Abstract
Ca(2+) elevations in Chinese hamster ovary cells stably expressing OX(1) receptors were measured using fluorescent Ca(2+) indicators fura-2 and fluo-3. Stimulation with orexin-A led to pronounced Ca(2+) elevations with an EC(50) around 1 nm. When the extracellular [Ca(2+)] was reduced to a submicromolar concentration, the EC(50) was increased 100-fold. Similarly, the inositol 1,4,5-trisphosphate production in the presence of 1 mm external Ca(2+) was about 2 orders of magnitude more sensitive to orexin-A stimulation than in low extracellular Ca(2+). The shift in the potency was not caused by depletion of intracellular Ca(2+) but by a requirement of extracellular Ca(2+) for production of inositol 1,4,5-trisphosphate. Fura-2 experiments with the "Mn(2+)-quench technique" indicated a direct activation of a cation influx pathway by OX(1) receptor independent of Ca(2+) release or pool depletion. Furthermore, depolarization of the cells to +60 mV, which almost nullifies the driving force for Ca(2+) entry, abolished the Ca(2+) response to low concentrations of orexin-A. The results thus suggest that OX(1) receptor activation leads to two responses, (i) a Ca(2+) influx and (ii) a direct stimulation of phospholipase C, and that these two responses converge at the level of phospholipase C where the former markedly enhances the potency of the latter.
Highlights
The recently described hypothalamic peptides called orexins [1] or hypocretins [2] mediate their effects through G proteincoupled receptors called OX1 and OX2 receptors [1]
To test whether the inhibitory effect of EGTA on the orexin-A response was caused by Ca2ϩ pool depletion by EGTA, 2 M thapsigargin was added to the cells in the presence of 1 mM and 140 nM extracellular Ca2ϩ (Fig. 1, D and E)
No changes in [Ca2ϩ]i, total inositol phosphates, or IP3 could be detected upon activation of the receptor with low concentration of orexin-A when the [Ca2ϩ]o was reduced immediately before the challenge
Summary
The recently described hypothalamic peptides called orexins [1] or hypocretins [2] mediate their effects through G proteincoupled receptors called OX1 and OX2 receptors [1] The peptides and their receptors are widespread in the hypothalamus, cortex, and brainstem [2,3,4,5]. The orexins cause robust increases in intracellular Ca2ϩ both in neurons cultured from rat medial and lateral hypothalamus [6] and spinal cord [7], and when studied using recombinant receptors [1]. This has led to the suggestion that the receptors are coupled to the Gq family G proteins. The results reveal the presence of a novel amplification mechanism at the level of phospholipase C that is dependent on activation of Ca2ϩ influx pathway upstream of phospholipase C
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