Calcium signalling through nucleotide receptor P2X1 in rat portal vein myocytes

Abstract

1. ATP-mediated Ca2+ signalling was studied in freshly isolated rat portal vein myocytes by means of a laser confocal microscope and the patch-clamp technique. 2. In vascular myocytes held at -60 mV, ATP induced a large inward current that was supported mainly by activation of P2X1 receptors, although other P2X receptor subtypes (P2X3, P2X4 and P2X5) were revealed by reverse transcription-polymerase chain reaction. 3. Confocal Ca2+ measurements revealed that ATP-mediated Ca2+ responses started at initiation sites where spontaneous or triggered Ca2+ sparks were not detected, whereas membrane depolarizations triggered Ca2+ waves by repetitive activation of Ca2+ sparks from a single initiation site. 4. ATP-mediated Ca2+ responses depended on Ca2+ influx through non-selective cation channels that activated, in turn, Ca2+ release from the intracellular store via ryanodine receptors (RYRs). Using specific antibodies directed against the RYR subtypes, we show that ATP-mediated Ca2+ release requires, at least, RYR2, but not RYR3. 5. Our results suggest that, in vascular myocytes, Ca2+ influx through P2X1 receptors may trigger Ca2+-induced Ca2+ release at intracellular sites where RYRs are not clustered.

Figure 1. Membrane currents activated in rat portal vein myocytes by external application of ATP

A, effects of 10 μm ATP, 10 μmαβ-MeATP and 100 μm UTP obtained from three different cells. B, pipette solution contained 2 mm GDPβS and the cell was dialysed with the pipette solution for 5 min before application of 10 μm ATP. C, intracellular application of 10 μg ml⁻¹ anti-P2X1 antibody for 7 min before application of 10 μm ATP. In A–C, the myocytes were held at −60 mV. D, typical Ba²⁺ currents elicited by depolarization to 10 mV from a holding potential of −40 mV and current-voltage relationships obtained in control conditions (•) and after intracellular application of 10 μg ml⁻¹ anti-P2X1 antibody for 7 min (○). Currents are expressed as a fraction of the maximal current (I/I_max) and are the means ±s.e.m. for 7–9 cells.

Figure 2. Expression of P2X receptors in rat portal vein myocytes

A, amplified DNA fragments of P2X receptors (lanes 1–7) from rat brain (a) and rat portal vein myocytes (b) were separated on a 2 % agarose gel and visualized by staining with ethidium bromide. Lane 8, RNA from brain and portal vein myocytes in the absence of reverse transcriptase served as a negative control. Numbers on the left indicate molecular size standards in base pairs (bp). For RNA purification and PCR conditions, see Methods. B, immunostaining of P2X receptor subtypes in portal vein myocytes. Myocytes were stained with anti-P2X1 receptor (a) or anti-P2X4 receptor antibody (b) and vizualization was realized with a donkey anti-rabbit IgG FITC-conjugated antibody. In the absence of primary antibodies or after inactivation of the antibodies by their antigen peptides, only a faint background fluorescence was observed (not shown). Typical confocal sections were performed above the nucleus and therefore appeared spherical. Both P2X1 (a) and P2X4 (b) receptor subtypes were distributed throughout the confocal sections with a marked staining of P2X1 receptor subtype at the cell periphery.

Figure 3. Increase in [Ca²⁺]_i evoked by increasing concentrations of ATP

A–C, Ca²⁺ responses shown as a linescan image (a) and spatial averaged fluorescence (b; F/F₀). Data are for a 2 μm region indicated by the vertical line to the left of the corresponding linescan image. Responses are to 0.1 μm (A), 1 μm (B) or 10 μm ATP (C). D, concentration-response curve for increasing concentrations of ATP, obtained by measuring peak [Ca²⁺]_i (Δ(F/F₀)) in a 2 μm region of the linescan images. Data are means ±s.e.m with the number of cells tested indicated in parentheses. Myocytes were loaded with fluo 3 via the patch pipette and held at −60 mV.

Figure 4. Localization of Ca²⁺ sparks and Ca²⁺ responses induced by ATP, membrane depolarization and Bay K8644

Aa, vascular myocyte stained with DI-8-Anepps (10 μm) and fluo 3 (60 μm) showing the plasma membrane (red) and cytosol (green). The dashed line corresponds to the scanned line. Ab, linescan image showing Ca²⁺ sparks. Ac, superimposition of Ca²⁺ sparks and DI-8-Anepps staining, illustrating the localization of the Ca²⁺ sparks close to the infolding of the plasma membrane shown in Aa. B, Ca²⁺ sparks triggered by application of 5 nm Bay K8644 and ATP-induced Ca²⁺ response in the same cell, shown as a linescan image (a) and spatial averaged fluorescence (b). Data are for the same 2 μm region indicated by the vertical line to the left of the corresponding linescan image. C, ATP-induced Ca²⁺ response and Ca²⁺ spark induced by a depolarization step from −60 to −10 mV in the same cell, shown as a linescan image (a) and spatial averaged fluorescence (b). Data are for the same 2 μm region indicated by the vertical line on the linescan image. Myocytes were loaded with fluo 3 via the patch pipette and held at −60 mV.

Figure 5. Effects of anti-RYR and anti-InsP₃R antibodies on ATP-induced Ca²⁺ responses

A, peak Ca²⁺ responses evoked by membrane depolarizations (−60 to 10 mV) in control conditions (C) and in the presence of increasing concentrations of anti-RYR antibody or 10 μg ml⁻¹ boiled anti-RYR antibody, each applied intracellularly for 7 min. Data are means ±s.e.m. with the number of cells tested indicated in parentheses. B, peak Ca²⁺ responses evoked by 10 μm noradrenaline in control conditions (C) and in the presence of increasing concentrations of anti-InsP₃R antibody or 10 μg ml⁻¹ boiled anti-InsP₃R antibody, each applied intracellularly for 7 min. Data are means ±s.e.m. with the number of cells tested indicated in parentheses. C, peak Ca²⁺ responses evoked by 10 μm ATP in control conditions (C) and in the presence of 10 μg ml⁻¹ anti-InsP₃R antibody, anti-RYR antibody or boiled anti-RYR antibody, each applied intracellularly for 7 min. Data are means ±s.e.m. with the number of cells tested indicated in parentheses. [Ca²⁺]_i was measured in a 2 μm region of the linescan image. Cells were obtained from three different batches. ⋆, values significantly different from controls (P < 0.05). Myocytes were loaded with fluo 3 via the patch pipette and held at −60 mV.

Figure 6. Effects of anti-RYR and anti-RYR2 antibodies on Bay K8644-induced Ca²⁺ sparks and on Ca²⁺ responses induced by membrane depolarization

A, mean number of Ca²⁺ sparks per linescan image evoked by external application of 5 nm Bay K8644 in control conditions (C) and in the presence of 10 μg ml⁻¹ anti-RYR antibody, anti-RYR2 antibody or boiled anti-RYR2 antibody, each applied intracellularly for 7 min. B, peak Ca²⁺ responses evoked by membrane depolarization (−60 to 10 mV) in control conditions (C) and in the presence of 10 μg ml⁻¹ anti-RYR antibody, anti-RYR2 antibody or boiled anti-RYR2 antibody, each applied intracellularly for 7 min. Data are means ±s.e.m. with the number of cells tested indicated in parentheses. [Ca²⁺]_i was measured in a 2 μm region of the linescan image. Cells were obtained from three different batches. ⋆, values significantly different from controls (P < 0.05). Myocytes were loaded with fluo 3 via the patch pipette and held at −60 mV.

Figure 7. Effects of anti-RYR and anti-RYR2 antibodies on Ca²⁺ responses induced by ATP and caffeine

A, peak Ca²⁺ responses evoked by 10 μm ATP in control conditions (C) and in the presence of 10 μg ml⁻¹ anti-RYR antibody, anti RYR2 antibody or boiled anti-RYR2 antibody, each applied intracellularly for 7 min. B, peak Ca²⁺ responses evoked by 10 mm caffeine in control conditions (C) and in the presence of 10 μg ml⁻¹ anti-RYR antibody, anti-RYR2 antibody or boiled anti-RYR2 antibody, each applied intracellularly for 7 min. Data are means ±s.e.m. with the number of cells tested indicated in parentheses. [Ca²⁺]_i was measured in a 2 μm region of the linescan image. Cells were obtained from three differents batches. ⋆, values significantly different from controls (P < 0.05). Myocytes were loaded with fluo 3 via the patch pipette and held at −60 mV.

Figure 8. Effects of anti-RYR3 antibody on Ca²⁺ responses induced by ATP and caffeine

A, peak Ca²⁺ responses evoked by 10 mm caffeine in 1.7 mm[Ca²⁺]_o (C1), 10 mm[Ca²⁺]_o (C2) and in the presence of 10 μg ml⁻¹ anti-RYR3 antibody or boiled anti-RYR3 antibody (applied intracellularly for 7 min). ⋆, values significantly different from controls in 1.7 mm[Ca²⁺]_o (P < 0.05). B, peak Ca²⁺ responses evoked by 10 μm ATP and 10 mm caffeine in control conditions (C) in 1.7 mm[Ca²⁺]_o and in the presence of 10 μg ml⁻¹ anti-RYR3 antibody (applied intracellularly for 7 min). Data are means ±s.e.m. with the number of cells tested indicated in parentheses. Cells are obtained from two different batches. Myocytes were loaded with fluo 3 via the patch pipette and held at −60 mV.