TY - JOUR
T1 - Characterization of phospholipase C activity of the plasma membrane and cytosol of an osteoblast-like cell line
AU - Suzuki, Y.
AU - Hruska, K. A.
AU - Reid, I.
AU - Alvarez, U. M.
AU - Avioli, L. V.
N1 - Funding Information:
From the *Division of Bone and Mineral Diseases and the fRenal Division, The Jewish Hospital of St. Louis, at Washington University Medical Center, St. Louis, Missouri. . These studies were supported by grants from the NIH AM32087 and AM09976. The authors thank Linda R. Halstead and Jim Harter for expert technical assistance and Helen Odie and Bernice Kaplan for secretarial assistance. Reprint requests: Louis V. Avioli, MD, Division of Bone and Mineral Diseases, The Jewish Hospital of St. Louis, 216 South Kingshighway, St. Louis, MO 63110.
PY - 1989
Y1 - 1989
N2 - The properties of phospholipase C (PL-C) in the plasma membranes (PM) and the cytosol of osteoblast-like osteosarcoma cells, UMR-106, were analyzed to see if separate enzymes or similar enzymes were involved in signalling, transduction, and arachidonate release. The cytosolic PL-C displayed substrate affinities in the order of phosphatidylinositol (PI) > phosphatidylinositol-4-phosphate (PIP) or phosphatidylinositol-4, 5-bisphosphate (PIP2). Hydrolysis of PI, PIP, and PIP2 by cytosolic PL-C was not affected by GTP or GTP(γ)S and other nucleotides. PI hydrolysis by PM and cytosolic PL-C was undetectable in the presence of 500 μM EGTA and displayed two activity plateaus at various concentrations of Ca2+. The Km for Ca2+ in the PL-C activity of the first plateau was 0.08 μM. Significant hydrolysis of PIP2 by cytosolic PL-C was observed in the absence of Ca2+. In contrast to the enzyme(s) predominant in the cytosol, the order of substrate affinities for PM PL-C was PIP2 > PIP > PI. Only PIP2 hydrolysis by PM PL-C was stimulated by both GTP and GTP(γ)S in a dose-dependent manner. PIP2 hydrolysis by PL-C of the PM was not observed in the absence of CA2+, serving to further discriminate this enzyme activity from that of the cytosol. PIP2 hydrolysis by PL-C of the PM also was biphasic in the dependence on Ca2+. At resting cytosolic Ca2+ levels, the Vmax of the high affinity activity already had been achieved. Guanine nucleotide stimulation of PIP2 hydrolysis by PM PL-C was characterized by increased maximum activity with an unchanged Km for Ca2+ or for PIP2. The pH optimum of PIP2 hydrolysis was similar between cytosolic and PM forms of PL-C. PIP2 hydrolysis with production of IP3 (PL-C activity) in UMR-106 cells treated with [2-3H]-myoinositol was stimulated by PTH, and this stimulation was not inhibited by pertussis toxin. These data suggest that UMR-106 cells possess at least two distinct PL-C activities, one predominant in the cytosol and activated by increasing cytosolic Ca2+ with PI as the substrate. The second enzyme, a GTP-activated PIP2-specific PL-C in the plasma membranes may play an important role in hormone-induced PIP2 hydrolysis mediated through guanine nucleotide regulatory proteins and may participate in the hormonal regulation of osteoblast cytosolic Ca2+ and bone remodeling functions.
AB - The properties of phospholipase C (PL-C) in the plasma membranes (PM) and the cytosol of osteoblast-like osteosarcoma cells, UMR-106, were analyzed to see if separate enzymes or similar enzymes were involved in signalling, transduction, and arachidonate release. The cytosolic PL-C displayed substrate affinities in the order of phosphatidylinositol (PI) > phosphatidylinositol-4-phosphate (PIP) or phosphatidylinositol-4, 5-bisphosphate (PIP2). Hydrolysis of PI, PIP, and PIP2 by cytosolic PL-C was not affected by GTP or GTP(γ)S and other nucleotides. PI hydrolysis by PM and cytosolic PL-C was undetectable in the presence of 500 μM EGTA and displayed two activity plateaus at various concentrations of Ca2+. The Km for Ca2+ in the PL-C activity of the first plateau was 0.08 μM. Significant hydrolysis of PIP2 by cytosolic PL-C was observed in the absence of Ca2+. In contrast to the enzyme(s) predominant in the cytosol, the order of substrate affinities for PM PL-C was PIP2 > PIP > PI. Only PIP2 hydrolysis by PM PL-C was stimulated by both GTP and GTP(γ)S in a dose-dependent manner. PIP2 hydrolysis by PL-C of the PM was not observed in the absence of CA2+, serving to further discriminate this enzyme activity from that of the cytosol. PIP2 hydrolysis by PL-C of the PM also was biphasic in the dependence on Ca2+. At resting cytosolic Ca2+ levels, the Vmax of the high affinity activity already had been achieved. Guanine nucleotide stimulation of PIP2 hydrolysis by PM PL-C was characterized by increased maximum activity with an unchanged Km for Ca2+ or for PIP2. The pH optimum of PIP2 hydrolysis was similar between cytosolic and PM forms of PL-C. PIP2 hydrolysis with production of IP3 (PL-C activity) in UMR-106 cells treated with [2-3H]-myoinositol was stimulated by PTH, and this stimulation was not inhibited by pertussis toxin. These data suggest that UMR-106 cells possess at least two distinct PL-C activities, one predominant in the cytosol and activated by increasing cytosolic Ca2+ with PI as the substrate. The second enzyme, a GTP-activated PIP2-specific PL-C in the plasma membranes may play an important role in hormone-induced PIP2 hydrolysis mediated through guanine nucleotide regulatory proteins and may participate in the hormonal regulation of osteoblast cytosolic Ca2+ and bone remodeling functions.
UR - http://www.scopus.com/inward/record.url?scp=0024504710&partnerID=8YFLogxK
U2 - 10.1097/00000441-198903000-00001
DO - 10.1097/00000441-198903000-00001
M3 - Article
C2 - 2923133
AN - SCOPUS:0024504710
SN - 0002-9629
VL - 297
SP - 135
EP - 144
JO - American Journal of the Medical Sciences
JF - American Journal of the Medical Sciences
IS - 3
ER -