RESUMEN

The mechanisms of activation of cytoplasmic phospholipase A2 (cPLA2) are complex and incompletely defined. In Chinese hamster ovary (CHO) cells, receptor stimulation of cPLA2 is due to the interaction of pathways involving the alpha subunits of at least two guanine-nucleotide-binding (G) proteins, G alpha i2 and G alpha q. Activation of cPLA2 is inhibited by pertussis toxin and G alpha i2 mutants. In addition, activation of phospholipase C via G alpha q results in increased intracellular calcium ([Ca2+]i) and activation of protein kinase C, both of which interact with and activate cPLA2. The present study was undertaken to analyze the mechanism of interaction of G alpha i2 with the phospholipase-C-stimulated pathway in the activation of cPLA2. We addressed this question using a dominant negative G alpha i2 mutant, [G203T]G alpha i2, in which Gly203 is mutated to Thr. [G203T]G alpha i2 inhibits ATP receptor activation of cPLA2. The effect of [G203T]G alpha i2 was specific to G alpha i2-activated pathways, as shown by its lack of effect on other purinergic receptor stimulated pathways: ATP stimulation of [Ca2+]i or mitogen-activated protein kinase phosphorylation is unaltered by [G203T]G alpha i2. We addressed the possibility that the activation of cPLA2 by Ca2+ and/or protein kinase C is dependent on G alpha i2. Activation of cPLA2 by the Ca2+ ionophore, ionomycin, was inhibited by 61 +/- 9% (n = 5) in [G203T]G alpha i2-expressing cells; however the ionomycin-induced [Ca2+]i rise was unaffected by [G203T]G alpha i2. Thus, [G203T]G alpha i2. specifically inhibits Ca2+ activation of cPLA2. In contrast, activation of cPLA2 via protein kinase C by phorbol 12-myristate 13-acetate was unaffected by [G203T]G alpha i2. Our results demonstrate that Ca2+ but not phorbol ester activation of cPLA2 in CHO cells is G alpha i2-dependent. The possibility is discussed that G alpha i2 is downstream of Ca2+ but upstream of protein kinase C activation of cPLA2.

Asunto(s)

Calcio/fisiología , Proteínas de Unión al GTP/fisiología , Fosfolipasas A/fisiología , Animales , Ácido Araquidónico/metabolismo , Secuencia de Bases , Células CHO , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Cricetinae , AMP Cíclico/biosíntesis , Activación Enzimática , Ionomicina/farmacología , Datos de Secuencia Molecular , Fosfolipasas A2 , Fosforilación , Acetato de Tetradecanoilforbol/farmacología

RESUMEN

Somatostatin (SST) receptors activate potassium channels, stimulate protein phosphatases, inhibit adenylate cyclase and close calcium channels. These multiple effects are controlled by guanine nucleotide binding (G) proteins of the pertussis toxin-sensitive Gi and Go types. In the present study we have identified the G proteins coupling with brain SST receptors. To this end, brain SST receptors were solubilized in G-protein coupled form. Binding of the SST analogue MK 678 to the solubilized receptor was completely inhibited by guanosine 5'-O-thiotriphosphate (IC50 = 100 nM), reflecting decreased receptor affinity for agonist following uncoupling of the receptor and G protein(s). Antibodies raised against specific COOH-terminal peptides of the G proteins Gi(1-3), Go, and Gz were used to probe for SST receptor-G protein coupling in this system. Antibodies binding to the COOH-terminal regions of Gi1 and Gi2 (antibody AS) and Gi3 (antibody EC) inhibited binding of 125I-MK 678 (75 pM) by 57 +/- 4% and 48 +/- 5%, respectively. The effects of these antibodies were concentration-dependent and additive, such that in combination AS and EC completely inhibited binding. Antibodies binding to the COOH-terminal region of Go (GO) and Gz (QN) did not affect binding of 125I-MK 678, indicating that neither Go nor Gz are associated with the brain SST receptor. Prelabeling of the receptor with 125I-MK 678 prior to addition of antibody induced the formation of a "locked conformation" of the agonist-bound receptor-G protein complex which was insensitive to antibody. In conclusion, Gi1 and/or Gi2 and Gi3 are coupled in approximately equal proportions to the brain 125I-MK 678-binding SST receptor, accounting for all of the G protein coupling of this receptor.

Asunto(s)

Encéfalo/metabolismo , Proteínas de Unión al GTP/metabolismo , Receptores de Neurotransmisores/metabolismo , Somatostatina/metabolismo , Toxina de Adenilato Ciclasa , Secuencia de Aminoácidos , Animales , Anticuerpos , Membrana Celular/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Cinética , Datos de Secuencia Molecular , Oligopéptidos/química , Oligopéptidos/inmunología , Toxina del Pertussis , Ratas , Receptores de Neurotransmisores/aislamiento & purificación , Receptores de Somatostatina , Factores de Virulencia de Bordetella/farmacología

RESUMEN

Cells of the pituitary tumour cell line GH(4) C(1) were exposed to epidermal growth factor, estradiol and insulin for 5 days, a treatment which resulted in 1) increased prolactin storage in secretory granules, 2) the loss of spontaneous [Ca(2+) ](1) oscillations, and 3) a selective reduction of the protein G(s) α, seen in immunoblots, cholera toxin labelling, and vasoactive intestinal peptide stimulation of adenylyl cyclase. In contrast, the glucocorticoid dexamethasone, which increases the expression of G(s) α, partially restored spontaneous [Ca(2+) ](1) oscillations and decreased prolactin storage. It is concluded that G(s) α levels in tumoral cells result in spontaneous electrical activity which may empty prolactin stores by the continuous activation of exocytosis.