Fingolimod inhibits glutamate release through activation of S1P1 receptors and the G protein ßγ subunit-dependent pathway in rat cerebrocortical nerve terminals.

Fingolimod, a sphingosine-1-phosphate (S1P) receptor modulator approved for treating multiple sclerosis, is reported to prevent excitotoxic insult. Because excessive glutamate release is a major cause of neuronal damage in various neurological disorders, the effect of fingolimod on glutamate release in rat cerebrocortical nerve terminals (synaptosomes) was investigated in the current study. Fingolimod decreased 4-aminopyridine (4-AP)-stimulated glutamate release and calcium concentration elevation. Fingolimod-mediated inhibition of 4-AP-induced glutamate release was dependent on extracellular calcium, persisted in the presence of the glutamate transporter inhibitor DL-TBOA or intracellular Ca2+-releasing inhibitors dantrolene and CGP37157, and was prevented by blocking vesicular transporters or N- and P/Q-type channels. Western blot and immunocytochemical analysis revealed the presence of S1P1 receptor proteins in presynaptic terminals. Fingolimod-mediated inhibition of 4-AP-induced glutamate release was also abolished by the sphingosine kinase inhibitor DMS, selective S1P1 receptor antagonist W146, Gi/o protein inhibitor pertussis toxin, and G protein ßγ subunit inhibitor gallein; however, it was unaffected by the adenylyl cyclase inhibitor SQ22536, protein kinase A inhibitor H89, and phospholipase C inhibitor U73122. These data indicate that fingolimod decreases glutamate release from rat cerebrocortical synaptosomes by suppressing N- and P/Q-type Ca2+ channel activity; additionally, the activation of presynaptic S1P1 receptors and the G protein ßγ subunit participates in achieving the effect.

On the selectivity of the Gαq inhibitor UBO-QIC: A comparison with the Gαi inhibitor pertussis toxin.

Gαq inhibitor UBO-QIC (FR900359) is becoming an important pharmacological tool, but its selectivity against other G proteins and their subunits, especially ßγ, has not been well characterized. We examined UBO-QIC's effect on diverse signaling pathways mediated via various G protein-coupled receptors (GPCRs) and G protein subunits by comparison with known Gαi inhibitor pertussis toxin. As expected, UBO-QIC inhibited Gαq signaling in all assay systems examined. However, other non-Gαq-events, e.g. Gßγ-mediated intracellular calcium release and inositol phosphate production, following activation of Gi-coupled A1 adenosine and M2 muscarinic acetylcholine receptors, were also blocked by low concentrations of UBO-QIC, indicating that its effect is not limited to Gαq. Thus, UBO-QIC also inhibits Gßγ-mediated signaling similarly to pertussis toxin, although UBO-QIC does not affect Gαi-mediated inhibition or Gαs-mediated stimulation of adenylyl cyclase activity. However, the blockade by UBO-QIC of GPCR signaling, such as carbachol- or adenosine-mediated calcium or inositol phosphate increases, does not always indicate inhibition of Gαq-mediated events, as the ßγ subunits released from Gi proteins following the activation of Gi-coupled receptors, e.g. M2 and A1Rs, may produce similar signaling events. Furthermore, UBO-QIC completely inhibited Akt signaling, but only partially blocked ERK1/2 activity stimulated by the Gq-coupled P2Y1R. Thus, we have revealed new aspects of the pharmacological interactions of UBO-QIC.

Cardioprotective effect of histamine H3-receptor activation: pivotal role of G beta gamma-dependent inhibition of voltage-operated Ca2+ channels.

We previously showed that activation of G(i/o)-coupled histamine H(3)-receptors (H(3)R) is cardioprotective because it attenuates excessive norepinephrine release from cardiac sympathetic nerves. This action is characterized by a marked decrease in intraneuronal Ca(2+) ([Ca(2+)](i)), as G alpha(i) impairs the adenylyl cyclase-cAMP-protein kinase A (PKA) pathway, and this decreases Ca(2+) influx via voltage-operated Ca(2+) channels (VOCC). Yet, the G(i/o)-derived betagamma dimer could directly inhibit VOCC, and the subsequent reduction in Ca(2+) influx would be responsible for the H(3)R-mediated attenuation of transmitter exocytosis. In this study, we tested this hypothesis in nerve-growth factor-differentiated rat pheochromocytoma cells (PC12) stably transfected with H(3)R (PC12-H(3)) and with the G betagamma scavenger beta-adrenergic receptor kinase 1 (beta-ARK1)-(495-689)-polypeptide (PC12-H(3)/beta-ARK1). Thus, we evaluated the effects of H(3)R activation directly on the following: 1) Ca(2+) current (I(Ca)) using the whole-cell patch-clamp technique; and 2) K(+)-induced exocytosis of endogenous dopamine. H(3)R activation attenuated both peak I(Ca) and dopamine exocytosis in PC12-H(3) but not in PC12-H(3)/beta-ARK1 cells. Moreover, a membrane permeable phosducin-like G betagamma scavenger also prevented the antiexocytotic effect of H(3)R activation. In contrast, the H(3)R-induced attenuation of cAMP accumulation and dopamine exocytosis in response to forskolin were the same in both PC12-H(3) and PC12-H(3)/beta-ARK1 cells. Our findings reveal that although G alpha(i) participates in the H(3)-mediated antiexocytotic effect when the adenylyl cyclase-cAMP-PKA pathway is stimulated, a direct G betagamma-induced inhibition of VOCC, resulting in an attenuation of I(Ca), plays a pivotal role in the H(3)R-mediated decrease in [Ca(2+)](i) and associated cardioprotective antiexocytotic effects. The discovery of this H(3)R-signaling step may offer new therapeutic approaches to cardiovascular diseases characterized by hyperadrenergic activity.