Fig. 1. Effect of osmotic stress on the synthesis of sulfolipids of MDCK cells. Confluent MDCK·P3 cells were incubated in the medium with the indicated amount of osmolytes for 1 day, and then the cells were labeled with ³⁵S-sulfuric acid (370 kBq/mL) for 24 h. The lipids were extracted and analyzed as described in Materials and methods. HPTLC was developed with chloroform/methanol/acetone/acetic acid/water (8:2:4:2:1, v/v). A. Autoradiogram of sulfolipids labeled with ³⁵S-sulfate. The lipid corresponding to 500 μg protein was applied to each lane. Lane 1; normal medium (300 mOsm/L), lane 2; 500 mOsm/L (addition of 100 mM NaCl), lane 3; hypotonic medium (150 mOsm/L), CHL-S; cholesterol sulfate B. Orcinol reagent detected standard sulfoglycolipids from rat kidney (lane 4). SM4s (galactosyl ceramide I³-sulfate) and SM3 (lactosyl ceramide II³-sulfate).

Fig. 2. Mass spectra of the sulfoglycolipid SX. The sulfolipid was obtained from the preparative TLC.

Fig. 3. Analysis of neutral glycolipids by 2-D TLC. Cells were cultured for 2 days as indicated. Neutral glycolipids were prepared and developed by HPTLC with chloroform/methanol/water (60:25:4, v/v) in the vertical direction and with 2-propanol/ammonium hydroxide/methyl acetate/water (15:2:1:3, v/v) in the horizontal direction. CONTROL (300 mOsm/L), HYPERTONIC (500 mOsm/L) by addition of NaCl, HYPOTONIC (150 mOsm/L). Detection was by orcinol reagent.

Fig. 4. Northern blot analysis of cerebroside sulfotransferase gene. Total RNA was prepared from the cells cultured for 24 h under osmotic stress, and northern blot analysis was performed as described in Materials and methods. A: normal culture (300 mOsm/L), B: hypotonic culture (200 mOsm/L), C: hypertonic culture (500 mOsm/L) CST; dog cerebroside sulfotransferase mRNA, GAPDH; dog glyceraldehyde-3-phosphate dehydrogenase mRNA.

Fig. 5. Summary of sulfoglycolipid metabolism as modified by osmotic stresses. The character size of the metabolites under osmotic stresses represents the content of the metabolite relative to the normal condition, and the thickness of the arrow shows the relative rates of synthesis and degradation. Deg; degradation. (1) Ceramide galactosyltransferase (EC 2.4.1.47), (2) Ceramide glucosyltransferase (EC 2.4.1.80), (3) Cerebroside sulfotransferase (EC 2.8.2.11).

Changes in the incorporation of ³⁵S-sulfate into sulfolipids by osmotic stress

Values (dpm/mg protein) shown are mean ± SD from triplicate samples. Significantly different from normal culture, P < 0.05.

Metabolic turnover of CMH

Values (dpm/mg protein) are mean ± SD from triplicate samples. Cells were labeled with [¹⁴C]-serine as described in Materials and methods. Pulse labeling for 24 h. Normal; normal medium (300 mOsm/L), Hypertonic; 500 mOsm/L (addition of 100 mM NaCl), Hypotonic; hypotonic medium (150 mOsm/L), Heat shock; cultured in the normal medium (300 mOsm/L) at 40 °C for 24 h. Chase experiment. After pulse labeling as described above in the normal medium for 24 h, cells were cultured for 6 h in the indicated media. Significantly different from normal culture, P < 0.05.

Effect of osmotic stress on ceramide content.

Values (μg/mg protein) are mean ± SD from triplicate samples. Cells were cultured under the indicated conditions for 7 h and 24 h. Ceramide was determined as described in Materials and methods. Heat shock at 40 °C in the normal medium was introduced as a positive control. Significantly different from normal culture, P < 0.05.

Effects of osmotic stress on ceramide turnover

Values (dpm/mg protein) are mean ± SD from triplicate samples. Cells were labeled with [¹⁴C]-serine as described in Materials and methods. Pulse labeling for 24 h. Normal; normal medium (300 mOsm/L), Hypertonic; 500 mOsm/L (addition of 100 mM NaCl), Hypotonic; hypotonic medium (150 mOsm/L), Heat shock; cultured in the normal medium (300 mOsm/L) at 40 °C for 24 h. Chase experiment. After pulse labeling as described above in the normal medium for 24 h, cells were cultured for 6 h in the indicated media. Significantly different from normal culture, P < 0.05.