Activation of Maxi-K Channels by Parathyroid Hormone and Prostaglandin E 2 in Human Osteoblast Bone Cells

Abstract

Patch clamp experiments were performed on two human osteosarcoma cell lines (MG-63 and SaOS-2 cells) that show an osteoblasticlike phenotype to identify and characterize the specific K channels present in these cells. In case of MG-63 cells, in the cell-attached patch configuration (CAP) no channel activity was observed in 2 mM Ca Ringer (control condition) at resting potential. In contrast, a maxi-K channel was observed in previously silent CAP upon addition of 50 nM parathyroid hormone (PTH), 5 nM prostaglandin E2 (PGE2) or 0.1 mM dibutyryl cAMP + 1 microM forskolin to the bath solution. However, maxi-K channels were present in excised patches from both stimulated and nonstimulated cells in 50% of total patches tested. A similar K channel was also observed in SaOS-2 cells. Characterization of this maxi-K channel showed that in symmetrical solutions (140 mM K) the channel has a conductance of 246 +/- 4.5 pS (n = 7 patches) and, when Na was added to the bath solution, the permeability ratio (PK/PNa) was 10 and 11 for MG-63 and SaOS-2 cells respectively. In excised patches from MG-63 cells, the channel open probability (Po) is both voltage- (channel opening with depolarization) and Ca-dependent; the presence of Ca shifts the Po vs. voltage curve toward negative membrane potential. Direct modulation of this maxi-K channel via protein kinase A (PKA) is very unlikely since in excised patches the activity of this channel is not sensitive to the addition of 1 mM ATP + 20 U/ml catalytic subunit of PKA. We next evaluated the possibility that PGE2 or PTH stimulated the channel through a rise in intracellular calcium. First, calcium uptake (45Ca2+) by MG-63 cells was stimulated in the presence of PTH and PGE2 an effect inhibited by Nitrendipine (10 microM). Second, whereas PGE2 stimulated the calcium-activated maxi-K channel in 2 mM Ca Ringer in 60% of patches studied, in Ca-free Ringer bath solution, PGE2 did not open any channels (n = 10 patches) nor did cAMP + forskolin (n = 3 patches), although K channels were present under the patch upon excision. In addition, in the presence of 2 mM Ca Ringer and 10 microM Nitrendipine in CAP configuration, PGE2 (n = 5 patches) and cAMP + forskolin (n = 2 patches) failed to open K channels present under the patch. As channel activation by phosphorylation with the catalytic subunit of PKA was not observed, and Nitrendipine addition to the bath or the absence of calcium prevented the opening of this channel, it is concluded that activation of this channel by PTH, PGE2 or dibutyryl cAMP + forskolin is due to an increase in intracellular calcium concentration via Ca influx.

Prostaglandin E2 Induces Interaction Between hSlo Potassium Channel and Syk Tyrosine Kinase in Osteosarcoma Cells

“…Interestingly, PGE 2 has been shown to increase the intracellular calcium concentration and, consequently, the opening of Ca 2ϩ -dependent potassium channels. (15) In this study, we focused on the Ca 2ϩ -dependent large conductance potassium channel, because of its expression in osteoblasts, and its ability to open in response to PGE 2 . We first verified that the product of the hslo gene, responsible for the BK conductance, was indeed expressed in the MG63 osteosarcoma cell line.…”

“…(12)(13)(14) This increase in intracellular Ca 2ϩ has been shown to trigger the opening of Ca 2 -dependent K ϩ channels of high conductance (BK). (15,16) It is noteworthy, in the context of bone remodeling, that this type of channel also has been shown to exhibit mechanosensitive properties in osteoblasts and odontoblasts. (16,17) In contrast to other voltage-dependent K ϩ channels, the pore-forming ␣-subunit of BK channels is encoded by a single gene slo (slowpoke), (18,19) that undergoes extensive, hormonally regulated, alternative RNA splicing.…”

The Action of Prostaglandins on Ion Channels

“…PGE 2 (100 nM) almost totally blocks the Ca 2+ -activated K + current which gives rise to a slow, Ca 2+ -dependent spike after-hyperpolarization in rabbit nodose neurons [118]. By contrast, 5 nM PGE 2 activates large conductance Ca 2+ -activated K + channels from otherwise silent patches in human osteoblast bone cells [75]. PGE 2 (10 M) also activates G protein-gated inwardly rectifying K + (GIRK) channels heterologously expressed in rat superior cervical ganglion [91].…”

“…The membrane permeant cAMP analogue dibutyryl cAMP acted similarly to PGE 2 . An increase of [Ca 2+ ] i due to Ca 2+ influx is most likely the reason for the activation of Ca 2+ -activated K + channels in human osteoblast bone cells by PGE 2 [75]. The effect is mimicked by cAMP + forskolin and disappears in Ca 2+ -free bath.…”

Effect of Voltage-Gated Calcium Channels (Cav) Blocker on Ovariectomy Induced Osteoporosis in Rats

“…7 Intracellular calcium concentration, at least part of which is decreased by calcium channel blockers (CCB'), has been shown to be increased by various bone regulatory factors, such as vitamin D 3 8,9 parathyroid hormone 8,10 and prostaglandin E 2 . 8,11 These factors also alter osteoblast differentiation 12,13 thus; it shows that signaling through the L-type calcium channel may be important for osteoblast functions. All these above facts suggests that involvement of calcium channels in the bone remodeling, so this study is undertaken to exploit their detail role and development of new strategies for treatment of osteoporosis.…”