The triakontatetraneuropeptide TTN increases [Ca²⁺]_i in rat astrocytes through activation of peripheral‐type benzodiazepine receptors

Abstract

Astrocytes synthesize a series of regulatory peptides called endozepines, which act as endogenous ligands of benzodiazepine receptors. We have recently shown that one of these endozepines, the triakontatetraneuropeptide TTN, stimulates DNA synthesis in astroglial cells. The purpose of the present study was to determine the mechanism of action of TTN on cultured rat astrocytes. Binding of the peripheral-type benzodiazepine receptor ligand [3H]Ro5-4864 to intact astrocytes was displaced by TTN, whereas its C-terminal fragment (TTN[17-34], the octadecaneuropeptide ODN) did not compete for [3H]Ro5-4864 binding. Microfluorimetric measurement of cytosolic calcium concentrations ([Ca2+]i) with the fluorescent probe indo-1 showed that TTN (10(-10) to 10(-6) M) provokes a concentration-dependent increase in [Ca2+]i in cultured astrocytes. Simultaneous administration of TTN (10(-8) M) and Ro5-4864 (10(-5) M) induced an increase in [Ca2+]i similar to that obtained with Ro5-4864 alone. In contrast, the effects of TTN (10(-8) M) and ODN (10(-8) M) on [Ca2+]i were strictly additive. Chelation of extracellular Ca2+ by EGTA (6 mM) or blockage of Ca2+ channels with Ni2+ (2 mM) abrogated the stimulatory effect of TTN. The calcium influx evoked by TTN (10(-7) M) or by Ro5-4864 (10(-5) M) was not affected by the N- and T-type calcium channel blockers omega-conotoxin (10(-6) M) and mibefradil (10(-6) M), but was significantly reduced by the L-type calcium channel blocker nifedipine (10(-7) M). Patch-clamp studies showed that, at negative potentials, TTN (10(-7) M) induced a sustained depolarization. Reduction of the chloride concentration in the extracellular solution shifted the reversal potential from 0 mV to a positive potential. These data show that TTN, acting through peripheral-type benzodiazepine receptors, provokes chloride efflux, which in turn induces calcium influx via L-type calcium channels in rat astrocytes.

Functional characterization and expression of PBR in rat gastric mucosa: stimulation of chloride secretion by PBR ligands

“…Furthermore, when calcium was removed from extracellular medium, PK 11195 induced an inversed current, possibly due to the calcium efflux from the cytosol. The effect of exogenous and endogenous PBR ligands on activation of L-type Ca 2ϩ channels has been previously reported (14,59). It was also shown that PBR ligands provoke chloride efflux related to calcium influx via L-type Ca 2ϩ channels in the rat astrocytes (14).…”

“…The effect of exogenous and endogenous PBR ligands on activation of L-type Ca 2ϩ channels has been previously reported (14,59). It was also shown that PBR ligands provoke chloride efflux related to calcium influx via L-type Ca 2ϩ channels in the rat astrocytes (14). On the other hand, it has been indicated that DBI elicits intracellular Ca 2ϩ oscillations in STC-1 cells inhibited by L-type Ca 2ϩ channel blockers (59).…”

“…5, 9, and 38). These include porphyrin transport and heme biosynthesis (49), cholesterol transport and steroidogenesis (39) or bile salts biosynthesis (51), apoptosis (21), and anion transport (3,14).…”

Peptide signaling during terminal differentiation ofDictyostelium

“…In mammals ACBP is processed to form diazepam binding inhibitor, which modulates the GABA A receptor in the brain and affects various peripheral organs (17)(18)(19). In the brain, ACBP is processed to an anionic peptide of 36 aa (triakontatetraneuropeptide, TTN) and its C-terminal 20-aa fragment (ODN) (Fig.…”

Pharmacological Characterization of the Receptor Mediating the Anorexigenic Action of the Octadecaneuropeptide: Evidence for an Endozepinergic Tone Regulating Food Intake

“…DBI and ODN act as inverse agonists of central-type benzodiazepine receptors (CBR) associated with the GABA A receptor complex (Guidotti et al, 1983;Tonon et al, 1989;Bormann, 1991;Louiset et al, 1993). TTN acts preferentially through peripheral-type benzodiazepine receptors (PBR) located either at the outer mitochondrial membrane or at the plasma membrane level Berkovich et al, 1990;Gandolfo et al, 2001). Finally, ODN can also activate a metabotropic receptor positively coupled to phospholipase C through a pertussis toxin-sensitive G-protein (Patte et al, 1995;Gandolfo et al, 1997).…”