Muscarinic receptor-operated Ca2+ influx in transfected fibroblast cells is independent of inositol phosphates and release of intracellular Ca2+.

Abstract

Receptor-mediated changes in cytoplasmic calcium concentrations occur either through release from intracellular calcium stores or by the opening of channels in the plasma membrane, allowing influx of calcium from the extracellular fluid. Carbachol, a muscarinic receptor agonist, stimulated both calcium influx and inositol 1,4,5-trisphosphate (InsP3)-mediated intracellular calcium release in A9 fibroblast cells expressing a m3 muscarinic receptor done. The calcium influx persisted even after pretreatment of cells with phorbol 12-myristate 13-acetate, which completely prevented the rise in inositol phosphates and intracellular calcium levels. The calcium influx was blocked by divalent cations but was not affected by inhibitors of voltage-dependent calcium channels or high potassium depolarization, indicating the presence of a receptor-operated and voltage-insensitive calcium channel in these cells. Calcium influx was not stimulated by the addition of cAMP analogs or arachidonic acid. To examine the possible involvement of G proteins in m3 receptor-activated calcium influx, two chimeric m2 and m3 muscarinic receptors were expressed in A9 cells in which the third cytoplasmic loop (the primary structural determinant in G protein coupling selectivity of muscarinic receptors) had been exchanged between the m2 receptor, which has no effect on calcium influx, and the m3 receptor. Calcium influx was found to be associated with a structural component of the m3 muscarinic receptor other than the third cytoplasmic loop.Receptor-stimulated increases in cytosolic free calcium generally occur in two phases, an initial rapid spike followed by a sustained phase. The initial spike is the result of release of calcium from intracellular stores mediated primarily by inositol 1,4,5-trisphosphate (InsP3) generated following receptormediated activation of phospholipase C (1). The sustained plateau phase follows influx of calcium from the extracellular medium and requires the continued presence of agonist. Receptor-mediated calcium influx is thought to occur either through the well-studied voltage-sensitive calcium channels or through receptor-operated calcium channels, about which little is currently known. Receptor-operated calcium channels have been identified in a variety of both excitable and nonexcitable cell types (2). Unlike the voltage-sensitive calcium channels, receptor-operated calcium channels have avoided direct measurement when electrophysiological techniques have been used possibly because of their low abundance or extremely low conductance. These channels can be indirectly activated by the second messengers InsP3 (3, 4), inositol 1,3,4,5-tetrakisphosphate (5), or intracellular calcium (6) and in some cells may be independent of secondmessenger regulation (7). Plasma membrane calcium channels responsible for the refilling ofintracellular calcium stores have been described that open following InsP3-mediated calcium release (8). It is not clear if the refilling channels and receptor-operated calcium channels are ide...

Differential Effects of the Gβ5-RGS7 Complex on Muscarinic M3 Receptor–Induced Ca2+ Influx and Release

“…It is well documented that activated M3R initiates both release from intracellular stores and influx across the plasma membrane (Felder et al, 1992;Parekh and Brading, 1992;Carroll and Peralta, 1998). In this study, we experimentally isolated these constituents of Ca 21 entry and found that the Gb5-RGS7 complex inhibits the release pathway (Fig.…”

“…Ca 21 release is mediated by the canonical pathway involving Gq, phospholipase C b (PLCb), and the second messenger inositol triphosphate, and the inhibitory action of Gb5-RGS7 on M3R-stimulated Ca 21 release may be explained by its competition with Gq for the receptor (Blin et al, 1995;Sandiford and Slepak, 2009). Elucidating the role of Gb5-RGS7 in M3R-stimulated Ca 21 influx is more complex as it involves multiple channels that can be expressed and regulated in a cell-and even species-specific manner (Felder et al, 1992;Singer-Lahat et al, 1996;Wuest et al, 2007). We began dissecting this pathway here by using G protein inhibitors and ion channel blockers (Figs.…”

Differential Effects of μ-Opioid Receptor Ligands on Ca2+Signaling

“…response, which seems to represent rapid influx of extracellular Ca 2ϩ (peak 1). Ca 2ϩ e influx before, or independent of, Ca 2ϩ i release had been shown to occur upon activation of muscarinic receptors and other GPCRs (Felder et al, 1992;Montero et al, 1994), including MOR in SH-SY5Y cells (Smart et al, 1995). However, a direct experimental demonstration of rapid induction of Ca 2ϩ influx by MOR was lacking.…”

“…This occurs either by stimulating influx of extracellular Ca 2ϩ e or release of intracellular Ca 2ϩ i stores via soluble second messengers (inositol trisphosphates), or both. Several GPCRs induce rapid Ca 2ϩ influx independent of intracellular Ca 2ϩ release (Felder et al, 1992;Montero et al, 1994). Smart et al (1995) reported that Ca 2ϩ influx preceded intracellular Ca 2ϩ release upon MOR stimulation in human SH-SY5Y neuroblastoma cells shown to express MOR and ␦-opioid receptors (Yu et al, 1986).…”

Endothelin‐1 increases the levels of mRNA and protein of muscarinic acetylcholine receptors and c‐fos mRNA in cerebellar granule cells

“…ET-1 is endogenously present in the CNS including the cerebellum [2,3] and m,-mAChR is the major mAChR subtype expressed in cerebellar granule cells [15,16]. Besides its robust effect in stimulating phosphoinositide hydrolysis [15], m3-mAChR mediates Ca*+ influx independent of inositol phosphates and intracellular Ca*' release [24]. Thus, ETl-induced expression of m,-mAChR protein may result in long-lasting changes in neuronal Ca*+ homeostasis and excitability.…”