Homologous Desensitization Research Articles

The origin and evolution of G protein-coupled receptor kinases.

G protein-coupled receptor (GPCR) kinases (GRKs) play key role in homologous desensitization of GPCRs. GRKs phosphorylate activated receptors, promoting high affinity binding of arrestins, which precludes G protein coupling. Direct binding to active GPCRs activates GRKs, so that they selectively phosphorylate only the activated form of the receptor regardless of the accessibility of the substrate peptides within it and their Ser/Thr-containing sequence. Mammalian GRKs were classified into three main lineages, but earlier GRK evolution has not been studied. Here we show that GRKs emerged at the early stages of eukaryotic evolution via an insertion of a kinase similar to ribosomal protein S6 kinase into a loop in RGS domain. GRKs in Metazoa fall into two clades, one including GRK2 and GRK3, and the other consisting of all remaining GRKs, split into GRK1-GRK7 lineage and GRK4-GRK5-GRK6 lineage in vertebrates. One representative of each of the two ancient clades is found as early as placozoan Trichoplax adhaerens. Several protists, two oomycetes and unicellular brown algae have one GRK-like protein, suggesting that the insertion of a kinase domain into the RGS domain preceded the origin of Metazoa. The two GRK families acquired distinct structural units in the N- and C-termini responsible for membrane recruitment and receptor association. Thus, GRKs apparently emerged before animals and rapidly expanded in true Metazoa, most likely due to the need for rapid signalling adjustments in fast-moving animals.

The Rates of Protein Synthesis and Degradation Account for the Differential Response of Neurons to Spaced and Massed Training Protocols

The sensory-motor neuron synapse of Aplysia is an excellent model system for investigating the biochemical changes underlying memory formation. In this system, training that is separated by rest periods (spaced training) leads to persistent changes in synaptic strength that depend on biochemical pathways that are different from those that occur when the training lacks rest periods (massed training). Recently, we have shown that in isolated sensory neurons, applications of serotonin, the neurotransmitter implicated in inducing these synaptic changes during memory formation, lead to desensitization of the PKC Apl II response, in a manner that depends on the method of application (spaced versus massed). Here, we develop a mathematical model of this response in order to gain insight into how neurons sense these different training protocols. The model was developed incrementally, and each component was experimentally validated, leading to two novel findings: First, the increased desensitization due to PKA-mediated heterologous desensitization is coupled to a faster recovery than the homologous desensitization that occurs in the absence of PKA activity. Second, the model suggests that increased spacing leads to greater desensitization due to the short half-life of a hypothetical protein, whose production prevents homologous desensitization. Thus, we predict that the effects of differential spacing are largely driven by the rates of production and degradation of proteins. This prediction suggests a powerful mechanism by which information about time is incorporated into neuronal processing.

Setting the Clock for Recirculating Lymphocytes

In their search for antigens, lymphocytes continuously shuttle among blood vessels, lymph vessels, and lymphatic tissues. Chemokines mediate entry of lymphocytes into lymphatic tissues, and sphingosine 1-phosphate (S1P) promotes localization of lymphocytes to the vasculature. Both signals are sensed through G protein-coupled receptors (GPCRs). Most GPCRs undergo ligand-dependent homologous receptor desensitization, a process that decreases their signaling output after previous exposure to high ligand concentration. Such desensitization can explain why lymphocytes do not take an intermediate position between two signals but rather oscillate between them. The desensitization of S1P receptor 1 (S1PR1) is mediated by GPCR kinase 2 (GRK2). Deletion of GRK2 in lymphocytes compromises desensitization by high vascular S1P concentrations, thereby reducing responsiveness to the chemokine signal and trapping the cells in the vascular compartment. The desensitization kinetics of S1PR1 allows lymphocytes to dynamically shuttle between vasculature and lymphatic tissue, although the positional information in both compartments is static.

Agonist-Promoted Homologous Desensitization of Human Airway Smooth Muscle Bitter Taste Receptors

Bitter taste receptors (TAS2Rs) were shown to be expressed in human airway smooth muscle (ASM). They couple to specialized [Ca(2+)](i) release, leading to membrane hyperpolarization, the relaxation of ASM, and marked bronchodilation. TAS2Rs are G-protein-coupled receptors, known to undergo rapid agonist-promoted desensitization that can limit therapeutic efficacy. Because TAS2Rs represent a new drug target for treating obstructive lung disease, we investigated their capacity for rapid desensitization, and assessed their potential mechanisms. The pretreatment of human ASM cells with the prototypic TAS2R agonist quinine resulted in a 31% ± 5.1% desensitization of the [Ca(2+)](i) response from a subsequent exposure to quinine. No significant change in the endothelin-stimulated [Ca(2+)](i) response was attributed to the short-term use of quinine, indicating a homologous form of desensitization. The TAS2R agonist saccharin also evoked desensitization, and cross-compound desensitization with quinine was evident. Desensitization of the [Ca(2+)](i) response was attenuated by a dynamin inhibitor, suggesting that receptor internalization (a G-protein coupled receptor kinase [GRK]-mediated, β-arrestin-mediated process) plays an integral role in the desensitization of TAS2R. Desensitization was insensitive to antagonists of the second messenger kinases protein kinase A and protein kinase C. Using intact airways, short-term, agonist-promoted TAS2R desensitization of the relaxation response was also observed. Thus these receptors, which represent a potential novel target for direct bronchodilators, undergo a modest degree of agonist-promoted desensitization that may affect clinical efficacy. Collectively, the results of these mechanistic studies, along with the multiple serines and threonines in intracellular loop 3 and the cytoplasmic tail of TAS2Rs, suggest a GRK-mediated mode of desensitization.

Protein Kinase Cζ Mediates μ-Opioid Receptor-induced Cross-desensitization of Chemokine Receptor CCR5

We have previously shown that the μ-opioid receptor (MOR) is capable of mediating cross-desensitization of several chemokine receptors including CCR5, but the biochemical mechanism of this process has not been fully elucidated. We have carried out a series of functional and biochemical studies and found that the mechanism of MOR-induced cross-desensitization of CCR5 involves the activation of PKCζ. Inhibition of PKCζ by its pseudosubstrate inhibitor, or its siRNA, or dominant negative mutants suppresses the cross-desensitization of CCR5. Our results further indicate that the activation of PKCζ is mediated through a pathway involving phosphoinositol-dependent kinase-1 (PDK1). In addition, activation of MOR elevates the phosphorylation level and kinase activity of PKCζ. The phosphorylation of PKCζ can be suppressed by a dominant negative mutant of PDK1. We observed that following MOR activation, the interaction between PKCζ and PDK1 is immediately increased based on the analysis of fluorescent resonance energy transfer in cells with the expression of PKCζ-YFP and PDK1-CFP. In addition, cells expressing PKCζ kinase motif mutants (Lys-281, Thr-410, Thr-560) fail to exhibit full MOR-induced desensitization of CCR5 activity. Taken together, we propose that upon DAMGO treatment, MOR activates PKCζ through a PDK1-dependent signaling pathway to induce CCR5 phosphorylation and desensitization. Because CCR5 is a highly proinflammatory receptor, and a critical coreceptor for HIV-1, these results may provide a novel approach for the development of specific therapeutic agents to treat patients with certain inflammatory diseases or AIDS.

Modeling Cell Gradient Sensing and Migration in Competing Chemoattractant Fields

Directed cell migration mediates physiological and pathological processes. In particular, immune cell trafficking in tissues is crucial for inducing immune responses and is coordinated by multiple environmental cues such as chemoattractant gradients. Although the chemotaxis mechanism has been extensively studied, how cells integrate multiple chemotactic signals for effective trafficking and positioning in tissues is not clearly defined. Results from previous neutrophil chemotaxis experiments and modeling studies suggested that ligand-induced homologous receptor desensitization may provide an important mechanism for cell migration in competing chemoattractant gradients. However, the previous mathematical model is oversimplified to cell gradient sensing in one-dimensional (1-D) environment. To better understand the receptor desensitization mechanism for chemotactic navigation, we further developed the model to test the role of homologous receptor desensitization in regulating both cell gradient sensing and migration in different configurations of chemoattractant fields in two-dimension (2-D). Our results show that cells expressing normal desensitizable receptors preferentially orient and migrate toward the distant gradient in the presence of a second local competing gradient, which are consistent with the experimentally observed preferential migration of cells toward the distant attractant source and confirm the requirement of receptor desensitization for such migratory behaviors. Furthermore, our results are in qualitative agreement with the experimentally observed cell migration patterns in different configurations of competing chemoattractant fields.

Agonist-Induced Desensitization/Resensitization of Human G Protein-Coupled Receptor 17: A Functional Cross-Talk between Purinergic and Cysteinyl-Leukotriene Ligands

G protein-coupled receptor (GPR) 17 is a P2Y-like receptor that responds to both uracil nucleotides (as UDP-glucose) and cysteinyl-leukotrienes (cysLTs, as LTD(4)). By bioinformatic analysis, two distinct binding sites have been hypothesized to be present on GPR17, but little is known on their putative cross-regulation and on GPR17 desensitization/resensitization upon agonist exposure. In this study, we investigated in GPR17-expressing 1321N1 cells the cross-regulation between purinergic- and cysLT-mediated responses and analyzed GPR17 regulation after prolonged agonist exposure. Because GPR17 receptors couple to G(i) proteins and adenylyl cyclase inhibition, both guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPγS) binding and the cAMP assay have been used to investigate receptor functional activity. UDP-glucose was found to enhance LTD(4) potency in mediating activation of G proteins and vice versa, possibly through an allosteric mechanism. Both UDP-glucose and LTD(4) induced a time- and concentration-dependent GPR17 loss of response (homologous desensitization) with similar kinetics. GPR17 homologous desensitization was accompanied by internalization of receptors inside cells, which occurred in a time-dependent manner with similar kinetics for both agonists. Upon agonist removal, receptor resensitization occurred with the typical kinetics of G protein-coupled receptors. Finally, activation of GPR17 by UDP-glucose (but not vice versa) induced a partial heterologous desensitization of LTD(4)-mediated responses, suggesting that nucleotides have a hierarchy in producing desensitizing signals. These findings suggest a functional cross-talk between purinergic and cysLT ligands at GPR17. Because of the recently suggested key role of GPR17 in brain oligodendrogliogenesis and myelination, this cross-talk may have profound implications in fine-tuning cell responses to demyelinating and inflammatory conditions when these ligands accumulate at lesion sites.

Rosiglitazone reverses salbutamol-induced β2-adrenoceptor tolerance in airway smooth muscle

β₂-Adrenoceptor agonists are important therapeutic agents in the treatment of asthma and chronic obstructive pulmonary disease. The regular use of these drugs has been associated with proasthmatic-like changes that limit their efficacy and increase the risk of severe adverse reactions. We investigated whether the peroxisome-proliferator-activated receptor (PPAR)γ agonist rosiglitazone modulated salbutamol-induced β₂-adrenoceptor desensitization in vivo and in vitro. An in vivo model of homologous β₂-adrenoceptor desensitization, established in guinea-pigs by administering salbutamol continuously, was used to study the ability of rosiglitazone to prevent β₂-adrenoceptor tolerance. In vitro experiments on human bronchial smooth muscle cells were performed to increase the clinical relevance of the study. In tracheal smooth muscle tissues from desensitized animals, we observed a decrease in the protective effect of salbutamol on carbachol-induced contraction, a hyperresponsiveness to cholinergic stimuli, a modest underexpression of β₂-adrenoceptor gene and a marked decrease in β-adrenoceptor number, relative to control values. Treatment with rosiglitazone preserved salbutamol relaxant activity, mitigated carbachol hyperresponsiveness and partially restored β₂-adrenoceptor binding sites in tracheal tissues from homologously desensitized animals. The highly selective PPARγ agonist, GW1929, reproduced the effect of rosiglitazone, in vivo. In vitro β₂-adrenoceptor desensitization decreased salbutamol-mediated cAMP production, without affecting forskolin responses and β₂-adrenoceptor expression. Rosiglitazone and 15-deoxy-Δ¹²(,)¹⁴-prostaglandin J₂ restored salbutamol sensitivity in homologously desensitized cells. These data suggest a potential pharmacodynamic interaction between PPARγ agonists and salbutamol on airway smooth muscle responsiveness, supporting the therapeutic potential of this combination in chronic airway disease.

Receptor desensitization and blockade of the suppressive effects of prostaglandin E2 and adenosine on the cytotoxic activity of human melanoma-infiltrating T lymphocytes

Previous studies document that PGE(2) and adenosine suppress production of inflammatory cytokines. The present study demonstrates for the first time that (1) PGE(2) and 2-chloroadenosine (CADO; a stable analog of adenosine) directly inhibit the cytolytic function of human tumor-infiltrating lymphocytes (TILs); (2) the combination PGE(2) and CADO have additive suppressive effects; and (3) the cooperative immunosuppressive actions of PGE(2) and CADO are mediated via EP2 receptors (EP2Rs) and A(2A) receptors (A(2A)Rs) and are due to amplification of cAMP production, activation of protein kinase A (PKA) and T cell receptor (TCR) inhibitor Csk leading to inhibition of Lck, ZAP-70 and Akt phosphorylation. (4) During ex vivo expansion, TILs undergo three stages of differentiation converting from TILs with high cytotoxic activity and relative resistance to combined EP2R/A(2A)R suppression (stage I) to TILs retaining high cytotoxicity and gaining sensitivity to combined suppression (stage II) and then to TILS that are less cytotoxic and very sensitive to combined suppression (stage III). (5) Finally, we find that pretreatment of TILs with non-inhibitory concentrations of EP2R agonists (such as PGE(2) or butaprost) or A(2A)R agonists (such as CADO or CGS21680) increases their cytotoxic activity and induces resistance to EP2R and A(2A)R inhibitory signaling (cross-resistance) due to homologous and heterologous desensitization and internalization of EP2Rs and A(2A)Rs, thus preventing their inhibitory signaling. We conclude that inducing resistance of TILs to the suppressive effects of PGE(2) and adenosine in the tumor microenvironment could represent a novel strategy for improving the efficacy of adoptive immunotherapy.

Metabotropic glutamate receptor mGlu2 is resistant to homologous agonist-induced desensitization but undergoes protein kinase C-mediated heterologous desensitization

To investigate the susceptibility of the group II metabotropic glutamate receptor mGlu2 to agonist-induced desensitization, the receptor was stably expressed in Chinese hamster ovary (CHO-mGlu2) or C6 glioma cells (C6-mGlu2). Exposure of CHO-mGlu2 cells to the group II mGlu receptor agonist (2 S,1′ S,2′ S)-2-(carboxycyclopropyl)glycine (LCCG-1; 10 μM) for up to 15 h did not affect the subsequent ability of LCCG-1 to inhibit forskolin-stimulated cAMP accumulation. Similarly, in C6-mGlu2 cells, prolonged exposure to LCCG-1 also did not affect the subsequent ability of LCCG-1 to inhibit cAMP formation. In contrast, exposure of CHO-mGlu2 cells to the protein kinase C activator phorbol myristate acetate (PMA) suppressed the ability of LCCG-1 to inhibit cAMP formation. Using an in vitro model of group II mGlu receptor activity, the hemisected neonatal rat spinal cord preparation, the ability of the selective group II agonist (2 R,4 R)-4-aminopyrrolidine-2,4-dicarboxylate ((2 R,4 R)-APDC) to depress the fast component of the dorsal root-evoked ventral root potential (fDR-VRP) did not desensitize when applied for up to 2 h. Together these results indicate that in contrast to most G protein-coupled receptors, the mGlu2 receptor is resistant to agonist-induced homologous desensitization, and that in vitro data suggests that resistance to desensitization is a physiologically relevant property of this mGlu receptor subtype.

The Protective Signaling of Metabotropic Glutamate Receptor 1 Is Mediated by Sustained, β-Arrestin-1-dependent ERK Phosphorylation

Metabotropic glutamate receptor 1 (mGlu1) is a G protein-coupled receptor that enhances the hydrolysis of membrane phosphoinositides. In addition to its role in synaptic transmission and plasticity, mGlu1 has been shown to be involved in neuroprotection and neurodegeneration. In this capacity, we have reported previously that in neuronal cells, mGlu1a exhibits the properties of a dependence receptor, inducing apoptosis in the absence of glutamate, while promoting neuronal survival in its presence (Pshenichkin, S., Dolińska, M., Klauzińska, M., Luchenko, V., Grajkowska, E., and Wroblewski, J. T. (2008) Neuropharmacology 55, 500-508). Here, using CHO cells expressing mGlu1a receptors, we show that the protective effect of glutamate does not rely on the classical mGlu1 signal transduction. Instead, mGlu1a protective signaling is mediated by a novel, G protein-independent, pathway which involves the activation of the MAPK pathway and a sustained phosphorylation of ERK, which is distinct from the G protein-mediated transient ERK phosphorylation. Moreover, the sustained phosphorylation of ERK and protective signaling through mGlu1a receptors require expression of beta-arrestin-1, suggesting a possible role for receptor internalization in this process. Our data reveal the existence of a novel, noncanonical signaling pathway associated with mGlu1a receptors, which mediates glutamate-induced protective signaling.

β-Arrestin 2-mediated heterologous desensitization of IGF-IR by prolonged exposure of SH-SY5Y neuroblastoma cells to a mu opioid agonist

Prolonged (12 h) exposure of SH-SY5Y neuroblastoma cells to the mu-opioid receptor (MOPr) agonist [D-Ala 2,N-Me-Phe 4,Gly 5-ol]-enkephalin (DAMGO) causes homologous desensitization as well as heterologous desensitization of the extracellular signal-regulated kinase 1/2 (ERK 1/2) phosphorylation induced by insulin-like growth factor (IGF)-I. Brief (15 min) but not prolonged exposure to DAMGO transregulates the insulin-like growth factor-I (IGF-I) receptor, as evidenced by its phosphorylation in the absence of IGF-I. Silencing of β-arrestin 2 uncouples the crosstalk between the two receptors, thus maintaining IGF-I-mediated receptor phosphorylation and ERK 1/2 activation even after prolonged DAMGO exposure. Furthermore, MOPr-induced activation of IGF-I receptor requires the tyrosine kinase c-Src.

Homologous desensitization of guanylyl cyclase A, the receptor for atrial natriuretic peptide, is associated with a complex phosphorylation pattern

Atrial natriuretic peptide (ANP), via its guanylyl cyclase A (GC-A) receptor and intracellular guanosine 3′,5′-cyclic monophosphate production, is critically involved in the regulation of blood pressure. In patients with chronic heart failure, the plasma levels of ANP are increased, but the cardiovascular actions are severely blunted, indicating a receptor or postreceptor defect. Studies on metabolically labelled GC-A-overexpressing cells have indicated that GC-A is extensively phosphorylated, and that ANP-induced homologous desensitization of GC-A correlates with receptor dephosphorylation, a mechanism which might contribute to a loss of function in vivo. In this study, tandem MS analysis of the GC-A receptor, expressed in the human embryonic kidney cell line HEK293, revealed unambiguously that the intracellular domain of the receptor is phosphorylated at multiple residues: Ser487, Ser497, Thr500, Ser502, Ser506, Ser510 and Thr513. MS quantification based on multiple reaction monitoring demonstrated that ANP-provoked desensitization was accompanied by a complex pattern of receptor phosphorylation and dephosphorylation. The population of completely phosphorylated GC-A was diminished. However, intriguingly, the phosphorylation of GC-A at Ser487 was selectively enhanced after exposure to ANP. The functional relevance of this observation was analysed by site-directed mutagenesis. The substitution of Ser487 by glutamate (which mimics phosphorylation) blunted the activation of the GC-A receptor by ANP, but prevented further desensitization. Our data corroborate previous studies suggesting that the responsiveness of GC-A to ANP is regulated by phosphorylation. However, in addition to the dephosphorylation of the previously postulated sites (Ser497, Thr500, Ser502, Ser506, Ser510), homologous desensitization seems to involve the phosphorylation of GC-A at Ser487, a newly identified site of phosphorylation. The identification and further characterization of the specific mechanisms involved in the downregulation of GC-A responsiveness to ANP may have important pathophysiological implications.Structured digital abstract•MINT-7713870, MINT-7713887: PMCA (uniprotkb:P20020) and GC-A (uniprotkb:P18910) colocalize (MI:0403) by fluorescence microscopy (MI:0416)

A role for inflammatory mediators in heterologous desensitization of CysLT1 receptor in human monocytes

Cysteinyl-leukotrienes (cysteinyl-LT) are rapidly generated at sites of inflammation and, in addition to their role in asthma, rhinitis, and other immune disorders, are increasingly regarded as significant inflammatory factors in cancer, gastrointestinal, cardiovascular diseases. We recently demonstrated that in monocyte/macrophage-like U937 cells, extracellular nucleotides heterologously desensitize CysLT(1) receptor (CysLT(1)R)-induced Ca(2+) transients. Given that monocytes express a number of other inflammatory and chemoattractant receptors, this study was aimed at characterizing transregulation between these different stimuli. We demonstrate that in U937 cells and in primary human monocytes, a series of inflammatory mediators activating G(i)-coupled receptor (FPR1, BLT(1)) desensitize CysLT(1)R-induced Ca(2+) response unidirectionally through activation of PKC. Conversely, PAF-R, exclusively coupled to G(q), cross-desensitizes CysLT(1)R without the apparent involvement of any kinase. Interestingly, G(s)-coupled receptors (beta(2)AR, H(1/2)R, EP(2/4)R) are also able to desensitize CysLT(1)R response through activation of PKA. Heterologous desensitization seems to affect mostly the G(i)-mediated signaling of the CysLT(1)R. The hierarchy of desensitization among agonists may be important for leukocyte signal processing at the site of inflammation. Considering that monocytes/macrophages are likely to be the major source of cysteinyl-LT in many immunological and inflammatory processes, shedding light on how their receptors are regulated will certainly help to better understand the role of these cells in orchestrating this complex network of integrated signals.

ANCA antigen proteinase 3 acts as a phagocytotic receptor of bacteria via PAR2 pathway in human neutrophils. (89.40)

Abstract The molecular mechanisms underlying the phagocytosis of bacteria by neutrophils are pooly understood. We previously reported the efficient uptake of Streptococcus sangunis by human neutrophils in the absence of opsonins. To characterize the phagocytosis receptor, protein lysates of neutrophils and HL-60 cells were subjected to affinity chromatography using epoxy beads immobilized with S. sangunis. Denaturing electrophoresis of the eluted proteins revealed several neutrophil-specific bands. One of these proteins was identified as proteinase 3 (PR3) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), which was confirmed by western blotting. PR3 is a serine proteinase of the azurophil granules and also co-expressed with NB1, a glycosylphosphatidylinositol (GPI)-linked receptor, on the surface of neutrophils. Enzymatic cleavage of GPI anchors or blocking of PR3 significantly reduced the ability of neutrophils to phagocytose S. sangunis. Protease activity was required for phagocytosis, and S. sanguinis and protease-activated receptor 2 (PAR2) agonist induced calcium mobilization in a homologous desensitization manner, indicating the involvement of PAR 2 in phagocytosis. Finally, exposure to S. sanguinis preferentially activated RhoA to Rac1 and Cdc42, which was blocked by an intracellular calcium chelator. Collectively, our data show that PR3 acts as a phagocytotic receptor via PAR2 and the Rho family GTPases in neutrophils.

TRPA1 Receptor Induced Relaxation of the Human Urethra Involves TRPV1 and Cannabinoid Receptor Mediated Signals, and Cyclooxygenase Activation

We studied whether TRPA1 agonists interact with sensory and inflammatory signals to relax human urethral smooth muscle. Urethral specimens were obtained perioperatively from 19 patients, and prepared for immunohistochemistry and functional experiments. The effects of allyl isothiocyanate, cinnamaldehyde and NaHS were studied in phenylephrine activated preparations combined with capsaicin, capsazepine, N omega-nitro-L-arginine, indomethacin or CP55940. TRPA1, cannabinoid 1 and cannabinoid 2 immunoreactivity was colocalized in nerve fibers of the human urethra. All TRPA1 agonists produced relaxation of phenylephrine contracted urethral preparations. Capsaicin increased relaxant responses to all TRPA1 agonists. It increased the mean +/- SEM -logIC50 of cinnamaldehyde and NaHS from 4.91 +/- 0.26 to 5.15 +/- 0.22 and 3.27 +/- 0.14 to 3.79 +/- 0.35, and the -logIC30 of allyl isothiocyanate from 3.11 +/- 0.24 to 3.41 +/- 0.26 (each p <0.05). Capsazepine in 5 preparations, indomethacin in 6 and CP55940 in 5 decreased cinnamaldehyde mediated relaxation by up to 39%, 88% and 89%, respectively. Nomega-nitro-L-arginine and urothelial removal had no effect on relaxation by cinnamaldehyde in 5 preparations. Relaxation to TRPA1 agonists in human urethral preparations seem to work in cooperation with TRPV1 mediated signals, are negatively coupled via cannabinoid receptor activation and involve cyclooxygenase products. Urothelial TRPA1 signals may not be important to regulate normal human urethral smooth muscle tone. This does not exclude a role in the initiation of afferent activity normally and in disease states.

Hypercholesterolemia modifies angiotensin II desensitisation and cross talk between α 1-adrenoceptor and angiotensin AT 1 receptor in rabbit aorta

This study characterised the effect of a hypercholesterolemic diet on the interactions of hormone receptors in the rabbit aorta, both in homologous desensitisation to angiotensin II and cross talk between α 1-adrenoceptors and angiotensin AT 1 receptors. Rabbits were fed either a normal chow or a diet containing 1% cholesterol for 6–7-weeks. Isometric contractions were measured in endothelium-intact or endothelium-removed aortic rings from control and hypercholesterolemic rabbits. Concentration response curves to angiotensin II or noradrenaline incubated with or without prazosin or losartan were performed. In another group, the resting potential was recorded at baseline and following angiotensin II or noradrenaline stimulation. Rabbits fed a hypercholesterolemic diet showed higher plasma levels of total cholesterol and LDL-cholesterol and impaired relaxation to acetylcholine. Homologous desensitisation to angiotensin II was found in endothelium-intact but not in endothelium-removed arteries. Cross talk between α 1-adrenoceptors and angiotensin AT 1 receptors was modified with respect to physiological conditions. In control rabbits, angiotensin II desensitised the noradrenaline response but noradrenaline did not modify the angiotensin II-response. However, in hypercholesterolemic rabbits, angiotensin II sensitised the noradrenaline-response and noradrenaline desensitised the angiotensin II-response. Furthermore, the resting potential remains hyperpolarised after noradrenaline stimulation in hypercholesterolemic rabbits. Modifications in homologous desensitisation to angiotensin II and cross talk between α 1-adrenoceptors and angiotensin AT 1 receptors suggest that hypercholesterolemia induces early tissue dysfunction by altering endothelial and smooth muscle cell regulatory properties. This may be one of the mechanisms by which hypercholesterolemia could be involved in the onset and progression of chronic vascular diseases such as hypertension and arteriosclerosis.

Histamine Develops Homologous Desensitization under Ca&lt;sup&gt;2+&lt;/sup&gt;-free Conditions with Increase in Basal Tone in Smooth Muscle of Guinea Pig Taenia Caeci

Histamine regulates a variety of physiological or pathophysiological processes via the activation of G(q/11) protein-coupled and Ca(2+)-mobilizing histamine H(1) receptors, including smooth muscle contraction. We have found that histamine induces progression from heterologous to homologous desensitization of contraction under normal physiological conditions in smooth muscle of guinea pig taenia caeci. In this study, we characterized the development of histamine-induced desensitization under Ca(2+)-free conditions and we found that histamine developed only a homologous phase of desensitization to histamine with an increase in EC(50) values for histamine and basal tone. In contrast, histamine treatment reduced EC(50) values for a muscarinic agonist, carbachol, and depolarizing high K(+). These results suggest that the failure of excess histamine to induce a normal Ca(2+) response under Ca(2+)-free conditions may lead to homologous desensitization to histamine with apparent hyper-reactivity of smooth muscles to cholinergic and depolarizing stimuli. We estimate that this characteristic of histamine to change smooth muscle contractility may be potentially involved in its physiological and pathophysiological aspects, including histamine-induced allergic conditions, depending on cellular circumstances.

The GnRH receptor and the response of gonadotrope cells to GnRH pulse frequency code. A story of an atypical adaptation of cell function relying on a lack of receptor homologous desensitization.

Brain control of the reproductive system is mediated through hypothalamic gonadotropin-releasing hormone (GnRH) which activates specific receptors (GnRHR) present at the surface of the pituitary gonadotropes to trigger secretion of the two gonadotropins LH and FSH. A unique feature of this system is the high dependence on the secretion mode of GnRH, which is basically pulsatile but undergoes considerable fluctuations in pulse frequency pattern in response to endogenous or external factors. How the physiological fluctuations of GnRH secretion that orchestrate normal reproduction are decoded by the gonadotrope cell machinery to ultimately control gonadotropin release and/or subunit gene transcription has been the subject of intensive studies during the past decades. Surprisingly, the mammalian GnRHR is unique among G protein-coupled receptor family as it lacks the carboxy-terminal tail usually involved in classical endocytotic process. Accordingly, it does not desensitize properly and internalizes very poorly. Both this atypical intrinsic property and post-receptor events may thus contribute to decode the GnRH signal. This includes the participation of a network of signaling pathways that differently respond to GnRH together with a growing amount of genes differentially sensitive to pulse frequency. Among these are two pairs of genes, the transcription factors EGR-1 and NAB, and the regulatory factors activin and follistatin, that function as intracellular autoregulatory feedback loops controlling respectively LHbeta and FSHbeta gene expression and hence, LH and FSH synthesis. Pituitary gonadotropes thus represent a unique model of cells functionally adapted to respond to a considerably fluctuating neuroendocrine stimulation, from short individual pulses to sustained GnRH as observed at the proestrus of ovarian cycle. Altogether, the data emphasize the adaptative reciprocal complementarity of hypothalamic GnRH neurones and pituitary gonadotropes to function as an original unit.

The GnRH receptor and the response of gonadotrope cells to GnRH pulse frequency code. A story of an atypical adaptation of cell function relying on a lack of receptor homologous desensitization.

The GnRH receptor and the response of gonadotrope cells to GnRH pulse frequency code. A story of an atypical adaptation of cell function relying on a lack of receptor homologous desensitization.