Effects of Parathyroid Hormone–Related Peptide on the Large Conductance Calcium–Activated Potassium Channel and Calcium Homeostasis in Vascular Smooth Muscle Cells

Abstract

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.

Keywords:

• smooth muscle cells
• parathyroid hormone–related peptide
• calcium–activate potassium channels
• calcium
• hyperpolarization