Agonist-dependent phosphorylation of the inositol 1,4,5-trisphosphate receptor: A possible mechanism for agonist-specific calcium oscillations in pancreatic acinar cells

Abstract

The properties of inositol 1,4,5-trisphosphate (IP3)-dependent intracellular calcium oscillations in pancreatic acinar cells depend crucially on the agonist used to stimulate them. Acetylcholine or carbachol (CCh) cause high-frequency (10-12-s period) calcium oscillations that are superimposed on a raised baseline, while cholecystokinin (CCK) causes long-period (>100-s period) baseline spiking. We show that physiological concentrations of CCK induce rapid phosphorylation of the IP3 receptor, which is not true of physiological concentrations of CCh. Based on this and other experimental data, we construct a mathematical model of agonist-specific intracellular calcium oscillations in pancreatic acinar cells. Model simulations agree with previous experimental work on the rates of activation and inactivation of the IP3 receptor by calcium (DuFour, J.-F., I.M. Arias, and T.J. Turner. 1997. J. Biol. Chem. 272:2675-2681), and reproduce both short-period, raised baseline oscillations, and long-period baseline spiking. The steady state open probability curve of the model IP3 receptor is an increasing function of calcium concentration, as found for type-III IP3 receptors by Hagar et al. (Hagar, R.E., A.D. Burgstahler, M.H. Nathanson, and B.E. Ehrlich. 1998. Nature. 396:81-84). We use the model to predict the effect of the removal of external calcium, and this prediction is confirmed experimentally. We also predict that, for type-III IP3 receptors, the steady state open probability curve will shift to lower calcium concentrations as the background IP3 concentration increases. We conclude that the differences between CCh- and CCK-induced calcium oscillations in pancreatic acinar cells can be explained by two principal mechanisms: (a) CCK causes more phosphorylation of the IP3 receptor than does CCh, and the phosphorylated receptor cannot pass calcium current; and (b) the rate of calcium ATPase pumping and the rate of calcium influx from the outside the cell are greater in the presence of CCh than in the presence of CCK.

Figure 1

Phosphorylation of type-III IP₃ receptor after stimulation by cholecystokinin in rat pancreatic acini: acini were prepared and samples were processed as detailed in the Methods. (A) In acini under control conditions, a single phosphorylated band was observed at ∼300 kD, corresponding to the type-III IP₃ receptor. On stimulation by CCK, the intensity of this band increased; the increase was detected at 10 pM CCK and reached a maximum at 100 pM CCK. These concentrations of CCK are physiologically relevant and induce oscillatory [Ca²⁺]_i signals. In the extreme right lane, samples were processed in an identical manner except that the immunoprecipitating antisera were omitted. (B) Pooled data (mean ± SEM) from at least three independent experiments, where samples were run in duplicate.

Figure 2

Phosphorylation of type-III IP₃ receptor after stimulation by carbachol in rat pancreatic acini. (A) Stimulation with the muscarinic agonist carbachol also results in increased phosphorylation of the type-III IP₃ receptor. Increased phosphorylation of the 300-kD protein could be detected at 1 μM, concentrations of carbachol that induce “peak-and-plateau” type Ca²⁺ responses. No phosphorylation could be detected at concentrations of CCh that induce [Ca²⁺]_i oscillations. (B) Pooled data (mean ± SEM) from at least three independent experiments, where samples were run in duplicate.

Figure 3

Phosphorylation of type-III IP₃ receptor in rat pancreatic acini after stimulation by agents that modulate second-messenger levels. (A) Increased phosphorylation of the IP₃ receptor could be detected from acini samples incubated with CPT-cAMP, a cell-permeable cAMP analogue, but not from samples incubated with agents known to increase [Ca²⁺]_i (30 μm CPA) or activate protein kinase C (10 nM TPA). (B) Pooled data (mean ± SEM) from at least three independent experiments, where samples were run in duplicate.

Figure 4

Diagram of the receptor states of the model of the IP₃ receptor. Phosphorylation of the receptor by PKA shunts the receptor through the I₂ state, thus shutting off the Ca²⁺ current, and leading to long-period oscillations.

Figure 5

Data, reproduced from Dufour et al. (1997), Figures 1 A and 2 A, showing the effects of Ca²⁺ and IP₃ on Ca²⁺ efflux through IP₃ receptors from the rat liver. For the Ca²⁺ dose response, the concentration of IP₃ was held at 10 μM and [Ca²⁺] was stepped up and held at the indicated concentration. For the IP₃ dose response, [Ca²⁺] was held fixed at 400 nM and [IP₃] was stepped up and held fixed at the indicated concentrations. The efflux through the receptor was then measured every 70 ms. Figure reprinted with permission.

Figure 6

Dose responses, calculated from the model. The same Ca²⁺ and IP₃ concentrations as in Fig. 5 were used, except that the whole range of concentrations was not covered.

Figure 7

Steady state open probability of the model IP₃ receptor, as a function of [Ca²⁺], for a range of constant IP₃ concentrations.

Figure 8

Typical ACh-induced oscillations in the model for [IP₃] = 0.66 μM.

Figure 9

Bifurcation diagram for ACh-induced model oscillations showing the values of [IP₃] for which oscillations occur. A dotted line denotes an unstable steady state, while a solid line denotes a stable steady state or a stable oscillation.

Figure 10

Typical CCK-induced oscillations in the model for [IP₃] = 0.6 μM.

Figure 11

Bifurcation diagram for CCK- induced model oscillations, showing the values of [IP₃] for which oscillations occur. A dotted line denotes an unstable steady state, while a solid line denotes a stable steady state, or a stable oscillation.

Figure 12

(A) The effect of reducing external Ca²⁺ in the model. ACh-induced oscillations are abolished by reducing to 0.35 μM s⁻¹ the Ca²⁺ influx from outside the cell. Oscillations are then restored by increasing the IP₃ concentration to 0.75 μM. (B) Experimental test of the prediction in A. Mouse pancreatic acinar cells were first exposed to 70 nM ACh in the presence of 1.2 mM Ca²⁺, which initiated oscillations. Reduction of external Ca²⁺ to 100 nM abolished the oscillations, but an increase in ACh concentration to 100 nM restored the oscillations, as predicted by the model.