Stimulated cAMP Production Research Articles

Adenosine relaxes vagina smooth muscle through the cyclic guanosine monophosphate- and cyclic guanosine monophosphate-dependent pathways.

In males, adenosine (ADO) is known to relax penile smooth muscles, although its role in the vagina is not yet fully elucidated. This study investigated the effect of ADO on vagina smooth muscle activity, using a validated female Sprague-Dawley rat model. Contractility studies, using noradrenaline-precontracted vaginal strips, tested the effects of ADORA1/3 antagonists and ADORA2A/2B antagonists and agonists. Increasing doses of ADO were tested after in vivo or in vitro treatment with Nω-nitro-L-arginine-methyl-ester hydrochloride (L-NAME) or with guanylate or adenylate cyclase inhibitors. Immunopositivity for ADORA2A and ADORA2B was assessed, and messenger RNA (mRNA) analysis was performed. Cyclic ADO monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) were quantified both in rat vagina smooth muscle cells (rvSMCs) and in vaginal tissues with increasing doses of ADO. Demonstrating ADO's role in the relaxing/contractile mechanism in distal vagina smooth muscle. All ADO receptors mRNAs were expressed in vaginal tissue, with a prevalent content of ADORA2B. A high expression of genes regulating ADO catabolism (ADK) and de novo synthesis (NT5E) was found. In vaginal strips, ADO induced relaxation with IC50 = 144.7μM and a flat pseudo-Hill coefficient value = -0.42, indicating an activity on heterogeneous receptors. Blocking ADORA1/3 shifted ADO response to the left and with a steeper slope. ADORA2A/2B agonists showed a higher potency than ADO in inducing relaxation. Immunolocalization confirmed the presence of ADORA2A/2B in vaginal musculature, in the blood vessels endothelium, and in the epithelium. ADO stimulation of vagina tissues induced a significant increase in cAMP and cGMP contents. Experiments on rvSMCs confirmed that ADO time- and dose-dependently stimulated cAMP production in these cells. However, ADORA2A/2B antagonists, although reducing the ADO-induced relaxation, did not completely block it. A similar inhibition was obtained by blocking adenylate cyclase. Overall, these findings suggest that ADO relaxation involves other pathways, eg, nitric oxide (NO)/cGMP. Accordingly, blocking NO formation through L-NAME substantially blunted ADO responsiveness, as it does the block of cGMP formation through 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one. Simultaneous incubation with cGMP and cAMP blockers completely inhibited ADO responsiveness. The study highlights ADO's role in regulating vaginal smooth muscle activity, suggesting its potential effect on the vagina. This is the first study on ADO in the vagina, although the results are preliminary and limited to the rat model. These results show that ADO acts as a vaginal relaxing modulator through selective activation of receptors involving not only cAMP but also cGMP.

Dopaminergic cAMP signaling in mouse olfactory bulb astrocytes

Cyclic AMP (cAMP) is an important second messenger in virtually all animal cell types, including astrocytes. In the brain, it modulates energy metabolism, development and synaptic plasticity. Dopamine receptors are G protein-coupled receptors that affect cAMP production by adenylyl cyclases. They are divided into two subgroups, D1-like receptors linked to Gs proteins stimulating cAMP production and D2-like receptors linked to Gi/o proteins inhibiting cAMP production. In the present study, we investigated the effect of dopamine receptor activation on cAMP dynamics in astrocytes of the mouse olfactory bulb, the brain region with the largest population of dopaminergic neurons. Using the genetically encoded cAMP sensor Flamindo2 we visualized changes in the cytosolic cAMP concentration and showed that dopamine application results in a transient increase in cAMP. This cAMP increase could be mimicked by the D1-like receptor agonist A 68930 and was inhibited by the D1-like receptor antagonist SCH 23390, whereas D2-like receptor ligands had no effect on the astrocytic cAMP concentration. Thus, olfactory bulb astrocytes express D1-like receptors that are linked to cAMP production.

Role of Orai1 and Ca2+-stimulated adenylyl cyclase 8 in the adverse cardiac remodeling after myocardial infarction

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Agencia Estatal de Investigación. Background It is well established that abnormalities in the regulation of the intracellular concentration of Ca2+ ([Ca2+]i) occurs in diseased heart such as under acute myocardial infarction (AMI). Recent studies have demonstrated that proteins related to the Store Operated Calcium Entry (SOCE), such as Orai1/2/3 and TRPC channels, play a role in the adverse cardiac remodelling. Previously, we demonstrated that the expression of Orai1 increased in heart subjected to ischemia and reperfusion (I/R) syndrome. However, the signalling pathway implicated in Orai1 regulation has not been addressed. Objective The aim of this study was to examine the molecular mechanism and signalling pathways involved in the of Orai1 upregulation after I/R in cardiomyocytes. Materials and Methods The experiments were performed in adult (ARVM) and neonatal rat ventricular cardiomyocytes (NRVM), in a rat model of myocardial I/R and in ventricular biopsies of patients with ischemic heart failure. Ca2+ studies were realized using microfluorimetric technique in FURA-2AM loaded cardiomyocytes. Immunofluorescence was used to investigate CREB activation using a specific phospho-CREB antibody and Orai1 and AC8 protein expression were analysed by Western Blot. This study was performed in accordance with the recommendations of the Royal Decree 53/2013 in agreement to the Directive 2010/63/EU of the European Parliament and approved by the local Ethics Committee on human Research and the Animal Research Committee. Results We found that under I/R diastolic level of [Ca2+]i was significantly increased in cardiac myocytes isolated from I/R hearts, which was inhibited by SOCE blockers. Moreover, we found that Orai1 and Ca2+-sensitive adenylyl cyclase 8 (AC8) are overexpressed in the heart isolated from I/R rat model, as compared to sham. Similarly, we found significant increase in the expression of Orai1 and AC8 in ventricle biopsies of post-infarction patients with HF. We also observed that I/R stimulated cAMP production, involving Orai1 and AC8 activation in rats, indicating that Ca2+ entry through Orai1 stimulated AC8. Moreover, I/R phosphorylated cAMP response element-binding protein (CREB) through PKA activation, but not via EPAC2 or ERK1/2. Interestingly, we found that CREB facilitated I/R-induced Orai1 upregulation and related exacerbated SOCE by its activation of Orai1 promoter under I/R. Finally, the intramyocardial administration of AAV9 holding AC8 shRNA decreased the expression of AC8, Orai1 and CREB, which restored diastolic [Ca2+]i. Conclusions Our results show that the activation of Orai1/AC8/CREB axis plays a key role in the I/R-induced Orai1 upregulation, which participated in diastolic [Ca2+]i mishandling related to the adverse cardiac remodelling.

Left Ventricular Dilation Resulting from the Absence of Either Rgs2 or Rgs5 Is Associated with Increased Immune Cell Infiltration in the Myocardium

Sustained sympathoexcitation is a hallmark of heart failure, with equally sustained activation of downstream signaling pathways involving Gs- and Gi/o-coupled β-adrenergic receptors (βAR) stimulating cAMP production and PKA activation. A critical mechanism controlling G protein-mediated βAR signaling is the activity of regulator of G protein signaling (RGS) proteins, including members of the B/R4 subfamily, which accelerate the termination of Gq/11 and Gi/o protein signaling. Previous studies by us and others reported that moderation of Gi/o activity separately by RGS2 and RGS5 of the R4 subfamily is key to maintaining normal ventricular rhythm. The loss of one or both R4 RGS proteins also results in left ventricular (LV) dilation and reduced ejection fraction. Here, we tested the hypothesis that the LV structural remodeling is due to the disruption of the coordinated activity of RGS2 and 5 resulting in sensitization of βAR signaling. To test this hypothesis, we challenged wild type (WT), Rgs2 KO ,Rgs5 KO, and Rgs2/5 double knockout (dbKO) adult male mice with sub-chronic infusion of isoproterenol (ISO, 30 mg/kg/day, 3 days) via a peritoneal osmotic minipump implantation. Rgs2 KO and Rgs5 KO mice showed increased LV chamber size at baseline, which was unchanged by ISO administration. In saline-treated animals, the loss of Rgs2 increased Rgs5 mRNA expression, while Rgs5 absence increased Rgs4 mRNA expression. ISO infusion induced concentric cardiac hypertrophy in WT mice, as evidenced by increased heart weight/tibia length (HW/TL) ratio and cardiomyocyte cross sectional area. While ISO infusion increased interstitial fibrosis in midventricular tissue and HW/TL ratio in dbKO mice, it did not affect cardiomyocyte cross sectional area or exacerbate LV dilation, despite increased expression of the cardiac fetal gene Nppa as well as the sarcomeric protein α-actinin and Ca2+/calmodulin-dependent kinase II (CaMKII). Cardiact tissue from saline-treated Rgs2 KO and Rgs5 KO mice had markedly increased CD45+ cells vs WT, while tissue from Rgs5 KO mice showed increased CD68+ cells, as revealed by immunohistochemistry. Taken together, these results indicate that ventricular remodeling seen in Rgs2 KO, Rgs5 KO, and Rgs2/5 dbKO mice is insensitive to sustained βAR stimulation but may be due to increased cardiac resident immune cell population. This work was supported by NIH R56 DK132859-01A1 and R01 HL139754, and Craig H. Neilsen Foundation Grant 382566 to Patrick Osei-Owusu. 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.

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.

Role of protein kinase A and A kinase anchoring proteins in buffering and compartmentation of cAMP signalling in human airway smooth muscle cells.

In human airway smooth muscle (hASM) cells, not all receptors stimulating cAMP production elicit the same effects. This can only be explained if cAMP movement throughout the cell is restricted, yet the mechanisms involved are not fully understood. Phosphodiesterases (PDEs) contribute to compartmentation of many cAMP responses, but PDE activity alone is predicted to be insufficient if cAMP is otherwise freely diffusible. We tested the hypothesis that buffering of cAMP by protein kinase A (PKA) associated with A kinase anchoring proteins (AKAPs) slows cAMP diffusion and that this contributes to receptor-mediated, compartmentalized responses. Raster image correlation spectroscopy (RICS) was used to measure intracellular cAMP diffusion coefficients and evaluate the contribution of PKA-AKAP interactions. Western blotting and immunocytochemistry were used to identify the AKAPs involved. RNA interference was used to down-regulate AKAP expression and determine its effects on cAMP diffusion. Compartmentalized cAMP responses were measured using fluorescence resonance energy transfer (FRET) based biosensors. Cyclic AMP movement was significantly slower than that of free-diffusion in hASM cells, and disrupting PKA-AKAP interactions significantly increased the diffusion coefficient. PKA associated with the outer mitochondrial membrane appears to play a prominent role in this effect. Consistent with this idea, knocking down expression of D-AKAP2, the primary mitochondrial AKAP, increased cAMP diffusion and disrupted compartmentation of receptor-mediated responses. Our results confirm that AKAP-anchored PKA contributes to the buffering of cAMP and is consequential in the compartmentation of cAMP responses in hASM cells.

Real-Time Determination of Intracellular cAMP Reveals Functional Coupling of Gs Protein to the Melatonin MT1 Receptor.

Melatonin is a neuroendocrine hormone that regulates the circadian rhythm and many other physiological processes. Its functions are primarily exerted through two subtypes of human melatonin receptors, termed melatonin type-1 (MT1) and type-2 (MT2) receptors. Both MT1 and MT2 receptors are generally classified as Gi-coupled receptors owing to their well-recognized ability to inhibit cAMP accumulation in cells. However, it remains an enigma as to why melatonin stimulates cAMP production in a number of cell types that express the MT1 receptor. To address if MT1 can dually couple to Gs and Gi proteins, we employed a highly sensitive luminescent biosensor (GloSensorTM) to monitor the real-time changes in the intracellular cAMP level in intact live HEK293 cells that express MT1 and/or MT2. Our results demonstrate that the activation of MT1, but not MT2, leads to a robust enhancement on the forskolin-stimulated cAMP formation. In contrast, the activation of either MT1 or MT2 inhibited cAMP synthesis driven by the activation of the Gs-coupled β2-adrenergic receptor, which is consistent with a typical Gi-mediated response. The co-expression of MT1 with Gs enabled melatonin itself to stimulate cAMP production, indicating a productive coupling between MT1 and Gs. The possible existence of a MT1-Gs complex was supported through molecular modeling as the predicted complex exhibited structural and thermodynamic characteristics that are comparable to that of MT1-Gi. Taken together, our data reveal that MT1, but not MT2, can dually couple to Gs and Gi proteins, thereby enabling the bi-directional regulation of adenylyl cyclase to differentially modulate cAMP levels in cells that express different complements of MT1, MT2, and G proteins.

An Orally Efficacious Thyrotropin Receptor Ligand Inhibits Growth and Metastatic Activity of Thyroid Cancers.

Thyroid-stimulating hormone (or thyrotropin) receptor (TSHR) could be a selective target for small molecule ligands to treat thyroid cancer (TC). We report a novel, orally efficacious ligand for TSHR that exhibits proliferation inhibitory activity against human TC in vitro and in vivo, and inhibition of metastasis in vivo. A35 (NCATS-SM4420; NCGC00241808) was selected from a sublibrary of >200 TSHR ligands. Cell proliferation assays including BrdU incorporation and WST-1, along with molecular docking studies were done. In vivo activity of A35 was assessed in TC cell-derived xenograft (CDX) models with immunocompromised (NSG) mice. Formalin-fixed, paraffin-embedded sections of tumor and lung tissues were observed for the extent of cell death and metastasis. A35 was shown to stimulate cAMP production in some cell types by activating TSHR but not in TC cells, MDA-T32, and MDA-T85. A35 inhibited proliferation of MDA-T32 and MDA-T85 in vitro and in vivo, and pulmonary metastasis of MDA-T85F1 in mice. In vitro, A35 inhibition of proliferation was reduced by a selective TSHR antagonist. Inhibition of CDX tumor growth without decreases in mouse weights and liver function showed A35 to be efficacious without apparent toxicity. Lastly, A35 reduced levels of Ki67 in the tumors and metastatic markers in lung tissues. We conclude that A35 is a TSHR-selective inhibitor of TC cell proliferation and metastasis, and suggest that A35 may be a promising lead drug candidate for the treatment of differentiated TC in humans.

Targeting cAMP in D1-MSNs in the nucleus accumbens, a new rapid antidepressant strategy

Studies have suggested that the nucleus accumbens (NAc) is implicated in the pathophysiology of major depression; however, the regulatory strategy that targets the NAc to achieve an exclusive and outstanding anti-depression benefit has not been elucidated. Here, we identified a specific reduction of cyclic adenosine monophosphate (cAMP) in the subset of dopamine D1 receptor medium spiny neurons (D1-MSNs) in the NAc that promoted stress susceptibility, while the stimulation of cAMP production in NAc D1-MSNs efficiently rescued depression-like behaviors. Ketamine treatment enhanced cAMP both in D1-MSNs and dopamine D2 receptor medium spiny neurons (D2-MSNs) of depressed mice, however, the rapid antidepressant effect of ketamine solely depended on elevating cAMP in NAc D1-MSNs. We discovered that a higher dose of crocin markedly increased cAMP in the NAc and consistently relieved depression 24 h after oral administration, but not a lower dose. The fast onset property of crocin was verified through multicenter studies. Moreover, crocin specifically targeted at D1-MSN cAMP signaling in the NAc to relieve depression and had no effect on D2-MSN. These findings characterize a new strategy to achieve an exclusive and outstanding anti-depression benefit by elevating cAMP in D1-MSNs in the NAc, and provide a potential rapid antidepressant drug candidate, crocin.

OR23-01 The Nonredundant Role Of Beta-Amyloid In Mediating Tonic PTH Secretion In Physiological And Pathological States

Abstract Disclosure: C. Tu: None. Z. Cheng: None. K.A. Pena: None. N. Szeto: None. J.A. Sosa: None. J. Vilardaga: None. J. Koh: None. W. Chang: None. Understanding the mechanisms driving parathyroid hormone (PTH) hypersecretion in primary hyperparathyroidism (PHPT) is essential for better management of this common endocrinopathy. Prior studies showed that reduced Ca2+-sensing receptor (CaSR) expression and the subsequent increases in heterodimerization of the CaSR with the type B γ-aminobutyric acid receptor 1 (GABAB1R) are causally linked to PTH hypersecretion in PHPT mouse models (Nat Metab 2:243-255). We further showed that expression of the putative GABAB1R ligands, amyloid precursor protein (APP) and its proteolytic product, β-amyloid (Aβ1-42), is significantly upregulated in adenomas of PHPT patients associated with vitamin D insufficiency/deficiency when compared to normal donor controls. The current study aims to delineate the actions of Aβ1-42 in promoting tonic PTH secretion and its interactions with vitamin D receptor (VDR) signaling in parathyroid glands (PTGs) in basal and PHPT states. We show that Aβ1-42 (200 nM) stimulated tonic PTH secretion in cultures of normal human PTGs without shifting the calcium/PTH secretion setpoint (Ca2+-setpoint). In contrast, conditional knockout (KO) of the App gene in the parathyroid cell (PTC) of PTCAppΔflox/Δflox mice significantly reduced serum PTH levels (KO: 64±13 pg/ml vs Control: 109±12 pg/ml; p<0.05, n=8) despite hypocalcemia, indicating hypoparathyroidism. In PTGs cultured from the PTCAppΔflox/Δflox mice, supplementation of Aβ1-42 dose-dependently (EC50=5.6 nM, p<0.001) increased PTH secretion without altering the Ca2+-setpoint. However, the stimulatory effects of Aβ1-42 on tonic PTH secretion were completely abrogated in the PTGs with concurrent deletions of App, Casr and Gabbr1 genes, supporting a direct action of Aβ1-42 on CaSR and/or GABAB1R. The latter notion is further supported by the ability of Aβ1-42 to stimulate cAMP production in cells co-expressing CaSR and GABAB1R. PTC-specific deletion of the Vdr gene in the PTCVdrΔflox/Δflox mice led to elevated serum PTH levels in vivo and increased tonic PTH secretion with unaffected Ca2+-setpoint in PTGs in vitro. Concurrent ablation of the App gene completely normalized serum PTH levels in the PTCVdrΔflox/Δflox mice and prevented PTH hypersecretion in their PTGs in culture. These findings support a critical role of the APP-derived Aβ1-42 in mediating tonic PTH secretion in the normal physiological state and suggest a new mechanism driving PTH hypersecretion in PHPT due to vitamin D deficiency. Presentation: Saturday, June 17, 2023

GPR114/ADGRG5 is activated by its tethered peptide agonist because it is a cleaved adhesion GPCR

Family B2 or adhesion G protein-coupled receptors (AGPCRs) are distinguished by variable extracellular regions that contain a modular protease, termed the GPCR autoproteolysis-inducing domain that self-cleaves the receptor into an N-terminal fragment (NTF) and a C-terminal fragment (CTF), or seven transmembrane domain (7TM). The NTF and CTF remain bound after cleavage through noncovalent interactions. NTF binding to a ligand(s) presented by nearby cells, or the extracellular matrix anchors the NTF, such that cell movement generates force to induce NTF/CTF dissociation and expose the AGPCR tethered peptide agonist. The released tethered agonist (TA) binds rapidly to the 7TM orthosteric site to activate signaling. The orphan AGPCR, GPR114 was reported to be uncleaved, yet paradoxically capable of activation by its TA. GPR114 has an identical cleavage site and TA to efficiently cleave GPR56. Here, we used immunoblotting and biochemical assays to demonstrate that GPR114 is a cleaved receptor, and the self-cleavage is required for GPR114 TA-activation of Gs and no other classes of G proteins. Mutagenesis studies defined features of the GPR114 and GPR56 GAINA subdomains that influenced self-cleavage efficiency. Thrombin treatment of protease-activated receptor 1 leader/AGPCR fusion proteins demonstrated that acute decryption of the GPR114/56TAs activated signaling. GPR114 was found to be expressed in an eosinophilic-like cancer cell line (EoL-1cells) and endogenous GPR114 was efficiently self-cleaved. Application of GPR114TA peptidomimetics to EoL-1cells stimulated cAMP production. Our findings may aid future delineation of GPR114 function in eosinophil cAMP signaling related to migration, chemotaxis, or degranulation.

Free fatty acid receptor 1 stimulates cAMP production and gut hormone secretion through Gq-mediated activation of adenylate cyclase 2

ObjectiveFree fatty acid receptor 1 (FFAR1) is highly expressed in enteroendocrine cells of the small intestine and pancreatic beta cells, where FFAR1 agonists function as GLP-1 and insulin secretagogues, respectively. Most efficacious are so-called second-generation synthetic agonists such as AM5262, which, in contrast to endogenous long-chain fatty acids are able to signal through both IP3/Ca2+ and cAMP pathways. Whereas IP3 signaling is to be expected for the mainly Gq-coupled FFAR1, the mechanism behind FFAR1-induced cAMP accumulation remains unclear, although originally proposed to be Gs mediated. Methods and resultsWhen stimulated with AM5262, we observe that FFAR1 can activate the majority of the Gα proteins, except - surprisingly - members of the Gs family. AM5262-induced FFAR1-mediated transcriptional activation through cAMP response element (CREB) was blocked by the specific Gq inhibitor, YM253890. Furthermore, in Gq-deficient cells no CREB signal was observed unless Gq or G11 was reintroduced by transfection. By qPCR we determined that adenylate cyclase 2 (Adcy2) was highly expressed and enriched relative to the nine other Adcys in pro-glucagon expressing enteroendocrine cells. Co-transfection with ADCY2 increased the FFAR1-induced cAMP response 4-5-fold in WT HEK293 cells, an effect fully inhibited by YM253890. Moreover, co-transfection with ADCY2 had no effect in Gq-deficient cells without reintroduction of either Gq or G11. Importantly, although both AM5262/FFAR1 and isoproterenol/β2 adrenergic receptor (β2AR) induced cAMP production was lost in Gs-deficient cells, only the β2AR response was rescued by Gs transfection, whereas co-transfection with ADCY2 was required to rescue the FFAR1 cAMP response. In situ hybridization demonstrated a high degree of co-expression of ADCY2 and FFAR1 in enteroendocrine cells throughout the intestine. Finally, in the enteroendocrine STC-1 and GLUTag cell lines AM5262-induced cAMP accumulation and GLP-1 secretion were both blocked by YM253890. ConclusionsOur results show that Gq signaling is responsible not only for the IP3/Ca2+ but also the cAMP response, which together are required for the highly efficacious hormone secretion induced by second-generation FFAR1 agonists - and that ADCY2 presumably mediates the Gq-driven cAMP response.

Homozygous Ser-1 to Pro-1 mutation in parathyroid hormone identified in hypocalcemic patients results in secretion of a biologically inactive pro-hormone

Like other secreted peptides, nascent parathyroid hormone (PTH) is synthesized with a pre- and a pro-sequence (25 and 6 amino acids, respectively). These precursor segments are sequentially removed in parathyroid cells before packaging into secretory granules. Three patients from two unrelated families who presented during infancy with symptomatic hypocalcemia were found to have a homozygous serine (S) to proline (P) change affecting the first amino acid of the mature PTH. Unexpectedly, biological activity of synthetic [P1]PTH(1-34) was indistinguishable from that of unmodified [S1]PTH(1-34). However, in contrast to conditioned medium from COS-7 cells expressing prepro[S1]PTH(1-84), medium from cells expressing prepro[P1]PTH(1-84) failed to stimulate cAMP production despite similar PTH levels when measured by an intact assay that detects PTH(1-84) and large amino-terminally truncated fragments thereof. Analysis of the secreted, but inactive PTH variant led to the identification of pro[P1]PTH(-6 to+84). Synthetic pro[P1]PTH(-6 to+34) and pro[S1]PTH(-6 to+34) had much less bioactivity than the corresponding PTH(1-34) analogs. Unlike pro[S1]PTH(-6 to+34), pro[P1]PTH(-6 to+34) was resistant to cleavage by furin suggesting that the amino acid variant impairs preproPTH processing. Consistent with this conclusion, plasma of patients with the homozygous P1 mutation had elevated proPTH levels, as determined with an in-house assay specific for pro[P1]PTH(-6 to+84). In fact, a large fraction of PTH detected by the commercial intact assay represented the secreted pro[P1]PTH. In contrast, two commercial biointact assays that use antibodies directed against the first few amino acid residues of PTH(1-84) for capture or detection failed to detect pro[P1]PTH.

Mitochondrial bound PKA contributes to cyclic-AMP compartmentation in human airway smooth muscle cells.

In human airway smooth muscle (hASM) cells, compartmentation of cAMP signaling is necessary to maintain the fidelity of responses by different G protein-coupled receptors sharing this common diffusible second messenger. Using FRET-based biosensors targeted to different subcellular locations, we found that the EP2 prostaglandin receptors (EP2Rs) stimulate cAMP production that is restricted to locations associated with non-lipid raft regions of the plasma membrane. Furthermore, inhibition of phosphodiesterase type 4 (PDE4) allowed EP2R production of cAMP to spread to subcellular locations associated with lipid-raft membrane domains. However, computational modeling has suggested that PDEs may only contribute to compartmentation if cAMP diffusion is slowed by some other means. To test this hypothesis, we used raster image correlation spectroscopy (RICS) to estimate the diffusion coefficient of fluorescently labeled cAMP (φ575-cAMP) in hASM cells and found that it is 3.9 ± 0.49 μm2/s, or nearly two orders of magnitude slower than the predicted rate of free diffusion. Disrupting protein kinase A (PKA) interactions with A kinase anchoring proteins (AKAPs) using the anchoring inhibitor Ht31 nearly doubled the diffusion coefficient of φ575-cAMP to 7.9 ± 1.5 μm2/s, suggesting that cAMP movement was being buffered by PKA anchored by one or more AKAPs. Confocal imaging demonstrated that φ575-cAMP colocalizes with mitochondria, and Western blot analysis found that D-AKAP2 is the primary mitochondrial AKAP found in hASM cells. Knockdown of D‑AKAP2 expression was achieved using adenovirus-mediated delivery of shRNA, and in knockdown cells EP2R activation was able to stimulate cAMP production in subcellular locations associated with lipid-raft domains, similar to effects previously seen with PDE4 inhibition. These results suggest that buffering of cAMP movement by PKA anchored to the outer membrane of mitochondria contributes to compartmentation of EP2R-mediated responses in hASM cells.

Role of mitochondrial pka in compartmentation of cAMP signaling in cardiac ventricular myocytes.

In cardiac ventricular myocytes, both β1 and β2-adrenergic receptors (βARs) produce an increase in the rate and force of contraction by stimulating cAMP production. However, only β1ARs enhance the rate of relaxation by regulating the activity of the SERCA2 pump found in the distal scaroplasmic reticulum. Previously we found that β1, but not β2AR production of cAMP could be detected by Epac2-αKAP, a FRET-based biosensor targeted to the the SERCA2 signaling complex, demonstrating that compartmentation of cAMP signaling contributes to the difference in functional responses. While inhibition of phosphodiesterase (PDE) activity was found to influence this behavior, in silico studies have suggested that intracellular cAMP must also be slowed by other mechanisms in order for this to occur. Consistent with this prediction, using raster image correlation spectroscopy (RICS), we found that the intracellular diffusion coefficient of fluorescently labeled cAMP is actually 30 fold slower than the rate of free diffusion. Furthermore, this slowing of diffusion was found to be due to the buffering effect of protein kinase A (PKA), specifically PKA anchored to the outer membrane of mitochondria. To directly test the hypothesis that this buffering effect contributes to cAMP compartmentation, we used adenoviral-mediated knockdown to target two key mitochondrial A kinase anchoring proteins (AKAPs), D-AKAP1 and D-AKAP2. Following knockdown of either AKAP alone, we found that the cAMP diffusion coefficient was significantly increased compared to control. Furthermore, knocking down the expression of D-AKAP1 enhanced β2AR stimulation of cAMP production detected by Epac2-αKAP. These results support the conclusion that buffering of cAMP mobility by PKA anchored to mitochondria influences cAMP dynamics and contributes to compartmentation of β2AR signaling in cardiac ventricular myocytes.

Adenylate cyclase activity of TIR1/AFB auxin receptors in plants.

The phytohormone auxin is the major coordinative signal in plant development1, mediating transcriptional reprogramming by a well-established canonical signalling pathway. TRANSPORT INHIBITOR RESPONSE 1 (TIR1)/AUXIN-SIGNALING F-BOX (AFB) auxin receptors are F-box subunits of ubiquitin ligase complexes. In response to auxin, they associate with Aux/IAA transcriptional repressors and target them for degradation via ubiquitination2,3. Here we identify adenylate cyclase (AC) activity as an additional function of TIR1/AFB receptors across land plants. Auxin, together with Aux/IAAs, stimulates cAMP production. Three separate mutations in the AC motif of the TIR1 C-terminal region, all of which abolish the AC activity, each render TIR1 ineffective in mediating gravitropism and sustained auxin-induced root growth inhibition, and also affect auxin-induced transcriptional regulation. These results highlight the importance of TIR1/AFB AC activity in canonical auxin signalling. They also identify a unique phytohormone receptor cassette combining F-box and AC motifs, and the role of cAMP as a second messenger in plants.

Subcellular β-Adrenergic Receptor Signaling in Cardiac Physiology and Disease.

Adrenergic receptors are critical regulators of cardiac function with profound effects on cardiac output during sympathetic stimulation. Chronic stimulation of the adrenergic system of the heart under conditions of cardiac stress leads to cardiac dysfunction, hypertrophy, and ultimately failure. Emerging data have revealed that G protein-coupled receptors in intracellular compartments are functionally active and regulate distinct cellular processes from those at the cell surface. β2 adrenergic receptors internalize onto endosomes in various cell types where they have recently been shown to continue to stimulate cAMP production to selectively regulate gene expression. Other studies have identified β1 adrenergic receptors at the nuclear envelope and the Golgi apparatus. Here, we discuss data on signaling by β1 and β2 adrenergic receptors in the heart and the possible influence of their subcellular locations on their divergent physiological functions in cardiac myocytes and in cardiac pathology. Understanding the relative roles of these receptors at these locations could have a significant impact on pharmacological targeting of these receptors for the treatment of heart failure and cardiac diseases.

Cyclic Adenosine Monophosphate (cAMP)-Dependent Phosphodiesterase Inhibition Promotes Bone Anabolism Through CD8+ T Cell Wnt-10b Production in Mice.

ABSTRACTCyclic adenosine monophosphate (cAMP)‐dependent phosphodiesterase (PDE) inhibitors such as pentoxifylline (PTX) suppress cAMP degradation and promote cAMP‐dependent signal transduction. PDE inhibitors increase bone formation and bone mass in preclinical models and are used clinically to treat psoriatic arthritis by targeting inflammatory mediators including activated T cells. T cell activation requires two signals: antigen‐dependent CD3‐activation, which stimulates cAMP production; and CD28 co‐stimulation, which downregulates cAMP‐signaling, through PDE activation. PDE‐inhibitors consequently suppress T cell activation by disrupting CD28 co‐stimulation. Interestingly, we have reported that when CD8+ T cells are activated in the absence of CD28 co‐stimulation, they secrete Wnt‐10b, a bone anabolic Wnt ligand that promotes bone formation. In the present study, we investigated whether the bone anabolic activity of the PDE‐inhibitor PTX, has an immunocentric basis, involving Wnt‐10b production by CD8+ T cells. When wild‐type (WT) mice were administered PTX, biochemical markers of both bone resorption and formation were significantly increased, with net bone gain in the axial skeleton, as quantified by micro‐computed tomography (μCT). By contrast, PTX increased only bone resorption in T cell knockout (KO) mice, causing net bone loss. Reconstituting T cell–deficient mice with WT, but not Wnt‐10b knockout (KO) CD8+ T cells, rescued bone formation and prevented bone loss. To study the role of cAMP signaling in Wnt‐10b expression, reverse‐transcription polymerase chain reaction (RT‐PCR) and luciferase‐reporter assays were performed using primary T cells. PDE inhibitors intensified Wnt‐10b promoter activity and messenger RNA (mRNA) accumulation in CD3 and CD28 activated CD8+ T cells. In contrast, inhibiting the cAMP pathway mediators protein kinase A (PKA) and cAMP response element‐binding protein (CREB), suppressed Wnt‐10b expression by T cells activated in the absence of CD28 co‐stimulation. In conclusion, the data demonstrate a key role for Wnt‐10b production by CD8+ T cells in the bone anabolic response to PDE‐inhibitors and reveal competing T cell–independent pro‐resorptive properties of PTX, which dominate under T cell–deficient conditions. Selective targeting of CD8+ T cells by PDE inhibitors may be a beneficial approach for promoting bone regeneration in osteoporotic conditions. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

FC018: The B1 H+-ATPASE (ATP6V1B1) Subunit is Required for Non-Type a Intercalated Cell Function During Alkalosis

Abstract BACKGROUND AND AIMS Non-type A intercalated cells (IC) in the connecting tubule (CNT) and cortical collecting duct (CCD) express the luminal Cl−/HCO3− exchanger pendrin and apical and/or basolateral vacuolar H+-ATPases containing the B1 subunit isoform. One of the main functions of non-type A ICs is the secretion of bicarbonate during metabolic alkalosis. Mutations in the B1 subunit (ATP6V1B1) in man cause distal renal tubular acidosis due to its importance in acid secretion by type A ICs. However, the function of the B1 isoform in non-type A ICs has remained elusive. In this work, we investigated the role of the B1 H+-ATPase subunit in non-type A intercalated in the control of alkalosis. METHOD Acid-base metabolism of Atp6v1b1 mice was studied during an alkali load (0.28 M NaHCO3 in drinking water and 2 mg deoxycorticosterone i.p for 4 days) or during a chronic furosemide treatment (20 mg i.p. injection daily for 9 days). RESULTS Induction of metabolic alkalosis by 0.28 M NaHCO3 resulted in a more pronounced alkalosis in ATP6v1b1−/− mice with increased blood bicarbonate, hypokalaemia and hypochloraemia despite a reduced lung minute volume. Determination of the relative abundance of the different subtypes of cells in the collecting duct system revealed a remodelling of CNT and CCD in the cortex with an increase of type A ICs and a compensatory increase of non-type A ICs in collecting ducts of the outer medullary in ATP6v1b1−/− kidneys. Total pendrin expression in the kidney and activity in non-type A cells of ex vivo microperfused CCD were reduced in ATP6v1b1−/− mice. Moreover, the stimulating effect of Isoproterenol, a β2-adrenergic receptor agonist stimulating cAMP production, on pendrin activity was blunted in non-type A ICs from ATP6v1b1−/− mice. Basolateral H+-ATPase activity in pendrin-expressing cells was strongly reduced, even though the expression of the B2 isoform was increased at their basolateral side. Moreover, the E1 and A H+-ATPase subunits of the V0 domain did only partially colocalize with the a4 H+-ATPase subunit of the V1 domain of H+-ATPases at the basolateral pole of ATP6v1b1−/− non-type A ICs, indicating impaired assembly of V0 and V1 H+-ATPase domains. Finally, when exposed to a chronic furosemide treatment, a treatment associated with a high risk to develop metabolic alkalosis in patients, ATP6v1b1−/− mice developed a more pronounced hypokalaemic hypochloraemic alkalosis with polyuria and loss of urine chloride compared with their wildtype littermates. In ATP6v1b1−/− kidneys, pendrin expression was drastically reduced as well as the water channel AQP2 and the α and γ subunits of the sodium channel ENaC. An increase of BUN and a decrease of kidney klotho expression were also observed in ATP6v1b1−/− mice which might indicate a worsening of their kidney function. CONCLUSION Thus, this study demonstrates for the first time that i) the B1 H+-ATPase subunit is critical for normal non-type A ICs function and protects against alkalosis. ii) Moreover, the B1 subunit is required for the assembly of complete and functional basolateral vacuolar H+-ATPases complexes in intercalated cells.

Expression of functional thyroid-stimulating hormone receptor in microglia

ObjectiveThe purpose of the present study was to clarify the expression of thyroid-stimulating hormone receptor (TSHR) in microglial cells, and to explore its function. Materials and methodsExpression of TSHR in microglia was determined by Western blot, immunocytofluorescence and double immunohistofluorescence. Cyclic adenosine 3′,5′-monophosphate (cAMP) production was measured after thyrotropin receptor stimulating antibody (TSAb) treatment. ResultsResults showed that TSHR protein was expressed and mainly located in the mouse microglia membrane. Moreover, TSAb stimulated cAMP production in mouse microglia (P<0.05). ConclusionsThis study demonstrated the presence of TSHR in microglial cells. Brain TSHR was able to respond specifically to TSAb stimulation, suggesting that TSHR expression is functional. As microglia are innate immune cells that maintain environmental stability in the central nervous system and play a key role in many neuroimmune diseases, expression of functional TSHR in microglia has important pathophysiological implications.