Receptor-mediated activation of phospholipase A2 via GTP-binding proteins: arachidonic acid and its metabolites as second messengers

Two independent signal transduction pathways regulate lymphocyte amiloride-sensitive sodium channels (ASSCs), one utilizing cAMP as a second messenger and the other utilizing a GTP-binding protein. This implies that two plasma membrane receptors play a role in the regulation of lymphocyte ASSCs. In this study, we tested the hypothesis that α1- and α2-adrenergic receptors independently regulate lymphocyte ASSCs via the two previously identified second messengers. Direct measurements indicated that norepinephrine increased lymphocyte cAMP and activated ASSCs. The α2-specific inhibitor, yohimbine, blocked this activation, thereby linking α2-adrenergic receptors to ASSC regulation via cAMP. The α1-specific ligand, terazosin, acted as an agonist and activated lymphocyte ASSCs but inhibited ASSC current that had been preactivated by norepinephrine or 8-(4-chlorophenylthio) (CPT)-cAMP. Terazosin had no effect on the lymphocyte whole cell ASSC currents preactivated by treatment with pertussis toxin. This finding indirectly links α1-adrenergic receptors to lymphocyte ASSC regulation via GTP-binding proteins. Terazosin had no direct inhibitory or stimulatory effects on α,β,γ-endothelial sodium channels reconstituted into planar lipid bilayers and expressed in Xenopus oocytes, ruling out a direct interaction between terazosin and the channels. These findings support the hypothesis that both α1- and α2-adrenergic receptors independently regulate lymphocyte ASSCs via GTP-binding proteins and cAMP, respectively.

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Relationship of GTP-binding proteins, phospholipase C, and PGE2 synthesis in rat glomerular mesangial cells

AJP Renal Physiology ◽

10.1152/ajprenal.1989.256.1.f171 ◽

1989 ◽

Vol 256 (1) ◽

pp. F171-F178 ◽

Cited By ~ 2

Author(s):

D. Schlondorff ◽

P. Singhal ◽

A. Hassid ◽

J. A. Satriano ◽

S. DeCandido

Keyword(s):

Arachidonic Acid ◽

Phospholipase C ◽

G Protein ◽

Binding Proteins ◽

Mesangial Cells ◽

Cdna Probe ◽

Ang Ii ◽

Glomerular Mesangial Cells ◽

Gtp Binding Proteins ◽

Gtp Binding

We evaluated the role of GTP-binding proteins in the activation of phospholipase C, release of arachidonic acid, and synthesis of prostaglandin (PG) E2 in response to platelet-activating factor (PAF) and angiotensin II (ANG II) in cultured rat mesangial cells. Pretreatment with pertussis toxin (PT) decreased PGE2 formation and arachidonic acid release in response to PAF and ANG II but not that to A 23187. PT pretreatment also inhibited formation of inositol trisphosphate (IP3) in response to ANG II or PAF but did not significantly alter the rise in intracellular calcium detected by fura-2. PT catalyzed ADP ribosylation of two proteins of molecular mass approximately 40 and 41 kDa. Further evidence for involvement of GTP-binding protein in phospholipase C activation was that GTP-gamma S stimulated IP3 generation. Immunoblots with antibodies directed against different inhibitory alpha subunits of GTP-binding proteins showed that the major 40-kDa PT substrate reacted with an antibody directed against a decapeptide of the G protein subunit alpha i2 that is also found in leukocytes. This was further confirmed by Northern blot that showed the existence of mRNA in mesangial cells that hybridized with a cDNA probe for G alpha i2. In addition lesser amounts of mRNA hybridized with a restriction fragment cDNA probe for G alpha i3, which corresponds to the 41-kDa substrate for PT ribosylation. These results show that phospholipase C activation by PAF and ANG II in mesangial cells involves a specific G protein, most likely G alpha i2.(ABSTRACT TRUNCATED AT 250 WORDS)

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