Pertussis Toxin-sensitive Gi Protein Research Articles

Endothelial dysfunction and vascular disease - a 30th anniversary update.

The endothelium can evoke relaxations of the underlying vascular smooth muscle, by releasing vasodilator substances. The best-characterized endothelium-derived relaxing factor (EDRF) is nitric oxide (NO) which activates soluble guanylyl cyclase in the vascular smooth muscle cells, with the production of cyclic guanosine monophosphate (cGMP) initiating relaxation. The endothelial cells also evoke hyperpolarization of the cell membrane of vascular smooth muscle (endothelium-dependent hyperpolarizations, EDH-mediated responses). As regards the latter, hydrogen peroxide (H2 O2 ) now appears to play a dominant role. Endothelium-dependent relaxations involve both pertussis toxin-sensitive Gi (e.g. responses to α2 -adrenergic agonists, serotonin, and thrombin) and pertussis toxin-insensitive Gq (e.g. adenosine diphosphate and bradykinin) coupling proteins. New stimulators (e.g. insulin, adiponectin) of the release of EDRFs have emerged. In recent years, evidence has also accumulated, confirming that the release of NO by the endothelial cell can chronically be upregulated (e.g. by oestrogens, exercise and dietary factors) and downregulated (e.g. oxidative stress, smoking, pollution and oxidized low-density lipoproteins) and that it is reduced with ageing and in the course of vascular disease (e.g. diabetes and hypertension). Arteries covered with regenerated endothelium (e.g. following angioplasty) selectively lose the pertussis toxin-sensitive pathway for NO release which favours vasospasm, thrombosis, penetration of macrophages, cellular growth and the inflammatory reaction leading to atherosclerosis. In addition to the release of NO (and EDH, in particular those due to H2 O2 ), endothelial cells also can evoke contraction of the underlying vascular smooth muscle cells by releasing endothelium-derived contracting factors. Recent evidence confirms that most endothelium-dependent acute increases in contractile force are due to the formation of vasoconstrictor prostanoids (endoperoxides and prostacyclin) which activate TP receptors of the vascular smooth muscle cells and that prostacyclin plays a key role in such responses. Endothelium-dependent contractions are exacerbated when the production of nitric oxide is impaired (e.g. by oxidative stress, ageing, spontaneous hypertension and diabetes). They contribute to the blunting of endothelium-dependent vasodilatations in aged subjects and essential hypertensive and diabetic patients. In addition, recent data confirm that the release of endothelin-1 can contribute to endothelial dysfunction and that the peptide appears to be an important contributor to vascular dysfunction. Finally, it has become clear that nitric oxide itself, under certain conditions (e.g. hypoxia), can cause biased activation of soluble guanylyl cyclase leading to the production of cyclic inosine monophosphate (cIMP) rather than cGMP and hence causes contraction rather than relaxation of the underlying vascular smooth muscle.

Inhibition of metabotropic glutamate receptor 5 induces cellular stress through pertussis toxin-sensitive Gi-proteins in murine BV-2 microglia cells.

BackgroundActivation of metabotropic glutamate receptor 5 (mGluR5) by (RS)-2-chloro-5-hydroxyphenylglycine (CHPG) was shown to suppress microglia activation and decrease the release of associated pro-inflammatory mediators. In contrast, the consequences of mGluR5 inhibition are less well understood. Here, we used BV-2 cells, retaining key characteristics of primary mouse microglia, to examine whether mGluR5 inhibition by 2-methyl-6-(phenylethynyl)-pyridine (MPEP) enhances cellular stress and production of inflammatory mediators.MethodsBV-2 cells were treated with MPEP, followed by determination of cellular stress using fluorescent dyes and high-content imaging. The expression of inflammatory mediators, endoplasmic reticulum (ER)-stress markers and phosphorylated AMPKα was analyzed by quantitative PCR, ELISA and Western blotting. Additionally, phospholipase C (PLC) activity, cellular ATP content and changes in intracellular free Ca2+ ([Ca2+]i) were measured using luminescence and fluorescence assays.ResultsTreatment of BV-2 microglia with 100 μM MPEP increased intracellular reactive oxygen species (ROS), mitochondrial superoxide, mitochondrial mass as well as inducible nitric oxide synthase (iNOS) and IL-6 expression. Furthermore, MPEP reduced cellular ATP and induced AMPKα phosphorylation and the expression of the ER-stress markers CHOP, GRP78 and GRP96. The MPEP-dependent effects were preceded by a rapid concentration-dependent elevation of [Ca2+]i, following Ca2+ release from the ER, mainly via inositol triphosphate-induced receptors (IP3R). The MPEP-induced ER-stress could be blocked by pretreatment with the chemical chaperone 4-phenylbutyrate and the Ca2+ chelator BAPTA-AM. Pretreatment with the AMPK agonist AICAR partially abolished, whilst the inhibitor compound C potentiated, the MPEP-dependent ER-stress. Importantly, the PLC inhibitor U-73122 and the Gi-protein inhibitor pertussis toxin (PTX) blocked the MPEP-induced increase in [Ca2+]i. Moreover, pretreatment of microglia with AICAR, BAPTA-AM, U-73122 and PTX prevented the MPEP-induced generation of oxidative stress and inflammatory mediators, further supporting a role for Gi-protein-mediated activation of PLC.ConclusionsThe results emphasize the potential pathophysiological role of mGluR5 antagonism in mediating oxidative stress, ER-stress and inflammation through a Ca2+-dependent pathway in microglia. The induction of cellular stress and inflammatory mediators involves PTX-sensitive Gi-proteins and subsequent activation of PLC, IP3R and Ca2+ release from the ER.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-014-0190-7) contains supplementary material, which is available to authorized users.

Role of High Endothelial Venule–Expressed Heparan Sulfate in Chemokine Presentation and Lymphocyte Homing

Lymphocyte homing to peripheral lymph nodes (PLNs) is mediated by multistep interactions between lymphocytes and high endothelial venules (HEVs). Heparan sulfate (HS) has been implicated in the presentation of chemokines on the surface of HEVs during this process. However, it remains unclear whether this cell surface presentation is a prerequisite for lymphocyte homing. In this study, we generated conditional knockout (cKO) mice lacking Ext1, which encodes a glycosyltransferase essential for HS synthesis, by crossing Ext1(flox/flox) mice with GlcNAc6ST-2-Cre transgenic mice expressing Cre recombinase in HEVs. Immunohistochemical studies indicated that HS expression was specifically eliminated in PLN HEVs but retained in other blood vessels in the cKO mice. The accumulation of a major secondary lymphoid tissue chemokine, CCL21, on HEVs was also abrogated without affecting CCL21 mRNA levels, indicating that HS presents CCL21 on HEVs in vivo. Notably, a short-term lymphocyte homing assay indicated that lymphocyte homing to PLNs was diminished in the cKO mice by 30-40%. Consistent with this result, contact hypersensitivity responses were also diminished in the cKO mice. The residual lymphocyte homing to PLNs in the cKO mice was dependent on pertussis toxin-sensitive Gi protein signaling, in which lysophosphatidic acid-mediated signaling was partly involved. These results suggest that chemokine presentation by HS on the surface of HEVs facilitates but is not absolutely required for lymphocyte homing.

GABA B receptors modulate depolarization-stimulated [ 3H]glutamate release in slices of the pars reticulata of the rat substantia nigra

GABA B receptors decrease the release of GABA from the striatal terminals within the pars reticulata of the substantia nigra by opposing the increase in the release caused by dopamine D 1 receptors. The dopamine D 1 receptors also increase the release of glutamate from subthalamic terminals in the pars reticulata. Because GABA B receptors decrease the glutamate release from these terminals, we have explored if the effect of GABA B receptors also opposed the effect of the dopamine D 1 receptors. The effect of baclofen, a selective GABA B-receptor agonist, was tested on the release of [ 3H]glutamate caused by highly (40 mM) concentrated K + solutions in slices of the pars reticulata. Baclofen decreased (the concentration causing 50% inhibition, IC 50, was 8.15 μM) the increase in the release of the [ 3H]glutamate caused by the dopamine D 1 receptors and it also decreased (IC 50 was 0.51 μM) this release in the absence of the activation of the dopamine D 1 receptors. The GABA B receptors appear then to inhibit glutamate release in two ways; one dependent on the activation of the dopamine D 1 receptors and the other independent of such activation. The protein kinase A-inhibitor H89 blocked the increase in the release of the [ 3H]glutamate caused by the dopamine D 1 receptors, though it did not block the dopamine D 1 receptor-independent baclofen inhibition of the release. This finding indicates that this inhibition was not via the protein kinase A signal-transduction pathway. N-ethylmaleimide, an alkylating agent that inactivates pertussis toxin-sensitive Gi proteins, eliminated both the dopamine D 1 receptor-dependent and -independent baclofen inhibition, showing that both were mediated by these proteins. The injection of baclofen into the pars reticulata of unanesthetized rats caused contralateral rotation, suggesting a reduced glutamate release from the subthalamic terminals, thereby stopping the inhibition of the premotor thalamic nuclei, causing locomotion. Our data suggest that GABA B receptors restrain the excitatory input from the subthalamic nucleus and stimulate motor behavior.

Regeneration of the Endothelium in Vascular Injury

The endothelium mediates relaxations (dilatations) of the underlying vascular smooth muscle cells. The endothelium-dependent relaxations are due to the release of non-prostanoid vasodilator substances. The best characterized endothelium-derived relaxing factor (EDRF) is nitric oxide (NO). The endothelial cells also release substances (endothelium-derived hyperpolarizing factor, EDHF) that cause hyperpolarization of the cell membrane of the underlying vascular smooth muscle. The release of EDRF from the endothelium can be mediated by both pertussis toxin-sensitive G(i) (alpha(2)-adrenergic activation, serotonin, thrombin) and insensitive G(q) (adenosine diphosphate, bradykinin) coupling proteins. The ability of the endothelial cell to release relaxing factors can be upregulated by impregnation with estrogens, exercise and antioxidants, and down-regulated by oxidative stress and increased presence of oxidized LDL. Following injury or apoptotic death, the endothelium regenerates. However, in regenerated endothelial cells, there is an early selective loss of the pertussis-toxin sensitive mechanisms of EDRF-release. Functional studies suggest that abnormal handling of LDL because of increased oxidative stress play a key role in this selective loss. Genomic analysis demonstrates the emergence of fatty acid binding protein-A (A-FBP) and metalloproteinase-7 (MMP7) in regenerated endothelial cells. The reduced release of NO resulting from the endothelial dysfunction in regenerated areas creates a locus minoris resistentiae which favors the occurrence of vasospasm and thrombosis as well as the initiation of atherosclerosis.

Interactions Between Megakaryocytes and Tumour Cells at the Bone Marrow Vascular Stem Cell Niche Promote Tumour Growth and Metastasis.

Abstract 470 Introduction:Specialized microenvironments, or niches, of the bone marrow (BM) maintain and regulate physiological haematopoietic stem/progenitor cells and have been implicated as areas of preferential engraftment and ‘sanctuary sites' for leukaemic cells and metastasizing cells of solid tumours. The cellular and molecular factors that comprise the BM vascular niche have not been well described. Megakaryocytes (MKs) reside in close association with the BM sinusoidal endothelium. The aim of this study was to investigate whether MKs play a role in the homing and engraftment of malignant cells in the BM. Methods:C57Bl/6 wild type and thrombopoietin (TPO)-/- mice (which have <10% of the normal number of MKs and platelets) received flank or tail vein injections of the syngeneic B16-F10 melanoma or EL4 lymphoma cell lines fluorescently labelled with mCherry or GFP. The MK-vascular niche was examined using citrulline, MECA32, and TSP1 immunostaining. MKs were cultured from the Lin-Sca1+cKit+-enriched fraction of BM cells from flushed femurs and tibias, using 50ng/ml TPO + 20ng/ml SCF. Costar transwell co-culture plates and Neuroprobe chemotaxis chambers were used; cellular proliferation was assessed using the CellTitre MTS assay. RNA was extracted from flushed BM cells and in vitro cell cultures using Qiagen RNeasy columns/Trizol and gene expression was analyzed by RT-qPCR. Results:In wild type mice, the majority of BM sinusoids were surrounded by one or more large MKs forming the MK-vascular niche, with MKs tightly abutting the vascular endothelium. In TPO-/- mice, MKs were largely absent from the BM, blood vessels appeared more tortuous and the mean vessel diameter in the BM was significantly larger than in wild type mice (P<0.01). Expression of the angiogenic regulatory proteins platelet factor 4 (PF4) and thrombospondin 1 (TSP1) was markedly lower in BM from TPO-/- mice than wild type; expression of VEGF and TGFb was also reduced. Together these findings suggest that MKs support the integrity of the vascular niche and that homeostasis of the niche may be disrupted in the absence of MKs. Wild type mice injected with either B16-F10 melanoma or EL4 lymphoma had increased numbers of MKs and a larger mean vessel diameter. Although there was no increase in platelet count, the mean platelet volume was significantly increased by day 18 (p=0.002), suggesting increased thrombopoiesis. Furthermore, there was a linear decrease in PF4 in response to tumour, reaching a 3-fold reduction by late-stage tumour growth. This finding was consistent with the increase in MK-vascular niches as PF4 normally acts as an autocrine inhibitor of megakaryopoiesis and inhibits endothelial cell proliferation and migration. Consistent with a modulatory effect of MKs and/or the MK-vascular niche on tumour phenotype, tumour growth in TPO-/- mice was markedly retarded and there was reduced metastasis to the BM and lung. To investigate the mechanism of these effects, MK-conditioned medium (MCM) was added to in vitro cultures of B16 cells. MCM significantly enhanced the proliferation rate of B16 melanoma cells (P<0.001). Further, MCM was highly chemotactic for B16 cells (P<0.001). This effect was found to be mediated by pertussis toxin-sensitive Gi-protein receptors and reduced but not entirely abrogated in the absence of TSP1 (using MCM generated from TSP1-/- mice). To investigate the interactions between tumour cells and MKs, MKs were cocultured with B16 cells. Coculture increased MK expression of proangiogenic factors VEGF and TGFb while cocultured B16 cells displayed increased expression of alpha integrins, a4, a5 and a6. Moreover, coculturing B16 cells with MKs prior to tail vein injection enhanced tumour cell engraftment in the lung. A pilot study of BM trephine biopsies from 8 patients with carcinoma (breast, lung, prostate, bladder and kidney) supported these preclinical findings. MKs in 3/3 patients with BM metastasis and 3/5 patients without BM metastasis showed a variable excess of MKs, some in loose clusters, with abnormal morphology and localization. Conclusions:These findings suggest that MKs contribute to the integrity and functionality of the BM vascular niche in homeostasis and in malignancy, and that cellular/molecular cross-talk between MKs and tumour cells at the vascular niche may promote metastasis. Targeting these interactions may be a useful as adjunctive therapy to prevent dissemination of cancer to the BM. Disclosures:No relevant conflicts of interest to declare.

Endothelial dysfunction and vascular disease

The endothelium can evoke relaxations (dilatations) of the underlying vascular smooth muscle, by releasing vasodilator substances. The best characterized endothelium-derived relaxing factor (EDRF) is nitric oxide (NO). The endothelial cells also evoke hyperpolarization of the cell membrane of vascular smooth muscle (endothelium-dependent hyperpolarizations, EDHF-mediated responses). Endothelium-dependent relaxations involve both pertussis toxin-sensitive G(i) (e.g. responses to serotonin and thrombin) and pertussis toxin-insensitive G(q) (e.g. adenosine diphosphate and bradykinin) coupling proteins. The release of NO by the endothelial cell can be up-regulated (e.g. by oestrogens, exercise and dietary factors) and down-regulated (e.g. oxidative stress, smoking and oxidized low-density lipoproteins). It is reduced in the course of vascular disease (e.g. diabetes and hypertension). Arteries covered with regenerated endothelium (e.g. following angioplasty) selectively loose the pertussis toxin-sensitive pathway for NO release which favours vasospasm, thrombosis, penetration of macrophages, cellular growth and the inflammatory reaction leading to atherosclerosis. In addition to the release of NO (and causing endothelium-dependent hyperpolarizations), endothelial cells also can evoke contraction (constriction) of the underlying vascular smooth muscle cells by releasing endothelium-derived contracting factor (EDCF). Most endothelium-dependent acute increases in contractile force are due to the formation of vasoconstrictor prostanoids (endoperoxides and prostacyclin) which activate TP receptors of the vascular smooth muscle cells. EDCF-mediated responses are exacerbated when the production of NO is impaired (e.g. by oxidative stress, ageing, spontaneous hypertension and diabetes). They contribute to the blunting of endothelium-dependent vasodilatations in aged subjects and essential hypertensive patients.

The Effect of Detergent on Human Mu-opioid Receptor (hMOR) Localization as a Function of Pretreatment with Agonist and Antagonist

Like many other seven transmembrane G-protein coupled receptors (GPCRs), the human mu-opioid receptor (hMOR) interacts with multiple members of the pertussis toxin-sensitive Gi and Go protein families, to regulate adenylyl cyclase, Ca2+ and K+ channels. Notably, opioid agonists represent the most powerful analgesic drugs for the clinical management of pain, through binding opioid receptors. However, not all agonists exert the same level of effect.Plasma membranes are organized into specialized micro-domains differing in composition, biological function and physical properties. In recent years, detergent resistant membranes (DRM) are thought to serve as molecular sorting platforms to concentrate signalling molecules (e.g. opioid receptors) based on membrane fractionation and cholesterol depletion experiments. It remains unclear whether membrane organization with detergent has an effect on hMOR localization.Here we track active hMOR and lipid composition in isopycnic membrane fractions in the presence and absence of CHAPS detergent. hMOR activity was assessed using a modified binding assay. The relative amount of lipid raft marker (flotillin-1), actin and G-proteins were assessed by Western blot analysis. The data show the effects of detergent on receptor distribution. Relocation of the hMOR receptor in the membrane indicates an additional level regulation at the cell membrane.

Endothelial Dysfunction The First Step Toward Coronary Arteriosclerosis

The endothelium causes relaxations of the underlying vascular smooth muscle, by releasing nitric oxide (NO). The endothelial cells also can evoke hyperpolarization of the vascular smooth muscle cells (endothelium-dependent hyperpolarizations, endothelium-derived hyperpolarizing factors-mediated responses). Endothelium-dependent relaxations involve both pertussis toxin-sensitive Gi and pertussis toxin-insensitive Gq coupling proteins. The endothelial release of NO is reduced in diabetes and hypertension. Arteries covered with regenerated endothelium lose the pertussis-toxin sensitive pathway for NO-release. This dysfunction favors vasospasm, thrombosis, penetration of macrophages, cellular growth and the inflammatory reaction leading to atherosclerosis. Endothelial cells also release endothelium-derived contracting factors (EDCF). Most endothelium-dependent contractions are mediated by vasoconstrictor prostanoids (endoperoxides and prostacyclin), which activate thromboxane-prostanoid (TP)-receptors of the underlying vascular smooth muscle cells. EDCF-mediated responses are augmented by aging, hypertension and diabetes. Thus, endothelial dysfunction is the first step toward coronary arteriosclerosis.

Lanthanum and its rapidly emerging role as an anti‐carcinogenic agent

To the Editor: I read with great interest the recent article by Wang et al. [2008]. The authors have clearly demonstrated that lanthanum enhances in vitro osteoblast differentiation via pertussis toxin-sensitive gi protein and ERK signaling pathway. Interestingly, lanthanum is rapidly emerging as a major player in the management of multiple other different systemic diseases especially in the field of oncology. For instance, H2L1, a bis-coumarin complex of lanthanum shows cytotoxic activity against BV-173 (chronic myeloid leukemia) cells [Kostova et al., 2005]. Similarly, when lanthanum complexes with coumarin-3-carboxylic acid it causes growth inhibition in Reh cells while exposure of BGC-823 (gastric cancer) tissue to lanthanum results in growth delay secondary to upregulation of MP12 and downregulation of RHOJ [Shi and Huang, 2005]. In fact, a new lanthanum derivative, KP772 is highly effective against cancer cells, especially multi drug resistant cancer cells [Heffeter et al., 2007]. Interestingly, lanthanum lasers such as the Nd: yttrium–lanthanum–fluoride laser are also highly effective in the management of tumors such as choroidal melanomas [Krause et al., 2003]. Oncologists need to note that drug resistance against lanthanum derivatives may develop easily [Heffeter et al., 2008]. Despite this, lanthanum appears to be a bright and highly promising new development in the field of oncology. Hopefully, the next few years will see an increase in the clinical application of lanthanum for the management of systemic malignancies as well as an increase in the number of studies involving lanthanum, which will hopefully help us in having a better understanding of its anti-carcinogenic properties.

Small Neutralizing Molecules to Inhibit Actions of the Chemokine CXCL12

The chemokine CXCL12 and the receptor CXCR4 play pivotal roles in normal vascular and neuronal development, in inflammatory responses, and in infectious diseases and cancer. For instance, CXCL12 has been shown to mediate human immunodeficiency virus-induced neurotoxicity, proliferative retinopathy and chronic inflammation, whereas its receptor CXCR4 is involved in human immunodeficiency virus infection, cancer metastasis and in the rare disease known as the warts, hypogammaglobulinemia, immunodeficiency, and myelokathexis (WHIM) syndrome. As we screened chemical libraries to find inhibitors of the interaction between CXCL12 and the receptor CXCR4, we identified synthetic compounds from the family of chalcones that reduce binding of CXCL12 to CXCR4, inhibit calcium responses mediated by the receptor, and prevent CXCR4 internalization in response to CXCL12. We found that the chemical compounds display an original mechanism of action as they bind to the chemokine but not to CXCR4. The highest affinity molecule blocked chemotaxis of human peripheral blood lymphocytes ex vivo. It was also active in vivo in a mouse model of allergic eosinophilic airway inflammation in which we detected inhibition of the inflammatory infiltrate. The compound showed selectivity for CXCL12 and not for CCL5 and CXCL8 chemokines and blocked CXCL12 binding to its second receptor, CXCR7. By analogy to the effect of neutralizing antibodies, this molecule behaves as a small organic neutralizing compound that may prove to have valuable pharmacological and therapeutic potential.

The ligand occupancy of endothelial protein C receptor switches the protease-activated receptor 1-dependent signaling specificity of thrombin from a permeability-enhancing to a barrier-protective response in endothelial cells

AbstractRecent studies have indicated that activated protein C (APC) may exert its cytoprotective and anti-inflammatory activities through the endothelial protein C receptor (EPCR)-dependent cleavage of protease-activated receptor 1 (PAR-1) on vascular endothelial cells. Noting that (1) the activation of protein C on endothelial cells requires thrombin, (2) relative to APC, thrombin cleaves PAR-1 with approximately 3 to 4 orders of magnitude higher catalytic efficiency, and (3) PAR-1 is a target for the proinflammatory activity of thrombin, it is not understood how APC can elicit a protective signaling response through the cleavage of PAR-1 when thrombin is present. In this study, we demonstrate that EPCR is associated with caveolin-1 in lipid rafts of endothelial cells and that its occupancy by the γ-carboxyglutamic acid (Gla) domain of protein C/APC leads to its dissociation from caveolin-1 and recruitment of PAR-1 to a protective signaling pathway through coupling of PAR-1 to the pertussis toxin–sensitive Gi-protein. Thus, when EPCR is bound by protein C, the PAR-1 cleavage-dependent protective signaling responses in endothelial cells can be mediated by either thrombin or APC. These results provide a new paradigm for understanding how PAR-1 and EPCR participate in protective signaling events in endothelial cells.

The Ligand Occupancy of Endothelial Protein C Receptor Switches the PAR-1 Dependent Signaling Specificity of Thrombin from a Disruptive to a Protective Response in Endothelial Cells.

It has been hypothesized that activated protein C (APC) exerts its cytoprotective and antiinflammatory activities through the endothelial protein C receptor (EPCR)-dependent cleavage of protease activated receptor 1 (PAR-1) on vascular endothelial cells. Noting thati.the activation of protein C on endothelial cells requires thrombin,ii.relative to APC, thrombin cleaves PAR-1 with ∼3–4-orders of magnitude higher catalytic efficiency, andiii.PAR-1 is a target for the proinflammatory activity of thrombin, it is not understood how APC can elicit a protective signaling response through the cleavage of PAR-1 when thrombin is present.In this study, we demonstrate that EPCR is associated with caveolin-1 in lipid rafts of endothelial cells and that its occupancy by the Gla-domain of protein C/APC leads to its dissociation from caveolin-1 and recruitment of PAR-1 to a protective signaling pathway through coupling of PAR-1 to the pertussis toxin sensitive Gi-protein. Thus, when EPCR is bound by protein C/APC, the PAR-1 cleavage-dependent protective signaling responses in endothelial cells can be mediated by either thrombin or APC. These results provide a new paradigm for understanding how PAR-1 and EPCR participate in protective signaling events in endothelial cells.

Oxytocin Receptors Differentially Signal via Gq and Gi Proteins in Pregnant and Nonpregnant Rat Uterine Myocytes: Implications for Myometrial Contractility

Oxytocin (OT) receptors are important regulators of myometrial contractility. By using the activity of large conductance Ca2+-activated K+ (BKCa) channels as readout, we analyzed OT signaling in cells from nonpregnant (NPM) and pregnant (PM) rat myometrium in detail. In nystatin-perforated whole-cell patches from NPM cells, which leave the intracellular integrity intact, OT transiently increased BKCa-mediated outward currents (Iout). This OT-evoked Iout was caused by the Ca2+ transients in response to the Gq/11-mediated activation of phospholipase C and was inhibited by activation of protein kinase A (PKA). In an open-access whole-cell patch (OAP), the OT-induced transient rise in Iout was disrupted whereas the regulation of BKCa by the cAMP/PKA cascade remained intact. OT counteracted the isoprenaline, i.e. the beta-adrenoceptor/Gs-mediated effect in NPM cells measured in OAP. In contrast, OT further enhanced the beta-adrenoceptor/Gs-mediated effect on BKCa activity in PM cells. All OT effects in the OAP were mediated by pertussis toxin-sensitive Gi proteins and PKA. By quantitative real-time PCR and overexpression of the recombinant protein, we demonstrate that an up-regulation of the Gbetagamma-stimulated adenylyl cyclase II during pregnancy is most likely responsible for this switch. By studying the OT-evoked Iout in nystatin-perforated whole-cell patches of PM cells, we further detected that the OT receptor/Gibetagamma-mediated coactivation of adenylyl cyclase II enhanced the beta-adrenoceptor/Gs-induced suppression of the OT-evoked Ca2+ transients and thus diminishes and self-limits OT-induced contractility. The differential regulation of the PKA-mediated suppression of OT-evoked Ca2+ transients and BKCa activity likely supports uterine quiescence during pregnancy.

Sphingosine 1-phosphate prevents platelet-activating factor-induced increase in hydraulic conductivity in rat mesenteric venules: pertussis toxin sensitive

Sphingosine 1-phosphate (S1P) is a biologically active lipid. In vitro, S1P tightens the endothelial barrier, as assessed by a rapid increase in electrical resistance and a decrease in solute permeability. We hypothesized that this activity of S1P would also occur in vivo. Hydraulic conductivity (Lp), an assessment of endothelial barrier function, was measured in individually perfused venules in rat mesenteries. S1P (1 microM) decreased basal Lp by 63% when basal Lp was between 3.6 and 4.1 x 10(-7) cm x s(-1) x cmH2O(-1) but showed no effect when basal Lp was below 2 x 10(-7) cm x s(-1) x cmH2O(-1). Under either condition, S1P blocked the sixfold increase in Lp induced by platelet-activating factor (PAF, 10 nM). Perfusion of venules with pertussis toxin (0.1 microg/ml), a specific inhibitor of the inhibitory G protein, Gi, for 3 h did not affect basal Lp or the increased Lp induced by PAF. Pertussis toxin, however, significantly attenuated the inhibitory action of S1P on the PAF-induced increase in Lp, indicating the involvement of the Gi protein. Measurement of endothelial cytoplasmic Ca2+ concentration ([Ca2+]i) in venules loaded with fura-2 AM showed that S1P alone transiently increased basal endothelial [Ca2+]i (from 89 nM to 193 nM) but had no effect on the magnitude and time course of the PAF-induced increase in endothelial [Ca2+]i. These results indicate that S1P functions in vivo to prevent the PAF-induced increase in microvessel permeability. The inhibitory action of S1P involves the pertussis toxin-sensitive Gi protein and is not mediated by prevention of the PAF-induced increase in endothelial [Ca2+]i.

A functional C5a anaphylatoxin receptor in a teleost species.

The anaphylatoxins are potent, complement-derived low m.w. proteins that bind to specific seven-transmembrane receptors to elicit and amplify a variety of inflammatory reactions. C5a is the most potent of these phlogistic peptides and is a strong chemoattractant for neutrophils and macrophages/monocytes. Although lower vertebrates possess complement systems that are believed to function similarly to those of mammals, anaphylatoxin receptors have not previously been characterized in any nonmammalian vertebrate. To study the functions of C5a in teleost fish, we generated recombinant C5a of the rainbow trout, Oncorhynchus mykiss (tC5a), and used fluoresceinated tC5a (tC5aF) and flow cytometry to identify the C5a receptor (C5aR) on trout leukocytes. Granulocytes/Macrophages present in cell suspensions of the head kidney (HKL), the main hemopoietic organ in teleosts, showed a univariate type of receptor expression, whereas those from the peripheral blood demonstrated either a low or high level of expression. The binding of tC5aF was inhibited by excess amounts of unlabeled tC5a or tC5a(desArg), demonstrating that sites other than the C-terminal of tC5a interact with the C5aR. Both tC5a and tC5a(desArg) were able to induce chemotactic responses in granulocytes in a concentration-dependent manner, but the desArg derivative was at least 10-fold less active. Homologous desensitization occurred after HKL were exposed to continuous or high concentrations of tC5a, with a loss of tC5aF binding and an 80% reduction in chemotactic responses toward tC5a. Pertussis toxin reduced the migration of HKL toward tC5a by 40%, suggesting only a partial involvement of pertussis toxin-sensitive G(i) proteins in tC5a-mediated chemotaxis.

Characterization of Adenosine A1 Receptors in Human Proximal Tubule Epithelial (HK-2) Cells

Adenosine A1 receptors (A1ARs) modulate various aspects of renal functions, such as hormone release, hemodynamics and tubular absorption. Here we set up to demonstrate the expression of A1ARs in an immortalized cell line (HK-2) derived from normal adult human proximal tubule. We also examined the mechanism whereby A1ARs signal in HK-2 cells and their potential role in renal physiology such as sodium-dependent phosphate transport. Ligand binding assay of A1ARs was performed using plasma membranes of HK-2 cells and a selective high-affinity A1AR radioligand [3H]DPCPX. HK-2 cells in 96-well plates were treated with various agents (forskolin, adenosine receptor agonists, and antagonists) to activate or inhibit adenylate cyclase. Intracellular cyclic AMP accumulation was measured using cAMP flashplates. mRNA levels of adenosine receptors in HK-2 cells was determined by real-time PCR technique. Sodium-dependent phosphate transport across cell membrane was measured after 15-minute incubation of phosphorus-33 in transport buffer with HK-2 cells at room temperature. In HK-2 cells, A1ARs were expressed at a density of 211 +/- 74 fmol/mg membrane proteins. [3H]DPCPX bound to A1ARs on HK-2 cell membranes with Kd of 8.3 +/- 2.2 nM. Activation of A1ARs inhibited isoproterenol-stimulated adenylate cyclase activity through pertussis toxin-sensitive Gi protein in HK-2 cells. Coexpression of adenosine A2a receptors at a seemingly lower level than A1ARs was revealed by synergistically activating adenylate cyclase with forskolin. Real-time RT-PCR further demonstrated the expression of both A1AR and A2aAR in HK-2 cells. Sodium-dependent phosphate transport was augmented by activation of A1ARs in HK-2 cells. A1ARs are expressed in human proximal tubule epithelial (HK-2) cells and modulate sodium-dependent phosphate transport.

Zona pellucida-induced acrosome reaction in human sperm: dependency on activation of pertussis toxin-sensitive G(i) protein and extracellular calcium, and priming effect of progesterone and follicular fluid.

In these studies, we aimed to characterize the effects of the physiological, homologous agonists of the acrosome reaction, i.e. the zona pellucida (ZP) and progesterone/follicular fluid, on human sperm. The specific aims of our studies were: (i) to examine the dependency of the solubilized ZP-induced acrosome reaction on G(i) protein activation and presence of extracellular calcium; and (ii) to determine whether progesterone/follicular fluid exert a priming or synergist effect on the solubilized ZP-induced acrosome reaction. Highly motile sperm from fertile donors were exposed to the agonists in a microassay and the acrosomal status of live sperm was determined by indirect immunofluorescence using PSA-FITC/Hoechst double-staining. Pretreatment with pertussis-toxin (100 ng/ml) and EGTA (2.5 mmol/l) significantly inhibited the ZP-induced acrosome reaction without affecting the spontaneous rate of exocytosis. Progesterone (1.25 microg/ml) and human follicular fluid (10%) exerted a priming, time-dependent effect on the ZP-induced acrosome reaction. These studies demonstrated that: (i) acrosomal exocytosis of capacitated human sperm triggered by the homologous ZP is dependent on the activation of G(i) proteins (pertussis toxin-sensitive) and the presence of extracellular calcium; and (ii) progesterone and follicular fluid exert a priming effect on the ZP-induced acrosome reaction.

Human dopamine D(3) receptors mediate mitogen-activated protein kinase activation via a phosphatidylinositol 3-kinase and an atypical protein kinase C-dependent mechanism.

The mitogen-activated protein kinase (MAPK) cascade is stimulated by both receptor tyrosine kinases and G protein-coupled receptors. We show that recombinant human dopamine D(3) receptors expressed in Chinese hamster ovary cells transiently activate MAPK via pertussis toxin-sensitive Gi and/or Go proteins. The involvement of D(3) receptors was confirmed by use of the D(3) agonists PD 128,907 and (+)-7-hydroxy-2-dipropylaminotetralin, which mimicked the response to dopamine (DA). Furthermore, haloperidol and the selective D(3) receptor antagonists S 14297 and GR 218,231 attenuated DA-induced MAPK activation; however, when tested alone, S 14297 weakly stimulated MAPK activity, suggesting partial agonist activity. The transduction mechanisms by which hD(3) receptors activate MAPK were explored with specific kinase inhibitors. Genistein and lavendustin A, inhibitors of tyrosine kinase activity, did not reduce DA-induced MAPK activation. In contrast, PD 98059, an inhibitor of MAPK kinase, and Ro 31-8220 and Gö 6983, inhibitors of protein kinase C (PKC), blocked DA-induced MAPK activation. However, MAPK activation was insensitive to PKC down-regulation by phorbol esters, indicating the involvement of an "atypical" PKC. Furthermore, MAPK activation involved phosphatidylinositol 3-kinase inasmuch as its inhibition by LY 294002 and wortmannin reduced DA-induced MAPK activation. In conclusion, this study demonstrates that stimulation of hD(3) receptors activates MAPK. This action is mediated via an atypical isoform of PKC, possibly involving cross-talk with products of phosphatidylinositol 3-kinase activation.

Galanin inhibits acetylcholine release from rat cerebral cortex via a pertussis toxin-sensitive Giprotein

Galanin has been implicated in various physiological functions including memory, feeding and pain perception. Using rat cerebral cortical slices and synaptosome preparations incubated with [3H]choline in Kreb’s-Ringer solution, galanin was shown to inhibit both spontaneous and K+-stimulated [3H]ACh release in a concentration-related manner [EC50= 35 nM]. The galanin-mediated inhibition on spontaneous and K+-stimulated [3H]ACh release was respectively regulated by pertussis toxin-sensitive Gαi3and Gαi1. These suggest that galanin is a negative modulator of cortical cholinergic function and most probably acting on presynaptic cholinergic terminals. Although galantide blocked the galanin-mediated inhibitory effect on [3H]ACh release, it mimicked galanin in blocking K+-stimulated [3H]ACh release, indicating that galantide may have a more complicated pharmacology than being a galanin receptor antagonist. In addition, we demonstrate that galanin and β-amyloid peptide1–42synergistically attenuated K+-evoked [3H]ACh release from synaptosomes prepared from rat cerebral cortex. Since galanin is increased in Alzheimer’s disease brain, our results suggest that galanin may be involved in cholinergic dysfunctions that occur in Alzheimer’s disease.