Complement receptor 2 mediates enhancement of human immunodeficiency virus 1 infection in Epstein-Barr virus-carrying B cells.

Although the CD4 glycoprotein is the primary receptor for HIV-1, recent reports have suggested that other molecules might be involved in the enhancement of HIV-1 infection. We investigated the possible role of the complement receptor 2 in enhancement of HIV-1 infection in CD4+ EBV-containing B cells by infecting such cells in the presence of sera from HIV sero-positive donors, with or without added human complement. A marked increase in production of viral p24 and infectious progeny virus was observed only when infection had been carried out in the presence of human complement. The addition of mAb to the human complement receptor 2 completely inhibited this enhancement. This mechanism was CD4 dependent, suggesting a cooperative effect between these two ligands in the potentiation of viral entry.

Differential regulation of p27(Kip1) expression by mitogenic and hypertrophic factors: Involvement of transcriptional and posttranscriptional mechanisms.

Platelet-derived growth factor-BB (PDGF-BB) acts as a full mitogen for cultured aortic smooth muscle cells (SMC), promoting DNA synthesis and cell proliferation. In contrast, angiotensin II (Ang II) induces cellular hypertrophy as a result of increased protein synthesis, but is unable to drive cells into S phase. In an effort to understand the molecular basis for this differential growth response, we have examined the downstream effects of PDGF-BB and Ang II on regulators of the cell cycle machinery in rat aortic SMC. Both PDGF-BB and Ang II were found to stimulate the accumulation of G(1) cyclins with similar kinetics. In addition, little difference was observed in the expression level of their catalytic partners, Cdk4 and Cdk2. However, while both factors increased the enzymatic activity of Cdk4, only PDGF-BB stimulated Cdk2 activity in late G(1) phase. The lack of activation of Cdk2 in Ang II-treated cells was causally related to the failure of Ang II to stimulate phosphorylation of the enzyme on threonine and to downregulate p27(Kip1) expression. By contrast, exposure to PDGF-BB resulted in a progressive and dramatic reduction in the level of p27(Kip1) protein. The time course of p27(Kip1) decline was correlated with a reduced rate of synthesis and an increased rate of degradation of the protein. Importantly, the repression of p27(Kip1) synthesis by PDGF-BB was associated with a marked attenuation of Kip1 gene transcription and a corresponding decrease in Kip1 mRNA accumulation. We also show that the failure of Ang II to promote S phase entry is not related to the autocrine production of transforming growth factor-beta1 by aortic SMC. These results identify p27(Kip1) as an important regulator of the phenotypic response of vascular SMC to mitogenic and hypertrophic stimuli.

The ERK1/2 mitogen-activated protein kinase pathway as a master regulator of the G1- to S-phase transition.

The Ras-dependent extracellular signal-regulated kinase (ERK)1/2 mitogen-activated protein (MAP) kinase pathway plays a central role in cell proliferation control. In normal cells, sustained activation of ERK1/ERK2 is necessary for G1- to S-phase progression and is associated with induction of positive regulators of the cell cycle and inactivation of antiproliferative genes. In cells expressing activated Ras or Raf mutants, hyperactivation of the ERK1/2 pathway elicits cell cycle arrest by inducing the accumulation of cyclin-dependent kinase inhibitors. In this review, we discuss the mechanisms by which activated ERK1/ERK2 regulate growth and cell cycle progression of mammalian somatic cells. We also highlight the findings obtained from gene disruption studies.

Phosphorylation of IkappaBalpha in the C-terminal PEST domain by casein kinase II affects intrinsic protein stability.

The NF-kappaB/Rel transcription factors participate in the activation of immune system regulatory genes and viral early genes including the human immunodeficiency virus type 1 long terminal repeat. NF-kappaB/Rel proteins are coupled to inhibitory molecules, collectively termed IkappaB, which are responsible for cytoplasmic retention of NF-kappaB. Cell activation leads to the phosphorylation and degradation of IkappaBalpha, permitting NG-kappaB/Rel translocation to the nucleus and target gene activation. To further characterize the signaling events that contribute to IkappaBalpha phosphorylation, a kinase activity was isolated from Jurkat T cells that specifically interacted with IkappaBalpha in an affinity chromatography step and phosphorylated IkappaBalpha with high specificity in vitro. By using an in-gel kinase assay with recombinant IkappaBalpha as substrate, two forms of the kinase (43 and 38 kDa) were identified. Biochemical criteria and immunological cross-reactivity identified the kinase activity as the alpha catalytic subunit of casein kinase II (CKII). Deletion mutants of IkappaBalpha delta1 to delta4) localized phosphorylation to the C-terminal PEST domain of IkappaBalpha. Point mutation of residues T-291, S-283, and T-299 dramatically reduced phosphorylation of IkappaBalpha by the kinase in vitro. NIH-3T3 cells that stably expressed wild-type IkappaBalpha (wtIkappaB), double-point-mutated IkappaBalpha (T291A, S283A), or triple-point-mutated IkappaBalpha (T291A, S283A, T299A) under the control of the tetracycline-responsive promoter were generated. Constitutive phosphorylation of the triple point mutant was eliminated in vivo, although tumor necrosis factor-inducible IkappaBalpha degradation was unaffected. In cell lines and in transiently transfected cells, mutation of the CKII sites in IkappaBalpha resulted in a protein with increased intrinsic stability. Together with results demonstrating a role for N-terminal sites in inducer-mediated phosphorylation and degradation of IkappaBalpha, these studies indicate that CKII sites in the C-terminal PEST domain are important for constitutive phosphorylation and intrinsic stability of IkappaBalpha.

Functional expression and growth factor activation of an epitope-tagged p44 mitogen-activated protein kinase, p44mapk.

Mitogen-activated protein kinases (MAPKs) or extracellular signal-regulated kinases (ERKs) are serine/threonine kinases of apparent Mr 42-44 kDa that are rapidly activated by a variety of extracellular signals in many cell types. This activation coincides with their phosphorylation on tyrosine and threonine residues, and these covalent modifications are required for full activity of the enzymes. They are thought to play a pivotal role in integrating and transmitting transmembrane signals for growth and differentiation. Here, we report the cloning, sequence, and functional expression in fibroblasts of the hamster p44 MAP kinase (p44mapk). The protein deduced from the nucleotide sequence of an almost full-length cDNA is 98.6% homologous to the rat p44mapk (ERK1). To distinguish the expression of the cloned cDNA from the endogenous p44mapk, we fused to the 5' end of the cDNA an initiating codon followed by an influenza hemagglutinin 9-residue peptide epitope (HAP). The chimeric kinase HAP/p44mapk, under transcriptional control of the cytomegalovirus promoter, was stably expressed in Chinese hamster lung fibroblasts in a functional form. We show that its basal activity, measured by phosphorylation of the substrate myelin basic protein, is activated severalfold (up to 25) by the mitogens alpha-thrombin, platelet-derived growth factor, and fetal calf serum. In addition, we report that in response to alpha-thrombin, this activation is rapid (6-fold in 1 min), biphasic (first peak at 5 min, second broader peak at 1-2 h), persistent (for greater than or equal to 4 h), and parallel to an increased phosphorylation on tyrosine.We conclude that the constructed and stably expressed chimera, HAP/p44mapk, has retained apparently all the hormonal regulation features of the endogenous form. This system now offers the possibility to study structure-function relationships and to determine the role of this kinase in growth control.

Derepressed hyphal growth and reduced virulence in a VH1 family-related protein phosphatase mutant of the human pathogen Candida albicans.

Mitogen-activated protein (MAP) kinases are pivotal components of eukaryotic signaling cascades. Phosphorylation of tyrosine and threonine residues activates MAP kinases, but either dual-specificity or monospecificity phosphatases can inactivate them. The Candida albicans CPP1 gene, a structural member of the VH1 family of dual- specificity phosphatases, was previously cloned by its ability to block the pheromone response MAP kinase cascade in Saccharomyces cerevisiae. Cpp1p inactivated mammalian MAP kinases in vitro and acted as a tyrosine-specific enzyme. In C. albicans a MAP kinase cascade can trigger the transition from the budding yeast form to a more invasive filamentous form. Disruption of the CPP1 gene in C. albicans derepressed the yeast to hyphal transition at ambient temperatures, on solid surfaces. A hyphal growth rate defect under physiological conditions in vitro was also observed and could explain a reduction in virulence associated with reduced fungal burden in the kidneys seen in a systemic mouse model. A hyper-hyphal pathway may thus have some detrimental effects on C. albicans cells. Disruption of the MAP kinase homologue CEK1 suppressed the morphological effects of the CPP1 disruption in C. albicans. The results presented here demonstrate the biological importance of a tyrosine phosphatase in cell-fate decisions and virulence in C. albicans.

ERK1/2-induced phosphorylation of R-Ras GTPases stimulates their oncogenic potential.

The Ras-related (R-Ras) isoforms TC21, R-Ras and M-Ras are members of the Ras superfamily of small GTPases. R-Ras family proteins are frequently overexpressed in human cancers, and expression of activated mutants of these GTPases is sufficient to induce cell transformation. Unlike Ras, few activating mutations of R-Ras proteins have been reported in human cancer, and very little is known about the regulation of their activity. In this study, we report that TC21 and R-Ras are phosphorylated on a conserved serine, Ser186 and Ser201, respectively, in intact cells. This residue is located in the C-terminal hypervariable region of the proteins and is not conserved in M-Ras. We show that the MAP kinases ERK1/2 phosphorylate TC21 and R-Ras on this C-terminal serine residue both in vitro and in vivo. Phosphorylation of R-Ras proteins does not affect their subcellular localization or stability but rather stimulates their activation. Phosphorylation-defective mutants of R-Ras and TC21 are compromised in their ability to promote cancer cell adhesion and migration/invasion, respectively. Importantly, we show that phosphorylation of TC21 and R-Ras potentiates their tumorigenic activity in immunodeficient mice. Our results identify a novel regulatory mechanism of the small GTPases TC21 and R-Ras that controls their oncogenic potential.

Primary structure, expression and chromosomal locus of a human homolog of rat ERK3.

We report the cloning and characterization of a human cDNA encoding a novel homolog of rat extracellular signal-regulated kinase 3 (ERK3). The cDNA encodes a predicted protein of 721 amino acids which shares 92% amino acid identity with rat ERK3 over their shared length. Interestingly, the human protein contains a unique extension of 178 amino acids at its carboxy terminal extremity. The human ERK3 protein also displays various degrees of homology to other members of the MAP kinases family, but does not contain the typical TXY regulatory motif between subdomains VII and VIII. Northern blot analysis revealed that ERK3 mRNA is widely distributed in human tissues, with the highest expression detected in skeletal muscle. The human ERK3 gene was mapped by fluorescence in situ hybridization to chromosome 15q21, a region associated with chromosomal abnormalities in acute nonlymphoblastic leukemias. This information should prove valuable in designing studies to define the cellular function of the ERK3 protein kinase.

Essential role of calcium in the regulation of MAP kinase phosphatase-1 expression.

Mitogen-activated protein (MAP) kinase phosphatase-1 (MKP-1) is a dual-specificity protein phosphatase encoded by an immediate-early gene responsive to growth factors and stress. The MKP-1 protein selectively inactivates MAP kinases in vitro by dephosphorylation of the regulatory Thr and Tyr residues. Little is known on the mechanisms that regulate MKP-1 gene expression. Here, we demonstrate that Ca2+ is both necessary and sufficient for the induction of MKP-1 gene expression. Treatment of Rat1 fibroblasts with the Ca2+ chelating agent BAPTA completely suppressed serum-induced MKP-1 expression in a dose- and time-dependent manner. The inhibitory effect of BAPTA was observed at the level of the protein and the mRNA. Importantly, Ca2+ chelation blocked the induction of MKP-1 expression in response to all stimuli tested and in different cell types. Increasing the intracellular concentration of Ca2+ with the ionophore A23187 was sufficient to induce MKP-1 mRNA and protein expression in rat fibroblasts. We also provide evidence that activation of MAP kinases is not an absolute requirement for induction of the MKP-1 gene. Exposure of rat fibroblasts to A23187 induced MKP-1 expression without activating the JNK and p38 MAP kinase pathways. Also, inhibition of the ERK pathway with the selective MEK inhibitor PD98059 did not interfere with serum-stimulated MKP-1 mRNA expression. These results will help define the regulatory mechanisms that govern MKP-1 gene transcription in target cells.

Heregulin selectively upregulates vascular endothelial growth factor secretion in cancer cells and stimulates angiogenesis.

The interaction between the erbB tyrosine kinase receptors and their ligands plays an important role in tumor growth via the regulation of autocrine and paracrine loops. We report the effect of heregulin beta1, the ligand for erbB-3 and erbB-4 receptors, on the regulation of vascular endothelial growth factor (VEGF) expression, using a panel of breast and lung cancer cell lines with constitutive erbB-2 overexpression or engineered to stably overexpress the erbB-2 receptor. We demonstrate that heregulin beta1 induces VEGF secretion in most cancer cell lines, while no significant effect was observed in normal human mammary and bronchial primary cells. Overexpression of erbB-2 receptor results in induction of the basal level of VEGF and exposure to heregulin further enhances VEGF secretion. This is associated with increased VEGF mRNA expression. In contrast, VEGF induction is significantly decreased in a T47D cell line where erbB-2 is functionally inactivated. Conditioned media from heregulin-treated cancer cells, but not from normal cells, stimulates endothelial cell proliferation; this paracrine stimulation is inhibited by co-exposure to a specific VEGF neutralizing antibody. Furthermore, heregulin-mediated angiogenesis is observed in the in vivo CAM assay. This study reports the first evidence of VEGF regulation by heregulin in cancer cells. Oncogene (2000) 19, 3460 - 3469

Cardioprotective effects of ramipril and losartan in right ventricular pressure overload in the rabbit: importance of kinins and influence on angiotensin II type 1 receptor signaling pathway.

BACKGROUND: The role of kinins in the cardioprotective effects of ACE inhibitors remains controversial. METHODS AND RESULTS: Right ventricular pressure overload in rabbits was produced by pulmonary artery banding for 21 days. Rabbits were untreated, or they received the ACE inhibitor ramipril with or without bradykinin B(1) and B(2) receptor blockers or the angiotensin (Ang) II type I (AT(1)) receptor blocker losartan. Pulmonary artery banding caused right ventricular hypertrophy, depressed papillary muscle contractility, and loss of Ang II contractile effects because of a signaling defect downstream of AT(1) receptors. Paradoxically, AT(1) receptor density and G protein alpha subunits alphaq and alphai1/2 increased. Inotropic responsiveness to the alpha-receptor agonist phenylephrine was normal. Ramipril preserved cardiac contractility, but this effect was attenuated by simultaneous use of kinin receptor blockers. Ramipril also maintained responsiveness to Ang II and prevented AT(1) receptor and G protein upregulation. The simultaneous use of a kinin receptor blocker attenuated but did not prevent upregulation in the AT(1) receptor and G protein. Losartan had no effect on baseline contractility, but it maintained cardiac inotropic responsiveness to Ang II, prevented upregulation of AT(1) receptors, but did not modify G protein upregulation. CONCLUSIONS: Pressure overload of the right ventricle decreases contractility, uncouples AT(1) receptors to downstream signaling pathways, and changes the expression of components of the AT(1) receptor signaling pathway. Ramipril attenuates these effects via kinins. Interventions that prevent local increases in Ang II or block AT(1) receptors also prevent decreased responsiveness of the AT(1) receptor in this model.

Biphasic and synergistic activation of p44mapk (ERK1) by growth factors: correlation between late phase activation and mitogenicity.

We have examined the phosphorylation and protein kinase activity of p44 mitogen-activated protein kinase (p44mapk) in growth factor-stimulated hamster fibroblasts using a specific antiserum. The activity of p44mapk was stimulated both by receptor tyrosine kinases and G protein-coupled receptors. Detailed kinetics revealed that alpha-thrombin induces a biphasic activation of p44mapk in CCL39 cells: a rapid phase appearing at 5-10 min was followed by a late and sustained phase still elevated after 4 h. Inactivation of alpha-thrombin with hirudin after 30 sec, which prevented DNA synthesis, did not alter the early p44mapk response but completely abolished the late phase. Pretreatment of the cells with pertussis toxin, which inhibits by more than 95% alpha-thrombin-induced mitogenicity, resulted in the complete loss of late phase activity, while the early peak was partially attenuated. Treatment of CCL39 cells with basic fibroblast growth factor also induced a strong activation of p44mapk. Serotonin, which is not a mitogen by its own, had no effect on late phase p44mapk activity, but synergized with basic fibroblast growth factor to induce late kinase response and DNA synthesis. Both early and late phase activation of p44mapk were accompanied by tyrosine phosphorylation of the enzyme. Together, the results indicate that there is a very close correlation between the ability of a growth factor to induce late and sustained p44mapk activation and its mitogenic potential. Therefore, we propose that sustained p44mapk activation is an obligatory event for growth factor-induced cell cycle progression.

Digoxigenin-labeled peptides for the immunological quantification of intracellular signaling proteins: application to the MAP kinase kinase isoform MEK2.

Two competitive enzyme immunoassays using digoxigenin-labeled peptides have been developed for the quantification of the protein kinase MEK2 in cell extracts. Rabbit polyclonal antibodies directed against either the amino-terminal or proline-rich amino acid sequences of MEK2 were used for the immunoconcentration of the protein. Anti-digoxigenin Fab fragments labeled with horseradish peroxidase allowed the detection of the immune complexes. Amino-terminal and proline-rich enzyme immunoassays exhibited a sensitivity level of 63 and 71 fmol/mL, respectively, and displayed a half-maximal saturation value of 1320 and 1780 fmol/mL. The intra- and inter-assay coefficients of variation for both assays assessed at three different concentrations of MEK2 were lower than 6% and 12%, respectively. The amount of MEK2 measured by the two methods demonstrated an excellent correlation with the expression level of the protein detected by immunoblot analyses when tested on different cell lysates.

Pharmacokinetic analysis of the plasma disappearance of ovine follitropin and analogues in the male rat.

Ovine follitropin (oFSH) and its chemically deglycosylated (DG) or the asialo analogue were injected as a single bolus (2.5 micrograms) to intact mature male rats and plasma levels measured by radioimmunoassay. Semi-logarithmic plots of the plasma disappearance curves were all biphasic. For all substances tested, the data were best fitted to a biexponential equation and the following parameters were calculated: the half-lives of the distribution and elimination phases and the metabolic clearance rate (MCR) at steady state. It was found that the distribution phase of oFSH was much slower than for deglycosylated (DG) alpha + beta recombinant, DG-oFSH and asialo-oFSH. The elimination half-lives for asialo-oFSH and DG alpha + beta recombinant were much shorter than for oFSH and DG-oFSH. oFSH had the lowest MCR, followed by DG alpha + beta, DG-oFSH and asialo-oFSH. This study clearly shows that sugar residues internal to sialic acid also play a significant part in the clearance of the hormone.

Cell cycle reentry of mammalian fibroblasts is accompanied by the sustained activation of p44mapk and p42mapk isoforms in the G1 phase and their inactivation at the G1/S transition.

Mitogen-activated protein (MAP) kinases are serine/threonine kinases that are rapidly activated in response to mitogenic stimuli. Here we examined the enzymatic activity and phosphorylation state of the individual p44mapk and p42mapk isoforms during early G1 and late G1 phase of the mammalian cell cycle. Release of fibroblast cells from early G1 block was accompanied by a rapid rise in the myelin basic protein (MBP) kinase activity of p44mapk and p42mapk, which declined slowly over several hours to reach negligible values as cells enter S phase. When cells were released from late G1 block, the activity of p44mapk and p42mapk increased transiently, and then rapidly declined to baseline values during G1 to S phase transition. Cells released at the G1/S boundary in a medium lacking growth factors entered S phase in the complete absence of MAP kinase activity. Unlike MAP kinases, the histone H1 kinase activity of p33cdk2 was elevated in late G1 arrested cells and continued to increase during S phase entry. The enzymatic activation of p44mapk and p42mapk in both early G1 and late G1 phase was accompanied by an increase in the phosphothreonine and phosphotyrosine content of the proteins. These findings suggest that the sustained activation of MAP kinases during G1 progression and their inactivation at the G1/S transition are two regulatory processes involved in the mitogenic response to growth factors.

Angiotensin II stimulates tyrosine phosphorylation of the focal adhesion-associated protein paxillin in aortic smooth muscle cells.

Treatment of vascular smooth muscle cells (SMC) with angiotensin II (AII) leads to an increase in the tyrosine phosphorylation of multiple cellular substrates. Here, we have demonstrated that AII stimulates tyrosine phosphorylation of the focal adhesion-associated protein paxillin in rat aortic SMC. AII-induced phosphorylation of paxillin was detectable within 1 min and was sustained up to 60 min. Preincubation with the AT1-selective antagonist losartan abolished this response. The stimulatory effect of AII on paxillin tyrosine phosphorylation was observed only in aortic SMC and not in other target cells such as adrenal zona glomerulosa cells, chromaffin cells, or hepatocytes. The effect of AII was dependent on the activation of phospholipase C. Chelation of intracellular calcium completely inhibited the ability of AII to stimulate paxillin tyrosine phosphorylation, while selective inhibition of protein kinase C partially attenuated the response. In contrast, treatment of the cells with pertussis toxin had no effect on AII-induced paxillin tyrosine phosphorylation. These findings identify paxillin as a new substrate for AII-stimulated tyrosine phosphorylation and suggest a role for cytoskeleton-associated proteins in the growth response of aortic SMC.

Repression of mitogen-activated protein kinases ERK1/ERK2 activity by a protein tyrosine phosphatase in rat fibroblasts transformed by upstream oncoproteins.

The observation that mitogen-activated protein (MAP) kinases ERK1 and ERK2 are constitutively activated in a number of oncogene-transformed cell lines has led to the hypothesis that prolonged activation of these enzymes is required for the transformation process. To investigate this question, we have examined the regulation of the ERK pathway in Rat1 fibroblasts transformed with activated c-Raf-1 (Raf22W), v-Ha-Ras, and v-Src. Expression of these oncoproteins had no effect on the enzymatic activity of ERK1 and ERK2 in either serum-starved or exponentially growing cells. Moreover, the stimulatory effect of serum on ERK1/ERK2 activity was substantially reduced or abrogated in these cells; this impairment was associated with a strong attenuation of c-fos gene induction. In contrast, expression of Raf22w, v-Ha-Ras, or v-Src resulted in the constitutive activation of the upstream kinases MEK1 and MEK2. Treatment of the cells with vanadate completely restored the activation of ERK1/ERK2 in oncogene-transformed cells, suggesting the involvement of a vanadate-sensitive tyrosine phosphatase. Northern blot analysis of VH1-like dual-specificity MAP kinase phosphatases did not reveal any significant difference in the mRNA expression pattern of these genes between parental and transformed Rat1 cells. Phosphoamino acid analysis indicated that ERK1 is phosphorylated on threonine, but not on tyrosine, in oncogene-transformed cells and that vanadate treatment restores tyrosine phosphorylation. We conclude from these results that ERK1/ERK2 activity is repressed by a single-specificity tyrosine phosphatase in oncogene-transformed rat fibroblasts.

Role of p70 S6 protein kinase in angiotensin II-induced protein synthesis in vascular smooth muscle cells.

Angiotensin II (AII) is a growth factor which induces cellular hypertrophy in cultured vascular smooth muscle cells (SMC). To understand the molecular basis of this action, we have examined the role of the 70-kDa S6 kinases (p70S6K) in the hypertrophic response to AII in aortic SMC. AII potently stimulated the phosphotransferase activity of p70S6K, which reached a maximal value at 15 min and persisted for at least 4 h. This response was completely abolished when the cells were incubated in the presence of the AT1-selective receptor antagonist losartan. The enzymatic activation of p70S6K was associated with increased phosphorylation of the enzyme on serine and threonine residues. The immunosuppressant drug rapamycin was found to selectively inhibit the activation of p70S6K by AII, but not the activation of mitogen-activated protein kinase or the induction of c-fos mRNA expression. Treatment of aortic SMC with rapamycin also potently inhibited AII-stimulated protein synthesis with a half-maximal concentration similar to that required for inhibition of p70S6K. These results provide strong evidence that p70S6K plays a critical role in the signaling pathways by which AII induces hypertrophy of vascular SMC.

Metabolism of isotretinoin. Biliary excretion of isotretinoin glucuronide in the rat.

The biliary metabolites of isotretinoin were examined after iv administration of 4-20-mg/kg doses to vitamin A-normal bile duct-cannulated rats. Analysis of bile by reverse phase high performance liquid chromatography showed that injection of isotretinoin is followed by a rapid excretion of metabolites in bile. Isotretinoin glucuronide was identified as the major metabolite in bile. A specific high performance liquid chromatography method based on the assay of generated isotretinoin in beta-glucuronidase-treated bile was developed for the determination of isotretinoin glucuronide in bile samples. The excretion rate of isotretinoin glucuronide increased rapidly to reach a maximum 55 min after dosing and then declined exponentially. After 330 min of collection, biliary excretion of isotretinoin glucuronide was almost complete, and the metabolite accounted for 34.8-37.9% of the dose. These results indicate that conjugation with glucuronic acid represents a major pathway for the metabolism of pharmacological doses of isotretinoin. The maximum excretion rate of isotretinoin glucuronide in bile increased in a linear manner with the dose of isotretinoin, and no delay was observed after the larger doses. These data suggest that glucuronidation and biliary excretion are not saturated at high pharmacological doses of isotretinoin.