Effect of urea and osmotic cell shrinkage on Ca²⁺ entry and contraction of vascular smooth muscle cells

Abstract

The present study was performed to elucidate the effects of urea on vascular smooth muscle cells (SMC). Addition of urea (20, 50, 100 mM) to physiological salt solution blunted the vasoconstrictory effect of phenylephrine (by 17, 25 and 30%, respectively) and of an increased extracellular K⁺ concentration (by 7, 14 and 19%, respectively) without affecting the basal tone of rabbit arterial rings. According to Fura-2 fluorescence in cultured SMC (A7r5), urea had no effect on basal intracellular calcium activity ([Ca²⁺]_i), but significantly blunted the increase of [Ca²⁺]_i following an increase of extracellular K⁺. Whole-cell patch-clamp studies revealed that the Ca²⁺ current through voltage-sensitive Ca²⁺ channels is significantly inhibited in the presence of urea. As evident from calcein fluorescence, addition of urea leads to sustained cell shrinkage. The effects of urea on vascular tone, [Ca²⁺]_i activity, voltage-gated Ca²⁺ channels and cell volume are mimicked by addition of raffinose or NaCl. However, the cell shrinkage induced by urea is sustained, whereas the addition of equiosmolar NaCl is only transient and followed by a regulatory cell volume increase. Moreover, hypertonic NaCl increases, whereas urea decreases, the transcription of cell-volume-regulated kinase hsgk. In conclusion, urea leads to sustained shrinkage of vascular smooth muscle cells, which is followed by inhibition of voltage-gated Ca²⁺ channels, a decrease of [Ca²⁺]_i and thus blunts the vasoconstrictory action of phenylephrine and increased extracellular K⁺ concentration.