PKC‐mediated inhibitory feedback of the cholecystokinin 1 receptor controls the shape of oscillatory Ca²⁺ signals

Abstract

Translation of extracellular hormonal input into cellular responses is often mediated by repetitive increases in cytosolic free Ca 2+ concentration ([Ca 2+ ] c ). Amplitude, duration and frequency of these so-called [Ca 2+ ] c oscillations then carry information about the nature and concentration of the extracellular signalling molecule. At present, there are different hypotheses concerning the induction and control of these oscillations. Here, we investigated the role of agonist-induced receptor phosphorylation in this process using Chinese hamster ovary cells stably expressing a variant of the cholecystokinin 1 receptor (CCK1R) lacking the four consensus sites for protein kinase C (PKC) phosphorylation and deficient in CCK-induced receptor phosphorylation (CCK1R-mt cells). In the presence of cholecystokinin-(26-33)-peptide amide (CCK-8), these cells displayed Ca 2+ oscillations with a much more pronounced bursting dynamics rather than the dominant spiking dynamics observed in Chinese hamster ovary cells stably expressing the wild-type CCK1R. The bursting behaviour returned to predominantly spiking behaviour following removal of extracellular Ca 2+ , suggesting that CCK-8-induced, PKC-mediated CCK1R phosphorylation inhibits Ca 2+ influx across the plasma membrane. To gain mechanistic insight into the underlying mechanism we developed a mathematical model able to reproduce the experimental observations. From the model we conclude that binding of CCK-8 to the CCK1R leads to activation of PKC which subsequently phosphorylates the receptor to inhibit the receptormediated influx of Ca 2+ across the plasma membrane. Receptor-specific differences in this feedback mechanism may, at least in part, explain the observation that different agonists evoke [Ca 2+ ] c oscillations with different kinetics in the same cell type.

Inhibition of Suicidal Erythrocyte Death by Chronic Hypoxia

“…An increase of cytosolic Ca 2+ could stimulate phosphatidylserine scrambling and induce eryptosis . The concentration of cytosolic Ca 2+ is regulated by many factors such as Gardos K( + ) channels (Foller et al, 2010), platelet activating factor (PAF) (Xu et al, 2009), prostaglandin (Muravyov and Tikhomirova, 2014), and protein kinase C Willems et al, 2015). Our results showed that the decreased intracellular Ca 2+ prevented phosphatidylserine scrambling in hypoxia.…”

Contributing mechanisms underlying desensitization of cholecystokinin-induced activation of primary nodose ganglia neurons

“…Previous studies of CCK signal transduction in vagal afferents indicate that PKA, PLA 2 , and PI 3 K are not required for CCK-induced calcium responses and that the prominent point of control is likely via PLC cleavage of PIP 2 and subsequent gating of transient receptor potential (TRP) channels and/or L-type voltage-activated calcium channels (15,48,49). Nonetheless, both CCK receptors and ion channel effectors are strongly regulated by site specific phosphorylation via PKC and PKA subtypes, suggesting these parallel pathways may shape the magnitude and time-dependent changes in CCK signaling (43,46).…”

“…In dissociated vagal afferent neurons, CCK concentration dependently evokes depolarizing currents and elevates cytosolic calcium from extracellular sources (14,25). While the magnitudes of these responses are a result of CCK receptor density and the number of associated ion channel effectors, the signal desensitization is a function of CCK concentration and subsequent receptor occupancy (43). The CCK1R has both high-and low-affinity binding sites (13,31,41) that are coupled to different signaling cascades in pancreatic acinar cells (9,15).…”

“…In vagal afferents, high-affinity (~10 pM) binding elicits an oscillating calcium signal, whereas low-affinity (Ͼ1 nM) binding produces a large phasic calcium peak followed by desensitization to a steady-state signal that often exhibits some oscillations (29). Previous studies of the CCK1R expressed in Chinese hamster ovary (CHO) cells suggests these oscillations emerge from a feedback loop in which PKC phosphorylates and inhibits the CCK1R (43), whereas in pancreatic acinar cells increased PKA activity reduced CCK signaling via PLA 2 (9) . While neither of these pathways are required for the acute stimulating effects of CCK on vagal afferent neurons, given the robust time-and concentration-dependent changes in CCK receptor function understanding, their contributions to this process should prove useful.…”

Tamoxifen

“…3 Regarding increased intracellular calcium ion concentration ([Ca 2+ ]i) in acutely ill bipolar patients and the suggested antimanic effect of calcium channel blockers, it is notable that activation of PKC inhibits the influx of calcium across the membrane, which is suggestive of the relation between modulated calcium signaling and antimanic effects of PKC inhibitors. 4,5 Currently, tamoxifen is the only relatively specific PKC inhibitor capable of crossing blood-brain barrier that has been available for human use. 6 In recent years, several controlled trials evaluated the efficacy of tamoxifen in the treatment of acute manic episodes, as either monotherapy or adjunct.…”