Calcium mobilization and spontaneous transient outward current characteristics upon agonist activation of P2Y2 receptors in smooth muscle cells

Abstract

A quantitative model is provided that links the process of metabotropic receptor activation and sequestration to the generation of inositol 1,4,5-trisphosphate, the subsequent release of calcium from the central sarcoplasmic reticulum, and the consequent release of calcium from subsarcolemma sarcoplasmic reticulum that acts on large-conductance potassium channels to generate spontaneous transient outward currents (STOCs). This model is applied to the case of STOC generation in vascular A7r5 smooth muscle cells that have been transfected with a chimera of the P2Y(2) metabotropic receptor and green fluorescent protein (P2Y(2)-GFP) and exposed to the P2Y(2) receptor agonist uridine 5'-triphosphate. The extent of P2Y(2)-GFP sequestration from the membrane on exposure to uridine 5'-triphosphate, the ensuing changes in cytosolic calcium concentration, as well as the interval between STOCs that are subsequently generated, are used to determine parameter values in the model. With these values, the model gives a good quantitative prediction of the dynamic changes in STOC amplitude observed upon activation of metabotropic P2Y(2) receptors in the vascular smooth muscle cell line.

FIGURE 1

Schematic diagram of the multicompartment vascular smooth muscle cell model. P2Y₂ receptors are present in the sarcolemma. The cell interior contains three main compartments: the cytosol (1), the central SR (3), and the peripheral SR (2), with the cytosol being divided into a main part and a smaller subsarcolemmal space lying between the peripheral SR and the sarcolemma. The central SR membrane contains IP₃Rs and the peripheral SR membrane contains RyRs; both have Ca²⁺ leaks and pumps. BK channels are in the sarcolemma in close apposition to the RyRs in the peripheral SR. J_ab denotes a Ca²⁺ current from compartment a to compartment b.

FIGURE 2

(a) Kinetic scheme for receptor-ligand binding, phosphorylation, and internalization. (b) Schematic diagram of the compartments in the model and the Ca²⁺ fluxes between them. (c) Kinetic scheme of the model for the bursting behavior of the ryanodine channels on the peripheral SR membrane. (There are four states: inactive, open, closed, and refractory, denoted by the A, B, C, and D, respectively.)

FIGURE 3

Transfection of A7r5 cells with P2Y₂-GFP. (A) Confocal images of the membranes of A7r5 cells shortly after transfection with P2Y₂-GFP (a), 12 h after transfection when the P2Y₂-GFP distribution is still diffuse (b), and 24 h after transfection when P2Y₂-GFP has formed clusters of ∼0.5–1 μm in diameter (c); the insert in c shows several clusters of P2Y₂ receptors at higher magnification of the area indicated by the white square. (B) Comparison between the localization of P2Y₂-GFP (a) and of anti-P2Y₂ antibodies (b) on the same A7r5 cell; higher magnification inserts are of the identical areas indicated by the open squares. Calibration bar is 10 μm.

FIGURE 4

P2Y₂-GFP in A7r5 cells under different experimental conditions. (A) Confocal images of A7r5 cells showing the clustering of P2Y₂-GFP in the membrane 24 h after transfection (a), and then the gradual loss of many of the clusters after exposure of the cells to 10 μM UTP for 1 min (b), 2 min (c), and 5 min (d); higher magnification inserts in a and d are taken from the solid-square regions and show dense clusters of P2Y₂-GFP in a that are largely lost in d. Note that in addition to the loss of many of the clusters, there is a gradual rounding-up of cells in the presence of the agonist. (B) Confocal images of P2Y₂-GFP as in A, but this time in the presence of monensin (5 μM); note no loss of P2Y₂-GFP fluorescence, but the cells still round up. (C) Confocal images of P2Y₂-GFP as in A, but this time in the presence of suramin (10 μM); note again no loss of P2Y₂-GFP fluorescence, and in this case the cells do not round up. Calibration bar is 10 μm.

FIGURE 5

Experimental and theoretical membrane P2Y₂ receptor fraction with respect to time. Experimental data show the time-course of P2Y₂-GFP in A7r5 cells (squares with mean ± SE for six cells) after application of 10 μM UTP at t = 0. The P2Y₂-GFP fluorescence has been taken to be proportional to the number of receptors in the membrane and has been scaled to become a percentage of the amount of receptor relative to that before agonist stimulation. The solid line is the theoretical time-course of the total surface receptor fraction, and the broken line is the time-course of the phosphorylated surface receptor fraction, both obtained by solving Eqs. 1 and 2.

FIGURE 6

Experimental and theoretical Ca²⁺ fluorescence with respect to time. Experimental data (squares = mean ± SE < size of squares; n = 4 cells) show the time-course of fluo3-AM fluorescence in an A7r5 cell after application of 10 μM UTP at t = 0. The solid line shows the theoretical time-course of the cytosolic Ca²⁺ fluorescence obtained by solving the model equations to determine [(Ca²⁺)_cyt] and calculating the corresponding fluo3-AM fluorescence using Eq. 20. The broken line shows the effect of including RyRs on the central SR.

FIGURE 7

Theoretical IP₃ concentration, [IP₃] (solid line), and amount of activated G-protein, [G], expressed as a percentage of [G_T] (dashed line), with respect to time for a step application of 10 μM UTP at t = 0.

FIGURE 8

Theoretical Ca²⁺ concentrations with respect to time for a step application of 10 μM UTP at t = 0. The time-course of the Ca²⁺ concentration in the cytosol, [(Ca²⁺)]_cyt (dashed line), and the domain Ca²⁺ in the subsarcolemmal space, [(Ca²⁺)_d] (solid line), are shown in A. Shown in B are the Ca²⁺ concentration in the central SR, [(Ca²⁺)_csr] (dashed line) and peripheral SR [(Ca²⁺)_psr] (solid line). Note the very different vertical axes scales in A.

FIGURE 9

Experimental membrane current with respect to time showing several bursts of STOCs upon application of 10 μM UTP. These currents are given on a compressed time-base for illustration purposes, and are not those analyzed in Fig. 10.

FIGURE 10

Experimental results (▵) for the time between bursts (A) and amplitudes of bursts (B) of STOCs after application of 10 μM of UTP to an A7r5 cell. In A the dashed line shows the average value of the intervals which is used to specify the bursting interval for the theoretical model. In B the solid line gives the theoretical amplitudes obtained by solving the model equations using the theoretical cytosolic Ca²⁺ fluorescence (Fig. 6, solid curve) as input; the crosses indicate the locations of the peaks. The experimental data-point is not given for burst 1 because of an artifact shortly after the addition of UTP. The broken line in B gives the corresponding theoretical results when the experimental cytosolic Ca²⁺ fluorescence (Fig. 6, dotted curve) is used as input (see the Appendix for modifications to the theory); the remaining parameters are as in Table 1, except that w_dom has been increased to 4 × 10⁻³.

FIGURE 11

Theoretical membrane current, I_BK, with respect to time upon application of 10 μM UTP at t = 0. The insert shows a closeup of the fourth burst of STOCs, this being the burst with the greatest amplitude.