Activation Of Second Messengers Research Articles

Energy management and mitochondrial dynamics in cerebral cortex during endotoxemia

Mitochondria play an essential role in inflammatory processes such as sepsis or endotoxemia, contributing to organ-cellular redox metabolism, emerging as the energy hub of the cell, and as an important center of action of second messengers. In this work, we aimed to elucidate the energy state, redox balance, and mitochondrial remodeling status in cerebral cortex in an experimental model of endotoxemia. Female Sprague-Dawley rats were subjected to a single dose of LPS (ip 8 mg kg−1 body weight) for 6 h. State 3 O2 consumption was observed increased, ATP production and P/O ratio were observed decreased, probably indicating an inefficient oxidative phosphorylation process. O2− production and both systemic and tissue NO markers were observed increased in treated animals. The existence of nitrated proteins suggests an alteration in the local redox balance and possible harmful effects over energetic processes. Increases in PGC-1α and mtTFA expression, and in OPA-1 expression, suggest an increase in de novo formation of mitochondria and fusion of pre-existing mitochondria. The observed elongation of mitochondria correlates with the occurrence of mild mitochondrial dysfunction and increased levels of systemic NO. Our work presents novel results that contribute to unravel the mechanism by which the triad endotoxemia-redox homeostasis-energy management interact in the cerebral cortex, leading to propose a relevant mechanism for future developing therapeutics with the aim of preserving this organ from inflammatory and oxidative damage.

Opioid Receptor Regulation of Neuronal Voltage-Gated Calcium Channels.

Neuronal voltage-gated calcium channels play a pivotal role in the conversion of electrical signals into calcium entry into nerve endings that is required for the release of neurotransmitters. They are under the control of a number of cellular signaling pathways that serve to fine tune synaptic activities, including G-protein coupled receptors (GPCRs) and the opioid system. Besides modulating channel activity via activation of second messengers, GPCRs also physically associate with calcium channels to regulate their function and expression at the plasma membrane. In this mini review, we discuss the mechanisms by which calcium channels are regulated by classical opioid and nociceptin receptors. We highlight the importance of this regulation in the control of neuronal functions and their implication in the development of disease conditions. Finally, we present recent literature concerning the use of novel μ-opioid receptor/nociceptin receptor modulators and discuss their use as potential drug candidates for the treatment of pain.

In vitro human stem cell derived cultures to monitor calcium signaling in neuronal development and function.

The development of the human brain involves multiple cellular processes including cell division, migration, and dendritic growth. These processes are triggered by developmental cues and lead to interactions of neurons and glial cells to derive the final complex organization of the brain. Developmental cues are transduced into cellular processes through the action of multiple intracellular second messengers including calcium. Calcium signals in cells are shaped by large number of proteins and mutations in several of these have been reported in human patients with brain disorders. However, the manner in which such mutations impact human brain development in vivo remains poorly understood. A key limitation in this regard is the need for a model system in which calcium signaling can be studied in neurons of patients with specific brain disorders. Here we describe a protocol to differentiate human neural stem cells into cortical neuronal networks that can be maintained as live cultures up to 120 days in a dish. Our protocol generates a 2D in vitro culture that exhibits molecular features of several layers of the human cerebral cortex. Using fluorescence imaging of intracellular calcium levels, we describe the development of neuronal activity as measured by intracellular calcium transients during development in vitro. These transients were dependent on the activity of voltage gated calcium channels and were abolished by blocking sodium channel activity. Using transcriptome analysis, we describe the full molecular composition of such cultures following differentiation in vitro thus offering an insight into the molecular basis of activity. Our approach will facilitate the understanding of calcium signaling defects during cortical neuron development in patients with specific brain disorders and a mechanistic analysis of these defects using genetic manipulations coupled with cell biological and physiological analysis.

Developing Inversely Responding Genetically Encoded Superresolution Reporters

Super resolution activity imaging relies on specialized biosensors to illuminate the activity of cellular components. Unlike FRET or FLIM based biosensing, the goal of this imaging mode is to specifically reveal the compartmentalized action of a small number of molecules within a limited region in the cell. This untangles global activity from the well-orchestrated local dynamics required by numerous orthogonal pathways. Such details are essential for a precise mechanistic understanding of cell signaling. We have recently reported a general methodology termed Fluorescent Fluctuation Increase by Contact (FLINC) to monitor enzyme activities below the diffraction limit by converting FRET-based biosensors into super-resolution compatible sensors. While FLINC's nanometer sensitivity, reversibility, and high spatial precision within living cells make it a promising platform, an important issue remains. Genetically encoded FRET biosensor often show a decrease in signal upon the activation of the gene of interest. The characteristic “increase” of FLINC makes it currently unable to help convert these so-called negative biosensors to super-resolution mode. We outline our strategies and describe the results in developing a new “negative FLINC” system. This will permit us to study the compartmentalized activity of enzymes and second messengers lacking a positive FRET-biosensor such as cAMP, phosphoinositides, and Src family kinases.

Oxidative Stress and Cancer.

Cancer is considered a major cause of death worldwide. The etiology of cancer is linked to environmental and genetic inheritance causes. Approximately 90 percent of all human cancers have an environmental cause (non-genetic inheritance) predominantly through lifestyle choices (smoking, diet, UV radiation) while the remaining due to infections and chemical exposure. Cancer is a multistage process that involves mutational changes and uncontrolled cell proliferation. Research has firmly established a causal and contributory role of oxidative stress and oxidative damage in cancer initiation and progression. The purpose of this article is to review the role that oxidative stress and reactive oxygen species play in the development of cancer. Both endogenous and exogenous sources of reactive oxygen species result in increased oxidative stress in the cell. Excess reactive oxygen fumed can result in damage to and modification of cellular macromolecules most importantly genomic DNA that can produce mutations. In addition, oxidative stress modulates gene expression of downstream targets involved in DNA repair, cell proliferation and antioxidants. The modulation of gene expression by oxidative stress occurs in part through activation or inhibition of transcription factors and second messengers. The role of single nuclear polymorphism for oxidative DNA repair and enzymatic antioxidants is important in determining the potential human cancer risk. oxidative stress and the resulting oxidative damage are important contributors to the formation and progression of cancer.

Role of melatonin on oxidative stress in traumatic brain injury

Oxidative stress occurs in the several physiological processes such as phagocytic activity and mitochondrial membrane functions. Oxidative stress is controlled by several enzymatic and non-enzymatic antioxidants. Traumatic brain injury is one of the most common causes of the mortalities. Secondary events occur after primary events like shearing of nerve cells and blood vessels, cause posttraumatic neurodegenerations with an increase in ROS and ROSmediated lipid peroxidation. Melatonin is a member of non-enzymatic antioxidant group. The protective effects of melatonin on traumatic brain injury have been shown in vivo and in vitro studies (Barlow et al. 2018). Also melatonin has been shown to counteract oxidative stress-induced pathophysiologic conditions like ischemia/reperfusion injury, neuronal excitotoxicity and chronic inflammation. Recently, it was reported that TBI-induced oxidative stress in experimental TBI was inhibited by the melatonin treatment (Senol and Nazıroğlu, 2014). In the oral presentation, I will review recent studies on traumatic brain injury in human and rodents. I concluded that the oxidative stress causes changes through activation of second messengers, which may lead to the pathology of TBI, althoughmelatonin has protective effects on the pathology. It seems to that the exact relationship between melatonin and TBI still remain to be determined.

Lysophosphatidylcholine Promotes Phagosome Maturation and Regulates Inflammatory Mediator Production Through the Protein Kinase A-Phosphatidylinositol 3 Kinase-p38 Mitogen-Activated Protein Kinase Signaling Pathway During Mycobacterium tuberculosis Infection in Mouse Macrophages.

Tuberculosis is caused by the infectious agent Mycobacterium tuberculosis (Mtb). Mtb has various survival strategies, including blockade of phagosome maturation and inhibition of antigen presentation. Lysophosphatidylcholine (LPC) is a major phospholipid component of oxidized low-density lipoprotein and is involved in various cellular responses, such as activation of second messengers and bactericidal activity in neutrophils. In this study, macrophages were infected with a low infectious dose of Mtb and treated with LPC to investigate the bactericidal activity of LPC against Mtb. In macrophages infected with Mtb strain, H37Ra or H37Rv, LPC suppressed bacterial growth; however, this effect was suppressed in bone marrow-derived macrophages (BMDMs) isolated from G2A (a G protein-coupled receptor involved in some LPC actions) knockout mice. LPC also promoted phagosome maturation via phosphatidylinositol 3 kinase (PI3K)–p38 mitogen-activated protein kinase (MAPK)-mediated reactive oxygen species production and intracellular Ca2+ release during Mtb infection. In addition, LPC induced increased levels of intracellular cyclic adenosine monophosphate (cAMP) and phosphorylated glycogen synthase kinase 3 beta (GSK3β) in Mtb-infected macrophages. Protein kinase A (PKA)-induced phosphorylation of GSK3β suppressed activation of NF-κB in LPC-treated macrophages during Mtb infection, leading to decreased secretion of pro-inflammatory cytokines and increased secretion of anti-inflammatory cytokines. These results suggest that LPC can effectively control Mtb growth by promoting phagosome maturation via cAMP-induced activation of the PKA–PI3K–p38 MAPK pathway. Moreover, LPC can regulate excessive production of pro-inflammatory cytokines associated with bacterial infection of macrophages.

CD44 Signaling Mediates High Molecular Weight Hyaluronan-Induced Antihyperalgesia.

We studied, in male Sprague Dawley rats, the role of the cognate hyaluronan receptor, CD44 signaling in the antihyperalgesia induced by high molecular weight hyaluronan (HMWH). Low molecular weight hyaluronan (LMWH) acts at both peptidergic and nonpeptidergic nociceptors to induce mechanical hyperalgesia that is prevented by intrathecal oligodeoxynucleotide antisense to CD44 mRNA, which also prevents hyperalgesia induced by a CD44 receptor agonist, A6. Ongoing LMWH and A6 hyperalgesia are reversed by HMWH. HMWH also reverses the hyperalgesia induced by diverse pronociceptive mediators, prostaglandin E2, epinephrine, TNFα, and interleukin-6, and the neuropathic pain induced by the cancer chemotherapy paclitaxel. Although CD44 antisense has no effect on the hyperalgesia induced by inflammatory mediators or paclitaxel, it eliminates the antihyperalgesic effect of HMWH. HMWH also reverses the hyperalgesia induced by activation of intracellular second messengers, PKA and PKCε, indicating that HMWH-induced antihyperalgesia, although dependent on CD44, is mediated by an intracellular signaling pathway rather than as a competitive receptor antagonist. Sensitization of cultured small-diameter DRG neurons by prostaglandin E2 is also prevented and reversed by HMWH. These results demonstrate the central role of CD44 signaling in HMWH-induced antihyperalgesia, and establish it as a therapeutic target against inflammatory and neuropathic pain.SIGNIFICANCE STATEMENT We demonstrate that hyaluronan (HA) with different molecular weights produces opposing nociceptive effects. While low molecular weight HA increases sensitivity to mechanical stimulation, high molecular weight HA reduces sensitization, attenuating inflammatory and neuropathic hyperalgesia. Both pronociceptive and antinociceptive effects of HA are mediated by activation of signaling pathways downstream CD44, the cognate HA receptor, in nociceptors. These results contribute to our understanding of the role of the extracellular matrix in pain, and indicate CD44 as a potential therapeutic target to alleviate inflammatory and neuropathic pain.

Synapses in the spotlight with syntheticoptogenetics.

Membrane receptors and ion channels respond to various stimuli and relay that information across the plasma membrane by triggering specific and timed processes. These include activation of second messengers, allowing ion permeation, and changing cellular excitability, to name a few. Gaining control over equivalent processes is essential to understand neuronal physiology and pathophysiology. Recently, new optical techniques have emerged proffering new remote means to control various functions of defined neuronal populations by light, dubbed optogenetics. Still, optogenetic tools do not typically address the activity of receptors and channels native to neurons (or of neuronal origin), nor gain access to their signaling mechanisms. A related method-synthetic optogenetics-bridges this gap by endowing light sensitivity to endogenous neuronal receptors and channels by the appending of synthetic, light-receptive molecules, or photoswitches. This provides the means to photoregulate neuronal receptors and channels and tap into their native signaling mechanisms in select regions of the neurons, such as the synapse. This review discusses the development of synthetic optogenetics as a means to study neuronal receptors and channels remotely, in their natural environment, with unprecedented spatial and temporal precision, and provides an overview of tool design, mode of action, potential clinical applications and insights and achievements gained.

Studying the Effects of Glucagon-Modulators on the Activity of Second Messengers in Subcellular Compartments of Pancreatic Islet Cells

The convergence of multiple paracrine, juxtacrine, and intracellular signals is necessary to achieve proper glucagon secretion from the α-cells of the pancreatic islet. Elevated or dysregulated secretion of glucagon severely impairs the glycemic control of diabetic patients. Thus it is important to clarify how all these stimuli co-operate and interact to stimulate and or inhibit glucagon secretion. Using hyperspectral fluorescence microscopy, we measure the effect of glucagon modulators, insulin, somatostatin, and ephrins simultaneously on the activity of two second messengers, calcium and cyclic adenosine monophosphate (cAMP), in islet cells. We target genetically encoded biosensors to the cytoplasm, to the plasma membrane or to the nucleus. We hypothesize that in response to different stimuli, second messengers’ activities change differently in various cell compartments. Individual stimuli or their combination result in various patterns of activities, which we can use to improve our current mathematical model of glucose-inhibited glucagon secretion. These studies utilize both cell line models and primary cultured islet cells. This combination of specimens allows us to determine the conditions under which we can rely on cell lines to mimic intact islets hormone output. Having a reliable cell model for glucagon secretion form α-cells will be very beneficial for drug screening studies.

Calcitriol (1,25-dihydroxyvitamin D3) increases L-type calcium current via protein kinase A signaling and modulates calcium cycling and contractility in isolated mouse ventricular myocytes

Calcitriol, the bioactive metabolite of vitamin D, exerts its effects through interaction with the nuclear vitamin D receptor (VDR) to induce genomic responses. Calcitriol may also induce rapid responses via plasma membrane-associated VDR, involving the activation of second messengers and modulation of voltage-dependent channels. VDR is expressed in cardiomyocytes, but the molecular and cellular mechanisms involved in the rapid responses of calcitriol in the heart are poorly understood. The aim of the present study was to analyze the rapid nongenomic effect of calcitriol on L-type calcium channels, intracellular Ca2+ ([Ca2+]i) transients, and cell contractility in ventricular myocytes. We used the whole-cell patch-clamp technique to record L-type calcium current (ICaL) and confocal microscopy to study global [Ca2+]i transients evoked by electrical stimulation and cell shortening in adult mouse ventricular myocytes treated with vehicle or with calcitriol. In some experiments, ICaL was recorded using the perforated patch-clamp technique. Calcitriol treatment of cardiomyocytes induced a concentration-dependent increase in ICaL density (Half maximal effective concentration (EC50) = 0.23 nM) and a significant increase in peak [Ca2+]i transients and cell contraction. The effect of calcitriol on ICaL was prevented by pretreatment of cardiomyocytes with the protein kinase A (PKA) inhibitor KT-5720 but not with the β-adrenergic blocker propranolol. The effect of calcitriol on ICaL was absent in myocytes isolated from VDR knockout mice. Calcitriol induces a rapid response in mouse ventricular myocytes that involves a VDR-PKA-dependent increase in ICaL density, enhancing [Ca2+]i transients and contraction.

Marked Sexual Dimorphism in the Role of the Ryanodine Receptor in a Model of Pain Chronification in the Rat.

Hyperalgesic priming, an estrogen dependent model of the transition to chronic pain, produced by agonists at receptors that activate protein kinase C epsilon (PKCε), occurs in male but not in female rats. However, activation of second messengers downstream of PKCε, such as the ryanodine receptor, induces priming in both sexes. Since estrogen regulates intracellular calcium, we investigated the interaction between estrogen and ryanodine in the susceptibility to develop priming in females. The lowest dose of ryanodine able to induce priming in females (1 pg) is 1/100,000th that needed in males (100 ng), an effect dependent on the activation of ryanodine receptors. Treatment of female rats with antisense to estrogen receptor alpha (ERα), but not beta (ERβ), mRNA, prevented the induction of priming by low dose ryanodine, and the ERα agonist, PPT, induced ryanodine receptor-dependent priming. In vitro application of ryanodine in low concentration (2 nM) to small DRG neurons cultured from females, significantly potentiated calcium release via ryanodine receptors induced by caffeine. This effect was only observed in IB4+ neurons, cultured in the presence of β-estradiol or PPT. Our results demonstrate a profound regulatory role of ERα in ryanodine receptor-dependent transition to chronic pain.

Evaluation of the impact of neutralizing antibodies on IFNβ response

IFNβ therapeutic action depends on a sequence of biological steps: i) the interaction between interferon beta (IFNβ) and its receptor (IFNAR) located at the cell surface of peripheral blood mononuclear cells; ii) activation of second messengers; iii) transcription of several genes containing specific ISRE regions (Interferon Stimulated Response Elements); and iv) synthesis of specific proteins.Although IFNβ therapy has improved treatment options of patients with multiple sclerosis (MS), the long-term efficacy of IFNβs can be compromised due to the development of neutralizing antibodies (NAbs).High titer NAbs develop in about 15% of patients; they abolish IFNβ biological activity and consequently the therapeutic action of IFNβ. Different IFNβ preparations carry different risks of developing NAbs, ranging from 3 to 28%. The risk of inducing NAbs must be considered in the selection of treatment.Guidelines for NAbs testing and the therapeutic decision in case of NAbs positivity have been established. NAbs positivity predicts MRI and clinical activity. Precocious identification of Nabs-positive patients and switch to alternative treatments can improve the percentage of responders and allow a better allocation of relevant economical resources.

Role of calcium channels in cellular antituberculosis effects: Potential of voltage-gated calcium-channel blockers intuberculosis therapy.

The immunity of human immune cells and their ability to inhibit Mycobacterium tuberculosis (MTB) are key factors in the anti-MTB effect. However, MTB modulates the levels and activity of key intracellular second messengers, such as calcium, to evade protective immune responses. Recent studies suggest that inhibiting L-type calcium channel in immune cells using either antibodies or small interfering RNA increases calcium influx, upregulates the expression of proinflammation genes, and reduces MTB burden. First, we will review the key factors in calcium-signaling pathway that may affect the immunity of immune cells to MTB infection. Second, we will focus on the role of calcium channels in regulating cellular immunity to MTB. Finally, we will discuss the possibility of using calcium-channel blockers as anti-MTB chemotherapy drugs to enhance chemotherapy effects, shorten treatment period, and overcome drug resistance.

Cellular insulin resistance disrupts hypothalamic mHypoA-POMC/GFP neuronal signaling pathways

POMC neurons play a central role in the maintenance of whole-body energy homeostasis. This balance requires proper regulation of POMC neurons by metabolic hormones, such as insulin. However, the heterogeneous cellular population of the intact hypothalamus presents challenges for examining the molecular mechanisms underlying the potent anorexigenic effects of POMC neurons, and there is currently a complete lack of mature POMC neuronal cell models for study. To this end, we have generated novel, immortalized, adult-derived POMC-expressing/α-MSH-secreting cell models, mHypoA-POMC/GFP lines 1-4, representing the fluorescence-activated cell-sorted POMC population from primary POMC-eGFP mouse hypothalamus. The presence of Pomc mRNA in these cell lines was confirmed, and α-MSH was detected via immunofluorescence. α-MSH secretion in the mHypoA-POMC/GFP-1 was found to increase in response to 10 ng/ml ciliary neurotrophic factor (CNTF) or 10 nM insulin as determined by enzyme immunoassay. Further experiments using the mHypoA-POMC/GFP-1 cell line revealed that 10 ng/ml CNTF increases Pomc mRNA at 1 and 2 h after treatment, whereas insulin elicited an increase in Pomc mRNA level and decreases in insulin receptor (Insr (Ir)) mRNA level at 4 h. Furthermore, the activation of IR-mediated downstream second messengers was examined by western blot analysis, following the induction of cellular insulin resistance, which resulted in a loss of insulin-mediated regulation of Pomc and Ir mRNAs. The development of these immortalized neurons will be invaluable for the elucidation of the cellular and molecular mechanisms that underlie POMC neuronal function under normal and perturbed physiological conditions.

Abstract 547: TGFß Response Coupled to Stress Signaling by TGRL Lipolysis in Vascular Endothelial Cells

Elevation of circulating triglyceride-rich lipoproteins (TGRL) in the postprandial state is associated with increased endothelial cell inflammation and dysfunction potentially contributing to atherosclerosis. Previous studies from our laboratory showed that lipolysis products of TGRL induced endothelial cell barrier permeability, up-regulation of cytokines and adhesion molecules, and apoptosis. Gene array studies implicated the Stress Associated Protein Kinase and c-Jun N-terminal kinase (SAPK/JNK) pathway and identified the transcription factor activating transcription factor 3 (ATF3) as highly induced in this process. Additional studies demonstrated activation of second messengers for transforming growth factor beta (TGFβ) signaling (SMAD2/SMAD4) in response to lipolysis product treatment. In the present study, we asked whether TGFβ signaling was implicated in the induction of apoptosis by lipolysis products and whether there was a linkage between TGFβ receptor activation and pro-inflammatory responses. siRNA knockdown of ATF3 transcription abrogated both IL-8 and E-selectin up-regulation in lipolysis product-treated human aortic endothelial cells (HAEC). In addition, ATF3 knockdown prevented activation of caspase 3/7 and induction of p53, markers of apoptosis. Inhibition of the TGFβ Type I activin receptor-like kinase (ALK) receptors (ALKs 4, 5 and 7) by SB 431542 (10 μM) also inhibited inflammatory up-regulation, ATF3 induction and apoptosis. Both ATF3 knockdown and TGFβ receptor blockade prevented c-Jun phosphorylation, a co-activating transcription factor with ATF3 of AP-1 transcription sites. Our findings implicate TGFβ signaling in the inflammatory and apoptotic responses to TGRL lipolysis and suggest a complex interaction between TGFβ receptors and the SAPK subset of MAP Kinases.

Nonequilibrium Thermodynamics of Cell Signaling

Signal transduction inside and across the cells, also called cellular signaling, is key to most biological functions and is ultimately related with both life and death of the organisms. The processes giving rise to the propagation of biosignals are complex and extremely cooperative and occur in a far-from thermodynamic equilibrium regime. They are also driven by activation kinetics strongly dependent on local energetics. For these reasons, a nonequilibrium thermodynamical description, taking into account not just the activation of second messengers, but also transport processes and dissipation is desirable. Here we present a proposal for such a formalism, that considers cells as small thermodynamical systems and incorporates the role of fluctuations as intrinsic to the dynamics in a spirit guided by mesoscopic nonequilibrium thermodynamics. We present also a minimal model for cellular signaling that includes contributions from activation, transport, and intrinsic fluctuations. We finally illustrate its feasibility by considering the case of FAS signaling which is a vital signal transduction pathway that determines either cell survival or death by apoptosis.

Transient decrease in nociceptor GRK2 expression produces long-term enhancement in inflammatory pain

In heterozygous mice, attenuation of G-protein-coupled receptor kinase 2 (GRK2) level in nociceptors is associated with enhanced and prolonged inflammatory hyperalgesia. To further elucidate the role of GRK2 in nociceptor function we reversibly decreased GRK2 expression using intrathecal antisense oligodeoxynucleotide (AS-ODN). GRK2 AS-ODN administration led to an enhanced and prolonged hyperalgesia induced by prostaglandin E2, epinephrine and carrageenan. Moreover, this effect persisted unattenuated 2weeks after the last dose of antisense, well after GRK2 protein recovered, suggesting that transient attenuation of GRK2 produced neuroplastic changes in nociceptor function. Unlike hyperalgesic priming induced by transient activation of protein kinase C epsilon (PKCε), (Aley et al., 2000; Parada et al., 2003b), the enhanced and prolonged hyperalgesia following attenuation of GRK2 is PKCε- and cytoplasmic polyadenylation element binding protein (CPEB)-independent and is protein kinase A (PKA)- and Src tyrosine kinase (Src)-dependent. Finally, rats treated with GRK2 AS-ODN exhibited enhanced and prolonged hyperalgesia induced by direct activation of second messengers, adenyl cyclase, Epac or PKA, suggesting changes downstream of G-protein-coupled receptors. Because inflammation can produce a decrease in GRK2, such a mechanism could help explain a predilection to develop chronic pain, after resolution of acute inflammation.

Renin- and Prorenin-Induced Effects in Rat Vascular Smooth Muscle Cells Overexpressing the Human (Pro)Renin Receptor

Renin/prorenin binding to the (pro)renin receptor ([P]RR) results in direct (angiotensin-independent) second-messenger activation in vitro, whereas in vivo studies in rodents overexpressing prorenin (≈400-fold) or the (P)RR do not support such activation. To solve this discrepancy, DNA synthesis, extracellular signal-regulated kinase 1/2 phosphorylation, and plasminogen-activator inhibitor 1 release were evaluated in wild-type and human (P)RR-overexpressing vascular smooth muscle cells after their incubation with 1 to 80 nmol/L of (pro)renin. Human prorenin (4 nmol/L, ie, ≈800-fold above normal) + angiotensinogen increased DNA synthesis in human (P)RR cells only in an angiotensin II type 1 receptor-dependent manner. Prorenin at this concentration also increased plasminogen-activator inhibitor 1 release via angiotensin. Prorenin alone at 4 nmol/L was without effect, but at 20 nmol/L (≈4000-fold above normal) it activated extracellular signal-regulated kinase 1/2 directly (ie, independent of angiotensin). Renin at concentrations of 1 nmol/L (≈2000-fold above normal) and higher directly stimulated DNA synthesis, extracellular signal-regulated kinase 1/2 phosphorylation, and plasminogen-activator inhibitor 1 release in wild-type and human (P)RR cells, and similar effects were seen for rat renin, indicating that they were mediated via the rat (P)RR. In conclusion, angiotensin generation depending on prorenin-(P)RR interaction may occur in transgenic rodents overexpressing prorenin several 100-fold. Direct (pro)renin-induced effects via the (P)RR require agonist concentrations that are far above the levels in wild-type and transgenic rats. Therefore, only prorenin (and not [P]RR) overexpression will result in an angiotensin-dependent phenotype, and direct renin-(P)RR interaction is unlikely to ever occur in nonrenin-synthesizing organs.

The Hinge Region of the TSH Receptor Stabilizes Ligand Binding and Determines Different Signaling Profiles of Human and Bovine TSH

The hinge region (HinR) is a variable structure of glycoprotein hormone receptors. Its amino acid composition and length is different for glycoprotein hormone receptors and connects the ligand binding domain with the serpentine domain. The role of the HinR of the receptors for TSH, follitropin (FSH), and LH/choriogonadotropin (LHCG) in receptor and signaling specificity is unknown. To investigate the role of the HinR for ligand binding, signal generation, and for the transmission of the signal towards the serpentine domain, we replaced the HinR of the TSH receptor (TSHR) by those of LHCG receptor and FSH receptor and introduced constitutively activating mutations and one mutation deficient for bovine (b)TSH binding in these chimeras. Functional characterization of the TSHR variants was carried out after transient transfection of COS-7 cells by determination of the cell surface expression, ligand binding, and recombinant human (rh)TSH or bTSH activation of second messengers. We show that the HinR of the TSHR stabilizes hormone binding regarding ligand affinity and retention time of the bound ligand as determined by dissociation experiments. Introduction of a constitutively activating extracellular loop mutation in these constructs lead to partially restored binding patterns. These findings indicate that the HinR-extracellular loop interface is besides signaling also important for bTSH binding. Furthermore, data for G protein signaling reveal that the activity of bTSH, but not of rhTSH, depends on the TSHR HinR, which was indicated by a significant right shift in the dose-response curves for G(s) and G(q) activation for TSHR chimeras harboring the LHCG receptor and FSH receptor HinR, respectively. Moreover, we identified different G protein signaling profiles for bTSH and rhTSH, which cannot be explained by the characterized HinR. For future studies regarding structure and function of the TSHR, it will be necessary to characterize TSHR variants with both or more ligands.