Pulsatile intracellular calcium release does not depend on fluctuations in inositol trisphosphate concentration

Abstract

MANY hormones, neurotransmitters and growth factors evoke in their target cells oscillations in the free internal Ca²⁺ concentration ([Ca²⁺]i) (ref. 1). In electrically non-excitable cells these fluctuations are due to periodic release of Ca²⁺ from intracellular reservoirs^1,2, stimulated by the internal messenger inositol trisphosphate (InsP₃) (refs 2–4). Most models at present invoke fluctuating levels of InsP³ as a key component in generating the oscillations in [Ca²⁺]i (refs 5 and 6). InsP₃ injected into intact cells evokes irregular and transient oscillatory Ca²⁺-dependent current responses^7–9, but the intracellular InsP₃ concentration is not constant in such experiments. Here we monitor changes in [Ca²⁺_i by measuring Ca²⁺-activated Cl^- current in single internally perfused mouse pancreatic acinar cells^4,10–12and show that acetylcholine (ACh), acting through muscarinic receptors, evokes regular and repetitive current pulses which are mimicked by InsP³ applied through a patch pipette¹³. To exclude the possibility that InsP³is periodically phosphorylated¹⁴ or degraded, we replaced it by the non-metabolizable InsP³ analogue inositol trisphosphorothioate (InsPS₃)^9,15,16, which also evokes regular pulses of Ca²⁺-activated Cl^- current. These effects are independent of external Ca²⁺, but abolished by high intracellular concentrations of a Ca²⁺-chelator. We conclude that repetitive pulses of intracellular Ca²⁺release occur even when the concentration of InsP³ is constant.