Soluble Adenylyl Cyclase Research Articles

Bicarbonate, carbon dioxide and pH sensing via mammalian bicarbonate-regulated soluble adenylyl cyclase.

Soluble adenylyl cyclase (sAC; ADCY10) is a bicarbonate (HCO3 -)-regulated enzyme responsible for the generation of cyclic adenosine monophosphate (cAMP). sAC is distributed throughout the cell and within organelles and, as such, plays a role in numerous cellular signalling pathways. Carbonic anhydrases (CAs) nearly instantaneously equilibrate HCO3 -, protons and carbon dioxide (CO2); because of the ubiquitous presence of CAs within cells, HCO3 --regulated sAC can respond to changes in any of these factors. Thus, sAC can function as a physiological HCO3 -/CO2/pH sensor. Here, we outline examples where we have shown that sAC responds to changes in HCO3 -, CO2 or pH to regulate diverse physiological functions.

Molecular and biochemical characterization of the bicarbonate-sensing soluble adenylyl cyclase from a bony fish, the rainbow troutOncorhynchus mykiss

Soluble adenylyl cyclase (sAC) is a -stimulated enzyme that produces the ubiquitous signalling molecule cAMP, and deemed an evolutionarily conserved acid-base sensor. However, its presence is not yet confirmed in bony fishes, the most abundant and diverse of vertebrates. Here, we identified sAC genes in various cartilaginous, ray-finned and lobe-finned fish species. Next, we focused on rainbow trout sAC (rtsAC) and identified 20 potential alternative spliced mRNAs coding for protein isoforms ranging in size from 28 to 186 kDa. Biochemical and kinetic analyses on purified recombinant rtsAC protein determined stimulation by at physiologically relevant levels for fish internal fluids (EC50 ∼ 7 mM). rtsAC activity was sensitive to KH7, LRE1, and DIDS (established inhibitors of sAC from other organisms), and insensitive to forskolin and 2,5-dideoxyadenosine (modulators of transmembrane adenylyl cyclases). Western blot and immunocytochemistry revealed high rtsAC expression in gill ion-transporting cells, hepatocytes, red blood cells, myocytes and cardiomyocytes. Analyses in the cell line RTgill-W1 suggested that some of the longer rtsAC isoforms may be preferentially localized in the nucleus, the Golgi apparatus and podosomes. These results indicate that sAC is poised to mediate multiple acid-base homeostatic responses in bony fishes, and provide cues about potential novel functions in mammals.

The sodium/proton exchanger SLC9C1 (sNHE) is essential for human sperm motility and fertility

Asthenozoospermia, defined by the absence or reduction of sperm motility, constitutes the most frequent cause of human male infertility. This pathological condition is caused by morphological and/or functional defects of the sperm flagellum, which preclude proper sperm progression. While in the last decade many causal genes were identified for asthenozoospermia associated with severe sperm flagellar defects, the causes of purely functional asthenozoospermia are still poorly defined. We describe here the case of an infertile man, displaying asthenozoospermia without major morphological flagellar anomalies and carrying a homozygous splicing mutation in SLC9C1 (sNHE), which we identified by whole-exome sequencing. SLC9C1 encodes a sperm-specific sodium/proton exchanger, which in mouse regulates pH homeostasis and interacts with the soluble adenylyl cyclase (sAC), a key regulator of the signalling pathways involved in sperm motility and capacitation. We demonstrate by means of RT-PCR, immunodetection and immunofluorescence assays on patient's semen samples that the homozygous splicing mutation (c.2748 + 2 T > C) leads to in-frame exon skipping resulting in a deletion in the cyclic nucleotide-binding domain of the protein. Our work shows that in human, similar to mouse, SLC9C1 is required for sperm motility. Overall, we establish a homozygous truncating mutation in SLC9C1 as a novel cause of human asthenozoospermia and infertility.

Primary biliary cholangitis: pathogenic mechanisms.

Purpose of reviewPrimary biliary cholangitis (PBC) is characterized by autoimmune damage of intrahepatic bile ducts associated with a loss of tolerance to mitochondrial antigens. PBC etiopathogenesis is intriguing because of different perplexing features, namely: a) although mitochondria are present in all cell types and tissues, the damage is mainly restricted to biliary epithelial cells (BECs); b) despite being an autoimmune disorder, it does not respond to immunosuppressive drugs but rather to ursodeoxycholic acid, a bile salt that induces HCO3− rich choleresis; c) the overwhelming female preponderance of the disease remains unexplained. Here we present an etiopathogenic view of PBC which sheds light on these puzzling facts of the disease.Recent findingsPBC develops in patients with genetic predisposition to autoimmunity in whom epigenetic mechanisms silence the Cl−/HCO3− exchanger AE2 in both cholangiocytes and lymphoid cells. Defective AE2 function can produce BECs damage as a result of decreased biliary HCO3− secretion with disruption of the protective alkaline umbrella that normally prevents the penetration of toxic apolar bile salts into cholangiocytes. AE2 dysfunction also causes increased intracellular pH (pHi) in cholangiocytes, leading to the activation of soluble adenylyl cyclase, which sensitizes BECs to bile salt-induced apoptosis. Recently, mitophagy was found to be inhibited by cytosolic alkalization and stimulated by acidification. Accordingly, we propose that AE2 deficiency may disturb mitophagy in BECs, thus, promoting the accumulation of defective mitochondria, oxidative stress and presentation of mitochondrial antigens to the immune cells. As women possess a more acidic endolysosomal milieu than men, mitophagy might be more affected in women in an AE2-defective background. Apart from affecting BECs function, AE2 downregulation in lymphocytes may also contribute to alter immunoregulation facilitating autoreactive T-cell responses.SummaryPBC can be considered as a disorder of Cl−/HCO3− exchange in individuals with genetic predisposition to autoimmunity.

Soluble adenylyl cyclase regulates the cytosolic NADH/NAD+ redox state and the bioenergetic switch between glycolysis and oxidative phosphorylation

The evolutionarily conserved soluble adenylyl cyclase (sAC, ADCY10) mediates cAMP signaling exclusively in intracellular compartments. Because sAC activity is sensitive to local concentrations of ATP, bicarbonate, and free Ca2+, sAC is potentially an important metabolic sensor. Nonetheless, little is known about how sAC regulates energy metabolism in intact cells. In this study, we demonstrated that both pharmacological and genetic suppression of sAC resulted in increased lactate secretion and decreased pyruvate secretion in multiple cell lines and primary cultures of mouse hepatocytes and cholangiocytes. The increased extracellular lactate-to-pyruvate ratio upon sAC suppression reflected an increased cytosolic free [NADH]/[NAD+] ratio, which was corroborated by using the NADH/NAD+ redox biosensor Peredox-mCherry. Mechanistic studies in permeabilized HepG2 cells showed that sAC inhibition specifically suppressed complex I of the mitochondrial respiratory chain. A survey of cAMP effectors revealed that only selective inhibition of exchange protein activated by cAMP 1 (Epac1), but not protein kinase A (PKA) or Epac2, suppressed complex I-dependent respiration and significantly increased the cytosolic NADH/NAD+ redox state. Analysis of the ATP production rate and the adenylate energy charge showed that inhibiting sAC reciprocally affects ATP production by glycolysis and oxidative phosphorylation while maintaining cellular energy homeostasis. In conclusion, our study shows that, via the regulation of complex I-dependent mitochondrial respiration, sAC-Epac1 signaling regulates the cytosolic NADH/NAD+ redox state, and coordinates oxidative phosphorylation and glycolysis to maintain cellular energy homeostasis. As such, sAC is effectively a bioenergetic switch between aerobic glycolysis and oxidative phosphorylation at the post-translational level.

The Calcium-Sensing Receptor Is Essential for Calcium and Bicarbonate Sensitivity in Human Spermatozoa.

The calcium-sensing receptor (CaSR) is essential to maintain a stable calcium concentration in serum. Spermatozoa are exposed to immense changes in concentrations of CaSR ligands such as calcium, magnesium, and spermine during epididymal maturation, in the ejaculate, and in the female reproductive environment. However, the role of CaSR in human spermatozoa is unknown. This work aimed to investigate the role of CaSR in human spermatozoa. We identified CaSR in human spermatozoa and characterized the response to CaSR agonists on intracellular calcium, acrosome reaction, and 3',5'-cyclic adenosine 5'-monophosphate (cAMP) in spermatozoa from men with either loss-of-function or gain-of-function mutations in CASR and healthy donors. CaSR is expressed in human spermatozoa and is essential for sensing extracellular free ionized calcium (Ca2+) and Mg2+. Activators of CaSR augmented the effect of sperm-activating signals such as the response to HCO3- and the acrosome reaction, whereas spermatozoa from men with a loss-of-function mutation in CASR had a diminished response to HCO3-, lower progesterone-mediated calcium influx, and were less likely to undergo the acrosome reaction in response to progesterone or Ca2+. CaSR activation increased cAMP through soluble adenylyl cyclase (sAC) activity and increased calcium influx through CatSper. Moreover, external Ca2+ or Mg2+ was indispensable for HCO3- activation of sAC. Two male patients with a CASR loss-of-function mutation in exon 3 presented with normal sperm counts and motility, whereas a patient with a loss-of-function mutation in exon 7 had low sperm count, motility, and morphology. CaSR is important for the sensing of Ca2+, Mg2+, and HCO3- in spermatozoa, and loss-of-function may impair male sperm function.

Hepatocyte-specific TAZ deletion downregulates p62/ Sqstm1 expression in nonalcoholic steatohepatitis

Nonalcoholic steatohepatitis (NASH) is characterized by inflammation, hepatocellular injury, and different degrees of fibrosis. Previous studies have indicated that the transcriptional coactivator with PDZ-binding motif TAZ (WWTR1) is correlated with the increased level of liver cholesterol which suppresses TAZ proteasomal degradation and promotes fibrotic NASH by activating soluble adenylyl cyclase -calcium-RhoA pathway. However, the exact mechanism by which TAZ promotes inflammatory and hepatocyte injury has not yet been fully addressed. Reportedly, p62/Sqstm1plays a pivotal role in inflammatory and hepatocyte injury during NASH development. Here, we demonstrated that p62/Sqstm1 was overexpressed in the livers of mouse NASH models in a TAZ-dependent manner. In addition, hepatocyte-specific TAZ deletion reduced p62/Sqstm1 both in vitro and in vivo. Strikingly, luciferase reporter data demonstrated that p62/Sqstm1 is a TAZ/TEAD target gene and can be transcriptionally regulated by TAZ, indicating that hepatocyte-specific TAZ deletion downregulates p62/Sqstm1 expression in NASH.

Adverse effects of Δ9-tetrahydrocannabinol on neuronal bioenergetics during postnatal development.

Ongoing societal changes in views on the medical and recreational roles of cannabis increased the use of concentrated plant extracts with a Δ9-tetrahydrocannabinol (THC) content of more than 90%. Even though prenatal THC exposure is widely considered adverse for neuronal development, equivalent experimental data for young age cohorts are largely lacking. Here, we administered plant-derived THC (1 or 5 mg/kg) to mice daily during P5–P16 and P5–P35 and monitored its effects on hippocampal neuronal survival and specification by high-resolution imaging and iTRAQ proteomics, respectively. We found that THC indiscriminately affects pyramidal cells and both cannabinoid receptor 1+ (CB1R)+ and CB1R– interneurons by P16. THC particularly disrupted the expression of mitochondrial proteins (complexes I–IV), a change that had persisted even 4 months after the end of drug exposure. This was reflected by a THC-induced loss of membrane integrity occluding mitochondrial respiration and could be partially or completely rescued by pH stabilization, antioxidants, bypassed glycolysis, and targeting either mitochondrial soluble adenylyl cyclase or the mitochondrial voltage-dependent anion channel. Overall, THC exposure during infancy induces significant and long-lasting reorganization of neuronal circuits through mechanisms that, in large part, render cellular bioenergetics insufficient to sustain key developmental processes in otherwise healthy neurons.

Bone Deterioration in Response to Chronic High-Altitude Hypoxia Is Attenuated by a Pulsed Electromagnetic Field Via the Primary Cilium/HIF-1α Axis.

Chronic high-altitude hypoxia induces irreversible abnormalities in various organisms. Emerging evidence indicates that hypobaric hypoxia markedly suppresses bone mass and bone strength. However, few effective means have been identified to prevent such bone deficits. Here, we assessed the potential of pulsed electromagnetic fields (PEMFs) to noninvasively resist bone deterioration induced by hypobaric hypoxia. We observed that exogenous PEMF treatment at 15 Hz and 20 Gauss (Gs) improved the cancellous and cortical bone mass, bone microstructure, and skeletal mechano-properties in rats subjected to chronic exposure of hypobaric hypoxia simulating an altitude of 4500 m for 6 weeks by primarily modulating osteoblasts and osteoblast-mediated bone-forming activity. Moreover, our results showed that whereas PEMF stimulated the functional activity of primary osteoblasts in hypoxic culture in vitro, it had negligible effects on osteoclasts and osteocytes exposed to hypoxia. Mechanistically, the primary cilium was found to function as the major electromagnetic sensor in osteoblasts exposed to hypoxia. The polycystins PC-1/PC-2 complex was identified as the primary calcium channel in the primary cilium of hypoxia-exposed osteoblastic cells responsible for the detection of external PEMF signals, and thereby translated these biophysical signals into intracellular biochemical events involving significant increase in the intracellular soluble adenylyl cyclase (sAC) expression and subsequent elevation of cyclic adenosine monophosphate (cAMP) concentration. The second messenger cAMP inhibited the transcription of oxygen homeostasis-related hypoxia-inducible factor 1-alpha (HIF-1α), and thus enhanced osteoblast differentiation and improved bone phenotype. Overall, the present study not only advances our understanding of bone physiology at high altitudes, but more importantly, proposes effective means to ameliorate high altitude-induced bone loss in a noninvasive and cost-effective manner. © 2023 American Society for Bone and Mineral Research (ASBMR).

Differential effects of bicarbonate on severe hypoxia- and hypercapnia-induced cardiac malfunctions in diverse fish species.

We tested in six fish species [Pacific lamprey (Lampetra richardsoni), Pacific spiny dogfish (Squalus suckleyi), Asian swamp eel (Monopterus albus), white sturgeon (Acipenser transmontanus), zebrafish (Danio rerio), and starry flounder (Platichthys stellatus)] the hypothesis that elevated extracellular [HCO3-] protects spontaneous heart rate and cardiac force development from the known impairments that severe hypoxia and hypercapnic acidosis can induce. Hearts were exposed in vitro to either severe hypoxia (~ 3% of air saturation), or severe hypercapnic acidosis (either 7.5% CO2 or 15% CO2), which reduced heart rate (in six test species) and net force development (in three test species). During hypoxia, heart rate was restored by [HCO3-] in a dose-dependent fashion in lamprey, dogfish and eel (EC50 = 5, 25 and 30mM, respectively), but not in sturgeon, zebrafish or flounder. During hypercapnia, elevated [HCO3-] completely restored heart rate in dogfish, eel and sturgeon (EC50 = 5, 25 and 30mM, respectively), had a partial effect in lamprey and zebrafish, and had no effect in flounder. Elevated [HCO3-], however, had no significant effect on net force of electrically paced ventricular strips from dogfish, eel and flounder during hypoxia and hypercapnia. Only in lamprey hearts did a specific soluble adenylyl cyclase (sAC) inhibitor, KH7, block the HCO3--mediated rescue of heart rate during both hypoxia and hypercapnia, and wasthe only species where we conclusively demonstrated sAC activity was involved in the protective effects of HCO3- on cardiac function. Our results suggest a common HCO3--dependent, sAC-dependent transduction pathway for heart rate recovery exists in cyclostomes and a HCO3--dependent, sAC-independent pathway exists in other fish species.

The Role of Zinc in Male Fertility.

Several studies proposed the importance of zinc ion in male fertility. Here, we describe the properties, roles and cellular mechanisms of action of Zn2+ in spermatozoa, focusing on its involvement in sperm motility, capacitation and acrosomal exocytosis, three functions that are crucial for successful fertilization. The impact of zinc supplementation on assisted fertilization techniques is also described. The impact of zinc on sperm motility has been investigated in many vertebrate and invertebrate species. It has been reported that Zn2+ in human seminal plasma decreases sperm motility and that Zn2+ removal enhances motility. Reduction in the intracellular concentration of Zn2+ during epididymal transit allows the development of progressive motility and the subsequent hyper activated motility during sperm capacitation. Extracellular Zn2+ affects intracellular signaling pathways through its interaction with the Zn2+ sensing receptor (ZnR), also named GPR39. This receptor was found in the sperm tail and the acrosome, suggesting the possible involvement of Zn2+ in sperm motility and acrosomal exocytosis. Our studies showed that Zn2+ stimulates bovine sperm acrosomal exocytosis, as well as human sperm hyper-activated motility, were both mediated by GPR39. Zn2+ binds and activates GPR39, which activates the trans-membrane-adenylyl-cyclase (tmAC) to catalyze cAMP production. The NHE (Na+/H+-exchanger) is activated by cAMP, leading in increased pHi and activation of the sperm-specific Ca2+ channel CatSper, resulting in an increase in [Ca2+]i, which, together with HCO3−, activates the soluble adenylyl-cyclase (sAC). The increase in [cAMP]i activates protein kinase A (PKA), followed by activation of the Src-epidermal growth factor receptor-Pphospholipase C (Src-EGFR-PLC) cascade, resulting in inositol-triphosphate (IP3) production, which mobilizes Ca2+ from the acrosome, causing a further increase in [Ca2+]i and the development of hyper-activated motility. PKA also activates phospholipase D1 (PLD1), leading to F-actin formation during capacitation. Prior to the acrosomal exocytosis, PLC induces phosphadidylinositol-4,5-bisphosphate (PIP2) hydrolysis, leading to the release of the actin-severing protein gelsolin to the cytosol, which is activated by Ca2+, resulting in F-actin breakdown and the occurrence of acrosomal exocytosis.

Uncovering the Cyclic AMP Signaling Pathway of the Protozoan Parasite Entamoeba histolytica and Understanding Its Role in Phagocytosis.

Second messenger signaling controls a surprisingly diverse range of processes in several eukaryotic pathogens. Molecular machinery and pathways involving these messengers thus hold tremendous opportunities for therapeutic interventions. Relative to Ca2+ signaling, the knowledge of a crucial second messenger cyclic AMP (cAMP) and its signaling pathway is very scant in the intestinal parasite Entamoeba histolytica. In the current study, mining the available genomic resources, we have for the first time identified the cAMP signal transduction pathway of E. histolytica. Three heptahelical proteins with variable G-protein-coupled receptor domains, heterotrimeric G-proteins (Gα, Gβ, and Gγ subunits), soluble adenylyl cyclase, cyclase-associated protein, and enzyme carbonic anhydrase were identified in its genome. We could also identify several putative candidate genes for cAMP downstream effectors such as protein kinase A, A-kinase anchoring proteins, and exchange protein directly activated by the cAMP pathway. Using specific inhibitors against key identified targets, we could observe changes in the intracellular cAMP levels as well as defect in the rate of phagocytosis of red blood cells by the parasite E. histolytica. We thus strongly believe that characterization of some of these unexplored crucial signaling determinants will provide a paradigm shift in understanding the pathogenicity of this organism.

Capacitation increases glucose consumption in murine sperm.

Mammalian sperm acquire fertilization capacity in the female reproductive tract in a process known as capacitation. During capacitation, sperm change their motility pattern (i.e., hyperactivation) and become competent to undergo the acrosome reaction. We have recently shown that, in the mouse, sperm capacitation is associated with increased uptake of fluorescently labeled deoxyglucose and with extracellular acidification suggesting enhanced glycolysis. Consistently, in the present work we showed that glucose consumption is enhanced in media that support mouse sperm capacitation suggesting upregulation of glucose metabolic pathways. The increase in glucose consumption was modulated by bicarbonate and blocked by protein kinase A and soluble adenylyl cyclase inhibitors. Moreover, permeable cyclic adenosine monophosphate (cAMP) agonists increase glucose consumption in sperm incubated in conditions that do not support capacitation. Also, the increase in glucose consumption was reduced when sperm were incubated in low calcium conditions. Interestingly, this reduction was not overcome with cAMP agonists. Despite these findings, glucose consumption of sperm from Catsper1 knockout mice was similar to the one from wild type suggesting that other sources of calcium are also relevant. Altogether, these results suggest that cAMP and calcium pathways are involved in the regulation of glycolytic energy pathways during murine sperm capacitation.

Molecular Mechanism Underlying the Action of Zona-pellucida Glycoproteins on Mouse Sperm.

Mammalian oocytes are enveloped by the zona pellucida (ZP), an extracellular matrix of glycoproteins. In sperm, stimulation with ZP proteins evokes a rapid Ca2+ influx via the sperm-specific, pH-sensitive Ca2+ channel CatSper. However, the physiological role and molecular mechanisms underlying ZP-dependent activation of CatSper are unknown. Here, we delineate the sequence of ZP-signaling events in mouse sperm. We show that ZP proteins evoke a rapid intracellular pHi increase that rests predominantly on Na+/H+ exchange by NHA1 and requires cAMP synthesis by the soluble adenylyl cyclase sAC as well as a sufficiently negative membrane potential set by the spem-specific K+ channel Slo3. The alkaline-activated CatSper channel translates the ZP-induced pHi increase into a Ca2+ response. Our findings reveal the molecular components underlying ZP action on mouse sperm, opening up new avenues for understanding the basic principles of sperm function and, thereby, mammalian fertilization.

Cyclic adenosine monophosphate (cAMP) signaling in melanocyte pigmentation and melanomagenesis.

The second messenger cyclic adenosine monophosphate (cAMP) regulates numerous functions in both benign melanocytes and melanoma cells. cAMP is generated from two distinct sources, transmembrane and soluble adenylyl cyclases (tmAC and sAC, respectively), and is degraded by a family of proteins called phosphodiesterases (PDEs). cAMP signaling can be regulated in many different ways and can lead to varied effects in melanocytes. It was recently revealed that distinct cAMP signaling pathways regulate pigmentation by either altering pigment gene expression or the pH of melanosomes. In the context of melanoma, many studies report seemingly contradictory roles for cAMP in tumorigenesis. For example, cAMP signaling has been implicated in both cancer promotion and suppression, as well as both therapy resistance and sensitization. This conundrum in the field may be explained by the fact that cAMP signals in discrete microdomains and each microdomain can mediate differential cellular functions. Here, we review the role of cAMP signaling microdomains in benign melanocyte biology, focusing on pigmentation, and in melanomagenesis.

Targeting the sAC-Dependent cAMP Pool to Prevent SARS-Cov-2 Infection.

An outbreak of the novel coronavirus (CoV) SARS-CoV-2, the causative agent of COVID-19 respiratory disease, infected millions of people since the end of 2019, led to high-level morbidity and mortality and caused worldwide social and economic disruption. There are currently no antiviral drugs available with proven efficacy or vaccines for its prevention. An understanding of the underlying cellular mechanisms involved in virus replication is essential for repurposing the existing drugs and/or the discovery of new ones. Endocytosis is the important mechanism of entry of CoVs into host cells. Endosomal maturation followed by the fusion with lysosomes are crucial events in endocytosis. Late endosomes and lysosomes are characterized by their acidic pH, which is generated by a proton transporter V-ATPase and required for virus entry via endocytic pathway. The cytoplasmic cAMP pool produced by soluble adenylyl cyclase (sAC) promotes V-ATPase recruitment to endosomes/lysosomes and thus their acidification. In this review, we discuss targeting the sAC-specific cAMP pool as a potential strategy to impair the endocytic entry of the SARS-CoV-2 into the host cell. Furthermore, we consider the potential impact of sAC inhibition on CoV-induced disease via modulation of autophagy and apoptosis.

Abstract 287: A nuclear cAMP microdomain suppresses tumor growth by Hippo Pathway inactivation

Abstract Cyclic AMP can both promote and suppress tumor growth; however, how this single second messenger can simultaneously have such disparate effects on cancer remains largely undefined. It is well appreciated that cAMP does not diffuse within a cell but is made in microdomains, which consist of a unique adenylyl cyclase, one or more phosphodiesterases, and cAMP effector proteins, but the role of specific cAMP microdomains in cancer is unclear. Examination of human tissue biopsies revealed that the nuclear cAMP microdomain, defined by the soluble adenylyl cyclase (sAC), is upregulated early in tumorigenesis, but is lost with melanoma invasion and metastasis. Further, we showed that loss of sAC led to the transformation of melanocytes and reintroduction of sAC, only into the nucleus, was sufficient to block tumorigenesis in vitro and in mice. Neither mitochondrial, nor cytoplasmic cAMP microdomains affected tumor cell growth. Activation of nuclear sAC led to significant changes in chromatin accessibility suggesting that this cAMP microdomain regulates numerous transcription factors. Specifically, activation of nuclear sAC led to reduced chromatin accessibility for the TEAD and SMAD family of transcription factors, the key effectors of the Hippo Pathway. Consistent with those data, nuclear sAC suppressed TEAD-dependent pro-cancer gene expression profiles (e.g., MYC) by inhibiting YAP, one of the main oncoproteins in this signaling cascade. Moreover, we showed that nuclear cAMP evokes the same YAP-dependent inhibition of the Hippo Pathway in a variety of cancers, suggesting tumor suppression by nuclear sAC is not limited to melanoma. These studies reveal that a cAMP microdomain wholly contained within the nucleus is a pivotal a tumor suppressor that acts via Hippo Pathway inactivation. Citation Format: Marek M. Drozdz, Ashley Doane, Garrett Desman, Jenny Wang, Michael Reilly, Kelsey Aguirre, Elsbeth Kane, Jedd Wolchok, Taha Merghoub, Olivier Elemento, Elena Piskounova, Jonathan Zippin. A nuclear cAMP microdomain suppresses tumor growth by Hippo Pathway inactivation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 287.

Bicarbonate suppresses mitochondrial membrane depolarization induced by conventional uncouplers

Bicarbonate has been known to modulate activities of various mitochondrial enzymes such as ATPase and soluble adenylyl cyclase. Here, we found that the ability of conventional protonophoric uncouplers, such as 2,4-dinitrophenol (DNP), carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) and carbonyl cyanide m-chlorophenyl hydrazone (CCCP), but not that of the new popular uncoupler BAM15, to decrease mitochondrial membrane potential was significantly diminished in the presence of millimolar concentrations of bicarbonate. Thus, the depolarizing activity of DNP and FCCP in mitochondria could be sensitive to the local concentration of bicarbonate in cells and tissues. However, bicarbonate could not restore the ATP synthesis suppressed by DNP or CCCP in mitochondria. Bicarbonate neither altered the depolarizing action of DNP and FCCP on proteoliposomes with reconstituted cytochrome c oxidase, nor affected the protonophoric activity of DNP and FCCP in artificial lipid membranes as measured with pyranine-loaded liposomes, thereby showing that the bicarbonate-induced reversal of the depolarizing action of DNP and FCCP on mitochondria did not result from direct interaction of bicarbonate with the uncouplers.

Cardioprotective effects of GLP-1(28-36a): A degraded metabolite or GLP-1's better half?

Molecular mechanism underlying glucagon-like peptide-1 exertion of cardioprotective effects in glucagon-like peptide-1 receptor-dependent and -independent manners. Glucagon-like peptide-1 (28-36a) enters coronary artery endothelial cells through macropinocytosis and binds to mitochondrial trifunctional protein-α, shifting substrate utilization to increase adenosine triphosphate production and modulating a adenosine triphosphate-sensor soluble adenylyl cyclase, thereby producing cyclic adenosine monophosphate and activating protein kinaseA to exert cytoprotection from oxidative injury.

Immunological characterization of two types of ionocytes in the inner ear epithelium of Pacific Chub Mackerel (Scomber japonicus)

The inner ear is essential for maintaining balance and hearing predator and prey in the environment. Each inner ear contains three CaCO3 otolith polycrystals, which are calcified within an alkaline, K+-rich endolymph secreted by the surrounding epithelium. However, the underlying cellular mechanisms are poorly understood, especially in marine fish. Here, we investigated the presence and cellular localization of several ion-transporting proteins within the saccular epithelium of the Pacific Chub Mackerel (Scomber japonicus). Western blotting revealed the presence of Na+/K+-ATPase (NKA), carbonic anhydrase (CA), Na+-K+-2Cl--co-transporter (NKCC), vacuolar-type H+-ATPase (VHA), plasma membrane Ca2+ ATPase (PMCA), and soluble adenylyl cyclase (sAC). Immunohistochemistry analysis identified two distinct ionocytes types in the saccular epithelium: Type-I ionocytes were mitochondrion-rich and abundantly expressed NKA and NKCC in their basolateral membrane, indicating a role in secreting K+ into the endolymph. On the other hand, Type-II ionocytes were enriched in cytoplasmic CA and VHA, suggesting they help transport HCO3- into the endolymph and remove H+. In addition, both types of ionocytes expressed cytoplasmic PMCA, which is likely involved in Ca2+ transport and homeostasis, as well as sAC, an evolutionary conserved acid-base sensing enzyme that regulates epithelial ion transport. Furthermore, CA, VHA, and sAC were also expressed within the capillaries that supply blood to the meshwork area, suggesting additional mechanisms that contribute to otolith calcification. This information improves our knowledge about the cellular mechanisms responsible for endolymph ion regulation and otolith formation, and can help understand responses to environmental stressors such as ocean acidification.