoforms in cytotrophoblast cells and syncytiotrophoblasts by reverse transcription-polymerase chain reaction (RT–PCR) using total RNA or purified mRNA. Our results demonstrate that all membrane and soluble AC mRNA isoforms are present in both cell types. Interestingly in syncytiotrophoblasts, AC4 and AC8 mRNA are highly expressed, while AC1, AC2 mRNA are less abundant when compared to cytotrophoblast cells. Additionally, the soluble AC is expressed in both trophoblast cells, but its expression is greatly reduced in differentiated cells, syncytiotrophoblasts. The presence of these AC proteins in both cells was confirmed by Western blotting. Taken together, these data help us to characterize the different AC isoforms in human cytotrophoblast cells and syncytiotrophoblasts, and demonstrate that the AC isoforms expression seems to be mainly modulated in groups 1 and 2. Moreover, the important decrease of the soluble AC isoform in syncytiotrophoblasts as compared to cytotrophoblast cells could suggest an important role of this AC in the extravillous trophoblast formation.

Expression of adenylyl cyclase V/VI mRNA and protein is upregulated in cyanotic infant human myocardium.

We have previously demonstrated that both basal and isoproterenol-stimulated activities of myocardial adenylyl cyclase were greater in cyanotic patients with tetralogy of Fallet (TOF) than those in acyanotic patients. However, it was not determined whether increased enzyme activity was related to a similar increase in adenylyl cyclase protein and mRNA expression. In the current study, we examined the mRNA and protein expression of cardiac adenylyl cyclase, types V and VI, in cyanotic and acyanotic patients with TOF. Ribonuclease protection assays and immunoblotting were performed on myocardial specimens obtained from cyanotic patients with TOF and acyanotic patients with TOF or ventricular septal defect. We demonstrated that in both cyanotic and acyanotic patients, there was more type V adenylyl cyclase mRNA than type VI. Types V and VI cardiac adenylyl cyclase mRNA were significantly increased in myocardium of the cyanotic group compared to the acyanotic group. Protein expression of both V and VI adenylyl cyclases was correspondingly upregulated in cyanotic patients compared to acyanotic patients. Our results indicate that gene and protein expression of cardiac adenylyl cyclases, types V and VI, is increased in the cyanotic myocardium. These results suggest that chronic hypoxemia may regulate the expression of adenylyl cyclase enzymes.

496 Adenylyl cyclase mRNA expression and effects of adenylyl cyclase cascade on contractility in unilateral pregnant rat myometrium

496 ADENYLYL CYCLASE MRNA EXPRESSION AND EFFECTS OF ADENYLYL CYCLASE CASCADE ON CONTRACTILITY IN UNILATERAL PREGNANT RAT MYOMETRIUM KIMISATO ASANO 1, TOSHIAKI OKAWA t, HIDENORI TAKAHASHI 1, SATOSHI HASHIMOTO 1, HIROSHI ANBE t, AKIRA SATO I, YURI VEDERNIKOV~, GEORGE SAADE 2, ROBERT GARFIELD 2, YOSUKE SUZUKI t , ISAO MATSUOKA3; ]Fukushima Medical University, Obstetrics and Gynecology, Fukushima; 2University of Texas Medical Branch, Obstetrics and Gynecology, Galveston, TX; 3Fukushima Medical University, Pharmacology, Fukushima OBJECTIVE: To determine the effect of gestation on adenylyl cyclase (AC) mRNA expression, and on the response to protein kinase C (PKC) and a2 adrenergic agonists in gravid and non-gravid uterine horns of pregnant rats. STUDY DESIGN: Non-pregnan t and day 17 p regnan t Wistar rats were used (n = 6 per group) . In the p regnan t animals, one of the oviducts was ligated before mating in order to result in pregnancy in only one uterine horn. mRNA levels of AC2, AC4, AC6 ad AC9 were determined in the uterine horns with (fetus+) and without (fetus-) fetuses by semi-quantitive reverse transcription-polynaerase chain react ion (RT-PCR). In addit ion, strips of myometr ium fi-om the fetus+ and fetussides were p repa red for isometric tension recording in F-a'ebs-Ringer solution (37°C). The strips were treated with PK-C st inmlant (phorbol myristate acetat: PMA; 1 ~tM) or an a 2 adrenerg ic agonis t (UK-14304; 1 IxM). The changes of contracti l i ty were determined as %change from basal contractile activity. RESULTS: mRNA levels of AC4, AC6 and AC9 were no t significantly different between fetus+ and fetussides on day 47 of gestation. AC2's mRNA levels on the fetusside was significantly lower than the fetus+ side, and were not significantly different than the mRNA levels in the uteri of non-pregnant animals. Uterine contractil i ty was significantly decreased by PMA and UK14304 on the fetus+ side, but was not affected on the fetusside. CONCLUSION: Pregnancy increases AC4, AC6 and AC9 mRNA expression in the uterus by systenfic factors, but the gestational increase in AC2 is dependent on the local presence of pups. AC2 inhibits uterine contractility via a PKC and an ~2 adrenergic receptor in preglmnt myometrium. O f the various adenylyl cyclases, AC2 appears to play the d o m i n a n t role in mainta ining uter ine quiescence in pregnancy. Local factors affecting AC2's expression or function may initiate term or pre term labor. SMFM Abstracts S217