Physiological Sensing of Carbon Dioxide/Bicarbonate/pH via Cyclic Nucleotide Signaling

Carbon dioxide (CO2) is produced by living organisms as a byproduct of metabolism. In physiological systems, CO2 is unequivocally linked with bicarbonate (HCO3−) and pH via a ubiquitous family of carbonic anhydrases, and numerous biological processes are dependent upon a mechanism for sensing the level of CO2, HCO3, and/or pH. The discovery that soluble adenylyl cyclase (sAC) is directly regulated by bicarbonate provided a link between CO2/HCO3/pH chemosensing and signaling via the widely used second messenger cyclic AMP. This review summarizes the evidence that bicarbonate-regulated sAC, and additional, subsequently identified bicarbonate-regulate nucleotidyl cyclases, function as evolutionarily conserved CO2/HCO3/pH chemosensors in a wide variety of physiological systems

O2/CO2-sensitive cyclic AMP-signalling pathway in peripheral chemoreceptors

RESUMO: O corpo carotídeo (CB) é um pequeno órgão sensível a variações na PaO2, PaCO2 e pH. As células tipo I (células glómicas) do corpo carotídeo, as unidades sensoriais deste órgão, libertam neurotransmissores em resposta às variações dos gases arteriais. Estes neurotransmissores atuam quer em recetores pré-sinápticos, localizados nas células tipo I, quer em recetores póssinápticos, localizados nas terminações do nervo do seio carotídeo, ou em ambos. A activação dos recetores pré-sinápticos modula a atividade do corpo carotídeo, enquanto que, a activação dos recetores pós-sinápticos, de carater excitatório, desencadeia um aumento da frequência de descarga das fibras do CSN, com subsequente despolarização dos neurónios do gânglio petroso, e posterior despolarização de um grupo específico de neurónios do centro respiratório central, desencadeando, como resposta final, hiperventilação. Estes recetores pré- e pós-sinápticos podem ser classificados em ionotrópicos ou metabotrópicos, estando os últimos acoplados a adenilatos ciclases transmembranares (tmAC). O mecanismo exato pelo qual as variações dos gases arteriais são detetadas pelo CB não se encontra ainda completamente elucidado, mas tem sido sugerido que alterações nos níveis de cAMP estejam associadas ao mecanismo de deteção de variações de O2 e CO2. Os níveis de cAMP podem ser regulados através da sua via de síntese, mediada por dois tipos de adenilatos ciclases: tmAC sensível aos eurotransmissores e adenilato ciclase solúvel (sAC)sensível a variações de HCO3/CO2, e pela sua via de degradação mediada por fosfodiesterases. A via de degradação do cAMP pode ser manipulada farmacologicamente, funcionando enquanto alvo terapêutico para o tratamento de patologias do foro respiratório (e.g. asma, hipertensão pulmonar, doença pulmonar obstructiva crónica e apneia do sono), que induzem um aumento da actividade do CB.O trabalho descrito nesta dissertação partiu da hipótese de que a actividade do CB é manipulada por fármacos, que interferem com a via de sinalização do cAMP, tendo sido nosso objectivo geral, investigar o papel do cAMP na quimiotransdução do CB de rato, e determinar se a actividade dos enzimas responsáveis pela via de sinalização do cAMP é ou não regulada por variações de O2/CO2. Assim, a relevância deste trabalho é a de estudar e identificar possíveis alvos moleculares (sAC, isoformas de tmAC e PDE) com potencial para serem usados no tratamento de patologias relacionadas com o controlo respiratório. A primeira parte do presente trabalho, centrou-se na caracterização farmacológica da PDE4 no CB e em tecidos não quimiorecetores (e.g. gânglio cervical superior e artérias carótidas), e na observação do efeito de hipóxia aguda na acumulação dos níveis de cAMP, induzidos pelos inibidores de PDE, nestes tecidos. A quantificação de cAMP foi efectuada por técnica imunoenzimática (EIA), tendo sido elaboradas curvas de dose-resposta para os efeitos de inibidores, não específicos (IBMX) e específicos para a PDE2 e PDE4 (EHNA, Rolipram e Ro 20-1724), nos níveis de cAMP acumulados, em situações de normóxia (20%O2/5%CO2) e hipóxia (5%O2/5%CO2). A caracterização das PDE no gânglio cervical superior foi aprofundada, utilizando-se a técnica de transferência de energia de ressonância por fluorescência (FRET) em culturas primárias de neurónios, na presença de inibidores não específicos (IBMX) e específicos para a PDE3 e PDE4 (milrinone e rolipram, respetivamente). Foram igualmente estudadas, através de RT-qPCR, as alterações na expressão de PDE3A-B e PDE4A-D, no gânglio cervical superior, em resposta a diferentes percentagens de oxigénio. Na segunda parte do trabalho investigou-se a via de síntese do cAMP no CB em resposta a variações na concentração de HCO3/CO2. Em concreto, o protocolo experimental centrou-se na caracterização da sAC, dado que a sua actividade é regulada por variações de HCO3/CO2. A caracterização da expressão e regulação da sAC, em resposta a variações de HCO3/CO2 ,foi efectuada no CB e em tecidos não quimioreceptores periféricos (e.g. gânglio cervical superior, petroso e nodoso) por qRT-PCR. A actividade deste enzima foi caracterizada indirectamente através da quantificação dos níveis de cAMP (quantificação por EIA), induzidos por diferentes concentrações de HCO3/CO2, na presença de MDL-12,33-A, um inibidore da tmAC. A expressão das isoformas da tmAC no CB e gânglio petroso foi determinada por RT-qPCR. Adicionalmente, estudámos a contribuição relativa da tmAC e sAC no mecanismo de sensibilidade ao CO2 no CB. Para o efeito foram estudadas as alterações: 1) nos níveis de cAMP (quantificado por EIA) na presença de diferentes concentrações de HCO3/CO2 e ao longo do tempo (5-30 min); 2) na ativação da proteína cinase A (PKA, FRET baseado em sensores) em células tipo I do CB; e 3) na frequência de descarga do CSN (registos) na presença e ausência de ativadores e inibidores da sAC,tmAC e PKA. Por último, foi caracterizada a expressão e actividade da sAC nos quimioreceptors centrais (locus ceruleus, rafe e medula ventro-lateral) através de técnicas de RT-qPCR e EIA. A expressão das isoformas da tmAC foi aprofundada no locus coeruleus através de RT-qPCR. Por fim, comparámos a contribuição da tmAC e sAC nos níveis de cAMP no locus coeruleus em condições de normocapnia e hipercapnia.O nosso trabalho teve os seguintes resultados principais: 1) PDE4 está funcional no corpo carotídeo, artérias carótidas e gânglio cervical superior de rato, embora a PDE2 só se encontre funcional neste último; 2) Os efeitos dos inibidores de PDE nos níveis de acumulação de cAMP foram exacerbados em situações de hipóxia aguda no CB e artérias carótidas, mas foram atenuados no gânglio cervical superior; 3) No gânglio cervical superior, diferentes tipos de células apresentaram uma caracterização específica de PDEs, sugerindo uma subpopulação de células neste gânglio com funções fisiológicas distintas; 4) Embora todas as isoformas de PDE4 e PDE3 estivessem presentes no gânglio, a PDE3a, PDE4b e a PDE4d foram as isoformas mais expressas. Por outro lado, incubações de gânglio cervical superior, em diferentes percentagens de oxigénio, não alteraram (não regularam) significativamente a expressão das diferentes isoformas de PDE neste órgão; 5) a sAC encontra-se expressa e funcional no CB e nos quimiorecetores centrais estudados (locus coeruleus, rafe e medula ventrolateral). A sAC apresenta maior expressão no CB comparativamente aos restantes orgãos estudados, exceptuando os testículos, orgão controlo. Variações de HCO3/CO2 de 0/0 para 24/5 aumentaram os níveis de cAMP no CB e quimiorecetores centrais, tendo sido o aumento mais significativo observado no CB. Concentrações acima dos 24mM HCO3/5%CO2 não induziram alterações nos níveis de cAMP, sugerindo que a actividade da sAC se encontra saturada em condições fisiológicas (normocapnia) e que este enzima não desempenha qualquer papel na deteção de situações de hipercapnia; 6) No CB, a expressão das isoformas tmAC1, tmAC4, tmAC6 e tmAC9 é mais elevada comparativamente à expressão da sAC; 7) Utilizamos diferentes inibidores da tmAC (MDL 12-330A, 500μM, 2’5’-ddADO, 30-300μM, SQ 22536, 200μM) e da sAC (KH7, 10-100μM) para estudar a contribuição relativa destes enzimas na acumulação do cAMP no CB. Tanto a tmAC como a sAC contribuem para a acumulação dos níveis de cAMP em condições de hipercapnia. Contudo, existe um maior efeito destes inibidores nas condições de 12 mM HCO3/2.5%CO2 do que em condições de normocapnia e hipercapnia, sugerindo um papel relevante destes enzimas na atividade do CB em situações de hipocapnia; 8) Não se observaram variações nos níveis de cAMP em resposta a diferentes concentrações de HCO3/CO2 ao longo do tempo (5-30 min). O efeito inibitório induzido por ddADO e KH7 foi sobreponível após 5 ou 30 minutos de incubação em todas as concentrações de HCO3/CO2 estudadas; 9) Por último, verificou-se um aumento na frequência da descarga do nervo do seio carotídeo entre as condições de normocapnia e hipercapnia acídica. Ao contrário do KH7 (10μM), o 2’5’-ddADO reduziu significativamente a frequência de descarga do nervo, quer em condições de normocapnia quer de hipercapnia acídica. Contudo, não se verificou aumento na frequência de descarga do nervo entre normocapnia e hipercapnia isohídrica, sugerindo que a sensibilidade à hipercapnia no CB é mediada por variações de pH. Em conclusão, os resultados decorrentes deste trabalho permitiram demonstrar que, embora os enzimas que medeiam a via de sinalização do cAMP possam ser bons alvos terapêuticos em condições particulares, a sua actividade não é específica para o CB. Os resultados sugerem ainda que o cAMP não é um mediador específico da transdução à hipercapnia neste orgão. Contudo, os nossos resultados demonstraram que os níveis de cAMP são mais elevados em condições fisiológicas, o que sugere que o cAMP possa ter uma função homeostática neste orgão. Por último, o presente trabalho demonstrou que os aumentos de cAMP descritos por outros em condições de hipercapnia, não são observáveis quando o pH se encontra controlado. ------------------ ABSTRACT: The work presented in this dissertation was aimed to establish how specific is cAMP-signaling pathways in the CB mainly in different CO2 conditions and how O2 concentrations alter/drives the manipulation of cAMP signaling in the CB. The experimental studies included in this thesis sought to investigate the role of cAMP in the rat CB chemotransduction mechanisms and to determine whether the enzymes that participate in cAMP signal transduction in the CB are regulated by O2/CO2. We characterized the enzymes involved in the cAMP-signaling pathway in the CB (sAC, tmAC, PDE) under different O2/CO2 conditions. Our results demonstrated that many of these enzymes are involved in CO2/O2 sensing and while they may be useful in treating conditions with alterations in CO2/O2 sensing,they will not be specific to chemoreception within the CB: 1) PDE4 is ubiquitously expressed in CB and non-chemoreceptor related tissues and their affinity to inhibitors change with O2 tensions in both CB and carotid arteries, and 2) sAC and tmAC are expressed in peripheral and central chemo- and non-chemoreceptor tissues and their effect on cAMP levels do not change between normocapnic and isohydric hypercapnic conditions. Our results provide evidence against a specific role of cAMP as a mediator for O2 and CO2 chemotransduction in the rat CB and emphasized the role of pH in CO2 sensitivity of the CB. Furthermore, our results demonstrate that cAMP levels are maintained higher under physiological conditions, supporting recent finding from our lab, which all together suggests that cAMP has a homeostatic function in this organ

The Influence of Carbon Dioxide on Cellular Cyclic Adenosine Monophosphate

Inorganic carbon is fundamental to the physiology of all organisms, however elevated CO2 is generally detrimental. Numerous class III adenylyl cyclases (AC) from both prokaryotic and mammalian organisms have been shown to respond to inorganic carbon in vitro, however, at present there is limited evidence in vivo. This thesis demonstrates in cellulo evidence that hypercapnia CO2 blunts agonist induced cAMP signalling. The eect of CO2 is independent of changes in intracellular and extracellular pH, independent of the mechanism used to activate the cAMP signalling pathway, and is independent of the cell line employed. Through a combination of pharmacological and genetic tools this eect of elevated CO2 on cAMP signalling is demonstrated to require Ca2+ ion release from IP3 receptors in the endoplasmic reticulum. Consistent with these ndings, CO2 caused an increase in cytoplasmic Ca2+ concentrations which require the presence of active IP3 receptors and is absent under comparable acidotic conditions. Physiological relevance for this signalling mechanism is demonstrated through activity of the sodium dependant proton exchanger NHE3. This transporter exhibits well-characterised inhibition by cAMP dependant protein kinase PKA to increase bicarbonaturia in vivo. Overall these results provide conclusive evidence of potentially profound eects of inorganic carbon on intracellular cell signalling, which could lead to signicant insight into the pathophysiology and treatment of numerous disorders including metabolic acidosis, reperfusion injuries, acute lung injury and obesity

Mechanisms for Taste Sensation of Carbonation

Carbonation, or the presence of carbon dioxide (CO2) dissolved in solution, is a commonly encountered feature of beverages in the contemporary human diet. While the popularity of carbonation may be attributed to its distinct sensory qualities, the specific orosensory pathways mediating CO2 detection in mammals have not previously been delineated. This dissertation describes the identification of specific cellular and molecular mechanisms that mediate taste sensation of carbonation, using the mouse as a model system. The mammalian gustatory system is sensitive to CO2, and these responses are sensitive to inhibition of carbonic anhydrases, enzymes that catalyze the interconversion of carbon dioxide with carbonic acid. Through gene expression profiling I discovered that the gene carbonic anhydrase IV (Car4), encoding an extracellular enzyme, is specifically expressed in acid sensing taste receptor cells (TRCs). Genetic ablation of the Car4 locus resulted in a major deficit in gustatory CO2 sensation that is stimulus specific, not affecting responses to acid. Ablation or silencing of acid sensing TRCs likewise produced a profound deficit in taste responses to CO2. These studies identified a primary pathway of the gustatory carbonation response, substantiating acid sensing TRC and the Car4 enzyme as key mediators. A smaller gustatory neural response to carbonation remains even in the absence of sour-sensing TRC and/or Car4. To identify additional carbonation sensing pathways, I applied an in vivo calcium-imaging assay to define the ensemble of primary gustatory neurons activated by CO2. These studies revealed that in addition to robust activation of sour sensing neurons, a secondary gustatory pathway for CO2 detection is mediated by subpopulations of bitter and sweet responsive neurons. I identified carbonic anhydrase VII (Car7) as an intracellular carbonic anhydrase specifically expressed by sweet, bitter and umami sensing TRC. Pharmacological and gene expression data support a role for Car7 in transducing the secondary CO2 sensing pathway. These studies suggested that carbonation acts as a complex gustatory stimulus, stimulating sour, sweet and bitter taste qualities simultaneously. The rules governing peripheral encoding of multi-modal taste stimuli are not well understood. To address this issue, I examined the peripheral gustatory response to binary mixtures of taste qualities. I found that most combinations of taste qualities are represented as a superimposition of the component responses. However, neural responses to attractive stimuli, including natural sugars, artificial sweeteners and umami tastants, are selectively suppressed by simultaneous co-stimulation with a sour (acidic) stimulus. Acid-mediated suppression of sweet is cell autonomous, occurring even in the absence of gustatory acid sensing. Remarkably, carbonation stimulates sour signaling without suppressing sweet taste response. These studies suggest that cross-modal interactions at the periphery modulate the sensory response to complex taste stimuli

Established and Potential Physiological Roles of Bicarbonate-Sensing Soluble Adenylyl Cyclase (sAC) in Aquatic Animals

Soluble adenylyl cyclase (sAC) is a recently recognized source of the signaling molecule cyclic AMP (cAMP) that is genetically and biochemically distinct from the classic G-protein-regulated transmembrane adenylyl cyclases (tmACs). Mammalian sAC is distributed throughout the cytoplasm and it may be present in the nucleus and inside mitochondria. sAC activity is directly stimulated by HCO3-, and sAC has been confirmed to be a HCO3- sensor in a variety of mammalian cell types. In addition, sAC can functionally associate with carbonic anhydrases to act as a de facto sensor of pH and CO2. The two catalytic domains of sAC are related to HCO3--regilated adenylyl cyclases from cyanobacteria, suggesting the cAMP pathway is an evolutionarily conserved mechanism for sensing CO2 levels and/or acid/base conditions. Reports of sAC in aquatic animals are still limited but are rapidly accumulating. In shark gills, sAC senses blood alkalosis and triggers compensatory H+ absorption. And in sea urchin sperm, sAC may participate in the initiation of flagellar movement and in the acrosome reaction. Bioinformatics and RT-PCR results reveal that sAC orthologs are present in most animal phyla. This review summarizes the current knowledge on the physiological roles of sAC in aquatic animals and suggests additional functions in which sAC may be involved

High Adenylyl Cyclase Activity and \u3cem\u3eIn Vivo\u3c/em\u3e cAMP Fluctuations in Corals Suggest Central Physiological Role

Corals are an ecologically and evolutionarily significant group, providing the framework for coral reef biodiversity while representing one of the most basal of metazoan phyla. However, little is known about fundamental signaling pathways in corals. Here we investigate the dynamics of cAMP, a conserved signaling molecule that can regulate virtually every physiological process. Bioinformatics revealed corals have both transmembrane and soluble adenylyl cyclases (AC). Endogenous cAMP levels in live corals followed a potential diel cycle, as they were higher during the day compared to the middle of the night. Coral homogenates exhibited some of the highest cAMP production rates ever to be recorded in any organism; this activity was inhibited by calcium ions and stimulated by bicarbonate. In contrast, zooxanthellae or mucus had \u3e1000-fold lower AC activity. These results suggest that cAMP is an important regulator of coral physiology, especially in response to light, acid/base disturbances and inorganic carbon levels

THE IDENTIFICATION OF NOVEL INORGANIC CARBON SENSITIVE ENZYMES

Adenylyl cyclase catalyses the formation of the second messenger adenosine-3’, 5’-monophosphate from adenosine triphosphate, and is involved in a number of diverse signalling pathways in eukaryotes and prokaryotes. Adenylyl cyclases are diverse in their structure and biochemistry, and have been grouped into six distinct Classes (I–VI). The Class III cyclase homology domain comprises the majority of prokaryotic and eukaryotic adenylyl cyclases, and has been further divided into 4 sub-Classes (a-d) based on active site polymorphisms. A number of Class IIIb adenylyl cyclases display elevated catalytic activity in the presence of inorganic carbon. Whether a response to inorganic carbon can be observed in enzymes which do not possess a Class IIIb cyclase homology domain remains to be established. Experiments were performed to investigate the response to inorganic carbon of a Class IIIa cyclase homology domain; mammalian transmembrane adenylyl cyclase. In vivo experiments demonstrated that the activity of mammalian transmembrane adenylyl cyclase was potentially regulated by inorganic carbon, and that this had a downstream effect on the cAMP response element binding protein. In vitro experiments performed on a transmembrane adenylyl cyclase demonstrated that the increase in activity in the presence of inorganic carbon occurred through an increase in kcat and increased metal affinity. Experiments were performed to test the response to inorganic carbon of several enzymes that share a structurally similar active site with the adenylyl cyclases; the polymerase I family of prokaryotic DNA polymerases, the polymerase β family of DNA polymerases, and the guanylyl cyclases. Initial in vitro experiments performed on T7 RNA polymerase demonstrated a response to inorganic carbon, however, it was discovered that this was likely due to a non-specific effect of pH. It was shown that inorganic carbon increased assay pH over time, and this warranted a re-design of the in vitro assay used to test the response of an enzyme to inorganic carbon. This new in vitro assay methodology was used to re-test T7 RNA polymerase, as well as test DNA polymerase β and several guanylyl cyclase, and demonstrated that these enzymes were non-responsive to inorganic carbon. Using this newly devised in vitro assay, experiments were performed to re-test the response of mammalian transmembrane adenylyl cyclase to inorganic carbon, and demonstrated that this enzyme was unlikely to be regulated by inorganic carbon. Furthermore, this new in vitro assay was used to re-test the response of several Class IIIb cyclase homology domains to inorganic carbon. Results demonstrated that mammalian soluble adenylyl cyclase was responsive to inorganic carbon, however, results provided evidence to suggest that two prokaryotic Class IIIb cyclase homology domains (CyaB1 from Anabaena PCC 7120 and CyaC from Spirulina platensis) were possibly non-responsive to inorganic carbon

Revisiting cAMP signaling in the carotid body

Chronic carotid body (CB) activation is now recognized as being essential in the development of hypertension and promoting insulin resistance; thus, it is imperative to characterize the chemotransduction mechanisms of this organ in order to modulate its activity and improve patient outcomes. For several years, and although controversial, cyclic adenosine monophosphate (cAMP) was considered an important player in initiating the activation of the CB. However, its relevance was partially displaced in the 90s by the emerging role of the mitochondria and molecules such as AMP-activated protein kinase and O(2)-sensitive K(+) channels. Neurotransmitters/neuromodulators binding to metabotropic receptors are essential to chemotransmission in the CB, and cAMP is central to this process. cAMP also contributes to raise intracellular Ca(2+) levels, and is intimately related to the cellular energetic status (AMP/ATP ratio). Furthermore, cAMP signaling is a target of multiple current pharmacological agents used in clinical practice. This review (1) provides an outline on the classical view of the cAMP-signaling pathway in the CB that originally supported its role in the O(2)/CO(2) sensing mechanism, (2) presents recent evidence on CB cAMP neuromodulation and (3) discusses how CB activity is affected by current clinical therapies that modify cAMP-signaling, namely dopaminergic drugs, caffeine (modulation of A(2A)/A(2B) receptors) and roflumilast (PDE4 inhibitors). cAMP is key to any process that involves metabotropic receptors and the intracellular pathways involved in CB disease states are likely to involve this classical second messenger. Research examining the potential modification of cAMP levels and/or interactions with molecules associated with CB hyperactivity is currently in its beginning and this review will open doors for future explorations

The roles of soluble adenylate cyclase in cell cycle control of endothelial cells

The soluble form of ADCYs, ADCY10, is ubiquitously expressed in the cytoplasm and distinct organelles including cell nucleus. In contrast to its membrane-associated isoforms (ADCY1-9) which are stimulated by G-protein-coupled receptors, ADCY10 is activated by bicarbonate (HCO3-) and can form cAMP in nearly all cell compartments. ADCY10 is involved in a variety of physiological as well as pathological processes including cell cycle control in tumor cells. However, the underlying mechanism is still unclear. Here the role of ADCY10 in cell cycle control and cell proliferation is studied in endothelial cells from human umbilical veins (HUVECs). The current study reveals that ADCY10 and α-Tubulin translocate and colocalize during mitosis suggesting a role of ADCY10 in cell division. In addition, FACS analysis demonstrated that ADCY10 plays a role in cell proliferation by modulating cell cycle control. Inhibition of ADCY10 by 0 mM HCO3- or 10 μM KH7 (specific ADCY10 inhibitor) induced cell accumulation in G2 phase rather than M phase determined by decreased mitotic indicator cyclin B1 level. Thus, ADCY10 inhibition leads to decreased cell proliferation. The known cAMP effectors, Epac and PKA, were assessed as possible downstream targets of ADCY10 in cell proliferation. It was shown that ADCY10 and Epac induce cell proliferation via ERK1/2-MAPK pathway. Inhibition of Epac was associated with suppressed cell proliferation. However, an arrest of cell cycle after Epac inhibition was observed in G0/G1 phases rather than S or G2/M phases. Thus, Epac inhibition causes a different arrest of cell cycle compared to ADCY10 inhibition. Regarding PKA, it was demonstrated that deficiency of PKA might play a role in either activation or inhibition of cell proliferation. However, direct inhibition of PKA by PKI and H-89 did not lead to cell accumulation in G2. This effect might be associated with broadened roles of PKA in different pathways. In contrast, direct stimulation of PKA under ADCY10 inhibition revealed that PKA is a downstream molecule of ADCY10 as a regulator of cell cycle transition from G2 to mitotic phase. However, the underlying pathway remains to be investigated. The cell cycle transition of G2/M phase is regulated by an auto-amplification loop of cyclin B1/CDK1, which is controlled by the kinase WEE1 and the phosphatase PP2A. WEE1 content was regulated via ADCY10 but was independent of PKA or Epac. Direct inhibition of PP2A showed a suppression of cell proliferation and induced cell cycle arrest in G2. These results were in accordance with those observed after the ADCY10. Furthermore, inhibition of ADCY10 had no effect on PP2A expression level but rather affected PP2A activity and was independent of Epac and PKA. Therefore, this data provides evidence that ADCY10 controls cell proliferation and cell cycle regulation via PP2A. Taken together, ADCY10 coordinates the cell cycle progression in a complex framework. Downstream of ADCY10, Epac promotes G1/S transition, whereas PKA mediates cell cycle transition of G2/M

An investigation of the response of a model class IIIA Adenyly Cyclase to carbon dioxide

Adenylyl cyclase catalyses the conversion of adenosine triphosphate into 3’,5'-cyclic adenosine monophosphate and pyrophosphate. Adenylyl cyclases are grouped into six distinct Classes based on amino acid sequence similarity. Mammalian adenylyl cyclases and numerous prokaryotic adenylyl cyclases are grouped into Class III. Class III is further sub-divided into four sub-Classes, IIIa, IIIb, IIIc, and IIId, based upon amino acid polymorphisms within the active site. Class IIIa adenylyl cyclases include the mammalian G- protein regulated transmembrane adenylyl cyclases and numerous prokaryotic adenylyl cyclases such as Rv1625c from Mycobacterium tuberculosis. Class IIIb adenylyl cyclases include the mammalian soluble adenylyl cyclase and numerous prokaryotic adenylyl cyclase such as CyaB1 from Anabaena PCC 7120. Class IIIb adenylyl cyclases are stimulated by inorganic carbon. New findings dispute whether the inorganic carbon stimulation of Class IIIb adenylyl cyclases is by HCO(_3) or CO(_2). Class IIIa adenylyl cyclases have previously been demonstrated to be non-responsive to HCO(_3) but have not been investigated with CO(_2) .Experiments were performed using the prokaryotic Class IIIa adenylyl cyclase Rv1625c(_204-443)- The specific activity of Rvl625c (_204-443) was obtained by measuring the conversion of [a-(32)P]ATP into [(^32)P]cAMP. Rv1625c (_204-443) was assayed for pH dependence, cation dependence, dose dependence, enzyme kinetics, and inorganic carbon-activating species in the presence or absence of inorganic carbon. The results showed that in vitro Rv1625c(_204-443) is stimulated by CO(_2) and not HCO(_3) below pH 7.5 with an apparent E.C.(_50) value of 13.2 ± 0.6 mM. Experiments were performed to investigate in vivo CO(_2) stimulation of Rv1625c(_204-443) transformed into Escherichia coli cells. The results showed that in vivo Rv1625c(_204-443) is stimulated by 10 % (v/v) CO(_2) in air. CO(_2) stimulation of Rv1625c(_204-443) was further investigated by identifying the conditions required for CO(_2) to bind to Rv1625c(_204-443). Numerous spectroscopic and biochemical techniques were used to identify conditions required for CO(_2) binding such as circular dichroism spectroscopy, Fourier transform infrared spectroscopy, HCO(_3) dependent luminescence probe spectroscopy, CO(_2) stimulation assays on Rv1625c(_204-443) mutant proteins, and CO(_2) binding assays. The results showed that metal is not required for CO(_2) binding. CO(_2) binds solely to the apoprotein. As CO(_2) does not require any cefaclors or substrate to bind to Rv1625c(_204-443), the elucidation of the CO(_2) binding site on Rv1625c(_204-443) was attempted. Numerous binding sites were investigated, such as amino acids in the active site with a primary or secondary amine side group (forming a potential carbamate with CO(_2)). Double and triple amino acid CO(_2) binding sites were also investigated by identifying the amino acids that conespond to the HCO(_3) binding site from mammalian soluble adenylyl cyclase. CO(_2) binding was also investigated by growing Rv1625c crystals, exposing the crystals to CO(_2) and X-ray diffraction studies on the crystals to obtain a diffraction data set. Unfortunately none of these techniques was able to identify the CO(_2_ binding site of Rv1625c.This surprising and novel finding that CO’_2’ binds and stimulates Rv1625c(_204-443) suggests that other Class IIIa adenylyl cyclases, including the heterotrimeric G-protein stimulated mammalian adenylyl cyclases are also stimulated by CO(_2) and further work needs to be done in this area