Calcium mobilization by activation of M(3)/M(5) muscarinic receptors in the human retinoblastoma

J Pharmacol Sci. 2007 Oct;105(2):184-92. doi: 10.1254/jphs.fp0070877.

Abstract

Activation of muscarinic acetylcholine receptors (mAChR) is one of the most important signal transduction pathways in the human body. In this study, we investigated the role of mAChR activation in relation to its subtypes in human retinoblastoma cell-lines (WERI-Rb-1) using Ca(2+) measurement, real-time PCR, and Western Blot techniques. Acetylcholine (ACh) produced prominent [Ca(2+)](i) transients in a repeated manner in WERI-Rb-1 cells. The maximal amplitude of the [Ca(2+)](i) transient was almost completely suppressed by 97.3 +/- 0.8% after atropine (1 microM) pretreatment. Similar suppressions were noted after pretreatments with thapsigargin (1 microM), an ER Ca(2+)-ATPase (SERCA) inhibitor, whereas the ACh-induced [Ca(2+)](i) transient was not affected even in the absence of extracellular calcium. U-73122 (1 microM), a PLC inhibitor, and xestospongin C (2 microM), an IP(3)-receptor antagonist, elicited 11.5 +/- 2.9% and 17.8 +/- 1.9% suppressions, respectively. The 50% inhibitory concentration of (IC(50)) values for blockade of a 100 microM ACh response by pirenzepine and 4-DAMP were 315.8 and 9.1 nM, respectively. Moreover, both M(3) and M(5) mAChRs were prominent in quantitative real-time-PCR. Taken together, the M(3)/M(5) subtypes appear to be the major contributor, leading to intracellular calcium mobilization from the internal store via an IP(3)-dependent pathway in the undifferentiated retinoblastoma cells.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetylcholine / administration & dosage
  • Acetylcholine / pharmacology
  • Blotting, Western
  • Calcium / metabolism*
  • Calcium Signaling
  • Cell Line, Tumor
  • Cholinergic Agents / administration & dosage
  • Cholinergic Agents / pharmacology
  • Dose-Response Relationship, Drug
  • Gene Expression
  • Humans
  • Receptor, Muscarinic M3 / metabolism*
  • Receptor, Muscarinic M5 / metabolism*
  • Retinoblastoma / metabolism*
  • Reverse Transcriptase Polymerase Chain Reaction

Substances

  • Cholinergic Agents
  • Receptor, Muscarinic M3
  • Receptor, Muscarinic M5
  • Acetylcholine
  • Calcium