Gender-related differences in expression and function of hepatic P-glycoprotein and multidrug resistance-associated protein (Mrp2) in rats

To clarify whether gender-related differences exist in the expression and function of hepatic P-glycoprotein- and/or multidrug resistance-associated protein (Mrp2), we measured the hepatobiliary excretion of doxorubicin and their protein levels in male and female Sprague-Dawley rats. When rats received a single intravenous injection of doxorubicin (5 mg/kg), a delay in the disappearance of doxorubicin from plasma was observed in male rats. When rats received a constant-rate infusion of doxorubicin, no significant gender-related differences in the apparent biliary clearance of doxorubicin based on the steady state plasma concentrations were observed between male and female rats. However, the net biliary clearance of doxorubicin based on the liver concentration, which represents the actual function of P-glycoprotein and/or Mrp2, was higher in female rats than in male rats. These results suggest that the actual function of the hepatobiliary transport of doxorubicin is greater in female than in male rats. Western blot analysis revealed that the expression of P-glycoprotein and Mrp2 in the liver of female rats was significantly higher than in male rats, similar to results of hepatobiliary excretion experiments. The expression of hepatic cytochrome P450 (CYP) 2B1, which is involved in the metabolism of doxorubicin, was significantly higher in male than in female rats. By pretreatment with testosterone (10 mg/day for 7 days), the actual biliary clearance of doxorubicin in female rats was nearly that of male rats. The protein levels of P-glycoprotein and Mrp2 in female rats were also lowered by treatment with testosterone so as to be nearer those in male rats. These results suggest that gender-related differences exist in P-glycoprotein- and Mrp2-mediated hepatobiliary transport and that these two transporters may be regulated by sex hormones.     

Identification of the apical membrane-targeting signal of the multidrug resistance-associated protein 2 (MRP2/MOAT)

The human canalicular multispecific organic anion transporter (cMOAT), known as the multidrug resistance-associated protein 2 (MRP2), is normally expressed in the liver and to a lesser extent in the kidney proximal tubules. In these tissues MRP2 specifically localizes to the apical membrane. The construction of MRP2 fused to the green fluorescent protein, and subsequent site-directed mutagenesis enabled the identification of a targeting signal in MRP2 that is responsible for its apical localization in polarized cells. The specific apical localization of MRP2 is due to a C-terminal tail that is not present in the basolaterally targeted MRP1. Deletion of three amino acids from the C-terminal of MRP2 (DeltaMRP2) causes the protein to be localized predominantly in the basolateral membrane in polarized Madin-Darby canine kidney cells. Interestingly, MRP2 expressed in a mouse leukemia cell line (L1210 cells) predominantly accumulates intracellularly with minimal cell membrane localization. In contrast, DeltaMRP2 was shown to predominantly localize in the cell membrane in L1210 cells. Increased transport of 2,4-dinitrophenyl glutathione from L1210 cells expressing DeltaMRP2 showed that the re-targeted protein retains its normal function.     

Short-term regulation of multidrug resistance-associated protein 3 in rat and human hepatocytes

The short-term regulation of multidrug resistance-associated protein 3 (Mrp3/MRP3) by cAMP and PKC was investigated in sandwich-cultured rat and human hepatocytes and isolated perfused rat livers. The modulator glucagon (500 nM) and the phorbol ester PMA (0.1 muM) were utilized to increase intracellular cAMP and PKC levels, respectively. In glucagon-treated rat hepatocytes, efflux of the Mrp3 substrate 5-(6)-carboxy-2',7'-dichlorofluorescein (CDF) increased approximately 1.5-fold, even in hepatocytes treated with the organic anion transporter (Oatp) inhibitor sulfobromophthalein (BSP). Confocal microscopy revealed more concentrated Mrp3 fluorescence in the basolateral membrane (less diffuse staining pattern) with glucagon treatment. PMA had no effect on Mrp3 activity or localization in sandwich-cultured rat hepatocytes. Glucagon and PMA treatment in isolated perfused rat livers resulted in a threefold increase (14 +/- 4.6 mul.min(-1).g liver(-1)) and a fourfold decrease (1.3 +/- 0.3 mul.min(-1).g liver(-1)) in CDF basolateral clearance compared with control livers (4.7 +/- 2.3 mul.min(-1).g liver(-1)), whereas CDF biliary clearance was not statistically different. In sandwich-cultured human hepatocytes, glucagon treatment resulted in a 1.3-fold increase in CDF efflux and a concomitant increase in MRP3 fluorescence in the basolateral membrane. In summary, cAMP and PKC appear to be involved in the short-term regulation of Mrp3/MRP3, as demonstrated by alterations in activity and localization in rat and human hepatocytes.     

Up-regulation of multidrug resistance-associated protein 2 (MRP2) expression in rat hepatocytes by dexamethasone.

Regulation of multidrug resistance-associated protein (MRP2) expression in response to dexamethasone (DEX) was analyzed using mainly primary rat hepatocytes. Enhanced levels of MRP2 mRNAs associated with increased amounts of a 190 kDa MRP2 were found in cultured DEX-treated hepatocytes; similarly, administration of DEX to rats (100 mg/kg, i.p.) led to a marked increase of hepatic amounts of MRP2 mRNAs. Maximal induction of MRP2 expression in DEX-treated primary hepatocytes was reached with 10(-5) M DEX, a concentration higher than that (10(-7) M) required for maximal up-regulation of tyrosine aminotransferase (TAT), a typical glucocorticoid receptor-regulated enzyme. In addition, the anti-glucocorticoid compound RU486 failed to inhibit MRP2 induction caused by DEX whereas it fully blocked that of TAT. These findings therefore demonstrate that DEX is a potent inducer of MRP2 expression in rat hepatocytes through a mechanism that seems not to involve the classical glucocorticoid receptor pathway.     

Involvement of multidrug resistance-associated protein 2 (ABCC2/Mrp2) in biliary excretion of micafungin in rats

The drug transporter, multidrug resistance-associated protein 2 (ABCC2/Mrp2), is known to play important roles in excretion of various drugs. In the present study, we investigated whether Mrp2 is involved in the transport of micafungin, a newly developed antifungal agent. When Sprague-Dawley rats received an intravenous injection of micafungin (1 mg/kg) in combination with cyclosporine, the cyclosporine significantly delayed the disappearance of micafungin from plasma and decreased the systemic clearance and volume of distribution at steady-state of micafungin to 54% and 65% of the corresponding control values, respectively. When Sprague-Dawley rats received a constant-rate infusion of micafungin, cyclosporine significantly decreased the steady-state biliary clearance of micafungin (~80%). A significant decrease in the biliary clearance of micafungin (~60%) was observed in Eisai hyperbilirubinemic rats, which have a hereditary deficiency in Mrp2. The present findings at least suggest that Mrp2 is involved mainly in the hepatobiliary excretion of micafungin in rats.     

Hypothalamic growth hormone-releasing factor (GRF) participates in the negative feedback regulation of growth hormone secretion

Effects of growth hormone (GH) excess on immunoreactive hypothalamic GH-releasing factor (GRF) and somatostatin (SRIF) were studied in rats. Hypothalamic GRF content significantly reduced after 7-day daily treatment with 160 micrograms of rat GH or after inoculation of GH-secreting rat pituitary tumors, MtT-F4 for 9 or 13 days and GH3 for 3 months. Basal and 59 mM K+-evoked release of GRF from incubated hypothalami diminished, more than the content, by 43-51% in MtT-F4 tumor- or by 67-83% in GH3 tumor-bearing rats. In contrast, there was a small but significant increase in content or release of SRIF in rats harboring the GH3 or MtT-F4 tumor, respectively. These results indicate the existence of a negative feedback loop via hypothalamic GRF as well as SRIF in control of GH secretion.     

Absolute quantification of multidrug resistance-associated protein 2 (MRP2/ABCC2) using liquid chromatography tandem mass spectrometry

The multidrug resistance-associated protein 2 (MRP2/ABCC2) plays an important role in hepatobiliary efflux of many drugs and drug metabolites and has been reported to account for dramatic interspecies differences in the aspects of pharmacokinetics. In the present study, an absolute quantification method was developed to quantitatively measure MRP2/ABCC2 using LC-MS/MS for detection of a selective tryptic peptide. A unique 16-mer tryptic peptide was identified by conducting capillary LC nanospray ESI-Q-TOF analysis of the immunoprecipitation-enriched samples of MRP2/ABCC2 following proteolysis with trypsin. The lower limit of quantification was established to be 31.25 pM with the linearity of the standard curve spanned to 2500 pM. Both the accuracy (relative error) and the precision (coefficient of variation) of the method were below 15%. Using this method, we successfully determined the absolute amount of MRP2/ABCC2 protein in MRP2/ABCC2 gene-transfected MDCK cells as well as the basal levels of canine Mrp2/Abcc2 protein in MDCK cells. Our findings also demonstrate that the sensitivity of this method exceeds the sensitivity of immunoblotting assay which was not able to detect the basal levels of canine Mrp2/Abcc2 in MDCK cells. The method could be directly applicable to many current research needs related to MRP2/ABCC2 protein.     

Vitamin D receptor-dependent regulation of colon multidrug resistance-associated protein 3 gene expression by bile acids

The multidrug resistance-associated protein 3 (MRP3) is a multispecific anion transporter that is capable of transporting a number of conjugated and unconjugated bile acids. Expression of the MRP3 gene is increased during pathological states associated with elevated bile acid concentrations indicating a role for this transporter in adaptive and homeostatic bile acid metabolism. Analysis of Mrp3 mRNA levels in various mouse tissues with known relevance and/or exposure to bile acids revealed the highest levels of basal expression in the colon followed in order by the liver, duodenum, jejunum, ileum, and kidney. Functional analysis of a murine Mrp3 promoter reporter construct revealed vitamin D receptor (VDR)-dependent activation by 1,25-dihydroxyvitamin D(3) (VD3), 9-cis-retinoic acid (RA), and the cholestatic secondary bile acid, lithocholic acid (LCA). Using a series of deletion constructs combined with sequence analysis, a candidate VDR response element (VDRE) was identified between -1028 and -1014 bp of the Mrp3 promoter. Activation of the Mrp3 promoter in response to VD3, RA, or LCA, as well as binding of VDR/RXR heterodimers, was attenuated substantially by mutation of this VDRE. Treatment of mice with VD3 or LCA demonstrated in vivo modulation of the Mrp3 gene in colon but not in the liver. Reduction of endogenous VDR expression in colon adenocarcinoma MCA-38 cells by siRNA transfection was associated with reduced constitutive and inducible expression of the Mrp3 gene. These data support a regulatory role for the VDR in the protection of colon cells from bile acid toxicity through regulation of the Mrp3 expression.     

ATP-dependent transport of bile salts by rat multidrug resistance-associated protein 3 (Mrp3)

We have previously shown that cloned rat multidrug resistance-associated protein 3 (Mrp3) has the ability to transport organic anions such as 17beta-estradiol 17-beta-D-glucuronide (E(2)17betaG) and has a different substrate specificity from MRP1 and MRP2 in that glutathione conjugates are poor substrates for Mrp3 (Hirohashi, T., Suzuki, H., and Sugiyama, Y. (1999) J. Biol. Chem. 274, 15181-15185). In the present study, the involvement of Mrp3 in the transport of endogenous bile salts was investigated using membrane vesicles from LLC-PK1 cells transfected with rat Mrp3 cDNA. The ATP-dependent uptake of [(3)H]taurocholate (TC), [(14)C]glycocholate (GC), [(3)H]taurochenodeoxycholate-3-sulfate (TCDC-S), and [(3)H]taurolithocholate-3-sulfate (TLC-S) was markedly stimulated by Mrp3 transfection in LLC-PK1 cells. The extent of Mrp3-mediated transport of bile salts was in the order, TLC-S > TCDC-S > TC > GC. The K(m) and V(max) values for the uptake of TC and TLC-S were K(m) = 15.9 +/- 4.9 microM and V(max) = 50.1 +/- 9.3 pmol/min/mg of protein and K(m) = 3.06 +/- 0.57 microM and V(max) = 161.9 +/- 21.7 pmol/min/mg of protein, respectively. At 55 nM [(3)H]E(2)17betaG and 1.2 microM [(3)H]TC, the apparent K(m) values for ATP were 1.36 and 0.66 mM, respectively. TC, GC, and TCDC-S inhibited the transport of [(3)H]E(2)17betaG and [(3)H]TC to the same extent with an apparent IC(50) of approximately 10 microM. TLC-S inhibited the uptake of [(3)H]E(2)17betaG and [(3)H]TC most potently (IC(50) of approximately 1 microM) among the bile salts examined, whereas cholate weakly inhibited the uptake (IC(50) approximately 75 microM). Although TC and GC are transported by bile salt export pump/sister of P-glycoprotein, but not by MRP2, and TCDC-S and TLC-S are transported by MRP2, but not by bile salt export pump/sister of P-glycoprotein, it was found that Mrp3 accepts all these bile salts as substrates. This information, together with the finding that MRP3 is extensively expressed on the basolateral membrane of human cholangiocytes, suggests that MRP3/Mrp3 plays a significant role in the cholehepatic circulation of bile salts.     

Phosphorylation of microtubule-associated protein 2 by MAP kinase primarily involves the projection domain

Chymotryptic digestion was used to localize the sites in microtubule-associated protein 2 which are preferentially phosphorylated in vitro by MAP kinase, an insulin-stimulated serine/threonine kinase which efficiently utilizes high molecular weight MAPs as substrates. MAP kinase phosphorylates sites in the projection domain almost exclusively; less than 6% of the phosphate incorporated by MAP kinase was found in the tubulin binding domain. This site specificity is in marked contrast to that of the catalytic subunit of cAMP dependent protein kinase, and most other protein kinases phosphorylating MAP-2, which extensively phosphorylate the tubulin binding domain.     

Hormonal regulation of hepatic P-enolpyruvate carboxykinase (GTP) during development.

Hepatic gluconeogenesis in the rat does not begin until birth. The enzyme P-enolpyruvate carboxykinase appears initially at birth and is the final enzyme in the gluconeogenic sequence to develop. The appearance of this enzyme in the cytosol of rat liver is caused by the stimulation of enzyme synthesis, probably due directly to an increase in the hepatic concentration of cAMP. Enzyme degradation does not begin until 36 hours after birth. Studies with fetal rats in utero have shown that dibutyryl cAMP or glucagon will stimulate P-enolpyruvate carboxykinase synthesis and that this effect can be blocked by insulin. Insulin is known to depress the synthesis of P-enolpyruvate carboxykinase in adult rat liver and in Reuber H-35 liver cells in culture. The glucocorticoids are without effect on the synthesis of the enzyme in fetal rat liver. Work by Girard et al. (J. Clin. Invest. 52: 3190, 1973) has established that the molar ratio of insulin to glucagon drops from 10 immediately after birth, to 1 after one hour. This is due to both a rise in glucagon and a fall in insulin concentrations at birth. These studies, together with our work on the synthesis of P-enolpyruvate carboxykinase, indicate that the sharp drop in the concentration of insulin may relieve the normal inhibition of enzyme synthesis. This would allow the initial stimulation of enzyme synthesis by the glucagon-mediated rise in the concentration of CAMP.     

Episodic growth hormone secretion in the domestic fowl (Gallus domesticus): alpha adrenergic regulation

In animals the secretion of GH is characterized by a series of spontaneous peaks. The situation in avian species, however, is unknown. The present study examines this in the domestic fowl. Blood samples were obtained via a remote catheter at 5-min intervals from young male chickens. An episodic pattern of GH secretion was observed having an interpulse interval (frequency) of approximately 1 h. Episodic GH secretion was altered by peripheral administration of drugs which influence adrenergic function. Spontaneous GH peaks were not observed following the administration of (1) bis-(4-methyl-1-homopiperazinyl-thiocarbonyl) disulfide (FLA63) or diethyldithiocarbamate (DDC) (which blocks norepinephrine (NE) and epinephrine (E) synthesis by inhibiting dopamine-beta-hydroxylase), (2) phenoxybenzamine (an alpha 1-adrenergic antagonist) and (3) clonidine (an alpha 2-adrenergic agonist). Neither alpha-methyl-p-tyrosine, which blocks dopamine (DA), NE and E synthesis, nor yohimbine, a predominantly alpha 2-antagonist could completely suppress episodic GH secretion. These data support a role for NE/E, acting via alpha adrenergic receptors, in the control of epidosic GH secretion in the domestic fowl.     

Hormonal regulation of expression of the endogenous and transfected human growth hormone gene

Expression of the endogenous human GH (hGH) gene in response to glucocorticoids, thyroid hormone, and insulin was studied in cultures of dispersed GH-secreting human pituitary adenomas. Results were compared to those obtained when the hGH gene was transfected into rat pituitary tumor cells (GC). In the human pituitary cells the glucocorticoid dexamethasone [(Dex) 10(-6) M] increased the release of GH and the levels of GH mRNA by 2 to 4-fold (P less than 0.05). T3 (10(-8) M) had no effect on GH mRNA but increased hGH release by 2- to 6-fold (P less than 0.01). Insulin (5 x 10(-9) M) alone had no significant effect on either hGH mRNA or protein, but blunted the effect of Dex. Among 11 of 18 GC cell clones transfected with the hGH gene with detectable hGH mRNA expression, Dex increased hGH mRNA levels in seven and T3 treatment reduced hGH mRNA levels in eight. Conversely, rat GH mRNA levels from the endogenous rat gene were increased by either Dex or T3 in all 18 clones. Insulin alone or in combination with T3 or Dex was found to increase hGH mRNA levels in some cell lines and to decrease hGH mRNA levels in others; these effects were correlated strongly (r = 0.88; P less than 0.001) with the influence of insulin on the endogenous rat GH gene, implying that individual cellular differences can simultaneously affect the insulin responsiveness of both genes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chaperone-mediated regulation of hepatic protein secretion by caloric restriction

Calorie restriction (CR) delays age-related physiological changes, reduces cancer incidence, and increases maximum life span in mammals. Here we show that CR decreased the expression of many hepatic molecular chaperones and concomitantly increased the rate and efficiency of serum protein secretion. Hepatocytes from calorie-restricted mice secreted twice as much albumin, 63% more alpha1-antitrypsin, and 250% more of the 31.5-kDa protein 2 h after their synthesis. A number of trivial explanations for these results, such as differential rates of protein synthesis and cell leakage during the assay, were eliminated. These novel results suggest that CR may promote the secretion of serum proteins, thereby promoting serum protein turnover. This may reduce the circulating level of damaging, glycoxidated serum proteins.     

The multidrug resistance-associated protein (MRP/ABCC) subfamily of ATP-binding cassette transporters in plants

In many different plant species, genes belonging to the multidrug resistance-associated protein (MRP, ABCC) subfamily of ABC transporters have been identified. Following the discovery of vacuolar transport systems for xenobiotic or plant-produced conjugated organic anions, plant MRPs were originally proposed to be primarily involved in the vacuolar sequestration of potentially toxic metabolites. Indeed, heterologous expression of different Arabidopsis MRPs in yeast demonstrates their activity as ATP-driven pumps for structurally diverse substrates. Recent analysis of protein-protein interactions and the characterization of knockout mutants in Arabidopsis suggests that apart from transport functions plant MRPs play additional roles including the control of plant transpiration through the stomata. Here, we review and discuss the diverse functions of plant MRP-type ABC transporters and present an organ-related and developmental analysis of the expression of Arabidopsis MRPs using the publicly available full-genome chip data.