Development of a limited sampling approach in pharmacokinetic studies: experience with the antiepilepsy drug tiagabine

To examine the mechanisms involved in the progression of mercury chloride (HgCl2)-induced acute tubular necrosis (ATN), we investigated the histopathological changes and the expression of inducible nitric oxide synthase (iNOS) mRNA and protein in renal cortices of rats at 20 hours after exposure to HgCl2. The expression of iNOS mRNA was significantly augmented in renal cortices of rats with HgCl2-induced acute renal failure (ARF). Likewise, the induction of iNOS protein was observed in damaged proximal tubule epithelial cells of rats with HgCl2-induced ARF. Pretreatment of rats with iNOS inhibitor aminoguanidine, however, suppressed the development of proximal tubule epithelial cell injury and prevented an increase in blood urea nitrogen and serum creatinine as well as resulting in a marked fall in iNOS mRNA and protein in rats with HgCl2-induced ARF. These observations indicate that the induction of iNOS may play a role in the progression of HgCl2-induced ATN through the exacerbation of proximal tubule epithelial cell damage.     

Hepatocyte growth factor-stimulated renal tubular mitogenesis: effects on expression of c-myc, c-fos, c-met, VEGF and the VHL tumour-suppressor and related genes

Hepatocyte growth factor (HGF/SF) is a potent renal proximal tubular cell (PTEC) mitogen involved in renal development. HGF/SF is the functional ligand for the c-met proto-oncogene, and germline c-met mutations are associated with familial papillary renal cell carcinoma. Somatic von Hippel-Lindau disease tumour-suppressor gene (VHL) mutations are frequently detected in sporadic clear cell renal cell carcinomas (RCC), and germline VHL mutations are the commonest cause of familial clear cell RCC. pVHL binds to the positive regulatory components of the trimeric elongin (SIII) complex (elongins B and C) and has been observed to deregulate expression of the vascular endothelial growth factor (VEGF) gene. HGF/SF has similarly been reported to up-regulate expression of the VEGF gene in non-renal experimental systems. To investigate the mechanism of HGF/SF action in PTECs and, specifically, to examine potential interactions between the HGF/c-met and the VHL-mediated pathways for renal tubular growth control, we have isolated untransformed PTECs from normal kidneys, developed conditions for their culture in vitro and used these cells to investigate changes in mRNA levels of the VHL, elongin A, B and C, VEGF, c-myc, c-fos and c-met genes after HGF/SF exposure. Significant elevations in the mRNA levels of VEGF, c-myc, c-fos, c-met and elongins A, B and C, but not VHL, were detected after HGF/SF stimulation of human PTECs (P < 0.02), with a consistent order of peak levels observed over successive replicates (c-fos at 1 h, VEGF at 2-4 h, c-myc, at 4 h, followed by c-met and all three elongin subunits at 8 h). This study highlights the spectrum of changes in gene expression observed in PTECs after HGF/SF stimulation and has identified possible candidate mediators of the HGF/SF-induced mitogenic response. Our evidence would suggest that the changes in PTEC VEGF expression induced by HGF/SF are mediated by a VHL-independent pathway.     

Temporal analysis of changes in neuronal c-fos mRNA levels induced by depletion of endoplasmic reticulum calcium stores: effect of clamping cytoplasmic calcium activity at resting levels

Activation of immediate early gene expression is a key event in stress-induced neuronal cell injury. To study whether changes in cytoplasmic calcium activity are necessary to activate neuronal immediate early gene expression, endoplasmic reticulum (ER) calcium stores of primary neurons were depleted by exposing cells to thapsigargin (Tg), an irreversible inhibitor of ER Ca2+-ATPase. Tg-induced rise in [Ca2+]i and the effect of loading neurons with the cell-permeable calcium chelator BAPTA-AM on this increase in [Ca2+]i were measured in fura-2-loaded cells by fluorescence microscopy. Changes in c-fos mRNA levels were evaluated by quantitative PCR. Tg treatment of neurons produced a pronounced rise in c-fos mRNA levels (approximately 10-fold more than DMSO) which peaked at 1 h after exposure. The Tg-induced rise in c-fos mRNA content was unchanged (hippocampal neurons) or even increased further (cortical neurons) by preloading cells with BAPTA before incubation with Tg. It is concluded that in neuronal cells an increase in cytoplasmic calcium activity is not a prerequisite for a rise in mRNA levels of c-fos. Thus, stress-induced changes in mRNA levels of immediate early genes of neurons may also result from disturbances in ER calcium homeostasis and not necessarily by an overload of cells with calcium ions. The results of the present series of experiments cast further doubt on the widely accepted hypothesis that the stress-induced cytoplasmic overload of neurons with calcium ions is the primary event triggering cell injury.     

4-hydroxynonenal specifically inhibits c-myb but does not affect c-fos expressions in HL-60 cells

4-Hydroxynonenal, an aldehyde produced from lipid peroxidation of cellular membranes, inhibits growth and induces differentiation of HL-60 human leukemic cell line. Since it is highly unstable in the culture medium, its effectiveness is increased when added repeatedly to the cell suspension. We have previously demonstrated that HNE inhibits c-myc but not N-ras expression in HL-60 cells. Here we investigate its effect on the expression of c-myb and c-fos, two early genes involved in the induction of myeloid and monocytic differentiation. Moreover, since c-fos is directly correlated with the intracellular level of cAMP, we also analysed the cAMP concentration after aldehyde treatment. HNE significantly inhibits c-myb expression during and after repeated treatments. A single administration of 1 microM HNE decreases c-myb mRNA at 1 hour whereas 10 microM HNE inhibits c-myb expression from 3 to 6 hours after treatment, and then the expression returns to the control level. By contrast, c-fos expression and intracellular cAMP concentration do not show any significant change after HNE treatments.     

Inhibition of luteinizing hormone secretion and expression of c-fos and corticotrophin-releasing factor genes in the paraventricular nucleus during insulin-induced hypoglycaemia in sheep

Insulin can act within the brain to stimulate ovine luteinizing hormone (LH) secretion, but insulin-induced hypoglycaemia inhibits LH via unknown brain sites, possibly involving corticotrophin-releasing factor (CRF). Castrate male sheep, with (E+) or without (E-) subcutaneous oestradiol implants, were blood sampled every 12 min for 8 h. Insulin (0.25 or 0.5 IU/kg) was injected at 4 h via the carotid artery or jugular vein. All treatments reduced LH output with no differences between dose rate nor route of administration, but sensitivity was greater in E+ than E-sheep. There was no evidence for an effect of insulin on LH 0-1 h postinjection; however, 1-3 h after insulin, when hypoglycaemia was established, LH pulses were inhibited in both E+ and E- sheep (P<0.001). Additional intravenous (i.v.) glucose injections given 1 h (20 mmol) and 2 h (10 mmol) after insulin (0.5 IU/kg) were each followed by an LH pulse within 30 min (75% response in both E+ and E-sheep). In a separate experiment, sheep were killed 2 h after i.v. insulin (0.5 IU/kg) or saline. In-situ hybridization revealed c-fos mRNA in the paraventricular nucleus (PVN), but not in any other hypothalamic nuclei nor in the hindbrain; and this was linked with increased CRF gene expression in the PVN. Similar c-fos and CRF gene expression was seen in insulin-treated sheep given additional i.v. glucose (20 and 10 mmol, respectively, 40 and 20 min ante mortem), but not in saline-treated controls. Therefore, insulin-induced hypoglycaemia inhibited LH secretion, with oestradiol potentiating the effect, and was associated with gonadal steroid-independent c-fos gene expression and increased CRF gene expression in the PVN. The ovine PVN may be involved in mediating insulin-induced hypoglycaemic inhibition of LH by a mechanism which might involve CRF.     

Effect of non-toxic mercury, zinc or cadmium pretreatment on the capacity of human monocytes to undergo lipopolysaccharide-induced activation

1. Metal salts can inhibit cell activity through direct toxicity to critical cellular molecules and structures. On the other hand, they can also change cell behaviour by inducing specific genes (including genes encoding members of the metallothionein [MT] gene family). Therefore, transition metals may affect cell functions either by acting as a toxin, or by transmitting or influencing signals controlling gene expression. 2. To explore the latter possibility, we measured the ability of low, non-toxic metal pretreatment to alter immune cell behaviour. We previously found that pretreatment of human monocytes with zinc induces metallothionein gene expression and alters their capacity to undergo a bacterial lipopolysaccharide-induced respiratory burst. We showed here that cadmium and mercury salts, at concentrations that exert no discernible toxicity, inhibit activation of human monocytic leukemia (THP-1) cells. CdCl2 1 microM, ZnCl2 20-40 microM or HgCl2 2 microM pretreatment for 20 h induced MT-2 mRNA and total MT protein accumulation and had no effect on proliferation potential or metabolic activity, but significantly inhibited the ability of subsequent lipopolysaccharide treatment to induce the oxidative burst, increased adhesion to plastic, and MT-2 and interleukin-1 beta (IL-1 beta) mRNA accumulation. 3. The phenomenon of metal-induced suppression of monocyte activation, at metal concentrations that have no effect on cell viability, has important implications for assessment of acceptable levels of human exposure to cadmium, zinc and mercury.     

Coronary pressure as a determinant of B-type natriuretic peptide gene expression in isolated perfused adult rat heart

The role of coronary flow in the regulation of ventricular B-type natriuretic peptide (BNP) gene expression was studied in isolated perfused rat heart preparation. The increase of coronary flow from 5 ml/min to 20 ml/min for 2 h resulted in a 132+/-6 mm Hg increase in aortic perfusion pressure. The changes in BNP mRNA and immunoreactive BNP (IR-BNP) levels in response to hemodynamic stress were compared to those of c-fos and adrenomedullin (ADM) gene expression. The increase of coronary flow resulted in 1.5-fold increases in the left ventricular BNP mRNA (P < 0.001) and IR-BNP (P < 0.05) levels in 2-month old rats. There was also a 1.5-fold (P < 0.05) increase in ventricular c-fos mRNA levels, whereas ADM mRNA levels decreased by 74% (P < 0.001) in the left ventricle. In 18-month old rats, the increase in coronary flow decreased left and right ventricular BNP mRNA levels by 18% (P < 0.05) and 39% (P < 0.001), respectively. There were no changes in IR-BNP peptide and c-fos mRNA levels, whereas ADM mRNA levels decreased by 46% (P < 0.001) in the left ventricles. The results show that increased aortic perfusion pressure results in differential expression of cardiac genes including up-regulation of ventricular BNP and c-fos gene expression and down-regulation of ADM gene expression. Furthermore, aging seems to elevate the threshold at which hemodynamic stress of the heart results in a response at BNP gene level.     

Induction of basic fibroblast growth factor mRNA by basic fibroblast growth factor in Müller cells

PURPOSE: To investigate the induction of basic fibroblast growth factor (bFGF) gene expression in cultured rat Müller cells by bFGF and to study the mechanism of induction. METHODS: Müller cells from 1- to 3-day-old Sprague-Dawley rats were isolated and cultured with Dulbecco's modified Eagle's medium with 10% fetal calf serum. Cultured cells were identified by immunocytochemistry using antibodies against vimentin, carbonic anhydrase II, and glutamine synthetase. Cells of passages 1 through 4 were treated with bFGF, the protein kinase C (PKC) inhibitor, H-7; calphostin C, or the PKC activator, PMA; and protein kinase A (PKA) inhibitor, H-89; as well as the adenylate cylase activator, forskolin; or the adenylate cyclase inhibitor, SQ22536. Northern blot analysis was performed to determine the mRNA expression of bFGF, ciliary neurotrophic factor (CNTF) and brain-derived neurotrophic factor (BDNF). RESULTS: Addition of bFGF to culture medium induced bFGF gene expression in a dose- and time-dependent manner. Induction of bFCF mRNA started at a bFGF concentration of 0.1 ng/ml. The bFGF mRNA level was elevated by 2-fold at 1 ng/ml of bFGF, 2.8-fold at 5 ng/ml, and reached a peak of 4-fold at 10 ng/ml and 3.7-fold at 50 ng/ml. At 10 ng/ml of bFGF, induction of bFGF mRNA was observed as early as 2 hours (2-fold) after treatment. The bFGF mRNA level continued to increase to 3.7-fold by 4 hours, and reached a maximum of 4.4-fold by 8 hours. A slow decline of the bFGF mRNA level was observed after 8 hours of bFGF treatment (3.5-fold by 12 hours, and 3-fold by 24 hours). This induction of bFGF gene expression was blocked by PKC inhibitors H-7 (30 microM). The PKC activator PMA (0.1 microM) also upregulated bFGF gene expression, but the effects of bFGF and PMA were not additive. An adenylate cyclase inhibitor, SQ22536 (100 microM), did not inhibit bFGF-induced bFGF gene expression. Although forskolin (5 microM), an adenylate cyclase activator, also upregulated the level of bFGF mRNA, the effects of forskolin and bFGF were additive. In addition, no inhibitory effect on bFGF-induced expression of bFGF mRNA was found using H-89 (1 microM). Exogenous bFGF did not alter the mRNA levels of CNTF and BDNF. CONCLUSIONS: These results indicate that bFGF induces bFGF gene expression in cultured rat Müller cells through PKC activation. The authors' findings raise the possibility that Müller cells in vivo also respond to available bFGF (for example, that released from the endogenous reservoirs in the case of injury) or to exogenous bFGF by producing more bFGF, which could in turn promote photoreceptor survival.     

Peripheral nerve stimulation increases Fos immunoreactivity without affecting type II Ca2+/calmodulin-dependent protein kinase, glutamic acid decarboxylase, or GABAA receptor gene expression in cat spinal cord

Expression patterns of the immediate early gene c-fos and of other genes including those for the alpha-subunit of type II Ca2+/calmodulin-dependent protein kinase (CaMKII alpha), 67-kDa glutamic acid decarboxylase (GAD), and the alpha 1-, beta 2-, and gamma 2-subunits of the GABAA receptor were described in the spinal cord of normal cats and following peripheral nerve stimulation. As revealed by in situ hybridization histochemistry, CaMKII alpha messenger RNA (mRNA) is normally distributed only in cells of Rexed's laminae I-IV, whereas GAD mRNA is expressed by subpopulations of cells in all laminae, with the heaviest hybridization signal found in laminae I-III and medial parts of laminae V and VI. The three GABAA receptor subunits have varying expression patterns in the laminae. All of them are expressed by many cells located in the base of the dorsal horn and the intermediate zone, but only the gamma 2-subunit is intensely expressed by motoneurons. Single-pulse, electrical stimulation of the sciatic or median and ulnar nerve of anesthetized cats at a pulse rate of 1/s for 6-8 h failed to induce observable changes in gene expression for CaMKII alpha, GAD, or for the three subunits of the GABAA receptor; although immunoreactivity for the protein products of c-fos (or c-fos-related genes) was markedly upregulated in some neurons of the dorsal horn and the intermediate zone. Therefore, under the present experimental conditions, upregulation of the immediate early gene c-fos (or c-fos-related genes) is not associated with changes in expression of late-effector genes potentially involved in central nervous system plasticity.     

Enhancement of radiation-induced hepatic microsomal epoxide hydrolase gene expression by oltipraz in rats

The effects of radiation exposure in conjunction with oltipraz, a chemopreventive agent, on the expression of the gene encoding hepatic microsomal epoxide hydrolase (mEH) were examined in rats. Rats exposed to a single dose of 3 Gy gamma rays exhibited timerelated changes in the hepatic mEH mRNA level. Whereas the mEH mRNA level was transiently decreased at 3 and 8 h after irradiation, the mRNA levels were increased 3- to 4-fold at 15 to 48 h postirradiation, returning to the level in untreated animals at 72 h. Treatment of rats with oltipraz resulted in 1- to 19-fold increases in hepatic mEH mRNA levels 24 h post-treatment at doses of 5-200 mg/kg. Although treatment with oltipraz at a dose of 30 mg/kg affected the mEH mRNA level minimally (i.e. approximately 2-fold), 3 Gy whole-body irradiation along with oltipraz treatment resulted in a 9-fold increase in the mEH mRNA level at 24 h post-treatment. Treatment of animals with both oltipraz and 3 Gy gamma radiation for 3 consecutive days resulted in a 7-fold increase in mEH mRNA, showing that the increases in mEH mRNA were enhanced by the combination treatment. In rats irradiated with 3 Gy for 5 consecutive days, however, the mEH mRNA level failed to increase due to cell injury. Studies were further designed to assess the effects of 0.5 Gy ionizing radiation and concomitant oltipraz treatment. RNA blot analysis showed that mEH mRNA levels failed to be significantly altered at 3, 8, 15, 24 and 48 h after a single dose of 0.5 Gy. Nonetheless, exposure of animals to 0.5 Gy daily for 3 to 5 consecutive days caused a 3-fold elevation in the hepatic mEH mRNA level. Furthermore, treatment of animals with both oltipraz (30 mg/kg/day) and 0.5 Gy of gamma rays resulted in an enhanced elevation in the mEH mRNA level at 24 h post-treatment compared to the individual treatment, resulting in a 7-fold relative increase. The enhanced expression of hepatic mEH mRNA by 0.5 Gy gamma radiation and oltipraz was also observed after treatment for 3 to 5 days (8- to 6-fold relative increases). Western immunoblot analyses showed that hepatic microsomes produced from the rats treated with 0.5 Gy daily for 3 to 5 days resulted in a approximately 2-fold induction of hepatic mEH and that rats exposed to radiation in combination with oltipraz showed 3-fold increases in the liver mEH protein. Thus the relative increase in mEH mRNA levels was consistent with the expression of the protein. These results demonstrate that ionizing radiation causes alterations in hepatic mEH gene expression with the induction of the protein and that the mEH gene expression is enhanced by oltipraz treatment.