Activation of the neurokinin-1 receptor in rat spinal astrocytes induces Ca 2+ release from IP 3-sensitive Ca 2+ stores and extracellular Ca 2+ influx through TRPC3

Abstract

Substance P (SP) plays an important role in pain transmission through the stimulation of the neurokinin (NK) receptors expressed in neurons of the spinal cord, and the subsequent increase in the intracellular Ca 2+ concentration ([Ca 2+] i) as a result of this stimulation. Recent studies suggest that spinal astrocytes also contribute to SP-related pain transmission through the activation of NK receptors. However, the mechanisms involved in the SP-stimulated [Ca 2+] i increase by spinal astrocytes are unclear. We therefore examined whether (and how) the activation of NK receptors evoked increase in [Ca 2+] i in rat cultured spinal astrocytes using a Ca 2+ imaging assay. Both SP and GR73632 (a selective agonist of the NK1 receptor) induced both transient and sustained increases in [Ca 2+] i in a dose-dependent manner. The SP-induced increase in [Ca 2+] i was significantly attenuated by CP-96345 (an NK1 receptor antagonist). The GR73632-induced increase in [Ca 2+] i was completely inhibited by pretreatment with U73122 (a phospholipase C inhibitor) or xestospongin C (an inositol 1,4,5-triphosphate (IP 3) receptor inhibitor). In the absence of extracellular Ca 2+, GR73632 induced only a transient increase in [Ca 2+] i. In addition, H89, an inhibitor of protein kinase A (PKA), decreased the GR73632-mediated Ca 2+ release from intracellular Ca 2+ stores, while bisindolylmaleimide I, an inhibitor of protein kinase C (PKC), enhanced the GR73632-induced influx of extracellular Ca 2+. RT-PCR assays revealed that canonical transient receptor potential (TRPC) 1, 2, 3, 4 and 6 mRNA were expressed in spinal astrocytes. Moreover, BTP2 (a general TRPC channel inhibitor) or Pyr3 (a TRPC3 inhibitor) markedly blocked the GR73632-induced sustained increase in [Ca 2+] i. These findings suggest that the stimulation of the NK-1 receptor in spinal astrocytes induces Ca 2+ release from IP 3-sensitive intracellular Ca 2+ stores, which is positively modulated by PKA, and subsequent Ca 2+ influx through TRPC3, which is negatively regulated by PKC.