Characterization of Acetylcholine-induced Increases in Cytosolic Free Calcium Concentration in Individual Rat Pancreatic β-cells

The intracellular free Ca²⁺ ion concentration (Ca²⁺]i) was measured using fura-2 microspec-trofluorimetry in individual rat pancreatic β-cells prepared by enzymatic digestion and fluorescence-activated cell sorting. The mean basal concentration of Ca²⁺]i in β-cells in the presence of 4.4 mM glucose and 1.8 mM Ca²⁺ was 112±1.6 nM (n=207). The action of acetylcholine (ACh) was concentration-dependent, and raising the concentration resulted in Ca²⁺]i spikes of increasing amplitude and duration in some, but not all of the β-cells. In addition, the β-cells demonstrated variable sensitivity to ACh. The increases in Ca²⁺]i were rapid, transient and were blocked by atropine at 10^-6M. A brief exposure to 50 mM K⁺ resulted in a transient increase in Ca²⁺]i similar to that induced by ACh, but resistant to atropine. A high concentration of ACh (100μL 10^-4M or 10^-3M) induced Ca²⁺]i oscillations in 11 out of 57 β-cells in the presence of 4.4 mM glucose. Using calcium channel blockers and Ca²⁺ free medium, the source of the increase in Ca²⁺]i was deduced to be from extracellular spaces. Changing the temperature from 22 to 37°C did not affect the action of ACh on Ca²⁺]i. These data strongly suggest that ACh exerted a direct action on Ca²⁺]i in normal rat pancreatic β-cells and support a role for Ca²⁺ as a second messenger in the action of ACh.