2014 年 37 巻 6 号 p. 922-925
We recently found that hepatic triglyceride lipase (HTGL) was released from primary cultured rat hepatocytes after treatment with prazosin, an antagonist of alpha-1 adrenoceptors. However, the details of prazosin-induced HTGL release remain uncertain. Here we investigated whether changes in cAMP levels in hepatocytes were related to HTGL release from prazosin-stimulated hepatocytes. When hepatocytes were treated with prazosin, cAMP levels during stimulated release of HTGL increased in a time- and dose-dependent manner. Stimulated release of HTGL was suppressed by the adenylate cyclase inhibitors MDL-12,330A and 2′,5′-dideoxyadenosine. Further, cAMP-dependent protein kinase A (PKA) activity in prazosin-stimulated hepatocytes also increased in a time- and dose-dependent manner. Moreover, prazosin-stimulated HTGL release was suppressed by the PKA inhibitors H-89 and KT5720. These results suggest that prazosin-stimulated HTGL release from hepatocytes was due to cAMP production and partly due to subsequent PKA activation in hepatocytes.
Prazosin (1-[4-amino-6,7-dimethoxy-quinazolin-2-yl]-4-[2-furoyl]piperazine) is a well-known antagonist of alpha-1 adrenoceptors, and it is used in pharmacotherapy for hypertension, prostatomegaly,1,2) and pheochromocytoma. Its pharmacological effects result from inhibition of alpha-1 adrenoceptors of vascular smooth muscles and the sphincter muscle of the urethra.3,4) Prazosin also has other effects, including increasing high density lipoprotein (HDL) cholesterol levels in hypertensive patients after long-term administration,5) improving posttraumatic stress disorder (PTSD) symptoms,6) and inducing prostate cancer cell apoptosis.7) In addition, prazosin administration results in increased lipoprotein lipase (LPL) activity8–10) and decreased 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase activity in serum.11)
We recently found that prazosin stimulated the release of hepatic triacylglyceride lipase (HTGL; EC 188.8.131.52) from primary cultured rat hepatocytes via the activation of phospholipase C (PLC).12) In this previous study, the region downstream to PLC was suggested to be highly involved in the Ca2+/calmodulin-dependent protein kinase-II rather than protein kinase C. However, other interactions may also have been involved.
HTGL hydrolyzes triacylglycerides (TG) in HDL and intermediate density lipoproteins, and it is considered to play an important role in lipid metabolism.13–16) However, effects of HTGL other than this remain to be investigated.
HTGL is synthesized and secreted when it is partially glycosylated in hepatocytes.17) It is also anchored to the plasma membranes of endothelial cells by electrostatic binding with heparan sulfate proteoglycans.18) However, details regarding the regulation of HTGL release from hepatocytes remain uncertain.
Thus, in this study, we investigated whether prazosin-stimulated HTGL release from hepatocytes was due to a mechanism involving increased cAMP levels and a subsequent increase in protein kinase A (PKA) activity.
[γ32P]ATP (111 TBq/mmol) was from PerkinElmer, Inc. Japan (Yokohama, Japan). Glycerol tri[1-14C]oleate (2.0 GBq/mmol) and a cAMP enzyme immunoassay system (RPN. 2251) were from GE Healthcare Japan (Tokyo, Japan). PKA assay kits (# 17–134) were from Millipore (Billerica, MA, U.S.A.). Prazosin, 2′,5′-dideoxyadenosine, H-89, KT5720, collagenase, and Williams’ medium E were from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). MDL-12,330A was from Sigma (St. Louis, MO, U.S.A.). All other chemicals used were of analytical grade.
Male Wistar rats (weight: 200–300 g) were fed a commercial laboratory chow ad libitum and fasted for 24 h before the experiments. Hepatocytes were isolated by in vitro collagenase perfusion and low speed centrifugation, with modifications.19) Kupffer cell contamination in hepatocyte preparations was confirmed to be <2% by peroxidase staining. Cell viability was determined by trypan blue exclusion and ranged from 85 to 95%. Hepatocytes were cultured as monolayers for 24 h in a plastic dish (1×105 cells/cm2) in Williams’ medium E, containing 10% fetal calf serum, 10 nM insulin, 10 nM dexamethasone, and 5 KIU/mL aprotinin, in a 5% CO2 atmosphere. After removing the medium by aspiration, hepatocyte monolayers were incubated for an additional 0–60 min in Williams’ medium E containing 2% bovine serum albumin with prazosin and other reagents. Hepatocytes were harvested and centrifuged at 50 ×g for 5 min to remove cellular debris. The supernatant was then used for HTGL activity assay.
HTGL activity was determined using glycerol tri[1-14C]oleate (3.7 MBq/mL) as the substrate,20) and it was expressed as pmol of free fatty acids (FFA) produced/min/106 cells.
Incubated hepatocytes (1×105 cells/cm2) were homogenized in ice-cold 5% trichloroacetic acid (TCA) and centrifuged at 10000×g for 20 min. Supernatants were extracted with H2O-saturated diethyl ether and then subjected to quantitative analysis for cAMP using a commercial cAMP enzyme immunoassay system (GE Healthcare), as previously described.21)
Hepatocytes incubated with prazosin were homogenized in 20 mM 3-(N-morpholino)propanesulfonic acid (MOPS) buffer (pH 7.2) that contained 25 mM β-glycerophosphate, 5 mM ethylene glycol tetraacetic acid (EGTA), 1 mM sodium orthovanadate, and 1 mM dithiothreitol using a Vibra-cell ultrasonic processor (model VC-130PB, Sonics, Newtown, CT, U.S.A.), and then centrifuged at 10000×g at 4°C for 20 min. The supernatant was used as an enzyme preparation to determine PKA activity using a PKA assay kit. Radioactivity was measured and expressed as nmol phosphate/min/106 cells.
Results are expressed as mean±standard error of the mean for three or four determinations from independent experiments using different hepatocyte preparations.
In a previous study, we found that prazosin stimulation increased HTGL release from primary cultured rat hepatocytes.12) In this study, we investigated whether this prazosin-stimulated HTGL release was due to a mechanism involving an increase in intracellular cAMP levels. We found that prazosin-stimulated HTGL release was markedly suppressed by the adenylate cyclase (AC) inhibitors 2′,5′-dideoxyadenosine (Fig. 1a) and MDL-12,330A22,23) (Fig. 1b). We also determined the time dependence of cAMP levels in hepatocytes incubated with 100 µM prazosin over a 45-min period. In hepatocytes, cAMP levels increased in a time-dependent manner with a maximum of a 1.5-fold higher level than the basal level (Fig. 2a). Moreover, cAMP levels increased in a dose-dependent manner on treating hepatocytes with up to 100 µM prazosin (Fig. 2b).
a) Hepatocytes were incubated for 60 min either with (●) or without (○) 100 µM prazosin in the presence of 2′,5′-dideoxyadenosine. b) Hepatocytes were incubated for 60 min either with (●) or without (○) 100 µM prazosin in the presence of MDL-12,330A.
a) Hepatocytes were incubated for 0–45 min either with (●) or without (○) 100 µM prazosin. b) Hepatocytes were incubated for 45 min with varying concentrations (0–100 µM) of prazosin. cAMP levels in hepatocytes were determined as described in Materials and Methods.
cAMP levels were measured in the presence of AC inhibitors to determine whether AC was involved in the prazosin-induced increase in cAMP levels. cAMP levels in prazosin-stimulated hepatocytes were found to be suppressed by the AC inhibitors MDL-12,330A and 2′,5′-dideoxyadenosine (Fig. 3).
a) Hepatocytes were incubated for 45 min either with (●) or without (○) 100 µM prazosin in the presence of 2′,5′-dideoxyadenosine (0–2 µM). b) Hepatocytes were incubated for 45 min either with (●) or without (○) 100 µM prazosin in the presence of MDL-12,330A (0–100 µM). cAMP levels in hepatocytes were determined as described in Materials and Methods.
Later, we investigated the release of HTGL in the presence of cAMP-dependent PKA inhibitors, as cAMP levels are increased due to PKA activity. Figure 4 shows prazosin-stimulated HTGL release in the presence of PKA inhibitors. HTGL release was suppressed by the PKA inhibitors H-89 and KT5720.24,25)
a) Hepatocytes were incubated for 60 min either with (●) or without (○) 100 µM prazosin in the presence of H-89. b) Hepatocytes were incubated for 60 min either with (●) or without (○) 100 µM prazosin in the presence of KT5720.
Thus, we investigated whether the release of HTGL involved PKA activity in hepatocytes with or without prazosin treatment. It was observed that intracellular PKA activity increased after treatment with 100 µM prazosin in a time-dependent manner for up to 60 min (Fig. 5a). In addition, PKA activation by prazosin occurred in a dose-dependent manner on treating hepatocytes with up to 100 µM prazosin (Fig. 5b).
a) Hepatocytes were incubated either with (●) or without (○) 100 µM prazosin for up to 60 min. b) Hepatocytes were incubated with varying concentrations (0–100 µM) of prazosin. PKA activity in hepatocytes was determined as described in Materials and Methods.
Several clinical reports have shown that prazosin administration alters lipid metabolism in vivo,5,26,27) although the detailed mechanism underlying this phenomenon remains unknown. In a previous study, we investigated the release of HTGL from rat hepatocytes after stimulation with prazosin.12) It was considered that PLC activation played an important role in HTGL release. However, elucidating the involvement of other protein kinases is currently in progress. PKA regulation of G-protein-mediated PLC has been previously reported by Liu and Simon.28) These interactions may have been involved in prazosin-stimulated HTGL release. However, the signaling mechanisms underlying prazosin-stimulated HTGL release still remain uncertain. In this study, we investigated whether prazosin-stimulated HTGL release was due to increased intracellular cAMP levels in hepatocytes.
It is well-known that increased cAMP levels result in AC activation.29) Our results suggested that HTGL release was due to increased cAMP levels mediated through AC in prazosin-stimulated hepatocytes (Figs. 1–3). We speculate that AC activation was caused by prazosin incorporation in hepatocytes. Prazosin or its metabolites may cause an increase in AC activity or a decrease in phosphodiesterase (PDE) activity. An increase in intracellular cAMP levels also promotes PKA activation.30) Our results also suggested that prazosin-stimulated HTGL release was due to increased PKA activity in hepatocytes (Figs. 4, 5). HTGL release by prazosin probably involves PKA regulation, although the underlying mechanism remains to be determined.
In another report, a cAMP analog (dibutyryl cAMP, 1 mM) was reported to suppress HTGL release from rat hepatocytes.31) We did not consider that a major change in HTGL release would occur with 1 mM dibutyryl cAMP. However, HTGL release was suppressed with 500 µM dibutyryl cAMP along with prazosin stimulation (data not shown). Thus, only moderate levels of cAMP seem to be necessary for HTGL release.
In contrast, intracellular cAMP levels may increase in hepatocytes on inhibition of phosphodiesterase activity. In fact, we observed that 3-isobutyl-1-methylxanthine, a PDE inhibitor,32) maintained HTGL release (data not shown). In addition, prazosin can inhibit PDE,33,34) which was confirmed by PDE distribution in hepatocytes and other cells.35) Therefore, the increase in intracellular cAMP levels due to prazosin was related not only to AC but also to PDE. It is possible that prazosin increases intracellular cAMP levels by activating AC and inhibiting PDE. However, an increase in cAMP levels is necessary to some extent, although not in excess amounts.
PKA is also involved in exocytosis.36,37) In this study, HTGL release from rat hepatocytes due to prazosin stimulation suggests that the release should be suppressed by PKA inhibitors, as PKA activity is closely related to secretion by hepatocytes.
In conclusion, our results suggest that prazosin-stimulated HTGL release from primary cultured rat hepatocytes is promoted by activating PKA to increase intracellular cAMP levels due to the effects of activating AC and inhibiting PDE. In addition, these effects of prazosin were probably due to prazosin uptake by hepatocytes and not due to alterations of alpha-1 adrenoceptors by prazosin. The mechanism underlying the effects of prazosin remains unclear after its uptake by hepatocytes, as almost all of it is metabolized by the cells. Thus, mechanisms other than alpha-1 adrenoceptor activation need to be investigated.