Receptor-specific Ca2+ oscillation patterns mediated by differential regulation of P2Y purinergic receptors in rat hepatocytes

Abstract

Extracellular agonists linked to inositol-1,4,5-trisphosphate (IP₃) formation elicit cytosolic Ca²⁺ oscillations in many cell types, but despite a common signaling pathway, distinct agonist-specific Ca²⁺ spike patterns are observed. Using qPCR, we show that rat hepatocytes express multiple purinergic P2Y and P2X receptors (R). ADP acting through P2Y1R elicits narrow Ca²⁺ oscillations, whereas UTP acting through P2Y2R elicits broad Ca²⁺ oscillations, with composite patterns observed for ATP. P2XRs do not play a role at physiological agonist levels. The discrete Ca²⁺ signatures reflect differential effects of protein kinase C (PKC), which selectively modifies the falling phase of the Ca²⁺ spikes. Negative feedback by PKC limits the duration of P2Y1R-induced Ca²⁺ spikes in a manner that requires extracellular Ca²⁺. By contrast, P2Y2R is resistant to PKC negative feedback. Thus, the PKC leg of the bifurcated IP₃ signaling pathway shapes unique Ca²⁺ oscillation patterns that allows for distinct cellular responses to different agonists.

Keywords: Biological sciences; Cell biology; Cellular physiology; Functional aspects of cell biology.

Graphical abstract

Figure 1

[Ca²⁺]_c oscillation profiles elicited by purine nucleotides in hepatocytes (A–D) Representative traces of typical ATP (A), ADP (B), UTP (C), and UDP-induced (D) [Ca²⁺]_c responses in hepatocytes loaded with fura-2. The duration of exposure and concentration of each nucleotide is indicated in the bars above each trace. (E) The indicated agonist was present continuously from the first addition arrow, followed by a maximal dose of ATP (100 μM) at the second arrow. At low agonist doses (1–2 μM), different strengths of Ca²⁺ response from No response (blue), Single spike (cyan), Discontinuous oscillations (green), Continuous Oscillations (orange), Sustained & oscillations (red) through to Sustained (dark red) can be elicited by each extracellular nucleotide. (F) Ordinal plot of the Ca²⁺ response strength in hepatocytes challenged with ATP, ADP, UTP, or UDP. (Data are from ≥50 cells in each of four independent experiments).

Figure 2

Purinergic P2 receptor gene expression in primary rat hepatocytes (A and B) RT-qPCR determination of mRNA expression levels of P2X and P2Y receptors from freshly isolated (A) and overnight (B) cultured hepatocytes. Purinergic receptor gene expression was normalized to Rpl0 expression. Data are mean ± S.E.M from 3 to 4 hepatocyte preparations in each case. See Table S1 for primer details.

Figure 3

P2X receptor activity does not contribute to [Ca²⁺]_c oscillations in hepatocytes Hepatocytes loaded with fura-2 were exposed to the indicated concentrations of ATP (in μM). The Gα_q protein inhibitor YM-254890 (1 μM) was present during the period indicated by the gray shading. (A) ATP (5 μM) induced a large [Ca²⁺]_c spike that was terminated by addition of YM-254890. Subsequent additions of increasing concentrations of ATP (10 and 100 μM) had no effect on [Ca²⁺]_c, whereas a high ATP dose (300 μM) caused a slow monophasic [Ca²⁺]_c increase. (B) Effect of increasing ATP concentrations in the presence of YM-254890, followed by addition of the P2X agonist BzATP (10 μM). (C) Dose response to BzATP in the presence of YM-254890. (D) Response to BzATP (10 μM) in the absence of YM-254890.

Figure 4

Role of P2Y1 and P2Y2 purinergic receptors (A and B) Normalized and color-mapped view of traces from 60 rat hepatocytes loaded with fura-2. Cells were challenged as indicated with 15 μM MRS-2500, an antagonist of P2Y1 receptor (A), and 15 μM AR-C 118925XX, an antagonist of P2Y2 receptor (B). The indicated concentration of each nucleotide was added at the arrows and remained present for the remainder of the experiment. A maximal dose of ATP (100 μM) was added at the end of the experiment. (C and D) Representative traces of treatment with MRS-2500 and AR-C 118925XX, as indicated by the shaded areas, and treatment with ADP and UTP (C) or UTP and ADP (D). (E and F) Representative traces for pretreatment with MRS-2500 (E) and AR-C 118925XX (F) followed by ATP stimulation.

Figure 5

Downregulation of PKC prolongs ADP- and UTP-induced Ca²⁺ spike duration in isolated rat hepatocytes Isolated hepatocytes were cultured overnight with PMA (1 μM) to downregulate conventional and novel PKC isoforms (PKC-DR), or with the inactive analog α-PMA (1 μM, CTR). The cells were loaded with fura-2 and then stimulated with ADP or UTP as indicated. (A, B, F and G) Representative traces for ADP- and UTP-induced [Ca²⁺]_c oscillations are shown for control (A and F) and PKC-DR hepatocytes (B and G). (C, D, H and I) Summary data show the effects of PKC-DR on Ca²⁺ spike width measured as full width at half maximum (FWHM) and oscillation frequency for ADP- (C and D) and UTP- (H and I) induced [Ca²⁺]_c oscillations. (E and J) The distribution of Ca²⁺ response patterns are also shown for ADP (E) and UTP (J). Blue and green symbols represent data from ADP- and UTP-induced [Ca²⁺]_c responses, respectively. Data are mean ± S.E.M. from ≥50 cells from at least three independent experiments. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; ∗∗∗∗p < 0.0001; Student’s t test.

Figure 6

Absence of extracellular Ca²⁺ differentially affects P2Y1 and P2Y2 receptor-dependent [Ca²⁺]_c oscillations Isolated hepatocytes cultured overnight were loaded with fura-2 and then stimulated with ADP and UTP (1–10 μM) in the presence (1.3 mM Ca²⁺) or absence (Ca²⁺free) of extracellular Ca²⁺. (A, B, D and E) Representative traces of typical ADP- (A and B) and UTP- (D and E) evoked [Ca²⁺]_c oscillations are shown. (C and F) Summary data of the effect of extracellular Ca²⁺ removal on Ca²⁺ spike width (FWHM) for ADP (C) and UTP (F). Blue and green symbols represent data from ADP- and UTP-induced Ca²⁺ spikes, respectively. Data are mean ± S.E.M. from ≥50 cells from at least three independent experiments. ∗∗∗, p < 0.001; Student’s t test.

Figure 7

Extracellular Ca²⁺ has no effect on [Ca²⁺]_c oscillation spike width following PKC downregulation Isolated hepatocytes were cultured overnight with PMA (1 μM) to downregulate conventional and novel PKC isoforms (PKC-DR). (A, B, D and E) The cells were then loaded with fura-2 and stimulated with ADP or UTP (10–15 μM) in the presence (1.3 mM Ca²⁺) or absence (Ca²⁺free) of extracellular Ca²⁺. Representative traces for ADP (A and B) and UTP (D and E) are shown under both experimental conditions. (C and F) Summary data of the effect of extracellular Ca²⁺ removal on Ca²⁺ spike width (FWHM) in PKC-DR hepatocytes is shown for ADP (C) and UTP (F). Blue and green symbols represent data from ADP- and UTP-induced [Ca²⁺]_c oscillations, respectively. Data are mean ± S.E.M. from ≥50 cells from at least three independent experiments; Student’s t test.

Figure 8

Effects of acute activation and inhibition of PKC on ADP- and UTP-induced [Ca²⁺]_c oscillations The effects of PMA (1 nM) and BIM (5 μM) on ADP- and UTP-induced [Ca²⁺]_c oscillations were analyzed in rat hepatocytes loaded with fura-2. After stimulation with the purinergic agonists, cells were treated with PMA or BIM as indicated by the shaded areas on the Ca²⁺ traces. (A, B, G, and H) Representative [Ca²⁺]_c oscillation responses are shown for ADP (A and B) and UTP (G and H). (C–L) The frequency and width of the Ca²⁺ spikes induced by ADP and UTP were calculated from 5-min periods in the absence or presence of the PKC modulators. Summary data are shown for oscillation frequency and spike width (FWHM) for ADP (C–F) and UTP (I–L). See Figure S1 for the effect of staurosporine on the spike width of ADP- and UTP-induced [Ca²⁺]_c oscillations. Blue and green symbols represent data from ADP- and UTP-induced [Ca²⁺]_c oscillations, respectively. Data are mean ± S.E.M. from ≥50 cells from at least three independent experiments. ∗∗∗∗, p < 0.0001; Student’s t test.