Compatible osmolytes modulate the response of porcine endothelial cells to hypertonicity and protect them from apoptosis

Abstract

Porcine pulmonary arterial endothelial cells accumulated myo-inositol and taurine, as well as betaine, during adaptation to hypertonic stress. The cells grew and maintained their normal morphology during culture in hypertonic (0.5 osmol (kg H(2)O)(-1)) medium that contained osmolytes such as betaine, myo-inositol or taurine at concentrations close to reported physiological values. The cells did not grow well in hypertonic medium depleted of potential compatible osmolytes. After a few days, cell density decreased by about 50 % and many cells rounded up and detached from the plates, their nuclei showing clear apoptotic morphology. The caspase-3 activity of the cells also increased dramatically under these conditions, but remained negligibly low when betaine and myo-inositol were added to the medium. Addition of betaine and myo-inositol to hypertonic medium depleted of compatible osmolytes increased the number of colonies remaining after 12 days of culture; with each solute at 30-100 micromol l(-1) the number increased about sixfold. In the absence of compatible osmolytes, increased mRNA levels and corresponding activities of betaine/gamma-aminobutyric acid transporter (BGT1) and sodium/myo-inositol transporter (SMIT) induced by hypertonicity remained high after 72 h incubation, whereas they were down regulated in the presence of betaine and myo-inositol. Similarly, the down regulation of the amino acid System A transporter (ATA2) was markedly slowed in the absence of compatible osmolytes. We conclude that these compatible osmolytes at concentrations close to physiological values enable the endothelial cells to adapt to hypertonic stress, protecting them from apoptosis, and also modulate the adaptation process.

Figure 1. Accumulation of betaine, taurine and myo-inositol by endothelial cells incubated in hypertonic medium

Endothelial cells were incubated in isotonic (0.3 osmol (kg H₂O)⁻¹) or hypertonic (0.5 osmol (kg H₂O)⁻¹) medium containing, separately, 0.1 mmol l⁻¹ radio-labelled betaine, taurine or myo-inositol and the cellular concentration of each solute was measured at the indicated times. Sucrose was used as the extra osmolyte. Mean values (±s.d.) of four measurements are shown. The final concentration values noted in the text were calculated with the use of measured cell volumes, which had returned to the control value of 7–8 μl mg⁻¹ of protein after a few hours of hypertonic incubation (Petronini et al. 2000). Open symbols, isotonic medium; closed symbols, hypertonic medium; •, betaine; ▴, myo-inositol; ▾, taurine.

Figure 2. Effect of deprivation of compatible osmolytes on the morphology of cells exposed to hypertonicity

A, cells were seeded and cultured for 24 h in isotonic (0.3 osmol (kg H₂O)⁻¹) medium and then incubated for a further 72 h in either (B) depleted hypertonic (0.5 osmol (kg H₂O)⁻¹) medium or (D) depleted hypertonic medium with added betaine and myo-inositol (each at 0.1 mmol l⁻¹). The cells were then photographed under phase contrast. Cells that had detached after 72 h of hypertonic incubation in the absence of compatible osmolytes (C) were treated with the fluorescent DNA stain Hoechst 33342. Magnification: A, B and D, × 100; C, × 400. All the cells in field C exhibit the typical chromatin condensation of apoptosis.

Figure 3. Effect of deprivation of compatible osmolytes on cell caspase-3 activity during hypertonic incubation

Cells were incubated for the times indicated in depleted hypertonic (0.5 osmol (kg H₂O)⁻¹) medium with or without added betaine and myo-inositol, each at 0.1 mmol l⁻¹. Caspase-3 activity was measured in cell extracts as described in the Methods section. Mean values (±s.d.) of three independent determinations are given. □, depleted of compatible osmolytes; ▪, with betaine and myo-inositol.

Figure 4. Effect of removal of compatible osmolytes on cell growth and survival under hypertonic conditions

Cells were seeded and cultured for 24 h in isotonic (0.3 osmol (kg H₂O)⁻¹) medium and then transferred to depleted hypertonic (0.5 osmol (kg H₂O)⁻¹) medium with or without added betaine and myo-inositol, each at 0.1 mmol l⁻¹. Culture in both media was continued for the indicated times and cell growth was estimated by measurement of cell protein. Mean values (±s.d.) of three independent measurements are given. ○, depleted of compatible osmolytes; •, with betaine and myo-inositol.

Figure 5. Effect of betaine and myo-inositol on the colony-forming efficiency of cells incubated in depleted hypertonic medium

Endothelial cells were seeded at a density of about 200 cells per well (area 9 cm²) in isotonic (0.3 osmol (kg H₂O)⁻¹) medium and cultured for 24 h. Then the medium was replaced with the depleted hypertonic (0.5 osmol (kg H₂O)⁻¹) one containing added betaine and myo-inositol at the indicated concentrations. Colonies were allowed to grow for 12 days and then the number of colonies larger than 50 cells was counted. Values are the means (±s.d.) of three independent determinations. (The maximum number of colonies possible was less than 200.)

Figure 6. Modulation of hypertonic induction of amino acid transport activity and accumulation of NPS

Initial rates of cell uptake of 2-[1-¹⁴C]methylaminoisobutyric acid (MeAIB) (A) and cell contents of ninhydrin-positive solutes (NPS) (B) were measured after the cells had been incubated for the indicated times in depleted hypertonic (0.5 osmol (kg H₂O)⁻¹) medium with or without added betaine and myo-inositol, each at 0.1 mmol l⁻¹. The extracellular concentration of MeAIB was 0.1 mmol l⁻¹. Mean values (±s.d.) of four measurements are given. ○, depleted of compatible osmolytes; •, with betaine and myo-inositol.

Figure 7. Modulation of hypertonic induction of BGT1 and SMIT transport activities induced by hypertonicity

The initial rates of uptake of GABA and myo-inositol (extracellular concentrations 0.1 mmol l⁻¹) by endothelial cells were measured after the cells had been incubated for the indicated times in depleted hypertonic (0.5 osmol (kg H₂O)⁻¹) medium with or without added betaine and myo-inositol, each at 0.1 mmol l⁻¹. Sucrose was used as the extra osmolyte. Mean values (±s.d.) of four measurements are given. Open symbols, depleted of compatible osmolytes; filled symbols, with betaine and myo-inositol; ▴, GABA; •, myo-inositol.

Figure 8. Modulation of expression of mRNA for ATA2, BGT1 and SMIT induced by hypertonicity

Endothelial cells were incubated in isotonic (0.3 osmol (kg H₂O)⁻¹) medium (C) and depleted hypertonic (0.5 osmol (kg H₂O)⁻¹) medium with (T+) or without (T-) added betaine and myo-inositol, each at 0.1 mmol l⁻¹. After the indicated times, total cellular RNA was extracted and analysed by Northern blotting for (A) ATA2, (B) BGT1 and (C) SMIT mRNA, as described under Methods. 28S rRNA was used for standardisation, the densitometric quantification of the blots being normalised to the levels of 28S rRNA.