This study was carried out to examine DAG formation and degradation of phospholipids in rat liver cells treated with the thyroid hormones. It was demonstrated that L-T4 induced a biphasic formation of DAG peaking at 15 sec and 1-2 min (Fig. ). Early phase was abolished by neomycin sulfate, a specific inhibitor of phosphoinositide hydrolysis (Table ), and accompanied by PtdIns(4,5)P2 level decrease (Fig. ). It has also been shown that L-T4 stimulates inositol 1,4,5-trisphosphate (Ins(l,4,5)P3) formations in [3H]inositol pre-labeled hepatocytes. Basal and hormone-induced Ins(l,4,5)P3 levels in liver cells were 840+25 and 1832+42 cpm/107 cells, respectively (time of treatment: 0.25 min, n = 6, P < 0.05).
Figure 1 Effect of L-T4 on [14C]DAG and [14C]phospholipid level in liver slices and isolated hepatocytes. [14C]sodium acetate labeled liver slices (A) and [14C]oleic acid labeled hepatocytes (B) were treated with 100 nM NaOH (control) or 10 nM L-T4 for indicated (more ...)
Effect of neomycin sulfate and propranolol on the L-T4-induced DAG formation in oleic acid pre-labeled hepatocytes.
Many cells produce DAG in a biphasic manner, involving an initial and usually transient rise in DAG that is correlated with phosphoinositide - specific PLC activation followed by a sustained increase in DAG derived from phosphatidylcholine (PC) hydrolysis [14
]. It is shown in the present study that the second phase of DAG production in liver slices (Fig. ) and isolated hepatocytes (Fig. ) coincided with hormone-stimulated PC content decrease. L-T4
- induced DAG formation in hepatocytes was concentration-dependent and highly specific as D-T4
failed to accumulate DAG (Fig. ). In rat hepatocytes, it has been reported that various regulatory factors such as phorbol esters, guanine nucleotide-binding proteins, hepatocyte growth factor, vasopressin and insulin caused PC-dependent PLD activation and DAG formation [15
]. PLD catalyzed hydrolytic cleavage of the terminal diester bound of phospholipids, resulting in the direct formation of PA and the respective base. The PA produced can be converted to DAG by the action of PAP. A unique property of PLD, which provides a specific assay for this enzyme, is the transphosphatidylation reaction to generate the corresponding phosphatidylalcohol in the presence of primary alcohol.
Figure 2 Dose-dependent action of thyroid hormone on DAG (A), choline and Peth (B) formation in hepatocytes. [14C]oleic acid pre-labeled cells were treated with 100 nM NaOH (control) or thyroid hormones for 2 min (A). The DAG level in control cells: 4707 ± (more ...)
In the present study, to determine the PLD activity, the formation of phosphatidylethanol (Peth) was measured in intact (Fig. ) and [14
C]oleic acid pre-labeled cells (Table ) in the presence of 1.5% ethanol. L-T4
stimulatory effect on Peth formation was specific and concentration-dependent (Fig. ). To determine if PC could be the substrate for the PLD in L-T4
- stimulated cells the effect of hormone on isolated plasma membranes has been investigated in the presence of [14
C-methyl]PC (Fig. ). It was found that L-T4
rapidly stimulated [14
C]PC degradation and free [14
C]choline accumulation. Besides, L-T4
increased the level of choline in intact liver cells in a concentration -dependent manner (Fig. ). Early work [19
] has shown that L-T3
rapidly induced choline and PA accumulation in liver slices. To assess the possibility of PA conversion to DAG in hormone-stimulated liver cells we have used propranolol, which is known to block the conversion of PA to DAG by inhibiting PAP. 100 nM propranolol was added 15 min prior to the hormone to [14
C]oleic acid pre-labeled cells. Propranolol completely abolished L-T4
(10 nM for 2 min) stimulatory effect on DAG accumulation (Table ). Inhibitor of polyphosphoinositide-specific PLC, neomycin sulfate, nullified the first rise of DAG production and decreased the second one in hormone stimulated cells. In rat hepatocytes, commonly used PKC inhibitors, 1,(5- isoquinolinesulphonyl)2methylpiperasine dihydrochloride (H7), Ro31-8425 and calphostin C, inhibited HGF-, vasopressin- and insulin -induced PC-specific PLD, suggesting that PLD activity is regulated by PKC [16
]. Phorbol esters are highly effective stimuli of PLD activity in intact cells [reviewed in 15
]. The results presented show that the pretreatment of [14
C]-labeled hepatocytes with protein kinase inhibitor - H7 decreased L-T4
effect on [14
C]Peth and [14C]DAG formation (Table ). Pre-incubation of liver cells with L-T4
leads to translocation of PKC from cytosol to membrane fraction and its activation. D-T4
had no effect on enzyme activity of the fractions investigated. The PKC activity in membranes isolated from control (100 nM NaOH-treated cells) and D-T4
(10 nM)-treated hepatocytes was 1040 ± 111 and 865 ± 61 pmol/mg protein/min, respectively, and 1593 ± 167 and 937 ± 120 pmol/mg protein/min in cytosol of control and hormone-treated cells, respectively. However, the enzyme activity in L-T4
-induced cells was 9498 ± 224 pmol/mg protein/min (for membrane; Pcontrol-L-T4
< 0,05, n = 4) and 633 ± 20 pmol/mg protein/min (for cytosol).
Effect of H7 on the L-T4-induced DAG and Peth formation in oleic acid pre-labeled hepatocytes.
Figure 3 Effect of L-T4 on [14C-methyl]PC metabolism in isolated liver cell plasma membranes. [14C-methyl]PC metabolism in isolated plasma membranes was investigated as described in "Materials and Methods". Results are mean ± S.E. of four individual experiments. (more ...)