Propofol and Aminophylline Antagonize Each Other During the Mobilization of Intracellular Calcium in Human Umbilical Vein Endothelial Cells

Propofol is a widely used, intravenous, general anesthetic that provides long-term sedation of patients in intensive-care units, with effects on the central nervous and cardiovascular systems as a modulator of intracellular calcium mobilization (1-3). Propofol suppresses the mobilization of intracellular calcium in myocytes, astrocytoma cells, pituitary cells, and endothelial cells (2-6).

We examined whether propofol affects the mobilization of intracellular calcium in human umbilical vein endothelial cells (HUVECs). Furthermore, we examined whether propofol gave influences on the mobilization of intracellular calcium by lysophosphatidic acid (LPA), which stimulates intracellular calcium mobilization (7, 8). We also tested whether other commonly used drugs in intensive-care units stimulated the mobilization of intracellular calcium. Aminophylline, a bronchodilator for asthmatics, stimulated the mobilization of intracellular calcium in HUVECs (9, 10). However, the interaction of propofol and aminophylline, which affects the mobilization of intracellular calcium, has never been studied in vitro. Therefore, we studied whether propofol and aminophylline affect the mobilization of intracellular calcium in HUVECs.

The fluorescence intensity in untreated HUVECs loaded with Fluo-4 was very low and distributed homogeneously throughout the cells (Fig. 1A). Fluo-4 fluorescence in cells treated with 5 µg/mL of LPA or 1,000 µM of aminophylline increased rapidly (Fig. 1C, D). Propofol (300 µM) blocked the increase induced by LPA (5 µg/mL) (Fig. 1B). HUVECs treated with LPA (5 µg/mL) showed a very rapid increase in [Ca²⁺]_i (Fig. 2A), but propofol blocked this increase dose-dependently at clinically relevant concentrations (10, 30 µM) (Fig. 2B).

Fig. 1
Fluorescence of intracellular calcium in human umbilical-vein endothelial cells (HUVECs) incubated with Fluo-4 and detected by a fluorescence spectrophotometer (confocal microscope). (A) Control group (resting cells) before treatment. (B) Preincubation with propofol (300 µM) blocks lysophosphatidic acid (LPA) signals. (C) Aminophylline (1,000 µM) treatment increases Fluo-4 fluorescence. (D) LPA (5 µg/mL) increases Fluo-4 fluorescence.

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Fig. 2
Propofol inhibits lysophosphatidic acid (LPA)-induced intracellular calcium ([Ca²⁺]_i) elevation. (A) Levels of [Ca²⁺]_i generated by propofol. Cells preincubated in propofol for 30 min and treated with LPA (5 µg/mL). Serum-starved HUVECs were loaded with 2 µM of Fluo-4 for 40 min. Results are expressed as relative fluorescence intensity (RFI). Each trace is of a single cell and is representative of at least three independent experiments. Intracellular Ca²⁺ was monitored by confocal microscopy. P10 µM→LPA (S): incubation with propofol (10 µM) for 30 min and then treatment with LPA (5 µg/mL); P30 µM→LPA (S): incubation with propofol (30 µM) for 30 min and then treatment with LPA (5 µg/mL); P100 µM→LPA (S): incubation with propofol (100 µM) for 30 min and then treatment with LPA (5 µg/mL); P1,000 µM→LPA (S): incubation with propofol (1,000 µM) for 30 min and then treatment with LPA (5 µg/mL). (B) Propofol inhibits LPA-induced [Ca²⁺]_i elevation in a dose-dependent manner. Values represent the mean peak intracellular calcium response. Results are expressed as fold-stimulation, determined by comparing RFIs before stimulation and expressed as mean±SD from three separate determinations. Each determination represents the mean of at least 10 cells.

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Aminophylline induced a very rapid, dose-dependent increase in [Ca²⁺]_i (Fig. 3), and propofol (300 µM) treatment decreased the concentration of [Ca²⁺]_i. Aminophylline (1,000 µM) rapidly increased Fluo-4 fluorescence in cells to a maximum three to four-fold higher than the control (Fig. 3). Propofol (10 µM) treatment for 30 min decreased [Ca²⁺]_i induced by LPA (5 µg/mL) and aminophylline (100 µM) (Fig. 4A). Propofol (30 µM) showed similar activity (Fig. 4B). Following incubation with propofol (30 µM) and aminophylline (100; 1,000 µM) for 30 min, the peak level of [Ca²⁺]_i following LPA (5 µg/mL) treatment was higher than propofol (30 µM) only (Fig. 5). Furthermore, HUVECs incubated with propofol (30 µM) and aminophylline (1,000 µM) for 30 min had peak [Ca²⁺]_i levels higher than propofol (30 µM) and aminophylline (100 µM) (Fig. 5).

Fig. 3
Representative trace of intracellular calcium ([Ca²⁺]_i) induced by drugs. (A) The elevation of [Ca²⁺]_i generated by aminophylline treatment in HUVECs. Serum-starved HUVECs were loaded with 2 µM of Fluo-4 for 40 min. Results are expressed as relative fluorescence intensity (RFI). Each trace is of a single cell and is representative of at least three independent experiments. Intracellular Ca²⁺ was monitored by confocal microscopy. A, aminophylline; P, propofol; LPA, lysophosphatidic acid; (s), treatment. (B) Levels of mean peak [Ca²⁺]_i generated by various concentrations of aminophylline in HUVECs. Aminophylline generates [Ca²⁺]_i elevation in a dose-dependent manner. Values represent the mean peak intracellular calcium response. Results are expressed as fold-stimulation, determined by comparing RFIs before stimulation and expressed as mean±SD from three separate determinations. Each determination represents the mean of at least 10 cells.

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Fig. 4
Levels of mean peak intracellular calcium ([Ca²⁺]_i) generated by lysophosphatidic acid (LPA) or aminophylline treatment after incubation with propofol (10, 30 µM) for 30 min. (A) Levels of mean peak intracellular calcium ([Ca²⁺]_i) generated by lysophosphatidic acid (LPA) or aminophylline treatment after incubation with propofol (10 µM) and for 30 min. LPA only: treatment with LPA (5 µg/mL); P10→LPA: incubation with propofol (10 µM) and treatment with LPA (5 µg/mLl); A100 only: treatment with aminophylline (100 µM); P10→A100: incubation with propofol (10 µM) and treatment with aminophylline (100 µM) Values represent the mean peak intracellular calcium response. Results are expressed as fold-stimulation, determined by comparing relative fluorescence intensities (RFI) before stimulation and expressed as mean±SD from three separate determinations. Each determination represents the mean of at least 10 cells. (B) Levels of mean peak intracellular calcium ([Ca²⁺]_i) generated by lysophosphatidic acid (LPA) or aminophylline treatment after incubation with propofol (30 µM) and for 30 min. LPA only: treatment with LPA (5 µg/mL); P30→LPA: incubation with propofol (30 µM) and treatment with LPA (5 µg/mL); A100 only: treatment with aminophylline (100 µM); P30→A100: incubation with propofol (30 µM) and treatment with aminophylline (100 µM) Values represent the mean peak intracellular calcium response. Results are expressed as fold-stimulation, determined by comparing relative fluorescence intensities (RFI) before stimulation and expressed as mean±SD from three separate determinations. Each determination represents the mean of at least 10 cells.

^*P<0.05 compared with treatment with LPA (5 µg/mL); ^†P<0.05 compared with treatment with aminophylline (100 µM).

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Fig. 5
Levels of mean peak intracellular calcium ([Ca²⁺]_i) generated by lysophosphatidic acid (LPA) treatment after incubation with propofol (30 µM) and aminophylline for 30 min. LPA (S): treatment with LPA (5 µg/mL); P30→LPA (S): incubation with propofol (30 µM) and treatment with LPA (5 µg/mL); P30+A100→LPA (S): incubation with propofol (30 µM) and aminophylline (100 µM) and treatment with LPA (5 µg/mL); P30+A1000→LPA (S): incubation with propofol (30 µM) and aminophylline (1,000 µM) and treatment with LPA (5 µg/mL).

Values represent the mean peak intracellular calcium response. Results are expressed as fold-stimulation, determined by comparing relative fluorescence intensities (RFI) before stimulation and expressed as mean±SD from three separate determinations. Each determination represents the mean of at least 10 cells.

^*P<0.05 compared with propofol (30 µM)-incubated cells; ^†P<0.05 compared with propofol (30 µM) and aminophylline (100 µM)-incubated cells.

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In this study, propofol decreased the concentration of [Ca²⁺]_i in HUVECs, whereas aminophylline (30-1,000 µM) increased mobilization of [Ca²⁺]_i in a concentration-dependent manner. Propofol (10-1,000 µM) suppressed the LPA-induced mobilization of [Ca²⁺]_i in a concentration-dependent manner. Propofol further prevented the aminophylline-induced increase of [Ca²⁺]_i at clinically relevant concentrations (10 and 30 µM). However, aminophylline reversed the inhibitory effect of propofol on the elevation of [Ca²⁺]_i by LPA.

Cells employ various mechanisms for regulating the concentration of free Ca²⁺ in the cytosol, which usually is maintained below 0.2 µM. Therefore, most investigators measure the effect of a substance on the increase and/or decrease of [Ca²⁺]_i mediated by that specific substance, rather than directly measuring [Ca²⁺]_i (5). In this study, we determined changes of [Ca²⁺]_i that occurred in response to LPA.

LPA has multiple cellular effects, including the stimulation of phospholipases, the mobilization of [Ca²⁺]_i, and the inhibition of adenylate cyclase (12). The extracellular addition of LPA increased [Ca²⁺]_i through mobilization of intracellular calcium stores as well as through the influx of extracellular Ca²⁺ (6). However, the ability of LPA to stimulate increases in [Ca²⁺]_i is mediated predominantly via the mobilization of calcium stores (13). This action is transduced by the production of inositol triphosphate (IP3) through the G-protein-mediated activation of phospholipase C (12). In this study, propofol decreased the concentration of [Ca²⁺]_i in HUVECs as in other cells (2-6). Also, propofol suppressed the increase of [Ca²⁺]_i by LPA in a concentration-dependent manner.

Propofol is a brief-acting, intravenous, anesthetic agent possessing significant cardiovascular effects (14). Systemic hypotension caused by a marked decrease in systemic vascular resistance is often observed in the clinical use of propofol, probably via a decrease in [Ca²⁺]_i. The negative inotropic and vasodilatory effects associated with propofol are likely due the voltage-gated influx of extracellular calcium being blocked, and to the decrease in availability of intracellular calcium (1, 4). Propofol affects [Ca²⁺]_i by inhibiting the mobilization of intracellular-calcium stores (6). It also blocks the voltage-gated influx of extracellular Ca²⁺ by acting as a calcium channel blocker (6). In endothelial cells, the activation of adenosine-triphosphate-sensitive potassium channels (K⁺ATP channels) is dependent upon L-type, voltage-gated calcium channels, and causes an increase in K⁺ efflux, membrane hyperpolarization and calcium influx (15). Propofol attenuates the effect of K⁺ATP channels by inhibiting the nitric oxide pathway (16). The mobilization of calcium from intracellular stores is related to the hydrolysis of inositol-4,5-biphosphate (PIP2) to diacylglycerol and IP3 (17). IP3 releases internal calcium in endothelial cells, therefore triggering the calcium cascade and its associated biological process, such as vasoconstriction (18). Propofol reduces the production of IP3 and can thus act as a calcium blocker preventing the entry of calcium into the vasodilatation pathway (18). It further inhibits the entry of capacitative calcium and modulates Na⁺-Ca²⁺ exchange activity via the protein kinase C-signaling pathway (19, 20). The activation of protein kinase C by phorbol esters can be inhibited by propofol (20). In the present study, propofol suppressed the LPA-induced mobilization of [Ca²⁺]_i in HUVECs in a dose-dependent manner, presumably via one of these mechanisms.

Aminophylline is a bronchodilating drug widely used for the treatment of asthma and chronic airway-obstructing diseases (21). Aminophylline exerts multiple pharmacological effects either through phosphodiesterase inhibition or via adenosine receptor blockage (22, 23). It furthermore activates cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) (24) and enhances calcium currents. Adenosine inhibits calcium channels through a pertussis-toxin-sensitive G protein (24). In the present study, aminophylline increased intracellular calcium in a concentration-dependent manner in HUVECs. Specifically, aminophylline probably enhanced calcium release from the sarcoplasmic reticulum, along with the influx of extracellular calcium.

In the present study, there are two possible cellular mechanisms of the opposing effects of propofol and aminophylline on intracellular calcium mobilization. First, propofol blocks the voltage-gated influx of extracellular Ca²⁺ by acting as a calcium channel blocker (6), but aminophylline induced an influx of Ca²⁺ by activating calcium channels (23, 24). Second, propofol reduces the production of IP3 and calcium release from intracellular stores (16, 17), but aminophylline induced Ca²⁺ release from intracellular stores, occurs via increased production of IP3 through G-protein-mediated activation (21, 25).

Aminophylline has a narrow therapeutic index (9), with side effects of nausea, headache, and diuresis. The misuse or abuse of aminophylline can cause life-threatening cardiovascular conditions, such as cardiac arrhythmias and seizures (9, 10). Overdoses of aminophylline are the most common cause of death due to hospital-related asthma (10). Serious toxicity has been reported when high, prolonged doses of aminophylline are administered through continuous intravenous infusion (26). Aminophylline changes intracellular calcium concentrations (27), and calcium plays a major role in aminophylline-induced toxicity and death (28).

Propofol is widely used in intensive care medicine, especially for the long-term sedation of critically ill patients (29). Side effects, such as hypotension, vasodilation, and propofol infusion syndrome, are possible when used clinically. These side effects act as calcium channel blockers that cause cardiovascular depression (1). Aminophylline reverses propofol-induced postoperative sedation (22).

In this study, propofol and aminophylline showed opposing effects on intracellular calcium mobilization in HUVECs. Although this may decrease drug-related side effects, it may also decrease therapeutic effects. Therefore, clinicians should give adequate doses when administered together. Further work is required to understand the mechanism of interaction of propofol and aminophylline on intracellular calcium mobilization in HUVECs and its effects in the other cells such as bronchial and neuronal cells.

In conclusion, propofol and aminophylline antagonize [Ca²⁺]_i mobilization at clinically relevant concentrations in HUVECs. Serious consideration shou ld be given to drug interactions on intracellular calcium mobilization when used together.