scholarly journals Lens Connexin Channels Show Differential Permeability to Signaling Molecules

2020 ◽  
Vol 21 (18) ◽  
pp. 6943
Author(s):  
Peter R. Brink ◽  
Virginijus Valiunas ◽  
Thomas W. White
Keyword(s):  
Gap Junction ◽  
Inositol Phosphates ◽  
Second Messengers ◽  
Cyclic Adenosine Monophosphate ◽  
Transgenic Animals ◽  
Adenosine Monophosphate ◽  
Signaling Molecules ◽  
Gap Junction Channels ◽  
Connexin Genes

Gap junction channels mediate the direct intercellular passage of small ions as well as larger solutes such as second messengers. A family of proteins called connexins make up the subunits of gap junction channels in chordate animals. Each individual connexin forms channels that exhibit distinct permeability to molecules that influence cellular signaling, such as calcium ions, cyclic nucleotides, or inositol phosphates. In this review, we examine the permeability of connexin channels containing Cx43, Cx46, and Cx50 to signaling molecules and attempt to relate the observed differences in permeability to possible in vivo consequences that were revealed by studies of transgenic animals where these connexin genes have been manipulated. Taken together, these data suggest that differences in the permeability of individual connexin channels to larger solutes like 3′,5′-cyclic adenosine monophosphate (cAMP) and inositol 1,4,5-trisphosphate (IP3) could play a role in regulating epithelial cell division, differentiation, and homeostasis in organs like the ocular lens.

scholarly journals Two Drosophila Innexins Are Expressed in Overlapping Domains and Cooperate to Form Gap-Junction Channels

2000 ◽  
Vol 11 (7) ◽  
pp. 2459-2470 ◽  
Author(s):  
Lucy A. Stebbings ◽  
Martin G. Todman ◽  
Pauline Phelan ◽  
Jonathan P. Bacon ◽  
Jane A. Davies
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Ectopic Expression ◽  
In Vivo Studies ◽  
Electrophysiological Properties ◽  
Gap Junction Channels ◽  
Overlapping Domains ◽  
In Vitro Data

Members of the innexin protein family are structural components of invertebrate gap junctions and are analogous to vertebrate connexins. Here we investigate two Drosophila innexin genes,Dm-inx2 and Dm-inx3 and show that they are expressed in overlapping domains throughout embryogenesis, most notably in epidermal cells bordering each segment. We also explore the gap-junction–forming capabilities of the encoded proteins. In pairedXenopus oocytes, the injection of Dm-inx2mRNA results in the formation of voltage-sensitive channels in only ∼ 40% of cell pairs. In contrast, Dm-Inx3 never forms channels. Crucially, when both mRNAs are coexpressed, functional channels are formed reliably, and the electrophysiological properties of these channels distinguish them from those formed by Dm-Inx2 alone. We relate these in vitro data to in vivo studies. Ectopic expression ofDm-inx2 in vivo has limited effects on the viability ofDrosophila, and animals ectopically expressingDm-inx3 are unaffected. However, ectopic expression of both transcripts together severely reduces viability, presumably because of the formation of inappropriate gap junctions. We conclude that Dm-Inx2 and Dm-Inx3, which are expressed in overlapping domains during embryogenesis, can form oligomeric gap-junction channels.

scholarly journals Pharmacological and genetic activation of cAMP synthesis disrupts cholesterol utilization in Mycobacterium tuberculosis

2021 ◽  
Author(s):  
Kaley M. Wilburn ◽  
Christine R. Montague ◽  
Bo Qin ◽  
Ashley K. Woods ◽  
Melissa S. Love ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Adenylyl Cyclase ◽  
Cholesterol Metabolism ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Camp Signaling ◽  
Camp Synthesis ◽  
Pharmacokinetic Properties ◽  
Bacterial Utilization

There is a growing appreciation for the idea that bacterial utilization of host-derived lipids, including cholesterol, supports Mycobacterium tuberculosis (Mtb) pathogenesis. This has generated interest in identifying novel antibiotics that can disrupt cholesterol utilization by Mtb in vivo. Here we identify a novel small molecule agonist (V-59) of the Mtb adenylyl cyclase Rv1625c, which stimulates 3’, 5’-cyclic adenosine monophosphate (cAMP) synthesis and inhibits cholesterol utilization by Mtb. Similarly, using a complementary genetic approach that induces bacterial cAMP synthesis independent of Rv1625c, we demonstrate that inducing cAMP synthesis is sufficient to inhibit cholesterol utilization in Mtb. Although the physiological roles of individual adenylyl cyclase enzymes in Mtb are largely unknown, here we demonstrate that the transmembrane region of Rv1625c is required for cholesterol metabolism. Finally, in this work the pharmacokinetic properties of Rv1625c agonists are optimized, producing an orally-available Rv1625c agonist that impairs Mtb pathogenesis in infected mice. Collectively, this work demonstrates a novel role for Rv1625c and cAMP signaling in controlling cholesterol metabolism in Mtb and establishes that cAMP signaling can be pharmacologically manipulated for the development of new antibiotic strategies.

scholarly journals Mel1c Mediated Monochromatic Light-Stimulated IGF-I Synthesis through the Intracellular Gαq/PKC/ERK Signaling Pathway

2019 ◽  
Vol 20 (7) ◽  
pp. 1682
Author(s):  
Shujie Ning ◽  
Zixu Wang ◽  
Jing Cao ◽  
Yulan Dong ◽  
Yaoxing Chen
Keyword(s):  
Signaling Pathway ◽  
Intracellular Signaling ◽  
Monochromatic Light ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Erk Signaling ◽  
Sham Operation ◽  
Intracellular Signaling Pathway ◽  
Igf I

Previous studies have demonstrated that monochromatic light affects plasma melatonin (MEL) levels, which in turn regulates hepatic insulin-like growth factor I (IGF-I) secretion via the Mel1c receptor. However, the intracellular signaling pathway initiated by Mel1c remains unclear. In this study, newly hatched broilers, including intact, sham operation, and pinealectomy groups, were exposed to either white (WL), red (RL), green (GL), or blue (BL) light for 14 days. Experiments in vivo showed that GL significantly promoted plasma MEL formation, which was accompanied by an increase in the MEL receptor, Mel1c, as well as phosphorylated extracellular regulated protein kinases (p-ERK1/2), and IGF-I expression in the liver, compared to the other light-treated groups. In contrast, this GL stimulation was attenuated by pinealectomy. Exogenous MEL elevated the hepatocellular IGF-I level, which is consistent with increases in cyclic adenosine monophosphate (cAMP), Gαq, phosphorylated protein kinase C (p-PKC), and p-ERK1/2 expression. However, the Mel1c selective antagonist prazosin suppressed the MEL-induced expression of IGF-I, Gαq, p-PKC, and p-ERK1/2, while the cAMP concentration was barely affected. In addition, pretreatment with Ym254890 (a Gαq inhibitor), Go9863 (a PKC inhibitor), and PD98059 (an ERK1/2 inhibitor) markedly attenuated MEL-stimulated IGF-I expression and p-ERK1/2 activity. These results indicate that Mel1c mediates monochromatic GL-stimulated IGF-I synthesis through intracellular Gαq/PKC/ERK signaling.

239 USE OF CYCLIC ADENOSINE MONOPHOSPHATE MODULATORS IN IN VITRO PRODUCTION OF BOVINE EMBRYOS

2015 ◽  
Vol 27 (1) ◽  
pp. 209
Author(s):  
T. Fanti ◽  
N. M. Ortega ◽  
R. Garaguso ◽  
M. J. Franco ◽  
C. Herrera ◽  
...  
Keyword(s):  
Phosphodiesterase Inhibitor ◽  
Basal Medium ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Control Group ◽  
Hatching Rate ◽  
Bovine Embryos ◽  
Oocyte Competence

In vitro embryo production systems (IVP) try to emulate and enhance molecular events that occur in in vivo reproductive systems in order to increase, not only the number of embryos generated, but also their quality. Despite advances, IVP processes are still inefficient compared with in vivo systems. Several studies have attributed this deficiency to a lack of oocyte competence due to spontaneous premature resumption of meiotic maturation in the oocyte following the removal from its follicular environment. Therefore, our objective was to increase oocyte competence avoiding premature resumption of meiosis by using cyclic adenosine monophosphate modulators. Cumulus-oocyte complexes (COC) were obtained from ovaries of slaughterhouses, washed, and randomly allocated in 2 culture systems. Oocytes in the control group (IVM) were cultured for a period of 24 h in basal medium TCM-199 with EGF (1 µg mL–1) supplemented with rhFSH (25 mIU mL–1). Oocytes in the biphasic in vitro maturation (b-IVM) group were cultured for 2 h in a basal medium supplemented with a phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX, 500 µM), and an activator of adenylate cyclase (forskolin, 100 µM). Subsequently, COC were washed and cultured in basal medium supplemented with cilostamide (20 µM) and rhFSH (25 mIU mL–1) for 24 h. Maturation rates were analysed and IVF was performed with a dose of 1 × 106 sperm cells mL–1 in IVF-SOF medium. The presumptive zygotes were cultured in continuous-single-culture medium (Irvine) supplemented with 8 mg mL–1 of BSA until they reached the blastocyst stage. No significant differences in maturation, cleavage, and cryotolerance were observed between b-IVM and IVM groups (P > 0.05; Table 1). This study showed that b-IVM produced a significant increase in IVP compared with the control (IVM) at Days 7 and 8 (P < 0.01). Blastocyst hatching rate was significant (P < 0.05) for both treatment and day of analysis. The b-IVM group yielded an increase of 10 and 7.5% at Days 7 and 8, respectively, of IVP. The biphasic maturation showed an improvement in quality regarding the control group, in the timing analysis of production, and hatching percentages, and these results show that the use of cyclic adenosine monophosphate modulators in the oocyte maturation process enhances oocyte competence, which is reflected in increased productivity and embryo quality. We propose this treatment as an alternative to the standard protocols currently used in IVP of bovine embryos. Table 1.Effect of treatment on maturation, cleavage, and cryotolerance

159 EFFECT OF 3-ISOBUTYL-1-METHYLXANTHINE (IBMX) ON THE GERMINAL VESICLE STAGE OF PORCINE OOCYTES DURING OOCYTE COLLECTION IN VITRO

2014 ◽  
Vol 26 (1) ◽  
pp. 193
Author(s):  
R. Appeltant ◽  
J. Beek ◽  
D. Maes ◽  
A. Van Soom
Keyword(s):  
Germinal Vesicle ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Cumulus Cells ◽  
Guanosine Monophosphate ◽  
Developmental Competence ◽  
Meiotic Arrest ◽  
Oocyte Collection

When using modern maturation conditions for in vitro maturation, pig oocytes yield ~20% blastocysts only. One problem is that cumulus cells, which are normally connected with the immature oocyte by cellular projections penetrating through the zona pellucida and with the oolemma via gap junctions, are prematurely losing these connections after the cumulus–oocyte complex is removed from the follicle. The oocyte possesses a type 3 phosphodiesterase, which degrades 3′,5′-cyclic adenosine monophosphate (cAMP), and this activity is inhibited by supply of 3′,5′-cyclic guanosine monophosphate (cGMP) to the oocyte via the cumulus cells. Consequently, cAMP levels, which are typically high during early stages of oocyte maturation in vivo, decrease, leading to spontaneous nuclear maturation and oocytes of low developmental competence. Therefore, the maintenance of these cumulus-oocyte connections is important to keep cAMP high and the oocyte under meiotic arrest. One way to prevent this drop in cAMP is using N6, 2′-o-dibutyryladenosine 3′,5′-cyclic monophosphate sodium (dbcAMP) that causes an arrest at germinal vesicle (GV) stage II (Funahashi et al. 1997 Biol. Reprod. 57, 49–53). Another option is collecting the oocytes in a medium containing the phoshodiesterase inhibitor, IBMX. The present study investigated the influence of IBMX on the progression of the GV of the oocyte after collection, just before the start of the maturation procedure. The GV stage was defined according to Sun et al. (2004 Mol. Reprod. Dev. 69, 228–234). In parallel with the findings on dbcAMP, we hypothesised an arrest at GV II by the presence of IBMX during collection. One group of oocytes were collected in HEPES-buffered TALP without IBMX (n = 375) and another group in the same medium containing 0.5 mM IBMX (n = 586). An average incubation time of 140 min was applied in both groups, and 3 replicates were performed. The proportions of oocytes before or at GV II and beyond GV II were compared in both groups using logistic regression analysis. The proportion of oocytes was included as dependent variable and group (IBMX addition or not) as independent variable. Replicate was also included in the model. The proportion of oocytes before or at GV II was not statistically significant between the group without and the group with IBMX (59.2 v. 58.7% respectively; P > 0.05). In conclusion, the use of IBMX during oocyte collection did not influence the state of the germinal vesicle of the oocyte during collection, indicating that IBMX did not cause a meiotic arrest in the oocytes during collecting in vitro.

Secondary Messenger Systems in PTSD

2016 ◽  
Author(s):  
Ulrike Schmidt
Keyword(s):  
Second Messengers ◽  
Cyclic Adenosine Monophosphate ◽  
Second Messenger ◽  
Adenosine Monophosphate ◽  
Cyclic Guanosine Monophosphate ◽  
Brain Regions ◽  
Guanosine Monophosphate ◽  
Intracellular Camp ◽  
Post Traumatic Stress ◽  
Secondary Messenger

Second messengers such as cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), inositoltriphosphate, and diacylglycerol (DAG) are a prerequisite for the signal transduction of extracellular receptors. The latter are central for cellular function and thus are implicated in the pathobiology of a variety of disorders, such as schizophrenia, bipolar disorder, major depression, and post-traumatic stress disorder (PTSD). This chapter focuses on the involvement of second messenger molecules and their regulators as direct targets in human and animal PTSD and aims to stimulate the underdeveloped research in this field. The synthesis of literature reveals that second messengers clearly play a central role in PTSD-associated brain regions and processes. In particular, pituitary adenylate cyclase-activating polypeptide (PACAP), an important regulator of intracellular cAMP levels, as well as protein kinase c, the major target of DAG, belong to the hitherto most promising PTSD candidate molecules directly involved in second messenger signaling.

Adenylate-cyclase Activity in Obsessive-compulsive Patients

2017 ◽  
Vol 41 (S1) ◽  
pp. S641-S642
Author(s):  
D. Marazziti ◽  
S. Baroni ◽  
F. Mucci ◽  
L. Palego ◽  
A. Piccinni
Keyword(s):  
Adenylate Cyclase ◽  
Second Messengers ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Obsessive Compulsive ◽  
Biological Assays ◽  
Compulsive Disorder ◽  
Healthy Control ◽  
Competing Interest

IntroductionA possible role of second messengers, such as cyclic adenosine monophosphate (cAMP) signalling, in the development of obsessive-compulsive disorder (OCD) has been recently postulated.AimsThe aim of the present study was to explore and to compare the adenylate cyclase (AC) activity in both basal conditions and after the stimulation by isoprenaline (ISO) in platelets of OCD patients and healthy control subjects. The AC activity was measured both in the absence and in the presence of α- and β- adrenoreceptor antagonists.Materials and methodsForty patients were included in the study and compared with healthy volunteers. Biological assays were carried out with a method developed by us.ResultsThe basal AC activity was similar in both groups. The addition of 10 μM ISO enhanced significantly (P < .05) platelet basal AC in both groups. A stimulatory response following ISO in all subjects even without α-antagonists was also observed.DiscussionNo difference in the basal AC activity in platelet membranes of healthy subjects and OCD patients was found. Our findings showed that there is an inhibitory component of ISO effect on platelet AC, due to the agonist interaction with α2 receptors, at its higher concentrations (>1 μM), as well as a condition of supersensitive β-receptors. Our study suggests the presence of cathecolamine system disturbances in OCD.Disclosure of interestThe authors have not supplied their declaration of competing interest.

scholarly journals A conserved CaM- and radial spoke–associated complex mediates regulation of flagellar dynein activity

2007 ◽  
Vol 179 (3) ◽  
pp. 515-526 ◽  
Author(s):  
Erin E. Dymek ◽  
Elizabeth F. Smith
Keyword(s):  
Mass Spectrometry ◽  
Sequence Similarity ◽  
Second Messengers ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Calcium Sensor ◽  
Wild Type ◽  
Radial Spoke ◽  
Radial Spokes ◽  
Dynein Arms

For virtually all cilia and eukaryotic flagella, the second messengers calcium and cyclic adenosine monophosphate are implicated in modulating dynein- driven microtubule sliding to regulate beating. Calmodulin (CaM) localizes to the axoneme and is a key calcium sensor involved in regulating motility. Using immunoprecipitation and mass spectrometry, we identify members of a CaM-containing complex that are involved in regulating dynein activity. This complex includes flagellar-associated protein 91 (FAP91), which shares considerable sequence similarity to AAT-1, a protein originally identified in testis as an A-kinase anchor protein (AKAP)– binding protein. FAP91 directly interacts with radial spoke protein 3 (an AKAP), which is located at the base of the spoke. In a microtubule sliding assay, the addition of antibodies generated against FAP91 to mutant axonemes with reduced dynein activity restores dynein activity to wild-type levels. These combined results indicate that the CaM- and spoke-associated complex mediates regulatory signals between the radial spokes and dynein arms.

scholarly journals Gap junction permeability: selectivity for anionic and cationic probes

2011 ◽  
Vol 300 (3) ◽  
pp. C600-C609 ◽  
Author(s):  
G. Kanaporis ◽  
P. R. Brink ◽  
V. Valiunas
Keyword(s):  
Gap Junction ◽  
Lucifer Yellow ◽  
Second Messengers ◽  
Small Interfering Rnas ◽  
Weak Charge ◽  
Axial Diameter ◽  
Gap Junction Channels ◽  
Charge Selectivity ◽  
Specific Permeability ◽  
A Charge

Gap junction channels formed by different connexins exhibit specific permeability to a variety of larger solutes including second messengers, polypeptides, and small interfering RNAs. Here, we report the permeability of homotypic connexin26 (Cx26), Cx40, Cx43, and Cx45 gap junction channels stably expressed in HeLa cells to solutes with different size and net charge. Channel permeability was determined using simultaneous measurements of junctional conductance and the cell-cell flux of a fluorescent probe. All four connexins allowed passage of both cationic and anionic probes, but the transfer rates were connexin dependent. The negatively charged probes [Lucifer yellow (LY; median axial diameter 9.9 Å, charge −2), carboxyfluorescein (CF; 8.2 Å; −2), and Alexa Fluor350 (AF350, 5.4 Å; −1)] exhibited the following permeability order: Cx43 > Cx45 > Cx26 > Cx40. In contrast, for the positively charged species permeability, the orders were as follows: Cx26 ≈ Cx43 ≈ Cx40 ≈ Cx45 for N, N, N-trimethyl-2-[methyl-(7-nitro-2,1,3-benzoxadiol-4-yl) amino] ethanaminium (NBD-m-TMA; 5.5 Å, +1) and Cx26 ≥ Cx43 ≈ Cx40 > Cx45 for ethidium bromide (10.3 Å, +1). Comparison of probe permeability relative to K+ revealed that Cx43 and Cx45 exhibited similar permeability for NBD-m-TMA and AF350, indicating weak charge selectivity. However, lesser transfer of CF and LY through Cx45 relative to Cx43 channels suggests stronger size-dependent discrimination of solute. The permeability of NBD-m-TMA for Cx40 and Cx26 channels was approximately three times higher than to anionic AF350 despite the fact that both have similar minor diameters, suggesting charge selectivity. In conclusion, these results confirm that channels formed from individual connexins can discriminate for solutes based on size and charge, suggesting that channel selectivity may be a key factor in cell signaling.

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