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Rat fat-cells have three types of adenosine receptors (Ra, Ri and P). Differential effects of pertussis toxin.

ABSTRACT: Activation of rat adipocyte R1 adenosine receptors by phenylisopropyladenosine (PIA) decreased cyclic AMP and lipolysis; this effect was blocked in cells from pertussis-toxin-treated rats. In contrast, the ability of 2',5'-dideoxyadenosine to decrease cyclic AMP was not affected by pertussis-toxin treatment. Addition of adenosine deaminase to the medium in which adipocytes from control animals were incubated resulted in activation of lipolysis. Interestingly, adipocytes from toxin-treated rats (which had an already increased basal lipolysis) responded in an opposite fashion to the addition of adenosine deaminase, i.e. the enzyme decreased lipolysis, which suggested that adenosine might be increasing lipolysis in these cells. Studies with the selective agonists N-ethylcarboxamidoadenosine (NECA) and PIA indicated that adenosine increases lipolysis and cyclic AMP accumulation in these cells and that these actions are mediated through Ra adenosine receptors. Adenosine-mediated accumulation of cyclic AMP was also observed in cells preincubated with pertussis toxin (2 micrograms/ml) for 3 h. In these studies NECA was also more effective than PIA. Our results indicate that there are three types of adenosine receptors in fat-cells, whose actions are affected differently by pertussis toxin, i.e. Ri-mediated actions are abolished, Ra-mediated actions are revealed and P-mediated actions are not affected.

SUBMITTER: Garcia-Sainz JA

PROVIDER: S-EPMC1152899 | biostudies-other | 1985 Dec

REPOSITORIES: biostudies-other

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A2B adenosine receptors inhibit superoxide production from mitochondrial complex I in rabbit cardiomyocytes via a mechanism sensitive to Pertussis toxin.

Project description:Background and purposeA(2B) adenosine receptors protect against ischaemia/reperfusion injury by activating survival kinases including extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase (PI3K). However, the underlying mechanism(s) and signalling pathway(s) remain undefined.Experimental approachHEK 293 cells stably transfected with human A(2B) adenosine receptors (HEK-A(2B) ) and isolated adult rabbit cardiomyocytes were used to assay phosphorylation of ERK by Western blot and cation flux through cAMP-gated channels by patch clamp methods. Generation of reactive oxygen species (ROS) by mitochondria was measured with a fluorescent dye.Key resultsIn HEK-A(2B) cells, the selective A(2B) receptor agonist Bay 60-6583 (Bay 60) increased ERK phosphorylation and cAMP levels, detected by current through cAMP-gated ion channels. However, increased cAMP or its downstream target protein kinase A was not involved in ERK phosphorylation. Pertussis toxin (PTX) blocked ERK phosphorylation, suggesting receptor coupling to G(i) or G(o) proteins. Phosphorylation was also blocked by inhibition of PI3K (with wortmannin) or of ERK kinase (MEK1/2, with PD 98059) but not by inhibition of NO synthase (NOS). In cardiomyocytes, Bay 60 did not affect cAMP levels but did block the increased superoxide generation induced by rotenone, a mitochondrial complex I inhibitor. This effect of Bay 60 was inhibited by PD 98059, wortmannin or PTX. Inhibition of NOS blocked superoxide production because NOS is downstream of ERK.Conclusion and implicationsActivation of A(2B) adenosine receptors reduced superoxide generation from mitochondrial complex I through G(i/o) , ERK, PI3K, and NOS, all of which have been implicated in ischaemic preconditioning.

| S-EPMC3130946 | biostudies-literature

Differential cholera-toxin- and pertussis-toxin-catalysed ADP-ribosylation of G-proteins coupled to formyl-peptide and leukotriene B4 receptors.

Project description:N-Formylmethionyl-leucyl-phenylalanine (fMet-Leu-Phe) and leukotriene B4 (LTB4) induce disparate second-messenger generation and functional responses in neutrophils and HL-60 granulocytes. Receptors for these chemoattractants couple to a common pool of G-proteins which are substrates for both pertussis-toxin- and cholera-toxin-catalysed ADP-ribosylation. The hypothesis that formyl-peptide and LTB4 receptors induce different receptor-specific conformations of activated G-proteins was tested. The ability of pertussis toxin and cholera toxin to ADP-ribosylate G(i) proteins coupled to formyl-peptide or LTB4 receptors in membranes isolated from HL-60 granulocytes was used to assess the conformational state of the alpha subunits. Cholera-toxin-catalysed ADP-ribosylation of alpha 40 (40 kDa alpha subunit) was inhibited by guanosine 5'-[beta gamma-imido]triphosphate and GDP in a concentration-dependent manner. Addition of fMet-Leu-Phe, but not LTB4, re-established cholera-toxin labelling of alpha 40 in the presence of either guanine nucleotide. In the absence of guanine nucleotides, fMet-Leu-Phe and C5a enhanced cholera-toxin-catalysed labelling of alpha 40, whereas LTB4 and platelet-activating factor had no effect. Preincubation with fMet-Leu-Phe, but not LTB4, inhibited pertussis-toxin labelling of alpha 40 in the presence of guanosine 5'-[gamma-thio]triphosphate and in the absence of guanine nucleotides. Preincubation with fMet-Leu-Phe or LTB4 enhanced pertussis-toxin labelling of alpha 40 in the presence of GDP. These data suggest that activated G(i) proteins coupled to formyl-peptide and LTB4 receptors exist in different conformations determined by the receptor with which they interact.

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Hydrodynamic properties of adenosine Ri receptors solubilized from rat cerebral-cortical membranes.

Project description:Adenosine Ri receptors and inhibitory guanine-nucleotide-regulatory components were solubilized from rat cerebral-cortical membranes with sodium cholate. (-)-N6-Phenylisopropyl[2,8-3H]adenosine [( 3H]PIA) binds with high affinity to the soluble receptors, which retain the pharmacological specificity of adenosine Ri receptors observed in membranes. The binding is regulated by bivalent cations and guanine nucleotides. Bivalent cations increase [3H]PIA binding by increasing both the affinity and the apparent number of receptors. Guanine nucleotides decrease agonist binding by increasing the dissociation of the ligand-receptor complex. Adenosine agonists stabilize the high-affinity form of the soluble receptor. The hydrodynamic properties of the adenosine receptor were determined with cholate extracts of membranes that were treated with [3H]PIA. Sucrose-gradient-centrifugation analysis indicates that the receptor has a sedimentation coefficient of 7.7 S. The receptor is eluted from Sepharose 6B columns with an apparent Stokes radius of 7.2 nm. Labelling of either sucrose-gradient or gel-filtration-column fractions with pertussis toxin and [32P]-NAD+ reveals that both the 41,000- and 39,000-Mr substrates overlap with the receptor activity. These studies suggest that the high-affinity adenosine-receptor-binding activity in the cholate extract represents a stable R1-N complex.

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| S-EPMC4734536 | biostudies-literature

Differential effects of suramin on the coupling of receptors to individual species of pertussis-toxin-sensitive guanine-nucleotide-binding proteins.

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| S-EPMC1148984 | biostudies-other

The History of Pertussis Toxin.

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| S-EPMC2815961 | biostudies-literature

Pertussis toxin exacerbates and prolongs airway inflammatory responses during Bordetella pertussis infection.

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