Role of Adenylyl Cyclase Type 7 in Functions of BV-2 Microglia.

To assess the role of adenylyl cyclase type 7 (AC7) in microglia's immune function, we generated AC7 gene knockout (AC7 KO) clones from a mouse microglial cell line, BV-2, using the CRISPR-Cas9 gene editing system. The ability of BV-2 cells to generate cAMP and their innate immune functions were examined in the presence or absence of ethanol. The parental BV-2 cells showed robust cAMP production when stimulated with prostaglandin-E1 (PGE1) and ethanol increased cAMP production in a dose-dependent manner. AC7 KO clones of BV-2 cells showed diminished and ethanol-insensitive cAMP production. The phagocytic activity of the parental BV-2 cells was inhibited in the presence of PGE1; AC7 KO BV-2 cells showed lower and PGE1-insensitive phagocytic activity. Innate immune activities of the parental BV-2 cells, including bacterial killing, nitric oxide synthesis, and expression of arginase 1 and interleukin 10 were activated as expected with small effects of ethanol. However, the innate immune activities of AC7 KO cells were either drastically diminished or not detected. The data presented suggest that AC7 has an important role in the innate immune functions of microglial cells. AC7's involvement in ethanol's effects on immune functions remains unclear. Further studies are needed.

The effect of alcohol on recombinant proteins derived from mammalian adenylyl cyclase.

The cyclic AMP (cAMP) signaling pathway is implicated in the development of alcohol use disorder. Previous studies have demonstrated that ethanol enhances the activity of adenylyl cyclase (AC) in an isoform specific manner; AC7 is most enhanced by ethanol, and regions responsible for enhancement by ethanol are located in the cytoplasmic domains of the AC7 protein. We hypothesize that ethanol modulates AC activity by directly interacting with the protein and that ethanol effects on AC can be studied using recombinant AC in vitro. AC recombinant proteins containing only the C1a or C2 domains of AC7 and AC9 individually were expressed in bacteria, and purified. The purified recombinant AC proteins retained enzymatic activity and isoform specific alcohol responsiveness. The combination of the C1a or C2 domains of AC7 maintained the same alcohol cutoff point as full-length AC7. We also find that the recombinant AC7 responds to alcohol differently in the presence of different combinations of activators including MnCl2, forskolin, and Gsα. Through a series of concentration-response experiments and curve fitting, the values for maximum activities, Hill coefficients, and EC50 were determined in the absence and presence of butanol as a surrogate of ethanol. The results suggest that alcohol modulates AC activity by directly interacting with the AC protein and that the alcohol interaction with the AC protein occurs at multiple sites with positive cooperativity. This study indicates that the recombinant AC proteins expressed in bacteria can provide a useful model system to investigate the mechanism of alcohol action on their activity.

Observation on the ultrastructure morphology of HeLa cells treated with ethanol: Statistical analysis.

It is estimated that 5.9% of all human deaths are attributable to alcohol consumption and that the harmful use of ethanol ranks among the top five risk factors for causing disease, disability, and death worldwide. Ethanol is known to disrupt phospholipid packing and promote membrane hemifusion at lipid bilayers. With the exception of mitochondria involved in hormone synthesis, the sterol content of mitochondrial membranes is low. As membranes that are low in cholesterol have increased membrane fluidity and are the most easily disordered by ethanol, we hypothesize that mitochondria are sensitive targets for ethanol damage. HeLa cells were exposed to 50 mM ethanol and the direct effects of ethanol on cellular ultrastructure were examined utilizing transmission electron microscopy. Our ultramicroscopic analysis revealed that cells exposed to ethanol harbor fewer incidence of apoptotic morphology; however, significant alterations to mitochondria and to nuclei occurred. We observed statistical increases in the amount of irregular cells and cells with multiple nuclei, nuclei harboring indentations, and nuclei with multiple nucleolus-like bodies. Indeed, our analysis revealed that mitochondrial damage is the most extensive type of cellular damage. Rupturing of cristae was the most prominent damage followed by mitochondrial swelling. Ethanol exposure also resulted in increased amounts of mitochondrial rupturing, organelles with linked membranes, and mitochondria localizing to indentations of nuclear membranes. We theorize that these alterations could contribute to cellular defects in oxidative phosphorylation and, by extension, the inability to generate regular levels of cellular adenosine triphosphate.

Role of an adenylyl cyclase isoform in ethanol's effect on cAMP regulated gene expression in NIH 3T3 cells.

Previous research has indicated that the cyclic AMP (cAMP) signal transduction system plays an important role in the predisposition to and development of ethanol abuse in humans. Our laboratory has demonstrated that ethanol is capable of enhancing adenylyl cyclase (AC) activity. This effect is AC isoform-specific; type 7 AC (AC7) is most enhanced by ethanol. Therefore, we hypothesized that the expression of a specific AC isoform will play a role on the effect of ethanol on cAMP regulated gene expression. We employed NIH 3T3 cells transfected with AC7 or AC3 as a model system. To evaluate ethanol's effects on cAMP regulated gene expression, a luciferase reporter gene driven by a cAMP inducing artificial promoter was utilized. Stimulation of AC activity leads to an increase in the reporter gene activity. This increase was enhanced in the presence of ethanol in cells expressing AC7, while cells expressing AC3 did not respond to ethanol. cAMP reporter gene expression was increased in the presence of 8-bromo-cAMP; this expression was not enhanced by ethanol. These observations are consistent with our hypothesis. The basal level of CREB phosphorylation was high and did not change by cAMP stimulation or in the presence of ethanol. However, there were significant changes in the TORC3 amount in nuclei depending on stimulation conditions. The results suggest that nuclear translocation of TORC3 plays a more important role than CREB phosphorylation in the observed changes in the cAMP driven reporter gene activity.

Real-time monitoring of intracellular cAMP during acute ethanol exposure.

BACKGROUND: In previous studies, we have shown that ethanol (EtOH) enhances the activity of stimulatory G protein (Gs)-stimulated membrane-bound adenylyl cyclase (AC). The effect is AC isoform specific, and the type 7 AC (AC7) is most responsive to EtOH. In this study, we employed a fluorescence resonance energy transfer (FRET)-based cyclic AMP (cAMP) sensor, Epac1-camps, to examine real-time temporal dynamics of EtOH effects on cAMP concentrations. To our knowledge, this is the first report on real-time detection of the EtOH effect on intracellular cAMP. METHODS: Hela cells were transfected with Epac1-camps, dopamine (DA) receptor D1a , and 1 isoform of AC (AC7 or AC3). Fluorescent images were captured using a specific filter set for cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and FRET, respectively, and FRET intensity was calculated on a pixel-by-pixel basis to examine changes in cAMP. RESULTS: During 2-minute stimulation with DA, the cytoplasmic cAMP level quickly increased and then decreased to a plateau, where the cAMP level was higher than the level prior to stimulation with DA. EtOH concentration dependently increased cytoplasmic cAMP in cells transfected with AC7, while EtOH did not have effect on cells transfected with AC3. Similar trends were observed for cAMP at the plasma membrane and in the nucleus during 2-minute stimulation with DA. Unexpectedly, when cells expressing AC7 were stimulated with DA or other Gs-coupled receptor's ligand plus EtOH for 5 seconds, EtOH reduced cAMP concentration. CONCLUSIONS: These results suggest that EtOH has 2 opposing effects on the cAMP-generating system in an AC isoform-specific manner, the enhancing effect on AC activity and the short-lived inhibitory effect. Thus, EtOH may have a different effect on cAMP depending on not only AC isoform but also the duration of exposure.

Effects of alcohols on recombinant adenylyl cyclase type 7 expressed in bacteria.

BACKGROUND: Our previous studies showed that ethanol enhanced the activity of adenylyl cyclase (AC) in an isoform-specific manner and that alcohol cutoff point of AC was isoform specific. Recently, we showed that 2,3-butanediol inhibited AC type 7 (AC7) activity in a stereoisomer-specific manner and that this inhibition was also AC isoform specific. These observations strongly suggest that a major target of alcohol action on cAMP signaling is AC. We hypothesized that alcohols exhibit their effect on AC activity by direct interaction with AC proteins. However, experimental systems employed in past studies such as intact cells and membrane preparations are too complex and do not allow us to unequivocally test this hypothesis. In attempt to bypass, these complications of the membrane-bound AC, we decided to study the effect of alcohols on AC recombinant proteins expressed in bacteria. METHODS: A recombinant AC, designated as AC7sol, consisting of the C(1a) and C(2) domains of the human AC7 was designed and expressed in bacteria. The activity of AC7sol was examined using lysate prepared from bacteria expressing AC7sol. RESULTS: The activity of AC7sol was stimulated by manganese or by the α subunit of G protein that stimulates AC (G(sα) ). Forskolin by itself did not stimulate the activity of AC7sol. However, in the presence of activated G(sα) , forskolin stimulated the activity of AC7sol. A series of n-alkanols including ethanol enhanced the manganese-stimulated activity of AC7sol. The alcohol cutoff point of AC7sol was pentanol. Ethanol and butanol increased V(max) and K(M) values of AC7sol. CONCLUSIONS: These results are consistent with our hypothesis and suggest that the enhancing effect of alcohols on AC activity is because of the increase in turnover number of AC. The current study demonstrates for the first time that the effect of alcohols requires only the C(1a) and C(2) domains of AC and no other domains of AC as well as no other mammalian proteins.

Neurotransplantation of stem cells genetically modified to express human dopamine transporter reduces alcohol consumption.

INTRODUCTION: Regulated neurotransmitter actions in the mammalian central nervous system determine brain function and control peripheral organs and behavior. Although drug-seeking behaviors, including alcohol consumption, depend on central neurotransmission, modification of neurotransmitter actions in specific brain nuclei remains challenging. Herein, we report a novel approach for neurotransmission modification in vivo by transplantation of stem cells engineered to take up the neurotransmitter dopamine (DA) efficiently through the action of the human dopamine transporter (hDAT). As a functional test in mice, we used voluntary alcohol consumption, which is known to release DA in nucleus accumbens (NAC), an event hypothesized to help maintain drug-seeking behavior. We reasoned that reducing extracellular DA levels, by engrafting into NAC DA-sequestering stem cells expressing hDAT, would alter alcohol intake. METHODS: We have generated a neural stem cell line stably expressing the hDAT. Uptake kinetics of DA were determined to select a clone for transplantation. These genetically modified stem cells (or cells transfected with a construct lacking the hDAT sequence) were transplanted bilaterally into the NAC of wild-type mice trained to consume 10% alcohol in a two-bottle free-choice test for alcohol consumption. Alcohol intake was then ascertained for 1 week after transplantation, and brain sections through the NAC were examined for surviving grafted cells. RESULTS: Modified stem cells expressed hDAT and uptaken DA selectively via hDAT. Mice accustomed to drinking 10% ethanol by free choice reduced their alcohol consumption after being transplanted with hDAT-expressing stem cells. By contrast, control stem cells lacked that effect. Histologic examination revealed surviving stem cells in the NAC of all engrafted brains. CONCLUSIONS: Our findings represent proof of principle suggesting that genetically engineered stem cells can be useful for exploring the role of neurotransmitters (or other signaling molecules) in alcohol consumption and potentially in other aspects of brain function.

Effects of straight chain alcohols on specific isoforms of adenylyl cyclase.

BACKGROUND: Our previous studies showed that the activity of adenylyl cyclase (AC) was enhanced by pharmacologically relevant concentrations of ethanol, that this enhancing effect of ethanol on AC activity was AC isoform specific, and that the alcohol cutoff effect for n-alkanol potentiation of AC activity was also AC isoform specific. Therefore, we hypothesized that within the cyclic AMP-generating system, AC is the target of ethanol's action and that alcohols interact directly with the AC molecules. To characterize the interaction between alcohols and AC proteins, the effects of a series of straight chain alcohols would be very valuable in understanding alcohol action at the molecular level. To our knowledge, straight chain alcohols other than n-alkanols and 1,Omega-diols have not been used extensively to study alcohol effects on the activity of AC or other proteins important in the alcohol research field. METHODS: The effects of a series of straight chain alcohols on D1A dopamine receptor-stimulated activity of AC isoforms type 6, 7, and 9 (AC6, AC7, and AC9) were examined in transfected Hela cells by a cAMP accumulation assay. RESULTS: In general, all 3 AC isoforms responded to a series of straight chain alcohols in a similar manner. The order of responsiveness is as follows: monoalcohol > diol > triol and tetraol. Within monoalcohols, 1-alcohols had larger effects than 2-alcohols. Two of 3 stereoisomers of 2,3-butanediol, [D-(-)-2,3-butanediol and meso-2,3-butanediol] showed similar enhancing effects on all 3 AC isoforms. However, the third stereoisomer, L-(+)-2,3-butanediol, inhibited AC7 activity, while it stimulated AC6 and AC9. CONCLUSION: The number and the position of hydroxyl groups in straight chain alcohols play an important role in the magnitude of the enhancement on AC activity. Regardless of AC isoforms, the most effective of the straight chain alcohols seems to be the 1-alcohol (n-alkanol) for a given chain length. We found that one of the stereoisomers of 2,3-butanediol had opposite effects on AC activity depending on the AC isoform. Overall, the results are consistent with the hypotheses and demonstrate that a series of straight chain alcohols can be a valuable tool to study AC-alcohol interactions.

Conversion of red fluorescent protein into a bright blue probe.

We used a red chromophore formation pathway, in which the anionic red chromophore is formed from the neutral blue intermediate, to suggest a rational design strategy to develop blue fluorescent proteins with a tyrosine-based chromophore. The strategy was applied to red fluorescent proteins of the different genetic backgrounds, such as TagRFP, mCherry, HcRed1, M355NA, and mKeima, which all were converted into blue probes. Further improvement of the blue variant of TagRFP by random mutagenesis resulted in an enhanced monomeric protein, mTagBFP, characterized by the substantially higher brightness, the faster chromophore maturation, and the higher pH stability than blue fluorescent proteins with a histidine in the chromophore. The detailed biochemical and photochemical analysis indicates that mTagBFP is the true monomeric protein tag for multicolor and lifetime imaging, as well as the outstanding donor for green fluorescent proteins in Förster resonance energy transfer applications.

Isoform-specific enhancement of adenylyl cyclase activity by n-alkanols.

BACKGROUND: Numerous studies suggest that cAMP signaling pathways play important roles in the development of and predisposition to alcoholism. Our previous study showed that cAMP generation by various isoforms of adenylyl cyclase (AC) exhibits a broad spectrum of responses to ethanol in the human embryonic kidney (HEK) 293 cell system overexpressing individual AC isoforms. These findings suggest that the target of ethanol's action in the cAMP-generating system is AC. However, it is unknown if the action of ethanol is direct or indirect. METHODS: The effect of a series of n-alkanols (ethanol to decanol) on dopamine (DA)-stimulated activity of AC isoforms type 6, 7, and 9 (AC6, AC7, and AC9) were examined in transfected HEK293 cells by cAMP accumulation assay. RESULTS: n-Alkanols increased DA-stimulated cAMP production in an AC isoform-specific manner, and displayed the alcohol cutoff phenomenon (defined as the carbon chain length beyond which there is no further increase in the potency of an ascending series of n-alkanols). The n-alkanol cutoffs for AC6, AC7, and AC9 are butanol (C4), pentanol (C5), and equal to or greater than decanol (C10), respectively. CONCLUSION: The results clearly indicate that, in the HEK293 expression system, the alcohol cutoff effect for n-alkanol potentiation of DA-stimulated AC activity is AC isoform specific. These results strongly suggest that alcohols interact directly with AC molecules.

A sex-specific role of type VII adenylyl cyclase in depression.

Major depression represents a complex mental disorder. The identification of biological markers that define subtypes of major depressive disorder would greatly facilitate appropriate medical treatments, as well as provide insight into etiology. Reduced activity of the cAMP signaling system has been implicated in the etiology of major depression. Previous work has shown low adenylyl cyclase activity in platelets and postmortem brain tissue of depressed individuals. Here, we investigate the role of the brain type VII isoform of adenylyl cyclase (AC7) in the manifestation of depressive symptoms in genetically modified animals, using a combination of in vivo behavioral experiments, gene expression profiling, and bioinformatics. We also completed studies with humans on the association of polymorphisms in the AC7 gene with major depressive illness (unipolar depression) based on Diagnostic and Statistical Manual of Mental Disorders IV criteria. Collectively, our results demonstrate a sex-specific influence of the AC7 gene on a heritable form of depressive illness.

Identification of ethanol responsive domains of adenylyl cyclase.

BACKGROUND: The activity of adenylyl cyclase (AC) is enhanced by pharmacologically relevant concentrations of ethanol. The enhancing effect of ethanol on AC activity is AC isoform-specific. Therefore, we hypothesized that within a cyclic AMP-generating system, AC is the target of ethanol's action and that ethanol-sensitive AC molecules contain structural elements modulated by ethanol. The structural elements are designated as "ethanol responsive domains." METHODS: By using a series of chimeric mutants, we searched regions of the AC molecule that are important for the ethanol effect. These chimeric mutants were derived from 3 isoforms of AC: AC7 (type 7), the most ethanol responsive isoform; AC3 (type 3), an isoform that is far less responsive to ethanol; and AC2 (type 2), an isoform that is homologous to AC7 but less responsive to ethanol. RESULTS: We identified 2 discrete regions of the AC molecule that are important for the enhancement of AC activity by ethanol. The first is the N-terminal 28-amino-acid (aa) region of the C(1a) domain. The second is the C-terminal region ( approximately 140 aa) of the AC molecule. Sequence differences in the N-terminal tail, 2 putative transmembrane domains, and the C(1b) domain are not important for ethanol's effect. CONCLUSIONS: The current study with mammalian ACs provides a new class of alcohol-responsive protein and possibly a new mechanism of alcohol action on cellular function. The identification of ethanol responsive domains will facilitate the elucidation of the mechanisms by which ethanol enhances the activity of AC.

Ethanol-induced phosphorylation and potentiation of the activity of type 7 adenylyl cyclase. Involvement of protein kinase C delta.

Ethanol can enhance G(salpha)-stimulated adenylyl cyclase (AC) activity. Of the nine isoforms of AC, type 7 (AC7) is the most sensitive to ethanol. The potentiation of AC7 by ethanol is dependent on protein kinase C (PKC). We designed studies to determine which PKC isotype(s) are involved in the potentiation of Galpha(s)-activated AC7 activity by ethanol and to investigate the direct phosphorylation of AC7 by PKC. AC7 was phosphorylated in vitro by the catalytic subunits of PKCs. The addition of ethanol to AC7-transfected HEK 293 cells increased the endogenous phosphorylation of AC7, as indicated by a decreased "back-phosphorylation" of AC7 by PKC in vitro. The potentiation of Galpha(s)-stimulated AC7 activity by either phorbol 12,13-dibutyrate or ethanol, in HEL cells endogenously expressing AC7, was not through the Ca(2+)-sensitive conventional PKCs. However, the potentiation of AC7 activity by ethanol or phorbol 12,13-dibutyrate was found to be reduced by the selective inhibitor of PKCdelta (rottlerin), a PKCdelta-specific inhibitory peptide (deltaV1-1), and the expression of the dominant negative form of PKCdelta. Immunoprecipitation data indicated that PKCdelta could bind and directly phosphorylate AC7. The results indicate that the potentiation of AC7 activity by ethanol involves phosphorylation of AC7 that is mediated by PKCdelta.