Calcium Signaling in Individual BC³H1 Cells: Speed of Calcium Mobilization and Heterogeneity

Abstract

Receptor/ligand binding on a cell surface may activate the calcium signal transduction cascade, resulting in the release of calcium from intracellular stores into the cytosol. Changes in intracellular free calcium, [Ca2+]i, following ligand stimulation have been linked to a variety of cell responses, from muscle contraction to hormone secretion. We have monitored changes in [Ca2+]i in single smooth muscle‐like BC3H1 cells following stimulation by the vasoconstrictor phenylephrine, using the fluorescent calcium probe, fura‐2, in a digital fluorescence imaging system. We find that not all cells respond to ligand stimulation with changes in [Ca2+]i. In addition, cells which respond to ligand stimulation exhibit considerable heterogeneity in the speed of calcium mobilization for a given ligand concentration. Both the population‐averaged speed for calcium mobilization and the fraction of cells which respond to ligand stimulation are increasing functions of the ligand concentration. In contrast, the magnitude of the ligand‐stimulated increase in [Ca2+]i from basal to peak levels in responding cells is independent of ligand concentration. We postulate that the heterogeneity seen in the ligand‐induced mobilization of calcium among single cells is a function of distinct differences between cells, such as number of receptors, size of the intracellular calcium store, or phospholipase C activity. We have developed a mathematical model, based on the calcium signal transduction cascade, to predict single‐cell calcium responses to ligand stimulation. We have systematically incorporated cell‐to‐cell parameter heterogeneity into the model by randomly selecting single‐cell parameter values from a Gaussian distribution. Model simulations predict both single‐cell and population‐averaged trends that we have observed experimentally. The results of this work suggest that increases in a population response may be the result of increased participation in the response as opposed to increases in the magnitudes of individual cell responses.

Atherosclerosis and calcium signalling in endothelial cells

“…The IP 3 production and decay rate constants, k 1 = 5.46 x 10-3 µM s-1 and k 2 = 0.2 s-1 , are chosen to maintain a comparable timescale for IPa production, and a comparable steady state con\:!entration of IP 3 , at physiological levels of ATP. These values are similar to those cited by Mahama and Linderman (1994).…”

“…This phenomenon is termed Ca 2 +-induced Ca 2 + release (CICR) (Wood and Gillespie, 1998). The increased concentration of cytosolic Ca 2 + can also act cooperatively to increase IP 3 production, via increased phospholipase C activity (Mahama and Linderman, 1994;Meyer and Stryer, 1988). Furthermore, release of Ca 2 + from stores appears to stimulates influx of Ca 2 + across the plasma membrane, a process termed capacitative calcium entry (CCE) (Putney et al, 2001).…”

“…where [X] denotes the concentration of substance X, k1 = kdn [ARA] (0) and Kc= k,:,.kd. In the case of IP3 generation in EC, we assume, as discussed above, that the production rate k1 is an increasing function of Gae, We use a saturating function of the Michaelis-Menten type: k1 (Gae)= k1K 1 C:~ac for constants, k1,K1 2: 0 (Mahama and Linderman, 1994). We hence have the following ODE for intracellular IP 3 concentration (i):…”

Diffusion-limited reactions in G-protein activation: Unexpected consequences of antagonist and agonist competition

“…First, our simulations here do not incorporate cell-tocell variation in parameter values. We have previously shown that such variation, particularly when threshold-like behavior is present in later steps in the activation pathway, leads to a flattening (smaller Hill coefficient) of the dose-response curve (Mahama and Linderman, 1994). In addition, large Hill coefficients in our simulations may also be due to particular assumptions about molecular movement and reaction (e.g., movement is constrained to a lattice and the entire circumference of a molecule is considered to be equally reactive).…”

“…This allows, even when binding has equilibrated, for the continual movement of ligands among surface receptors. In previous theoretical and experimental studies, when comparing cases of equal occupancy of receptors by agonist, increased movement of agonist (shorter half-life of the receptor-agonist complex) resulted in increased G-protein activation, as receptors newly occupied by agonist have access to Gproteins close by (Mahama and Linderman, 1994;Shea et al, 1997;Stickle and Barber, 1993). Such movement has also been predicted to partially decouple G-protein activation from receptor phosphorylation .…”