A Novel Follicle-Stimulating Hormone-Induced Gαh/Phospholipase C-δ1 Signaling Pathway Mediating Rat Sertoli Cell Ca²⁺-Influx

Abstract

FSH is known to activate Gs/cAMP signaling pathway in Sertoli cells (SCs) to support spermatogenesis. However, the molecular mechanism of FSH-induced Gs/cAMP-independent Ca2+-influx in SCs is not clear. In this study, FSH indeed induced an immediate and dose-dependent intracellular Ca2+-elevation in rat SCs. In the presence of EDTA (2.5 mm) or in the absence of extracellular Ca2+, the FSH-induced intracellular Ca2+-elevation was abolished. The confocal microscopic observation of Ca2+ image revealed that the SC cellular Ca2+ level was gradually increased after 50 sec of FSH treatment. Dantrolene, a blocker of intracellular Ca2+ release, did not affect this FSH-induced intracellular Ca2+ elevation. The pretreatment of rat SCs with phosphatidylinositol-phospholipase C (PLC)-specific inhibitor, U73122 (3 and 10 microm), inhibited the FSH-induced Ca2+-influx in a dose-dependent manner, but treatment with Gs-specific inhibitor, NF449 (0.1 and 0.3 microm), did not. On the other hand, the activation of G alpha h was immediately induced by FSH in the rat SCs within 5 sec of treatment. The translocation of PLC-delta1 from cytosol to cell membrane and the formation of G alpha h /PLC-delta1 complexes occurred within 5 and 10 sec, respectively, of FSH exposure. The intracellular inositol 1,4,5-triphosphate (IP3) production was also detected after 30 sec of FSH treatment. The synthetic peptide of PLC-delta1 (TIPWNSLKQGYRHVHLL), not Gs inhibitor, predominantly inhibited the FSH-induced PLC-delta1 translocation, formation of G alpha h /PLC-delta1 complex, intracellular IP3 production, and Ca2+ influx. In contrast, the peptide did not interfere with FSH-induced intracellular cAMP accumulation. In conclusion, the FSH-induced immediate Ca2+ influx is unambiguously mediated by an alternative G alpha h /PLC-delta1/IP3 pathway that is distinct from the Gs/cAMP pathway in rat SCs.

Putative primary involvement of Arabidopsis phosphoinositide-specific phospholipase C1 within abscisic acid-induced stomatal closing

“…Despite some differences, the molecular structures of mammalian PLCδ1 (Essen et al 1996) and AtPLC1 (Mueller-Roeber and Pical 2002) are related: in particular, they do not contain a regulatory carboxyterminal region thought to be needed for coupling PI-PLC to G protein. However, PLCδ1 is tightly coupled to Gα h (Lin et al 2006). Then, assuming that the inhibitory property of U73122 depends on the molecular mechanisms that couple G proteins to PI-PLC isoforms, the fact that inhibition of AtPLC1 would be optimal at a considerably lower U73122 concentration than those reported for blocking Gα h -PLCδ1 coupling (Lin et al 2006) might corroborate that a putative Gα-like proteinAtPLC1 coupling exists, which differs from Gα h -PLCδ1 coupling.…”

“…However, PLCδ1 is tightly coupled to Gα h (Lin et al 2006). Then, assuming that the inhibitory property of U73122 depends on the molecular mechanisms that couple G proteins to PI-PLC isoforms, the fact that inhibition of AtPLC1 would be optimal at a considerably lower U73122 concentration than those reported for blocking Gα h -PLCδ1 coupling (Lin et al 2006) might corroborate that a putative Gα-like proteinAtPLC1 coupling exists, which differs from Gα h -PLCδ1 coupling.…”

“…Tested in the 1 -10 µM range, U73122, but not U73343, had been shown in animal systems to interfere with PI-PLC activity through inhibiting its positive regulation by G protein α subunits (Gαs) (Thompson et al 1991, Lin et al 2006. Coupling of Gα q or Gα h  to the PLCβ or PLCδ1 isoforms, respectively, had been demonstrated.…”

“…First, upon light stimulation of the seventransmembrane-span (7TMS) receptor, rhodopsin, Gα q is activated and directly binds to PLCβ in the compound eye of Drosophila (Bähner et al 2000). Second, folliclestimulating hormone induces rat sertoli cell Ca 2+ influx through a signalling pathway that implicates both the activation of Gα h   , the translocation of PLCδ1 from the cytosol to the plasma membrane and the formation of a Gα h /PLCδ1 complexe (Lin et al 2006). Likewise, although our study tested a thousand times lower concentration of U73122, the fact that U73122, but not U73343, substantially inhibited ABA stomatal closing only in the absence of Dex suggested G protein-coupled AtPLC1 activity within guard cell ABA signalling.…”

Novel pathways in gonadotropin receptor signaling and biased agonism

“…The FSH-R has been demonstrated to trigger pertussis toxin-sensitive pathways in certain circumstances depending on the nature and structure of the ligand bound [32] or the developmental stage of the host cells [33]. The FSH-R has also been shown to activate the inositol trisphosphate or IP3 signaling pathway, particularly at high doses of agonist [34,35]. Similarly, the LH-R has been reported to not only activate adenylate cyclase activity but also to stimulate IP3 production [36] and to couple to the Gi protein [37,38].…”