Membrane hyperpolarization is not required for sustained muscarinic agonist-induced increases in intracellular Ca2+ in arteriolar endothelial cells - PubMed (original) (raw)

Membrane hyperpolarization is not required for sustained muscarinic agonist-induced increases in intracellular Ca2+ in arteriolar endothelial cells

Kenneth D Cohen et al. Microcirculation. 2005 Mar.

Abstract

Objective: Hyperpolarization modulates Ca2+ influx during agonist stimulation in many endothelial cells, but the effects of hyperpolarization on Ca2+ influx in freshly isolated arteriolar endothelial cells are unknown. Therefore, the purpose of the present study was to characterize agonist-induced Ca2+ transients in freshly isolated arteriolar endothelial cells and to test the hypothesis that membrane hyperpolarization augments agonist-induced Ca2+ influx into these cells.

Methods: Arterioles were removed from hamster cremaster muscles and arteriolar endothelial cells were enzymatically isolated. Endothelial cells were loaded with Fura 2-AM and the Fura 2 ratio measured photometrically as an index of intracellular Ca2+. The cells were then stimulated with the muscarinic, cholinergic agonist, methacholine, and the resulting Ca2+ transients were measured.

Results: Methacholine (1 microM) increased the endothelial cell Fura 2 ratio from a baseline of 0.81 +/- 0.02 to an initial peak of 1.17 +/- 0.05 (n = 17) followed by a sustained plateau of 1.12 +/- 0.07. The plateau phase of the Ca2+ transient was inhibited by removal of extracellular Ca2+ (n = 12, p < .05), or the nonselective cation channel blockers Gd3+ (30 microM; n = 7, p < .05) or La3+ (50 microM; n = 7, p < .05) without significant effect on the baseline or peak (p > .05). The initial peak of methacholine-induced Ca2+ transients was inhibited by the IP3-receptor antagonist xestospongin D (10 microM, n = 5, p < .05). The methacholine-induced Ca2+ transients were accompanied by endothelial cell hyperpolarization of approximately 14-18 mV, as assessed by experiments using the potentiometric dye, di-8-ANEPPS as well as by patch-clamp experiments. However, inhibition of hyperpolarization by blockade of Ca2+-activated K+ channels with charybdotoxin (100 nM) and apamin (100 nM) (n = 5), or exposure of endothelial cells to 80 or 145 mM KCl (both n = 7) had no effect on the plateau phase of methacholine-induced Ca2+ transients (p > .05).

Conclusions: Freshly isolated arteriolar endothelial cells display agonist-induced Ca2+ transients. For the muscarinic agonist, methacholine, these Ca2+ transients result from release of Ca2+ from intracellular stores through IP3 receptors, followed by sustained influx of extracellular Ca2+. While these changes in intracellular Ca2+ are associated with endothelial cell hyperpolarization, the methacholine-induced, sustained increase in intracellular Ca2+ appears to be independent from this change in membrane potential. These data suggest that arteriolar endothelial cells may possess a novel Ca2+ influx pathway, or that the relationship between intracellular Ca2+ and Ca2+ influx is more complex than that observed in other endothelial cells.

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Figures

Figure 1

Figure 1

Arteriolar endothelial cell tubes take up and hydrolyze calcein-AM while excluding ethidium homodimer-1. (A) Transmitted light image of an endothelial cell tube from a hamster cremaster arteriole. Cells were incubated in calcein-AM (1 μM) and ethidium homodimer-1 (1 μM) for 15 min. The bar in this panel represents 50 μm. (B) Fluorescent image of the same endothelial cell tube as in (A) that has taken up and cleaved calcein-AM (excitation wavelength = 494 nm, emission wavelength = 520 nm). (C) Fluorescent image of the same endothelial cell tube as in panels (A) and (B) demonstrating exclusion of ethidium homodimer-1 (excitation wavelength = 528 nm, emission wavelength = 617 nm). (D) Fluorescent image of the same endothelial cell tube as in (A)–(C) after exposure to 0.1% Triton X-100 to permeabilize the cell membranes and demonstrate positive ethidium homodimer-1 staining (excitation wavelength = 528 nm, emission wavelength = 617 nm).

Figure 2

Figure 2

Agonist-induced Ca2+ transients in isolated arteriolar endothelial cell tubes are similar to responses of endothelial cells in an intact arteriole. Representative traces of the Fura 2 ratio in isolated arteriolar endothelial cells in response to 1 μM methacholine in isolated arteriolar endothelial cells (A) and in endothelial cells from an intact arteriole (B). Representative traces of the Fura 2 ratio in isolated arteriolar endothelial cells in response to 100 nM substance P in isolated arteriolar endothelial cells (C) and in endothelial cells from an intact arteriole (D). Methacholine (MCh) or substance P (sub P) application is denoted by a line in each figure.

Figure 3

Figure 3

Removal of [Ca2+]o inhibits methacholine-induced sustained increases in intracellular Ca2+ in isolated arteriolar endothelial cell tubes. (A) Representative traces of the Fura 2 ratio in endothelial cell tubes in response to methacholine in the presence (solid line) and absence (dotted line) of [Ca2+]o. (B) Summary data (n = 12) of the baseline, peak and plateau Fura 2 ratio values in response to methacholine in the presence (open bars) and absence (closed bars) of extracellular Ca2+. (C) Summary data (n = 5) of the baseline, peak, and plateau Fura 2 values in response to methacholine under control conditions (open bars), in the absence of [Ca2+]o (closed bars) and in the absence of [Ca2+]o but now in the presence of 10 μM XSP D (gray bars). *Significantly different from control at that same component of the Ca2+ transient; #significantly different from 0 [Ca2+]o ( p < .05 for both).

Figure 4

Figure 4

The inorganic cation channel blockers, La3+ and Gd3+, block the methacholine-induced sustained increases in intracellular Ca2+ in isolated arteriolar endothelial cells. Summary data of the baseline, peak and plateau Fura 2 values in isolated arteriolar endothelial cells in response to methacholine under control conditions (open bars) and in the presence of 30 μM Gd3+ (A; closed bars; n = 7) and 50 μM La3+ (B; closed bars; n = 7).

Figure 5

Figure 5

The combination of charybdotoxin and apamin did not inhibit the methacholine-induced Ca2+ transients in isolated arteriolar endothelial cells (n = 5). Summary data of the baseline, peak and plateau Fura 2 values in isolated arteriolar endothelial cells in response to methacholine under control conditions (open bars) and in the presence of 100 nM charybdotoxin and apamin (closed bars)

Figure 6

Figure 6

Charybdotoxin and apamin inhibit methacholine-induced K+ currents. (A) Current–voltage plots of whole endothelial cell K+ currents under control conditions (open circles) and in response to 1 μM methacholine (closed circles). (B) Current–voltage plots of whole endothelial cell K+ currents in the presence of 100 nM charybdotoxin and apamin (open circles) and in the presence of charybdotoxin, apamin and 1 μM methacholine (closed circles). *Significantly different from currents in the absence of methacholine (p < .05).

Figure 7

Figure 7

High [K+]o solutions did not inhibit the methacholine-induced Ca2+ transients. Summary data of the baseline, peak, and plateau Fura 2 values in isolated arteriolar endothelial cells in response to methacholine under control conditions (open bars) and in the presence of 80 mM [K+]o (A; closed bars; n = 7) and 145 mM [K+]o (B; closed bars; n = 6).

Figure 8

Figure 8

Methacholine hyperpolarizes freshly isolated arteriolar endothelial cells and this hyperpolarization is inhibited by high [K+]o. Summary data of the di-8-ANEPPS ratio in arteriolar endothelial cells corresponding (in time) to the plateau of the Fura 2 ratio changes in response to methacholine during exposure of arteriolar endothelial cells to 80 or 145 mM [K+]o, as indicated. *Significant change from baseline (p < .05); #significantly different from methacholine alone (p < .05).

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