Endothelin-1 increases intracellular Ca²⁺in rabbit pulmonary artery smooth muscle cells through phospholipase C

Abstract

In freshly isolated rabbit pulmonary artery smooth muscle cells, endothelin (ET)-1 induced a transient increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) followed by a return to the initial [Ca(2+)](i). This response was not abolished by the voltage-dependent Ca(2+) channel blocker nicardipine or removal of Ca(2+) from the bath solution but was inhibited by ryanodine and thapsigargin. This finding suggested that the increase in [Ca(2+)](i) induced by ET-1 was attributable to release of Ca(2+) from ryanodine- and inositol 1,4,5-trisphosphate-sensitive intracellular Ca(2+) stores. The transient increase in [Ca(2+)](i) induced by ET-1 was also inhibited by pretreatment with antagonists of ET type A and B (ET(A) and ET(B)) receptors (BQ-123 and BQ-788, respectively). Furthermore, the ET(B) receptor agonist IRL-1620 induced an increase in [Ca(2+)](i) that was followed by a sustained increase in [Ca(2+)](i); the sustained increase in [Ca(2+)](i) was blocked by nicardipine. Using the nystatin-perforated patch-clamp technique, we found that IRL-1620 caused an increase in Ca(2+) current that was inhibited by addition of ET-1. ET-1 did not inhibit Ca(2+) current when cells were pretreated with BQ-123. These results suggested that when both receptor types are activated, the opposing responses lead to abolition of the sustained [Ca(2+)](i) increases induced by ET(B) receptor activation. Western blot analysis confirmed expression of ET(A) and ET(B) receptors. Finally, U-73122 inhibited the ET-1-induced [Ca(2+)](i) increase, indicating that phospholipase C was involved in modulation of the ET-1-induced [Ca(2+)](i) increase in rabbit pulmonary artery smooth muscle cells.

Voltage-independent calcium channels mediate lipopolysaccharide-induced hyporeactivity to endothelin-1 in the rat aorta

“…Since removal of external Ca 2ϩ abolishes this difference in basal [Ca 2ϩ ] i levels, it is unlikely to result from an impairment of [Ca 2ϩ ] i storage (7,15), but probably from enhanced Ca 2ϩ influx through VOCCs (40) or other Ca 2ϩ channels. The ET-1-induced biphasic increase in Ca 2ϩ levels, comprising transient and sustained components, has been previously shown (18,42 , as previously demonstrated (20,42). Since LPS treatment had no effect on the transient phase increase in [Ca 2ϩ ] i , it is unlikely that Ca 2ϩ mobilization from intracellular stores was affected under our experimental conditions.…”

“…Protein samples (40 g/lane) were then subjected to SDS-PAGE on a 7.5% (for MLC20 and CPI-17) or 15% (for ROK␣, PKC, and MYPT1) gels, transferred to nitrocellulose membrane (Whatman) by semidry transfer blot (Transblot SD cell, Bio-Rad), and blocked for 1 h with Tris-buffered saline-Nonidet P-40 containing 5% nonfat milk. Blots were subsequently incubated overnight at 4°C with gentle shaking with one of the following rabbit polyclonal antibodies: anti-phospho-MLC 20 (Ser 19 ) antibody (1:1,000 dilution, Cell Signaling Technologies), anti-phospho-ROK␣ (The 396 ) antibody (2 g/ml dilution, Abcam, Cambridge, UK), anti-PKC antibody (1:200 dilution, Santa Cruz Biotechnology), anti-phospho-MYPT1 (Thr 850 ) antibody (1 g/ml dilution, Upstate), or anti-phospho-CPI-17 (Thr 38 ) antibody (2 g/ml dilution, Upstate). Blots were then incubated for 1 h with horseradish peroxidase-conjugated secondary antibodies (1:10,000 dilution in Tris-buffered saline-Nonidet P-40 with 2% milk, Dako Cytomation, Glostrup, Denmark), and immunoreactive protein bands were detected using ECL Advance kit (Amersham Biosciences) and visualized on an X-ray film (Fujifilm, Tokyo, Japan).…”

“…An increase in [Ca 2ϩ ] i , either through the release of Ca 2ϩ from internal stores or the influx of Ca 2ϩ through voltage-operated Ca 2ϩ channels (VOCCs), receptor-operated Ca 2ϩ channels, and store-operated Ca 2ϩ channels, leads to phosphorylation of the 20-kDa regulatory myosin light chain (MLC 20 ) by MLC kinase (16,33). MLC 20 is dephosphorylated by MLC phosphatase (MLCP) (34). MLCP activity is inhibited by phosphorylation of myosin phosphatase-targeting subunit (MYPT1) (37) or the smooth muscle-specific MLCP inhibitory protein CPI-17 (19).…”

Mechanosensitive nonselective cation channel facilitation by endothelin-1 is regulated by protein kinase C in arterial myocytes

“…Moreover, it has been argued that ET-1 dependent signaling is critical for hypoxia-induced pulmonary vasoconstriction and chronic hypoxia-induced pulmonary hypertension [12,18,19]. In vascular myocytes, stimulation of the two known ET-1 receptors, ET A and ET B [20], increases cytosolic Ca 2+ concentration ([Ca 2+ ] c ) via various ways which include phospholipase C (PLC)/InsP 3 -dependent release of Ca 2+ from intracellular stores and the activation of Ca 2+ permeable nonselective cation channels [20][21][22][23][24][25][26].…”

Endothelium-Dependent Vasoconstriction in Isolated Vessel Grafts: A Novel Mechanism of Vasospasm?

“…ETA receptors of smooth muscle induce the phospholipase C pathway without requir-10 Endothelium-dependent vasoconstrictions ing calcium influx [22]. As YC-1 induced contractions were attenuated by nifedipine, but not by antagonizing ET-1A, direct stimulation of smooth muscle by ET-1 appears unlikely.…”