Effects of parathyroid hormone-related peptide on the large conductance calcium-activated potassium channel and calcium homeostasis in vascular smooth muscle cells.

Aim: To demonstrate the impact of the parathyroid hormone-related peptide (PTHrP) on the large conductance calcium-activated potassium (BKCa) channels in vascular smooth muscle cells (VSMC) and hyperpolarization of the cell membrane and its dependence on calcium.

Materials And Methods: VSMC were isolated from rat aorta and further subcultured. Four experiments were conducted in calcium-release measurements and each of them consisted of a control group, PTHrP, chemical substance, and PTHrP + chemical substance. Chemical substances used were: iberiotoxin, xestospongin C, xestospongin D, and thapsigargin, respectively. Fura-2 imaging was used to determine changes in calcium release of VSMC. In membrane-potential experiments, groups were designed similarly to the Fura-2 imaging experiments: iberiotoxin, BAPTA, and xestospongin D were added, in respective order. Changes in the membrane potential were examined using the fluorescence dye (DiBAC).

Results: Given in a dose between 0.01 and 1.0 μmol/L, PTHrP caused a concentration-dependent decrease in fluorescence intensity, with a maximum effect at 0.5 μmol/L. The decrease, therefore, demonstrated a PTHrP-induced hyperpolarization of the VSMC. The effect was blocked by use of iberiotoxin (100 nmol/L), a highly selective inhibitor of BKCa. Furthermore, when the calcium chelator BAPTA (10 μmol/L) was added, there was a significant reduction in PTHrP-induced hyperpolarization. Use of PTHrP (0.5 μmol/L) also decreased the fluorescence intensity of the indicator for intracellular calcium, Fura-2AM (a membrane-permeable derivative of Fura 2). This effect was re-blocked by use of iberiotoxin. Xestospongin C (3 μmol/L) and xestospongin D (6 μmol/L), both inhibitors of the inositol 1,4,5 trisphosphate-triggered calcium release, inhibited the effects of PTHrP. Additionally, thapsigargin (1 μmol/L), a sarcoplasmic/endoplasmic reticulum Ca2+-ATPase inhibitor, inhibited the effect of PTHrP.

Conclusion: The results of our study show that PTHrP induces hyperpolarization and activates BKCa in VSMC. The activation of BKCa channels is calcium dependent; activation is linked to the inositol 1,4,5 trisphosphate-triggered calcium release and is also dependent on the endo/sarcoplasmic reticulum calcium pump.