Changes In cAMP Concentration Research Articles

The Effect of Bacterial AHL on the Cyclic Adenosine Monophosphate Content in Plants According to High-Performance Liquid Chromatography.

Cyclic adenosine monophosphate (cAMP) is an important second messenger in cells, mediating various stimulation signals such as the growth and development of organisms and stress and participating in regulating various biological processes of cells. This article explores the quantitative determination of cAMP in plants using High-Performance Liquid Chromatography (HPLC) and applies this method to analyzing the changes in cAMP content during the process of plant response to the bacterial quorum sensing signal N-acyl homoserine lactone (AHL). Research has shown that the optimal detection conditions for HPLC are as follows: the chromatographic column is Venusil MP C18 (2), the mobile phase is methanol-water (0.1% trifluoroacetic acid) (v:v, 10:90), the detection wavelength is 259 nm, the column temperature is 35 °C, and the flow rate is 0.8 mL/min. The precision of the standard sample of this method is 98.21%, the precision of the sample is 98.87%, and the recovery rate is 101.067%. The optimal extraction conditions for cAMP in Arabidopsis are to use 15% methanol ultrasonic extraction for 10 min, followed by a 40 °C water bath for 4 h. Bacterial AHL signal processing can significantly stimulate an increase in cAMP levels in Arabidopsis leaves and roots. The establishment of HPLC detection methods for the cAMP content in plants is of great significance for in-depth research on the signal transduction mechanisms of plant-bacterial interactions.

A high affinity switch for cAMP in the HCN pacemaker channels

Binding of cAMP to Hyperpolarization activated cyclic nucleotide gated (HCN) channels facilitates pore opening. It is unclear why the isolated cyclic nucleotide binding domain (CNBD) displays in vitro lower affinity for cAMP than the full-length channel in patch experiments. Here we show that HCN are endowed with an affinity switch for cAMP. Alpha helices D and E, downstream of the cyclic nucleotide binding domain (CNBD), bind to and stabilize the holo CNBD in a high affinity state. These helices increase by 30-fold cAMP efficacy and affinity measured in patch clamp and ITC, respectively. We further show that helices D and E regulate affinity by interacting with helix C of the CNBD, similarly to the regulatory protein TRIP8b. Our results uncover an intramolecular mechanism whereby changes in binding affinity, rather than changes in cAMP concentration, can modulate HCN channels, adding another layer to the complex regulation of their activity.

Evidence of Noisy Oscillations of cAMP under Nutritional Stress Condition in Budding Yeast

The Ras/cAMP/PKA pathway regulates responses to nutrients’ availability and stress in budding yeast. The cAMP levels are subjected to negative feedback, and we have previously simulated a dynamic model of this pathway suggesting the existence of stable oscillatory states depending on the symmetrical and opposed activity of the RasGEF (Cdc25) and RasGAPs (Ira proteins). Noisy oscillations related to the activity of this pathway were reported by looking at the nuclear localization of the transcription factor Msn2, and sustained oscillations of the nuclear accumulation of Msn2 under the condition of limiting glucose were observed. We were able to reproduce the periodic accumulation of Msn2-GFP protein in a yeast cell under the condition of limiting glucose, and we also detected oscillations of cAMP. We used a sensor based on a fusion protein between YFP-Epac2-CFP expressed in yeast cells. The FRET between CFP and YFP is controlled by the cAMP concentration. This sensor allows us to monitor changes in cAMP concentrations in a single yeast cell over a long time. Using this method, we were able to detect noisy oscillations of cAMP levels in single yeast cells under conditions of nutritional stress caused by limiting glucose availability.

Pharmacokinetic/Pharmacodynamic Assessment of Selective Phosphodiesterase Inhibitors in a Mouse Model of Autoimmune Hepatitis.

Autoimmune hepatitis (AIH) is a life-threatening disorder currently treated with nonspecific immunosuppressive drugs. It is postulated that phosphodiesterase (PDE) inhibitors, as agents exerting anti-inflammatory and immunomodulatory activities, may constitute a possible treatment of autoimmune disorders. This study develops a pharmacokinetic/pharmacodynamic (PK/PD) model to assess the effects of PDE-selective inhibitors, namely, cilostazol (PDE3), rolipram (PDE4), and BRL-50481 (PDE7), in a mouse model of AIH. The pharmacokinetics of the PDE inhibitors (PDEi) were assessed in male BALB/c mice after intraperitoneal administration. In pharmacodynamic studies, mice received PDEi and AIH was induced in these animals by intravenous injection of concanavalin A (ConA). Serum drug concentrations, tumor necrosis factor α (TNFα), interleukin 17 (IL-17), and aminotransferase activities were quantified. The PK/PD analysis was performed using ADAPT5 software. The PK/PD model assumes inhibition of cAMP hydrolysis in T cells by PDEi, ConA-triggered formation of TNFα and IL-17, suppression of TNFα and IL-17 production by cAMP, and stimulatory effects of TNFα and IL-17 on the hepatic release of aminotransferases. Selective blockage of PDE4 leads to the highest inhibition of cAMP degradation in T cells and amelioration of disease outcomes. However, inhibition of both PDE3 and PDE7 also contribute to this effect. The proposed PK/PD model may be used to assess and predict the activities of novel PDEi and their combinations in ConA-induced hepatitis. A balanced suppression of different types of PDE appears to be a promising treatment option for AIH; however, this hypothesis warrants testing in humans based on translation of the PK/PD model into clinical settings. SIGNIFICANCE STATEMENT: A novel PK/PD model of PDE inhibitor effects in mice with ConA-induced autoimmune hepatitis was developed involving a mechanistic component describing changes in cAMP concentrations in mouse T cells. According to model predictions, inhibition of PDE4 in T cells causes the highest cAMP elevation in T cells, but suppression of PDE3 and PDE7 also contribute to this effect. A balanced inhibition of PDE3, PDE4, and PDE7 appears to be a promising treatment strategy for AIH.

Ion Channel-Based Reporters for cAMP Detection.

In the last 20years tremendous progress has been made in the development of single cell cAMP sensors. Sensors are based upon cAMP binding proteins that have been modified to transduce cAMP concentrations into electrical or fluorescent readouts that can be readily detected using patch clamp amplifiers, photomultiplier tubes, or cameras. Here, we describe two complementary approaches for the detection and measurement of cAMP signals near the plasma membrane of cells using cyclic nucleotide (CNG) channel-based probes. These probes take advantage of the ability of CNG channels to transduce small changes in cAMP concentration into ionic flux through channel pores that can be readily detected by measuring Ca2+ and/or Mn2+ influx or by measuring ionic currents.

Placentas associated with female neonates from pregnancies complicated by urinary tract infections have higher cAMP content and cytokines expression than males.

The cAMP pathway is involved in important biological processes including immune regulation and hormone signaling. At the feto-maternal unit, cAMP participates in placental function/physiology and the establishment of immunoendocrine networks. Low cAMP in male fetuses cord blood has been linked to poorer perinatal outcomes; however, cAMP placental content and its relationship with immune factors and fetal sex in an infectious condition have not been investigated. Sex-dependent changes in cAMP content and its association with cytokines and antimicrobial peptides expression were studied in human placentas collected from normal pregnancies and with urinary tract infections (UTI). Radioimmunoassay was used to quantify cAMP in placental tissue, while immune markers expression was studied by qPCR. Additionally, cAMP effect on antimicrobial peptides expression was studied in cultured trophoblasts challenged with lipopolysaccharide, to mimic an infection. In UTI, placentas from female neonates had higher cAMP tissue content and increased expression of TNFA, IL1B, and IL10 than those from males, where IFNG was more elevated. While cAMP negatively correlated with maternal bacteriuria and IFNG, it positively correlated with the antimicrobial peptide S100A9 expression in a sex-specific fashion. In cultured trophoblasts, cAMP significantly stimulated β-defensin-1 while reduced the lipopolysaccharide-dependent stimulatory effect on β-defensin-2, β-defensins-3, and S100A9. Our results showed higher cAMP content and defense cytokines expression in placentas associated with female neonates from pregnancies complicated by UTI. The associations between cAMP and bacteriuria/immune markers, together with cAMP's ability to differentially regulate placental antimicrobial peptides expression, suggest a dual modulatory role for cAMP in placental immunity.

Synthesis and Biological Activity of the Pyridine-hexacyclic-steroid Derivative on a Heart Failure Model.

Several drugs with inotropic activity have been synthesized; however, there is very little information on biological activity exerted by steroid derivatives in the cardiovascular system. The aim of this research was to prepare a steroid-pyridine derivative to evaluate the effect it exerts on left ventricular pressure and characterize its molecular interaction. The first stage was carried out through the synthesis of a steroid-pyridine derivative using some chemical strategies. The second stage involved the evaluation of the biological activity of the steroid-pyridine derivative on left ventricular pressure using a model of heart failure in the absence or presence of the drugs, such as flutamide, tamoxifen, prazosin, metoprolol, indomethacin, and nifedipine. The results showed that steroid-pyridine derivative increased left ventricular pressure in a dose-dependent manner (0.001-100 nM); however, this phenomenon was significantly inhibited only by nifedipine at a dose of 1 nM. These results indicate that positive inotropic activity produced by the steroid-pyridine derivative was via calcium channel activation. Furthermore, the biological activity exerted by the steroid-pyridine derivative on the left ventricle produces changes in cAMP concentration. It is noteworthy that positive inotropic activity produced by this steroid-pyridine derivative involves a different molecular mechanism compared to other positive inotropic drugs. Therefore, this steroid could be a good candidate for the treatment of heart failure.

PKA functions in metabolism and resistance to obesity: lessons from mouse and human studies.

Both direct and indirect evidence demonstrate a central role for the cAMP-dependent protein kinase (PKA) signaling pathway in the regulation of energy balance and metabolism across multiple systems. However, the ubiquitous pattern of PKA expression across cell types poses a challenge in pinpointing its tissue-specific regulatory functions and further characterizing its many downstream effects in certain organs or cells. Mouse models of PKA deficiency and over-expression and studies in living cells have helped clarify PKA function in adipose tissue (AT), liver, adrenal, pancreas, and specific brain nuclei, as they pertain to energy balance and metabolic dysregulation. Limited studies in humans suggest differential regulation of PKA in AT of obese compared to lean individuals and an overall dysregulation of PKA signaling in obesity. Despite its complexity, under normal physiologic conditions, the PKA system is tightly regulated by changes in cAMP concentrations upstream via adenylate cyclase and downstream by phosphodiesterase-mediated cAMP degradation to AMP and by changes in PKA holoenzyme stability. Adjustments in the PKA system appear to be important to the development and maintenance of the obese state and its associated metabolic perturbations. In this review we discuss the important role of PKA in obesity and its involvement in resistance to obesity, through studies in humans and in mouse models, with a focus on the regulation of PKA in energy expenditure, intake behavior, and lipid and glucose metabolism.

Biphasic Effect of Sildenafil on Energy Sensing is Mediated by Phosphodiesterases 2 and 3 in Adipocytes and Hepatocytes.

Sirt1 (Sirtuin 1), AMPK (AMP-activated protein kinase), and eNOS (endothelial nitric oxide synthase) modulate hepatic energy metabolism and inflammation and play a major role in the development of NASH. Cyclic nucleotide phosphodiesterases (PDEs) play an important role in signal transduction by modulating intracellular levels of cyclic nucleotides. We previously found the PDE5 inhibitor sildenafil to synergize with leucine and leucine-metformin combinations in preclinical studies of NASH and obesity. However, efficacy is diminished at higher sildenafil concentrations. Herein, we have successfully modeled the U-shaped sildenafil dose-response in vitro and utilized this model to assess potential mechanisms of this dose-response relationship. Adipocytes and liver cells were treated with leucine (0.5 mM) and different concentrations of sildenafil (1 nM to 100 µM). cAMP, cGMP, and P-AMPK protein expression were used to demonstrate the biphasic response for increasing concentrations of sildenafil. The reversal with higher sildenafil levels was blunted by PDE2 inhibition. These data indicate that sildenafil-mediated increases in cGMP inhibits PDE3 at lower concentrations, which increases cAMP. However, further increases in cGMP from higher sildenafil concentrations activate PDE2 and consequently decrease cAMP, which demonstrates crosstalk between cAMP and cGMP via PDE2, PDE3, and PDE5. These changes in cAMP concentration are further reflected in downstream effects, including AMPK activation.

Activities of Adenylate Cyclase and Changes in cAMP Concentration in Root Cells of Pea Seedlings Infected with Mutualists and Phytopathogens

Work was carried out on pea (Pisum sativum L.) seedling roots to assess the attachment of the nitrogen-fixing symbiotic bacteria Rhizobium leguminosarum bv. vicea (Rlv) and the bacterial phytopathogens—specific Pseudomonas syringae pv. pisi (Psp) and nonspecific Clavibacter michiganensis ssp. sepedonicus (Cms). Different root zones were examined: (I) the meristem, 2 mm from the root tip; (II) the root hair-free zone, 27 mm; (III) the zone of root hair anlages, 712 mm; (IV) the young root hair zone, 1217 mm; and (V) the zone of root hair that completed the growth, 1722 mm. It was found earlier that the zones differed in their susceptibility to Rlv. In the present work, reactions of particular components of the adenylate cyclase signaling system (ACSS) were estimated, i.e., concentration of cAMP and activities of transmembrane adenylate cyclase (tAC) and soluble adenylate cyclase (sAC) in these zones after different times post inoculation (5, 15, 120, and 360 min). It was revealed that the degree of activation of particular components of ACSS did not depend on the sorption rate of differently specialized bacteria. Upon contact with Rlv, the character of changes in tAC and sAC activities was almost the same in different root zones and resembled the dynamics of the cAMP content. Inoculation with Psp changed the cAMP level similarly to that with Rlv, but the dynamics of tAC and sAC was opposite to each other in most cases. Inoculation with Cms, in spite of the absence of its attachment, elevated the cAMP content and activated tAC and sAC. It is suggested that the above-mentioned changes in ACSS is associated with exometabolites of Rlv, Psp, and Cms, which activate the PAMP-induced immunity of the pea seedling cells. The uniform dynamics of cAMP in different root zones upon the exposure to Rlv and Psp seems to reflect the specific reaction and, presumably, fulfills different functions—regulatory with Rlv and defensive with Psp. Upon short-term contact with Cms, the cAMP dynamics in the same root zones displayed a nonspecific character that might be related to the rate of adsorption of exopolysaccharides by the root hair. The systemic response of ACSS was observed in the hypocotyls of the seedlings exposed to any of the three organisms.

Responsiveness of pituitary to galanin throughout the reproductive cycle of male European sea bass (Dicentrarchus labrax)

The neuropeptide galanin (Gal) is a putative factor regulating puberty onset and reproduction through its actions on the pituitary. The present study investigated the pituitary responsiveness to galanin and the patterns of galanin receptors (Galrs) expression throughout the reproductive cycle of two years old male European sea bass (Dicentrarchus labrax), an important aquaculture species. Quantitative analysis of pituitary and hypothalamus transcript expression of four galr subtypes revealed differential regulation according to the testicular developmental stage, with an overall decrease in expression from the immature stage to the mid-recrudescence stage. Incubation of pituitary cells with mammalian 1–29Gal peptide induced significant changes in cAMP concentration, with sensitivities that varied according to the testicular development stages. Furthermore 1–29Gal was able to stimulate both follicle stimulating hormone (Fsh) and luteinizing hormone (Lh) release from pituitary cell suspensions. The magnitude of the effects and effective concentrations varied according to reproductive stage, with generalized induction of Fsh and Lh release in animals sampled in January (full spermiation). The differential expression of galrs in pituitary and hypothalamus across the reproductive season, together with the differential effects of Gal on gonadotropins release in vitro strongly suggests the involvement of the galaninergic system in the regulation the hypothalamus-pituitary-gonad axis of male sea bass. This is to our knowledge the first clear evidence for the involvement of galanin in the regulation of reproduction in non-mammalian vertebrates.

Microparticle Delivery of Cyclic AMP to Pulmonary Microvascular Endothelial Cells

Microparticles (MPs) are important for the exchange of information between cells. MPs are extracellular vesicles that range in size from 0.1 to 1 μm in diameter. They are constitutively released from many cell types and are thought to play a role in vascular homeostasis. Further, the number and content of MPs are altered in pathologies involving inflammation and vascular cell activation. MPs carry intracellular constituents from parent cells that affect target cell function. However, the mechanisms of delivery of the MP payload to target cells are unknown. Understanding the payload delivery system between MPs and target cells is the focus of this project.We recently discovered that cyclic adenosine monophosphate (cAMP), a ubiquitous second messenger, is found in MPs released from pulmonary microvascular endothelial cells (PMVECs). Additionally, MPs isolated from PMVECs treated with β adrenergic agonists and phosphodiesterase (PDE) inhibitors have increased cAMP levels. This suggests that different levels of cAMP are packaged in MPs from the parent cell. MPs containing maximal cAMP were therefore used to study the delivery of MP payload to target cells.We developed mathematical models of MP payload delivery to subcellular compartments of target PMVECs to better understand how the cAMP signaling pathway may be altered by MP‐mediated cAMP delivery. Simulations indicate that if the cAMP contained in one MP were released into a near‐membrane compartment of a target cell, cAMP levels within that compartment would be sufficient to activate protein kinase A (PKA) and Exchange protein activated by cAMP (Epac) for several minutes. Release of cAMP from a single MP released into the larger volume of the bulk cytoplasm would have little effect on target cell signaling. However, several MPs are often internalized by a target cell and their combined cAMP content would sufficiently elevate bulk cytosolic cAMP levels to activate PKA.To experimentally test the predictions of this model, MPs were collected from PMVECs treated with rolipram (a PDE type 4 inhibitor; 10 μM, 5 min) and isoproterenol (a β adrenergic agonist; 1 μM, 10 min). These MPs were added to cells transfected with a cytosolic cAMP FRET sensor. Time lapse hyperspectral image stacks were acquired using a Nikon A1R confocal microscope. Custom hyperspectral analysis scripts in MatLab (MathWorks) were used to unmix fluorescent spectra and quantify cAMP concentration changes throughout the target cell. Preliminary results indicate that MPs significantly increased cAMP levels in target cells compared to control. Together, these data indicate that MPs can deliver cAMP as a functional payload. Future studies will determine whether the MP‐delivered cAMP is sufficient to trigger sustained responses in target cells and contribute to MP‐mediated signaling events.Support or Funding InformationThis work was supported by NIH T32HL076125, P01HL066299, S10RR027535, and AHA 11SDG7390037.

5 Dimensional Hyperspectral FRET Imaging and Analysis Approaches for Measuring cAMP Gradients

cAMP is a second messenger that regulates numerous cellular and physiological functions. A growing body of evidence suggests that cAMP signals are localized resulting in the formation of spatial cAMP gradients. Recently, Forster Resonance energy transfer (FRET) imaging has become a standard method for assessing changes in cAMP concentration. However, many FRET probes have low signal-to-noise ratios and limited dynamic range, making measurements of cAMP difficult. Our previous work suggested that hyperspectral imaging and analysis allows quantitative measurement of FRET efficiencies in 3D (x, y, and t) within single cells. However, traditional (x, y, and t) approaches of measuring FRET often lead to misinterpretation of data. Here, we present data demonstrating that hyperspectral imaging approaches can be used for 5D (x, y, z, λ and t) assessment of FRET efficiency in living cells allowing the study of spatially-localized cAMP signals. We utilized H188-2 (cAMP reporter) and AKAR4 (PKA activity reporter) FRET biosensors that were transfected into PMVECs and HEK 293 cells, respectively. Time lapse hyperspectral Z-stack images were acquired using a Nikon A1R confocal microscope. We used custom MATLAB scripts to calculate FRET efficiencies from image data. These approaches allowed the measurement of spatial cAMP gradients and PKA activity induced by agonist treatment. We found that cAMP gradients developed from the apical to the basolateral side of PMVECs when treated with isoproterenol, while gradients developed from both the basolateral and apical side when treated with PGE1. In addition, we observed an increase in localized phosphorylation upon treatment with isoproterenol and PGE1 in HEK293 cells. Thus, these studies demonstrate the feasibility for simultaneously studying cAMP gradients and PKA activity in cells.This work was supported by AHA 16PRE27130004, NIH P01HL066299, NIH S10RR027535, and the Abraham Mitchell Cancer Research Fund.

Exchange protein directly activated by cAMP (EPAC) regulates neuronal polarization through Rap1B

Acquisition of neuronal polarity is a complex process involving cellular and molecular events. The second messenger cyclic adenosine monophosphate (cAMP) is involved in axonal specification through activation of protein kinase A (PKA). However, an alternative cAMP‐dependent mechanism involves the exchange protein directly activated by cAMP (EPAC), which also responds to physiological changes in cAMP concentration, promoting activation of the small Rap GTPases. Here we present evidence that EPAC signaling contributes to axon specification and elongation. In primary rat hippocampal neurons, EPAC isoforms were differentially expressed during axon specification. Furthermore, 8‐pCPT, an EPAC pharmacological activation and genetic manipulations of EPAC in neurons induced supernumerary axons, indicative of Rap1b activation. Moreover, 8‐pCPT–treated neurons expressed ankyrin G and other markers of mature axons such as synaptophysin and axonal accumulation of vGLUT1. In contrast, pharmacological inhibition of EPAC delayed neuronal polarity. Genetic manipulations to inactivate EPAC1 using either shRNA or neurons derived from EPAC1 knock‐out mice lead to axon elongation and polarization defects. Interestingly, multiaxonic neurons generated by 8‐pCPT treatments in wild type neurons were not found in EPAC1 knock‐out mice neurons. Altogether these results propose that EPAC signaling is an alternative and complementary mechanism for cAMP‐dependent axon determination.

Immunomodulatory Effects Mediated by Dopamine.

Dopamine (DA), a neurotransmitter in the central nervous system (CNS), has modulatory functions at the systemic level. The peripheral and central nervous systems have independent dopaminergic system (DAS) that share mechanisms and molecular machinery. In the past century, experimental evidence has accumulated on the proteins knowledge that is involved in the synthesis, reuptake, and transportation of DA in leukocytes and the differential expression of the D1-like (D1R and D5R) and D2-like receptors (D2R, D3R, and D4R). The expression of these components depends on the state of cellular activation and the concentration and time of exposure to DA. Receptors that are expressed in leukocytes are linked to signaling pathways that are mediated by changes in cAMP concentration, which in turn triggers changes in phenotype and cellular function. According to the leukocyte lineage, the effects of DA are associated with such processes as respiratory burst, cytokine and antibody secretion, chemotaxis, apoptosis, and cytotoxicity. In clinical conditions such as schizophrenia, Parkinson disease, Tourette syndrome, and multiple sclerosis (MS), there are evident alterations during immune responses in leukocytes, in which changes in DA receptor density have been observed. Several groups have proposed that these findings are useful in establishing clinical status and clinical markers.

Antipyretic Effects of Eupatorium chinense and Its Mechanism

ObjectiveTo investigate the antipyretic effect of Eupatorium chinense and its mechanism. MethodsThe content of arginine vasopressin (AVP) in ventral septal area (VSA) and blood plasma, and cyclic adenosine monophosphate (cAMP) levels of hypothalamus and blood plasma were determined by enzyme linked immunosorbent assay. ResultsThe body temperature (Tb) was decreased at 1 h after administration of E. chinense (3 and 6 g/kg) and Aspirin (0.3 g/kg) respectively, which was significantly different from the temperature of fever model group. The antipyretic effect of Aspirin and E. chinense lasted for longer time. Aspirin (0.3 g/kg) and E. chinense (3 and 6 g/kg) reduced the level of cAMP in hypothalamus of fever rats and increased AVP content in plasma. The changes in cAMP content in plasma of all drug treatment groups were not obvious. ConclusionE. chinense has strong antipyretic effect and may affect the production of AVP and cAMP in fever rats.

Channel-based reporters for cAMP detection.

In the last 15 years, tremendous progress has been made in the development of single-cell cAMP sensors. Sensors are based upon cAMP-binding proteins that have been modified to transduce cAMP concentrations into electrical or fluorescent readouts that can be readily detected using patch clamp amplifiers, photomultiplier tubes, or cameras. Here we describe two complementary approaches for the detection and measurement of cAMP signals near the plasma membrane of cells. These probes take advantage of the ability of cyclic nucleotide-gated (CNG) channels to transduce small changes in cAMP concentrations into ionic flux through channel pores that can be readily detected by measuring Ca(2+) and/or Mn(2+) influx or by measuring ionic currents.

Soluble adenylyl cyclase of sea urchin spermatozoa

Fertilization, a key step in sexual reproduction, requires orchestrated changes in cAMP concentrations. It is notable that spermatozoa (sperm) are among the cell types with extremely high adenylyl cyclase (AC) activity. As production and consumption of this second messenger need to be locally regulated, the discovery of soluble AC (sAC) has broadened our understanding of how such cells deal with these requirements. In addition, because sAC is directly regulated by HCO3− it is able to translate CO2/HCO3−/pH changes into cAMP levels. Fundamental sperm functions such as maturation, motility regulation and the acrosome reaction are influenced by cAMP; this is especially true for sperm of the sea urchin (SU), an organism that has been a model in the study of fertilization for more than 130years. Here we summarize the discovery and properties of SU sperm sAC, and discuss its involvement in sperm physiology. This article is part of a Special Issue entitled: The role of soluble adenylyl cyclase in health and disease.

A Luminescent Biosensor with Increased Dynamic Range for Intracellular cAMP

The second messenger cAMP is a key mediator of signal transduction following activation of G-protein coupled receptors. Investigations on Gs-coupled receptors would benefit from a second messenger assay that allows continuous monitoring of kinetic changes in cAMP concentration over a broad dynamic range. To accomplish this, we have evolved a luminescent biosensor for cAMP to better encompass the physiological concentration ranges present in living cells. When compared to an immunoassay, the evolved biosensor construct was able to accurately track both the magnitude and kinetics of cAMP change using a far less labor intensive format. We demonstrate the utility of this construct to detect a broad range of receptor activity, together with showing suitability for use in high-throughput screening.

Regulation of Inositol 1,4,5-Trisphosphate Receptors by cAMP Independent of cAMP-dependent Protein Kinase

In HEK cells stably expressing type 1 receptors for parathyroid hormone (PTH), PTH causes a sensitization of inositol 1,4,5-trisphosphate receptors (IP3R) to IP3 that is entirely mediated by cAMP and requires cAMP to pass directly from type 6 adenylyl cyclase (AC6) to IP3R2. Using DT40 cells expressing single subtypes of mammalian IP3R, we demonstrate that high concentrations of cAMP similarly sensitize all IP3R isoforms to IP3 by a mechanism that does not require cAMP-dependent protein kinase (PKA). IP3 binding to IP3R2 is unaffected by cAMP, and sensitization is not mediated by the site through which ATP potentiates responses to IP3. In single channel recordings from excised nuclear patches of cells expressing IP3R2, cAMP alone had no effect, but it increased the open probability of IP3R2 activated by a submaximal concentration of IP3 alone or in combination with a maximally effective concentration of ATP. These results establish that cAMP itself increases the sensitivity of all IP3R subtypes to IP3. For IP3R2, this sensitization results from cAMP binding to a novel site that increases the efficacy of IP3. Using stably expressed short hairpin RNA to reduce expression of the G-protein, Gαs, we demonstrate that attenuation of AC activity by loss of Gαs more substantially reduces sensitization of IP3R by PTH than does comparable direct inhibition of AC. This suggests that Gαs may also specifically associate with each AC·IP3R complex. We conclude that all three subtypes of IP3R are regulated by cAMP independent of PKA. In HEK cells, where IP3R2 selectively associates with AC6, Gαs also associates with the AC·IP3R signaling junction.