cAMP Analog Research Articles

CAMP/PKA-mediated in vitro angiogenesis: A novel interplay between PKA, RhoA/ROCK, and VEGFR2 signalling

Abstract Background and Aims Others and we have previously demonstrated that cAMP/PKA signalling stabilise endothelial cell (EC) barrier function via modulating the activities of Rho GTPases, mainly RhoA and Rac1. In the present study we analysed whether cAMP/PKA also modulates EC angiogenesis capacity and whether Rho GTPases are involved in this function. Methods The study was carried out on cultured human umbilical vein ECs (HUVECs). RhoA and Rac1 activities were genetically manipulated by lentiviral-mediated over expression of dominant negative (DN) and constitutive active (CA) forms of RhoA and Rac1. Pharmacologically, specific activation and inhibition of PKA was achieved by using cAMP analogue N6-Benzoyl-cAMP (5 mM) and PKI (1 µM), respectively. Pharmacologically, Rac1 was inhibited using NSC 23766 (10 µM) and RhoA was activated using Rho activator I (Cytoskeleton Inc.). In vitro angiogenesis was analysed by 3-D spheroid assay and matrigel tube formation assay. Results Pharmacologically, specific activation of PKA induced EC migration (Wound healing assay), tube formation, and 3-D spheroids and potentiated the VEGF-induced in vitro angiogenesis. These pro-angiogenesis activities were abrogated by highly specific cell-permeable peptide inhibitor (PKI) of PKA. Activation of cAMP/PKA-signalling resulted in an increased VEGFR2 and Akt phosphorylation, increased Rac1 activity, and a strong inhibition of RhoA/Rock pathway. Pharmacological activation of RhoA but not inhibition of Rac1 abrogated PKA-induced VEGFR2 phosphorylation, tube formation and 3-D spheroids. Lentiviral mediated over-expression of constitutive active form of RhoA but not Rac1 abrogated PKA-induced VEGFR2 and Akt phosphorylation and angiogenesis. Moreover, pharmacological inhibition of Akt also abrogated PKA-mediated VEGFR2 phosphorylation and angiogenesis. Likewise, over expression of either dominant negative RhoA or pharmacological inhibitors of Rho kinase (ROCK) promoted basal and potentiated PKA-mediated VEGFR2 and Akt phosphorylation and angiogenesis. Conclusion We describe for the first time a novel interplay between PKA, RhoA/ROCK, Akt, and VEGFR2 signalling and angiogenesis. Inhibition of RhoA/ROCK plays an important role in mediating PKA-induced angiogenesis and activation of RhoA/ROCK signalling may offer an important target to intervene therapeutic angiogenesis.

THE effect of P2X7 receptor activation on functional responses of human left internal mammary artery.

The Purinoreceptor 7 (P2X7R) has become a promising drug target in many cardiovascular diseases, including coronary artery disease, since prolonged activation of P2X7R could promote vascular dysfunction, atherosclerosis, and thrombosis. Thus, we aimed to study the effects of P2X7R activation on vascular relaxation responses of the human left internal mammary artery (LIMA). Sections of redundant human LIMA were cut into 3-mm wide rings,, suspended in 20-mL organ baths containing physiologic salt solution, and attached to an isometric force transducer connected to a computer-based data acquisition system. Long-term (60min) incubation with specific P2X7R agonist Bz-ATP caused significant reductions in relaxation responses of LIMA to ATP and acetylcholine, which were reversed by selective P2X7R antagonists Brilliant Blue G or AZ11645373, whereas there were no changes in relaxation responses to endothelium-independent vasodilators isoprenaline, cAMP analog 8-Br-cAMP, and nitric oxide donor sodium nitroprusside. The impairment in relaxant responses of LIMA to endothelium-dependent vasodilators following activation of P2X7R for the long-term may contribute to postoperative LIMA vasospasm and hypertension. Modulation of P2X7R activity with selective agents may represent a new potential therapeutic approach in patients undergoing coronary artery bypass grafting surgery.

Cyclic Adenosine Monophosphate Signaling in Chronic Kidney Disease: Molecular Targets and Therapeutic Potentials.

Chronic kidney disease (CKD) is characterized by a steady decline in kidney function and affects roughly 10% of the world's population. This review focuses on the critical function of cyclic adenosine monophosphate (cAMP) signaling in CKD, specifically how it influences both protective and pathogenic processes in the kidney. cAMP, a critical secondary messenger, controls a variety of cellular functions, including transcription, metabolism, mitochondrial homeostasis, cell proliferation, and apoptosis. Its compartmentalization inside cellular microdomains ensures accurate signaling. In kidney physiology, cAMP is required for hormone-regulated activities, particularly in the collecting duct, where it promotes water reabsorption through vasopressin signaling. Several illnesses, including Fabry disease, renal cell carcinoma, nephrogenic diabetes insipidus, Bartter syndrome, Liddle syndrome, diabetic nephropathy, autosomal dominant polycystic kidney disease, and renal tubular acidosis, have been linked to dysfunction in the cAMP system. Both cAMP analogs and phosphodiesterase inhibitors have the potential to improve kidney function and reduce kidney damage. Future research should focus on developing targeted PDE inhibitors for the treatment of CKD.

Extracellular cAMP elicits contraction of rat vas deferens: Involvement of ecto-5′-nucleotidase and adenosine A1 receptors

AimsIt is well established that intracellular cAMP contributes to the relaxation of vas deferens smooth muscle. In many tissues, intracellular cAMP is actively transported to the extracellular space, where it exerts regulatory functions, via its metabolite adenosine. These actions take place through the cAMP conversion to adenosine by ectoenzymes, a process called “extracellular cAMP-adenosine pathway”. Herein, we investigated whether, in addition to ATP, extracellular cAMP might be an alternative source of adenosine, influencing the contraction of vas deferens smooth muscle. Main methodsThe effects of cAMP, 8-Br-cAMP and adenosine were analyzed in the isometric contractions of rat vas deferens. cAMP efflux was analyzed by measuring extracellular cAMP levels after exposure of vas deferens segments to isoproterenol and forskolin in the presence or absence of MK-571, an inhibitor of MRP/ABCC transporters. Key findingsWhile 8-Br-cAMP, a cell-permeable cAMP analog, induced relaxation of KCl-precontracted vas deferens, the non-permeant cAMP increased the KCl-induced contractile response, which was mimicked by adenosine, but prevented by inhibitors of ecto-5′-nucleotidase or A1 receptors. Our results also showed that isoproterenol and forskolin increases cAMP efflux via an MRP/ABCC transporter-dependent mechanism, since it is inhibited by MK-571. SignificanceOur data show that activation of β-adrenoceptors and adenylyl cyclase increases cAMP efflux from vas deferens tissue, which modulates the vas deferens contractile response via activation of adenosine A1 receptors. Assuming that inhibition of vas deferens contractility has been proposed as a strategy for male contraception, the extracellular cAMP-adenosine pathway emerges as a potential pharmacological target that should be considered in studies of male fertility.

The Role of Aquaporin 4 in Lacrimal Gland Ductal Fluid Secretion in Mice.

Earlier reports highlighted the predominant presence of aquaporin 4 (AQP4) in the duct cells of rabbit lacrimal glands (LGs). Whereas significant alterations in AQP4 mRNA levels have been observed in experimental dry eye and during pregnancy, the impact of AQP4 in LG ductal fluid production remains unclear. In our recent work, the role of AQP4 in LG ductal fluid secretion was investigated utilizing wild type (WT) and AQP4 knock out (KO) mice. Tear production was assessed in both WT and KO animals. Immunostaining was used to identify AQP4 protein. Duct segments were harvested from LGs of WT and KO mice. Fluid secretion and filtration permeability (Pf) were quantified using video-microscopy. Ductal tear production, elicited by a cell-permeable cAMP analogue (8-bromo cAMP), carbachol, vasoactive intestinal peptide (VIP), and phenylephrine (PHE), were assessed in both WT and KO ducts. A higher expression of AQP4 protein was noted in the duct cells from WT mice when compared to acinar cells. Pf did not show notable alterations between WT and AQP4 KO ducts. Carbachol elicited comparable secretory responses in ducts from both WT and KO animals. However, 8-bromo cAMP, VIP, and PHE stimulation resulted in decreased secretion in ducts from AQP4 KO LGs. Our findings underscore the functional relevance of AQP4 in the fluid production of mouse LG ducts. AQP4 seems to play different roles in fluid secretions elicited by different secretagogues. Specifically, cAMP-mediated, and adrenergic agonist-related secretions were reduced in AQP4 KO ducts.

Internalized β2-Adrenergic Receptors Inhibit Subcellular Phospholipase C-Dependent Cardiac Hypertrophic Signaling

Chronic overstimulation of Gs-coupled b-adrenergic receptor (b-AR) signaling by catecholamines, induces pathological cardiac hypertrophy and ultimately heart failure. b1-ARs and b2-ARs are the two major subtypes of b-ARs present in the human heart and mediate the activity of sympathetic nervous system. It is known that b1-ARs and b2-ARs elicit significantly different or even opposite effects on cardiac functions such as contractility and heart failure, though they share overall sequence and structural homology, and both stimulate cAMP production. The molecular mechanisms underlying these distinct biological effects have not been fully elucidated. Previously, our laboratory demonstrated that a cAMP/EPAC/phospholipase Ce (PLCe)-mediated signaling pathway at the Golgi apparatus is important for regulation of cardiac hypertrophy. We also showed that this pathway is selectively stimulated by activation of b1-ARs localized at Golgi apparatus but not those at the plasma membrane (PM). Access to intracellular b1-ARs by catecholamines was mediated by the organic cation transporters (OCTs), and blockade of OCT3 inhibited catecholamine stimulated cardiomyocyte hypertrophy. In my current study using adult ventricular cardiac myocytes, I found that the activation of endosomal b2-ARs opposes stimulation of the prohypertrophic EPAC/PLCe pathway at the Golgi apparatus by either Angiotensin II (ATII), or the activation of Golgi localized b1-ARs by Dobutamine (Dob). b2-AR-dependent inhibition is at the level of PLCe since activation of b2-AR with salmeterol (Sal) blocks direct activation of EPAC/PLCe by the EPAC selective cAMP analog 8-(4-chlorophenylthio)-2′- O-methyl-cAMP (CPTOMe)-AM, and blocks PLCe activation by ATII which activates PLCe through a pathway that does not rely on EPAC. Blockade of internalization of b2-ARs with Dyngo-4A abolished this inhibitory signaling. Downstream of b2-ARs, I found that b2-AR-dependent PLCe inhibition is not mediated by PKA. Rather, blockade of Gi with PTX, or Gbg with gallein, inhibited the ability of b2-ARs to block Golgi PLCe activity. Additionally, inhibition of ERK activation by PD0325901 abolished b2-AR-mediated inhibition of Golgi PLCe activity. This supports a model where b2ARs internalized from the plasma membrane activate Gi releasing Gbg subunits leading to ERK activation and inhibition of PI hydrolysis at the Golgi apparatus, thereby inhibiting hypertrophic signaling by PLCe. In addition, we also showed PKD and HDAC phosphorylation downstream of PLCe signaling are significantly attenuated by b2ARs activation in mouse hearts. Consistent with the results from mice, b2ARs activation by Sal blunted cellular hypertrophy induced by b1-ARs activation by Dob in vitro measured by cell area and the expression of the hypertrophic marker, atrial natriuretic factor (ANF). Importantly, inhibition endosomal Gbg using a highly selective Gbg inhibitor (GRK2CT) fused to an endosomal targeting sequence (FYVE domain) prevent Sal mediated protective signaling in hypertrophy measurements in vitro. This indicates the inhibitory signaling downstream of b2ARs is indeed driven by endosomal Gbg. This study reveals a novel potential mechanism for b2-AR antagonism of the Epac/PLCe pathway that may contribute to the known protective effects of b2AR signaling on the development of heart failure. Elucidation of a mechanism for the anti-hypertrophic versus hypertrophic signaling balance from b1-ARs and b2-ARs gives critical insights into the development of new strategies for treatment of heart failure by targeting bAR subtypes. This work is supported by grant R35 GM127303-01 (A.V.S.). Preprint: https://www.biorxiv.org/content/10.1101/2023.06.07.544153v1. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Activating astrocytic α2A adrenoceptors in hippocampus reduces glutamate toxicity to attenuate sepsis-associated encephalopathy in mice

BackgroundGlutamate metabolism disorder is an important mechanism of sepsis-associated encephalopathy (SAE). Astrocytes regulate glutamate metabolism. In septic mice, α2A adrenoceptor (α2A-AR) activation in the central nervous system provides neuroprotection. α2A-ARs are expressed abundantly in hippocampal astrocytes. This study was performed to determine whether hippocampal astrocytic α2A-AR activation confers neuroprotection against SAE and whether this protective effect is astrocyte specific and achieved by the modulation of glutamate metabolism. MethodsMale C57BL/6 mice with and without α2A-AR knockdown were subjected to cecal ligation and puncture (CLP). They were treated with intrahippocampal guanfacine (an α2A-AR agonist) or intraperitoneal dexmedetomidine in the presence or absence of dihydrokainic acid [DHK; a glutamate transporter 1 (GLT-1) antagonist] and/or UCPH-101 [a glutamate/aspartate transporter (GLAST) antagonist]. Hippocampal tissue was collected for the measurement of astrocyte reactivity, GLT-1 and GLAST expression, and glutamate receptor subunit 2B (GluN2B) phosphorylation. In vivo real-time extracellular glutamate concentrations in the hippocampus were measured by ultra-performance liquid chromatography tandem mass spectrometry combined with microdialysis, and in vivo real-time hippocampal glutamatergic neuron excitability was assessed by calcium imaging. The mice were subjected to the Barnes maze and fear conditioning tests to assess their learning and memory. Golgi staining was performed to assess changes in the hippocampal synaptic structure. In vitro, primary astrocytes with and without α2A-AR knockdown were stimulated with lipopolysaccharide (LPS) and treated with guanfacine or dexmedetomidine in the presence or absence of 8-bromo- cyclic adenosine monophosphate (8-Br-cAMP, a cAMP analog). LPS-treated primary and BV2 microglia were also treated with guanfacine or dexmedetomidine. Astrocyte reactivity, PKA catalytic subunit, GLT-1 an GLAST expression were determined in primary astrocytes. Interleukin-1β, interleukin-6 and tumor necrosis factor-alpha in the medium of microglia culture were measured. ResultsCLP induced synaptic injury, impaired neurocognitive function, increased astrocyte reactivity and reduced GLT-1 and GLAST expression in the hippocampus of mice. The extracellular glutamate concentration, phosphorylation of GluN2B at Tyr-1472 and glutamatergic neuron excitability in the hippocampus were increased in the hippocampus of septic mice. Intraperitoneal dexmedetomidine or intrahippocampal guanfacine administration attenuated these effects. Hippocampal astrocytes expressed abundant α2A-ARs; expression was also detected in neurons but not microglia. Specific knockdown of α2A-ARs in hippocampal astrocytes and simultaneous intrahippocampal DHK and UCPH-101 administration blocked the neuroprotective effects of dexmedetomidine and guanfacine. Intrahippocampal administration of DHK or UCPH-101 alone had no such effect. In vitro, guanfacine or dexmedetomidine inhibited astrocyte reactivity, reduced PKA catalytic subunit expression, and increased GLT-1 and GLAST expression in primary astrocytes but not in primary astrocytes that received α2A-AR knockdown or were treated with 8-Br-cAMP. Guanfacine or dexmedetomidine inhibited microglial reactivity in BV2 but not primary microglia. ConclusionsOur results suggest that neurocognitive protection against SAE after hippocampal α2A-AR activation is astrocyte specific. This protection may involve the inhibition of astrocyte reactivity and alleviation of glutamate neurotoxicity, thereby reducing synaptic injury. The cAMP/protein kinase A (PKA) signaling pathway is a potential cellular mechanism by which activating α2A-AR modulates astrocytic function.

Pharmacological Evidence That Dictyostelium Differentiation-Inducing Factor 1 Promotes Glucose Uptake Partly via an Increase in Intracellular cAMP Content in Mouse 3T3-L1 Cells

Differentiation-inducing factor 1 (DIF-1) isolated from the cellular slime mold Dictyostelium discoideum can inhibit mammalian calmodulin-dependent cAMP/cGMP phosphodiesterase (PDE1) in vitro. DIF-1 also promotes glucose uptake, at least in part, via a mitochondria- and AMPK-dependent pathway in mouse 3T3-L1 fibroblast cells, but the mechanism underlying this effect has not been fully elucidated. In this study, we investigated the effects of DIF-1 on intracellular cAMP and cGMP levels, as well as the effects that DIF-1 and several compounds that increase cAMP and cGMP levels have on glucose uptake in confluent 3T3-L1 cells. DIF-1 at 20 μM (a concentration that promotes glucose uptake) increased the level of intracellular cAMP by about 20% but did not affect the level of intracellular cGMP. Neither the PDE1 inhibitor 8-methoxymethyl-3-isobutyl-1-methylxanthine at 10–200 μM nor the broad-range PDE inhibitor 3-isobutyl-1-methylxanthine at 40–400 μM had any marked effects on glucose uptake. The membrane-permeable cAMP analog 8-bromo-cAMP at 200–1000 μM significantly promoted glucose uptake (by 20–25%), whereas the membrane-permeable cGMP analog 8-bromo-cGMP at 3–100 μM did not affect glucose uptake. The adenylate cyclase activator forskolin at 1–10 μM promoted glucose uptake by 20–30%. Thus, DIF-1 may promote glucose uptake by 3T3-L1 cells, at least in part, via an increase in intracellular cAMP level.

N-acetyl cysteine turns EPAC activators into potent killers of acute lymphoblastic leukemia cells

Today, the majority of pediatric B cell precursor acute lymphoblastic leukemia (BCP-ALL, hereafter ALL) patients survive their disease, but many of the survivors suffer from life-limiting late effects of the treatment. ALL develops in the bone marrow, where the cells are exposed to cAMP-generating prostaglandin E2. We have previously identified the cAMP signaling pathway as a putative target for improved efficacy of ALL treatment, based on the ability of cAMP signaling to reduce apoptosis induced by DNA damaging agents. In the present study, we have identified the antioxidant N-acetyl cysteine (NAC) as a powerful modifier of critical events downstream of the cell permeable cAMP analogue 8-(4-chlorophenylthio) adenosine-3', 5'- cyclic monophosphate (8-CPT). Accordingly, we found NAC to turn 8-CPT into a potent killer of ALL cells in vitro both in the presence and absence of DNA damaging treatment. Furthermore, we revealed that NAC in combination with 8-CPT is able to delay the progression of ALL in a xenograft model in NOD-scid IL2Rγnull mice. NAC was shown to rely on the ability of 8-CPT to activate the guanine-nucleotide exchange factor EPAC, and we demonstrated that the ALL cells are killed by apoptosis involving sustained elevated levels of calcium imposed by the combination of the two drugs.. Taken together, we propose that 8-CPT in the presence of NAC might be utilized as a novel strategy for treating pediatric ALL patients, and that this powerful combination might be exploited to enhance the therapeutic index of current ALL targeting therapies.

Role of the cAMP-PKA-NF-κB pathway in Mucin1 over-expression in A549 cells during Respiratory syncytial virus infection

Respiratory syncytial virus (RSV) is the most common pathogen associated with acute lower respiratory tract infections in infants and young children worldwide. RSV commonly presents as bronchiolitis in young children; however, it can sometimes progress to pneumonia, respiratory failure, apnoea and even death. Although mucin1 (MUC1), a type of transmembrane glycoprotein present on airway epithelial surfaces, plays a crucial anti-inflammatory role in airway infections; however, its roles in RSV-associated acute lower respiratory tract infections have rarely been explored. In this study, we first revealed very high MUC1 protein levels in the exacerbation phase in sputum samples from children with RSV bronchiolitis. Because MUC1 is the downstream target of tumour necrosis factor-alpha (TNF-α) in RSV-infected A549 cells, we observed the inhibition of NF-κB activity, main downstream signalling of TNF-α and remarkably reduced levels of MUC1 in RSV-infected and TNF-α treated A549 cells. Furthermore, the cyclic adenosine monophosphate (cAMP) analogue (dbcAMP) downregulated the protein levels of p-IκBα and MUC1 in TNF-α-treated A549 cells. By contrast, a protein kinase A inhibitor (KT5720) up-regulated the levels of those proteins. dbcAMP and KT5720 had the same effects on MUC1 protein levels in RSV-infected A549 cells. In conclusion, we found that the cAMP-PKA-NF-κB pathway may play a role in the regulation of MUC-1 over-expression during RSV infection.

Lipid mediator resolvin D2 inhibits ATP currents in rat primary sensory neurons.

Resolvin D2 (RvD2), an endogenous lipid mediator derived from docosahexaenoic acid, has been demonstrated to have analgesic effects. However, little is known about the mechanism underlying RvD2 in pain relief. Herein, we demonstrate that RvD2 targeted the P2X3 receptor as an analgesic. The electrophysiological activity of P2X3 receptors was suppressed by RvD2 in rat dorsal root ganglia (DRG) neurons. RvD2 pre-application dose-dependently decreased α,β-methylene-ATP (α,β-meATP)-induced inward currents. RvD2 remarkably decreased the maximum response to α,β-meATP, without influencing the affinity of P2X3 receptors. RvD2 also voltage-independently suppressed ATP currents. An antagonist of the G protein receptor 18 (GPR18), O-1918, prevented the RvD2-induced suppression of ATP currents. Additionally, intracellular dialysis of the Gαi/o -protein antagonist pertussis toxin (PTX), the PKA antagonist H89, or the cAMP analog 8-Br-cAMP also blocked the RvD2-induced suppression. Furthermore, α,β-meATP-triggered depolarization of membrane potential along with the action potential bursts in DRG neurons were inhibited by RvD2. Lastly, RvD2 attenuated spontaneous nociceptive behaviors as well as mechanical allodynia produced by α,β-meATP in rats via the activation of the peripheral GPR18. These findings indicated that RvD2 inhibited P2X3 receptors in rat primary sensory neurons through GPR18, PTX-sensitive Gαi/o -proteins, and intracellular cAMP/PKA signaling, revealing a novel mechanism that underlies its analgesic effects by targeting P2X3 receptors.

ATOH8 Expression Is Regulated by BMP2 and Plays a Key Role in Human Endometrial Stromal Cell Decidualization.

During the secretory phase of the menstrual cycle, elongated fibroblast-like mesenchymal cells in the uterine endometrium begin to transdifferentiate into polygonal epithelioid-like (decidual) cells. This decidualization process continues more broadly during early pregnancy, and the resulting decidual tissue supports successful embryo implantation and placental development. This study was carried out to determine if atonal basic helix-loop-helix transcription factor 8 (ATOH8) plays a role in human endometrial stromal fibroblast (ESF) decidualization. ATOH8 messenger RNA and protein expression levels significantly increased in human ESF cells undergoing in vitro decidualization, with the protein primarily localized to the nucleus. When ATOH8 expression was silenced, the ability of the cells to undergo decidualization was significantly diminished. Overexpression of ATOH8 enhanced the expression of many decidualization markers. Silencing the expression of ATOH8 reduced the expression of FZD4, FOXO1, and several known FOXO1-downstream targets during human ESF cell decidualization. Therefore, ATOH8 may be a major upstream regulator of the WNT/FZD-FOXO1 pathway, previously shown to be critical for human endometrial decidualization. Finally, we explored possible regulators of ATOH8 expression during human ESF decidualization. BMP2 significantly enhanced ATOH8 expression when cells were stimulated to undergo decidualization, while an ALK2/3 inhibitor reduced ATOH8 expression. Finally, although the steroids progesterone plus estradiol did not affect ATOH8 expression, the addition of cyclic adenosine monophosphate (cAMP) analogue alone represented the major effect of ATOH8 expression when cells were stimulated to undergo decidualization. Our results suggest that ATOH8 plays a crucial role in human ESF decidualization and that BMP2 plus cAMP are major regulators of ATOH8 expression.

THU633 Atonal BHLH Transcription Factor 8 (ATOH8) Functions In Decidualization

Abstract Disclosure: R. Thapa: None. B. Bany: None. ATOH8 is a member of the vast basic helix-loop-helix (bHLH) protein family that is widely known to play a role in various physiological and pathological processes. However, the functional significance of ATOH8 expression in the endometrium during implantation and in the first trimester of pregnancy in women is still lacking. In the mid to late secretory phase of the menstrual cycle, elongated fibroblast-like mesenchymal cells in the uterine endometrium begin to trans-differentiate into polygonal epithelioid-like (decidual) cells. During pregnancy, this process continues more broadly and supports successful embryo implantation and early placental development. We employed an established in vitro model using uterine fibroblast (HuF) cells derived from the decidua parietalis of term patients to investigate the function of ATOH8 during in vitro decidualization. All experiments were carried out using cells isolated from at least six independent patients (N=6). We measured the expression of classical decidualization marker genes and the characteristic shape change from small fibroblast-like cells to large epitheloid-like decidual cells to assess decidualization. Treating cells with steroids plus a membrane-permeable cAMP analog (EPC) for 48h caused in vitro decidualization of these cells, while treatment with vehicle alone had no effect. We discovered that ATOH8 expression was dramatically upregulated during this decidualization at both the mRNA and protein levels, with the protein primarily localized to the nucleus. The functional role of ATOH8 during decidualization was investigated using lentivirus-mediated shRNA knockdown or overexpression of ATOH8. When ATOH8 expression was silenced, we discovered that the ability of the cells to undergo decidualization was greatly and significantly diminished. On the other hand, overexpression of ATOH8 enhanced some aspects of decidualization. The addition of recombinant BMP2 alone had little effect on ATOH8 expression. However, in the presence of EPC it significantly enhanced ATOH8 expression. This effect of BMP2 on ATOH8 expression was inhibited by preincubating the cells with 500 nM DMH-1 (an ALK2/3 inhibitor) for one hr prior to inducing decidualization. Steroids alone did not affect ATOH8 or BMP2 expression. However, the addition of cAMP alone represented the major effect of EPC on ATOH8 expression while having a slight impact on BMP2 expression. A robust increase in BMP2 expression required the presence of both steroids and cAMP together. Our results indicate that ATOH8 plays a crucial role in human endometrial stromal cell decidualization. Further, our results suggest that ALK2/3 receptors are most likely involved in mediating the effect of BMP2 on increasing ATOH8 expression. Keywords: ATOH8, Decidualization, Embryo Implantation Presentation: Thursday, June 15, 2023

Prohibitin-1 is an ACTH-Regulated Protein in Human and Mouse Adrenocortical Cells and Plays a Role in Corticosteroid Production

Cell-intrinsic early events involved in different trophic hormone-induced steroidogenesis in their respective steroidogenic cell type are very similar. For example, the activation of the cAMP-PKA signaling pathway in response to trophic hormone stimulation and, subsequently, cholesterol transport to the mitochondria to initiate steroidogenesis is common to them. Recently, we have found that an evolutionarily conserved protein, prohibitin-1 (PHB1), is regulated by Luteinizing Hormone (LH) in murine Leydig cells and plays a role in interconnected cell signaling and mitochondrial steps pertaining to testosterone production. Among the primary steroidogenic tissues, PHB1 expression levels are highest in the adrenal cortex (The Human Protein Atlas); however, its regulation and role in adrenocortical cells are virtually unknown. We investigated the regulation and the role of PHB1 in adrenocortical cells in vitro using human HAC15 and mouse Y-1 cell culture models. It was found that Adrenocorticotrophic Hormone (ACTH) stimulation upregulates PHB1 levels in adrenocortical cells in a time-dependent manner. A similar effect on PHB1 levels was also observed in response to dibutyryl-cAMP stimulation, a cell-permeable analogue of cAMP (the second messenger for ACTH action). Moreover, manipulating PHB1 levels in adrenocortical cells affected mitochondria, lysosomes, and lipid droplet characteristics, modulated phospho-PKA and phospho-ERK1/2 levels, and altered corticosteroid production. This finding suggests that ACTH regulates PHB1 in adrenocortical cells and plays a role in corticosteroid production, which was previously unknown.

Epac induces ryanodine receptor-dependent intracellular and inter-organellar calcium mobilization in mpkCCD cells.

Arginine vasopressin (AVP) induces an increase in intracellular Ca2+ concentration ([Ca2+]i) with an oscillatory pattern in isolated perfused kidney inner medullary collecting duct (IMCD). The AVP-induced Ca2+ mobilization in inner medullary collecting ducts is essential for apical exocytosis and is mediated by the exchange protein directly activated by cyclic adenosine monophosphate (Epac). Murine principal kidney cortical collecting duct cells (mpkCCD) is the cell model used for transcriptomic and phosphoproteomic studies of AVP signaling in kidney collecting duct. The present study examined the characteristics of Ca2+ mobilization in mpkCCD cells, and utilized mpkCCD as a model to investigate the Epac-induced intracellular and intra-organellar Ca2+ mobilization. Ca2+ mobilization in cytosol, endoplasmic reticulum lumen, and mitochondrial matrix were monitored with a Ca2+ sensitive fluorescent probe and site-specific Ca2+ sensitive biosensors. Fluorescence images of mpkCCD cells and isolated perfused inner medullary duct were collected with confocal microscopy. Cell permeant ligands of ryanodine receptors (RyRs) and inositol 1,4,5 trisphosphate receptors (IP3Rs) both triggered increase of [Ca2+]i and Ca2+ oscillations in mpkCCD cells as reported previously in IMCD. The cell permeant Epac-specific cAMP analog Me-cAMP/AM also caused a robust Ca2+ mobilization and oscillations in mpkCCD cells. Using biosensors to monitor endoplasmic reticulum (ER) luminal Ca2+ and mitochondrial matrix Ca2+, Me-cAMP/AM not only triggered Ca2+ release from ER into cytoplasm, but also shuttled Ca2+ from ER into mitochondria. The Epac-agonist induced synchronized Ca2+ spikes in cytosol and mitochondrial matrix, with concomitant declines in ER luminal Ca2+. Me-cAMP/AM also effectively triggered store-operated Ca2+ entry (SOCE), suggesting that Epac-agonist is capable of depleting ER Ca2+ stores. These Epac-induced intracellular and inter-organelle Ca2+ signals were mimicked by the RyR agonist 4-CMC, but they were distinctly different from IP3R activation. The present study hence demonstrated that mpkCCD cells retain all reported features of Ca2+ mobilization observed in isolated perfused IMCD. It further revealed information on the dynamics of Epac-induced RyR-dependent Ca2+ signaling and ER-mitochondrial Ca2+ transfer. ER-mitochondrial Ca2+ coupling may play a key role in the regulation of ATP and reactive oxygen species (ROS) production in the mitochondria along the nephron. Our data suggest that mpkCCD cells can serve as a renal cell model to address novel questions of how mitochondrial Ca2+ regulates cytosolic Ca2+ signals, inter-organellar Ca2+ signaling, and renal tubular functions.

Exposure to a phthalate mixture disrupts ovulatory progesterone receptor signaling in human granulosa cells in vitro†.

Exposure to phthalates disrupts ovarian function. However, limited studies have investigated the effects of phthalate mixtures on ovulation, especially in women. Human granulosa cells were used to test the hypothesis that exposure to a phthalate mixture (PHTmix) disrupts progesterone (P4)/progesterone receptor (PGR) signaling, which is a crucial pathway for ovulation. In addition, progestin and cyclic adenosine 3', 5'-monophosphate (cAMP) supplementation were tested as methods to circumvent phthalate toxicity. Granulosa cells from women undergoing in vitro fertilization were acclimated in culture to regain responsiveness to human chorionic gonadotropin (hCG; clinical luteinizing hormone analogue). Granulosa cells were treated with or without hCG, and with or without PHTmix (1-500μg/ml; dimethylsulfoxide = vehicle control) for 0.5-36h. In the supplementation experiments, cells were treated with or without R5020 (stable progestin), and with or without 8-Br-cAMP (stable cAMP analogue). Exposure to hCG + PHTmix decreased P4 levels and mRNA levels of steroidogenic factors when compared to hCG. This was accompanied by decreased mRNA levels of PGR and downstream P4/PGR ovulatory mediators (ADAM metallopeptidase with thrombospondin type 1 motif 1 (ADAMTS1), C-X-C motif chemokine receptor 4 (CXCR4), pentraxin 3 (PTX3), and regulator of G protein signaling 2 (RGS2)) in the hCG + PHTmix groups compared to hCG. Exposure to hCG + PHTmix 500μg/ml decreased cAMP levels and protein kinase A activity compared to hCG. Supplementation with progestin in the hCG + PHTmix 500μg/ml group did not rescue toxicity, while supplementation with cAMP restored PGR levels and downstream P4/PGR mediator levels to hCG levels. These findings suggest that phthalate mixture exposure inhibits P4/PGR signaling in human granulosa cells via decreased steroidogenesis, cAMP levels, and protein kinase A activity. Restored P4/PGR signaling with cAMP supplementation provides a potential cellular target for intervention of phthalate-induced ovulatory dysfunction in women.

Luteal Lipid Droplets: A Novel Platform for Steroid Synthesis.

Progesterone is an essential steroid hormone that is required to initiate and maintain pregnancy in mammals and serves as a metabolic intermediate in the synthesis of endogenously produced steroids, including sex hormones and corticosteroids. Steroidogenic luteal cells of the corpus luteum have the tremendous capacity to synthesize progesterone. These specialized cells are highly enriched with lipid droplets that store lipid substrate, which can be used for the synthesis of steroids. We recently reported that hormone-stimulated progesterone synthesis by luteal cells requires protein kinase A-dependent mobilization of cholesterol substrate from lipid droplets to mitochondria. We hypothesize that luteal lipid droplets are enriched with steroidogenic enzymes and facilitate the synthesis of steroids in the corpus luteum. In the present study, we analyzed the lipid droplet proteome, conducted the first proteomic analysis of lipid droplets under acute cyclic adenosine monophosphate (cAMP)-stimulated conditions, and determined how specific lipid droplet proteins affect steroidogenesis. Steroidogenic enzymes, cytochrome P450 family 11 subfamily A member 1 and 3 beta-hydroxysteroid dehydrogenase (HSD3B), were highly abundant on lipid droplets of the bovine corpus luteum. High-resolution confocal microscopy confirmed the presence of active HSD3B on the surface of luteal lipid droplets. We report that luteal lipid droplets have the capacity to synthesize progesterone from pregnenolone. Lastly, we analyzed the lipid droplet proteome following acute stimulation with cAMP analog, 8-Br-cAMP, and report increased association of HSD3B with luteal lipid droplets following stimulation. These findings provide novel insights into the role of luteal lipid droplets in steroid synthesis.

Meglumine cyclic adenylate improves cardiovascular hemodynamics and motor-function in a rat model of acute T4 thoracic spinal cord injury.

Animal experimental study. Spinal cord injury (SCI) at or above the T6 level causes cardiovascular dysfunction. Maintaining cAMP levels with cAMP analogs can facilitate neurological recovery. In the present study, the effects of meglumine cyclic adenylate (MCA), a cAMP analog and approved cardiovascular drug, on cardiovascular and neurological recovery in acute T4-SCI in rats were investigated. Hospital in Kunming, China. Eighty rats were randomly allocated to five groups, and groups A-D received SCI: (A) a group administered MCA at 2 mg/kg/d iv qd, (B) a group administered dopamine at 2.5 to 5 μg/kg/min iv to maintain mean arterial pressure above 85 mm Hg, (C) a group administered atropine at 1 mg/kg iv bid, (D) a group receiving an equal volume of saline iv qd for 3 weeks after SCI and (E) a group undergoing laminectomy only. The cardiovascular and behavioral parameters of the rats were examined, and spinal cord tissues were processed for hematoxylin and eosin staining, Nissl staining, electron microscopy, and analysis of cAMP levels. Compared with dopamine or atropine, MCA significantly reversed the decrease in cAMP levels in both myocardial cells and the injured spinal cord; improved hypotension, bradycardia and behavioral parameters at 6 weeks; and improved spinal cord blood flow and histological structure at 7 days post-SCI. The regression analysis suggested spinal cord motor-function improved as decreased heart rate and mean arterial pressure were stopped post-SCI. MCA may be an effective treatment for acute SCI by sustaining cAMP-dependent reparative processes and improving post-SCI cardiovascular dysfunction. N/A.

Protein Kinase A Is Responsible for the Presynaptic Inhibition of Glycinergic and Glutamatergic Transmissions by Xenon in Rat Spinal Cord and Hippocampal CA3 Neurons.

The effects of a general anesthetic xenon (Xe) on spontaneous, miniature, electrically evoked synaptic transmissions were examined using the "synapse bouton preparation," with which we can clearly evaluate pure synaptic responses and accurately quantify pre- and postsynaptic transmissions. Glycinergic and glutamatergic transmissions were investigated in rat spinal sacral dorsal commissural nucleus and hippocampal CA3 neurons, respectively. Xe presynaptically inhibited spontaneous glycinergic transmission, the effect of which was resistant to tetrodotoxin, Cd2+, extracellular Ca2+, thapsigargin (a selective sarcoplasmic/endoplasmic reticulum Ca2+-ATPase inhibitor), SQ22536 (an adenylate cyclase inhibitor), 8-Br-cAMP (membrane-permeable cAMP analog), ZD7288 (an hyperpolarization-activated cyclic nucleotide-gated channel blocker), chelerythrine (a PKC inhibitor), and KN-93 (a CaMKII inhibitor) while being sensitive to PKA inhibitors (H-89, KT5720, and Rp-cAMPS). Moreover, Xe inhibited evoked glycinergic transmission, which was canceled by KT5720. Like glycinergic transmission, spontaneous and evoked glutamatergic transmissions were also inhibited by Xe in a KT5720-sensitive manner. Our results suggest that Xe decreases glycinergic and glutamatergic spontaneous and evoked transmissions at the presynaptic level in a PKA-dependent manner. These presynaptic responses are independent of Ca2+ dynamics. We conclude that PKA can be the main molecular target of Xe in the inhibitory effects on both inhibitory and excitatory neurotransmitter release. SIGNIFICANCE STATEMENT: Spontaneous and evoked glycinergic and glutamatergic transmissions were investigated using the whole-cell patch clamp technique in rat spinal sacral dorsal commissural nucleus and hippocampal CA3 neurons, respectively. Xenon (Xe) significantly inhibited glycinergic and glutamatergic transmission presynaptically. As a signaling mechanism, protein kinase A was responsible for the inhibitory effects of Xe on both glycine and glutamate release. These results may help understand how Xe modulates neurotransmitter release and exerts its excellent anesthetic properties.

Role of the adenylate cyclase/cyclic AMP pathway in oxytocin-induced lacrimal gland myoepithelial cells contraction

The aim of these studies was to investigate the involvement of the second messenger 3′,5′-cyclic adenosine monophosphate (cAMP) and its downstream effectors in oxytocin (OXT)-mediated lacrimal gland myoepithelial cell (MEC) contraction. Lacrimal gland MEC were isolated and propagated from alpha-smooth muscle actin (SMA)-GFP mice. RNA and protein samples were prepared to analyze G protein expression by RT-PCR and western blotting; respectively. Changes in intracellular cAMP concentration were measured using a competitive ELISA kit. To increase intracellular cAMP concentration, the following agents were used: forskolin (FKN, a direct activator of adenylate cyclase), 3-isobutyl-1-methylxanthine (IBMX, an inhibitor of the phosphodiesterase that hydrolyzes cAMP), or a cell permeant cAMP analog, dibutyryl (db)-cAMP. In addition, inhibitors and selective agonists were used to investigate the role of cAMP effector molecules, protein kinase A (PKA) and exchange protein activated by cAMP (EPAC) in OXT-induced MEC contraction. MEC contraction was monitored in real time and changes in cell size were quantified using ImageJ software. The adenylate cyclase coupling G proteins, Gαs, Gαo, and Gαi, are expressed in lacrimal gland MEC at both the mRNA and protein levels. OXT increased intracellular cAMP in a concentration-dependent manner. FKN, IBMX and db-cAMP significantly stimulated MEC contraction. Preincubation of cells with either Myr-PKI, a specific PKA inhibitor or ESI09, an EPAC inhibitor, resulted in almost complete inhibition of both FKN- as well as OXT-stimulated MEC contraction. Finally, direct activation of PKA or EPAC using selective agonists triggered MEC contraction. We conclude that cAMP agonists modulate lacrimal gland MEC contraction via PKA and EPAC activation which also play a major role in OXT induced MEC contraction.