In this article, we review the use of stably transfected cells to study the regulation of receptor structure and function by chronic drug treatments and compare results from these cells to results obtained from other systems, including neuronal cultures and intact animals. We focus on the γ-aminobutyric acid type A (GABAA) receptor complex. Sedative/hypnotic drugs such as benzodiazepines, barbiturates and alcohol that potentiate GABAA receptor function produce tolerance and dependence. Chronic treatment of GABAA receptor preparations from brain and neuronal cultures with GABAA agonists, as well as these other three classes of drugs, results in regulation of several properties of the receptor. Drug treatments may regulate levels of binding sites, allosteric binding interactions, receptor function, levels of receptor subunit mRNA and levels of receptor subunit protein. Some or all of these effects may comprise the molecular mechanisms of tolerance to these GABAA-modulatory drugs. The use of cells stably transfected with neurotransmitter receptors provides a homogeneous population that can be cultured under controlled conditions. As most preparations contain mixed populations of GABAA receptor subunits, stably transfected cells offer the advantage of the expression of receptors with a defined subunit composition. We conclude that chronic drug treatments regulate allosteric coupling and function of GABAA receptors in stably transfected cells. This regulation does not appear to be due to decreases in the expression of α1- or β1-receptor subunits or to expression of subunits other than α1, β1, γ2L. Therefore, it is unlikely to be due to changes in receptor subunit composition and probably represents post-translational changes. The rapid regulation of allosteric coupling and function by drug treatment of the stably transfected cells should provide insights to the mechanisms of coupling between GABAA and benzodiazepine receptors as well as tolerance and dependence of benzodiazepines and ethanol.
The severity of naloxone-precipitated withdrawal in rats infused intravenously with morphine at the rates of 2.5, 5 and 10 mg/kg/hr over various time periods was investigated. Plasma morphine concentration reached a constant and rate-dependent level at 1 hr after the start of morphine infusion, and this level was maintained until the termination of infusion. Naloxone (2.0 mg/kg, s.c.) was challenged 18 hr after infusion was stopped, and the withdrawal was evaluated by plasma corticosterone (PCS) increase, diarrhea and body weight loss. The incidence of naloxone-precipitated withdrawal signs was related to both the infusion rate and duration of morphine infusion. The duration of morphine infusion (ET50) needed to elicit naloxone-precipitated PCS increase and diarrhea in 50% of the rats was inversely related to the morphine infusion rates, but the total amount of infused morphine (EA50) that elicited naloxone-precipitated withdrawals in 50% of rats was the same at all infusion rates. These results suggest that the total amount of morphine infused may play an important role in the development of acute physical dependence on morphine rendered by continuous intravenous morphine infusion for 1-8 hr.
The possibility that hyperlipidemia and an increase of mononuclear cells in the glomeruli could participate in the pathogenesis of minimal change glomerulopathy was evaluated in puromycin aminonucleoside (PAN) nephrosis in rats. Significant increases in intraglomerular CD4-, IL-2-receptor(R)- and ED-1-positive cells were found in PAN rats. Urinary protein excretion and mononuclear cells in the glomeruli of 1% cholesterol diet-fed rats significantly increased, compared with standard diet feeding. Moreover, administration of a subnephrogenic dose of PAN in cholesterol diet-fed rats substantially increased urinary protein excretion and mononuclear cells in the glomeruli. Additionally, antihyperlipidemia agents and immunosuppressive agents prevented urinary protein excretion and increases of CD4-, IL-2R- and ED-1-positive cells in the glomeruli of PAN nephrotic rats. Monoclonal antibodies directed against these cells also prevented urinary protein excretion. These results suggest that CD4-, IL-2R- and ED-1-positive cells and hyperlipidemia are involved in the progression, but not the pathogenesis, of PAN.
FR129169 (FR) (N-(1, 2-diphenylethyl)-2-octyloxyphenylacetamide) has been found to inhibit acyl-CoA: cholesterol acyltransferase (ACAT) activities in intestinal microsomes of rats and rabbits and the liver homogenate of rats with IC50 values of around 1.0 x 10-7 M. The inhibitory activity was 2-3 times more potent than that of CI 976 (CI). When FR in a dose of 10 mg/kg/day was administered as a dietary admixture, plasma cholesterol levels were normalized in rats fed a high cholesterol diet, but lower doses of FR had no effect. Similar results were obtained in the rats treated with CI. The ex vivo study where hepatic ACAT activity was measured after oral dosing of the two inhibitors revealed that ACAT activity was significantly reduced in rats treated with FR in a dose of 10 mg/kg/day, while CI reduced the activity at lower doses such as 0.1 and 1 mg/kg/day. Since FR was not orally absorbed, it is speculated that the inhibitory activity of FR on hepatic ACAT in the ex vivo study results from the reduction of plasma cholesterol levels. These results suggest that FR exerted cholesterol-lowering activity mainly through inhibition of intestinal ACAT activity. The significance of intestinal ACAT inhibition by FR for therapeutic treatment of hypercholesterolemia is discussed.
The effect of FK506 (tacrolims hydrate), an immunosuppressive agent produced by Streptomyces tsukubaensis, on crescentic-type anti-glomerular basement membrane (GBM) nephritis in rats was investigated. When rats were treated with FK506 from 1 or 20 days after the anti-GBM serum injection, FK506 inhibited the increase in urinary protein excretion. Histological observation demonstrated that FK506 suppressed glomerular alterations. In the FK506-treated rats, antibody production and rat-IgG and C3 deposits on the GBM were significantly less than those in the nephritic control group. FK506 treatment suppressed the accumulation of ED-1-positive cells, CD4-positive cells, CD8-positive cells, interleukin-2 (IL-2)-receptor-positive cells, leukocyte-function-associated antigen-1 (LFA-1)-positive cells and intercellular adhesion molecule-1 (ICAM-1)-expression in nephritic glomeruli. However, in the in vitro study, FK506 failed to inhibit the up-regulated ICAM-1 expression on endothelial cells in response to tumor necrosis factor (TNF)-α. On the other hand, IL-2 production from the spleen cells isolated from nephritic rats treated with FK506 was lower than that in the nephritic control rats. These results suggest that FK506 is effective against crescentic-type anti-GBM nephritis and that the antinephritic mechanisms of FK506 is due to the inhibition of intraglomerular accumulation and activation of leukocytes through the suppression of ICAM-1 expression and IL-2 production.
Effects of butein on crescentic-type anti-glomerular basement membrane (GBM) nephritis in rats were investigated. When rats were treated with butein from 1 day after i.v. injection of anti-GBM serum, it inhibited the elevation of protein excretion into urine. In the butein-treated rats, cholesterol content in plasma was lower than that of the nephritic control rats. Histological observation demonstrated that this agent suppressed the incidence of crescent formation, adhesion of capillary wall to Bowman''s capsule and fibrinoid necrosis in the glomeruli. Furthermore, butein suppressed the accumulation of leukocytes, including CD4-positive cells and CD8-positive cells in the glomeruli. However, butein failed to suppress the production of the antibody against rabbit γ-globulin and the deposition of rat-IgG on the GBM. These results suggest that butein may be a useful medicine against rapidly progressive glomerulonephritis, which is characterized by severe glomerular lesions with diffuse crescents.
The antitumor effect of CGP41251 (4''-N-benzoyl staurosporine), a selective protein kinase C (PKC) inhibitor, was examined on two kinds of human non-small cell lung cancer (NSCLC) cell lines (adenocarcinoma: A549 and squamous cell carcinoma: NCI-H520). CGP41251 at 0.5 or 1.0μM inhibited the proliferation of these tumor cell lines significantly; However, at 0.1μM, it did not show any significant inhibition. Cell cycle analysis indicated that CGP41251 at 0.5 or 1.0μM arrested the cell cycle progression at the G2/M phase up to 24 hr, but 0.1μM did not. It seems that the antiproliferative action of CGP41251 against human NSCLC is related to G2/M accumulation. In NCI-H520, CGP41251 caused DNA re-replication without mitosis. In a nude mice xenograft, CGP41251 at a dose of 200 mg/kg showed antitumor activity against these cell lines. Histopathologically, expansion of central necrosis was observed, although no destruction of tumor nests was seen by CGP41251 administration. In both tumor tissues, the PKC activity of the particulate fraction was significantly decreased by CGP41251 treatment. From these results, it is thought that the antitumor activity of CGP41251 against human NSCLS is accompanied by the decrease of PKC activity in the particulate fraction. Moreover, the G2/M arrest of the cell cycle induced by CGP41251 might be important for the growth inhibitory action of this compound.
To elucidate mechanisms involved in analgesia induced by effects of electro-acupuncture (EAP), effects of EAP on evoked potentials and release of substance P (SP) following tooth pulp stimulation (ST) in the superficial layers of the trigeminal nucleus caudalis (Vc-I-II) were studied in the rabbit. The potentials evoked by ST were composed of two main components with conduction velocity of ca. 30 m/sec (fast component) and ca. 12 m/sec (late component). The late component was significantly inhibited by morphine (10 mg/kg, i.v.) or CP-96, 345 (5 mg/kg, i.v.), an SP antagonist. This inhibitory effect of morphine was antagonized by naloxone (1 mg/kg, i.v.) or methysergide (5 mg/kg, i.v.). In addition, the late component was significantly inhibited by EAP, which was observed in ca. 70% of the rabbits examined. This EAP-induced inhibitory effect was antagonized by naloxone (1 mg/kg, i.v.) or methysergide (5 mg/kg, i.v.), but not by prazosin (5 mg/kg, i.v.) and yohimbine (1 mg/kg, i.v.). The stimulus-evoked SP release was inhibited by EAP, which was significantly antagonized by pretreatment with naloxone (1 mg/kg, i.v.) or methysergide (5 mg/kg, i.v.). These results suggest that one of the mechanisms of analgesia induced by EAP is due to inhibition of the stimulus-evoked SP release in the Vc-I-II through activation of the descending serotonergic systems linking up with opioidergic systems.
The agonistic action of fenamates on the α1-adrenoceptor-activated cationic current (Icat) in rabbit portal vein smooth muscle was investigated with the whole-cell patch clamp technique. At −50 mV, the fenamates (100-500 μM) increased Icat dose-dependently, up to several fold. This increase was not accompanied by changes in the reversal potential and strongly inhibited by 500 μM Cd2+ or 10 mM procaine. The enhancing effect of fenamates was also observed on the cationic current activated by intracellularly applied GTPγS. These results suggest that fenamates may be useful as a new class of activator for receptor-operated cation channels in smooth muscle.
Mice were given the extract of cultured Cordyceps sinensis (Cs) (200 mg/kg daily, p.o.) for 3 weeks. In vivo phosphorus-31 nuclear magnetic resonance (NMR) spectra of the liver were acquired at weekly intervals using a surface coil. From 1 to 3 weeks, a consistent increase in the ATP/inorganic phosphate ratio, which represents the high energy state, was observed in the Cs extract-treated mice. The intracellular pH of the Cs extract-treated mice was not significantly different from that of the control mice. No steatosis, necrosis, inflammation or fibrosis were observed in the liver specimens from Cs extract-treated mice.
Neurons can form a synaptic network in culture and show spontaneous oscillation of intracellular Ca2+ concentration ([Ca2+]i). In the present study, spontaneous oscillation of [Ca2+]i, was characterized in cultured hippocampal neurons. The oscillation was blocked completely by tetrodotoxin, 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX) and nicardipine, while DL-2-amino-5-phosphonovaleric acid (APV) showed only a partial depression of the increase in [Ca2+]i. These results suggest that the oscillation in [Ca2+]i is mainly mediated by non-N-methyl-D-aspartate (NMDA) type glutamatergic transmission. The oscillation of [Ca2+]i may be a good model for analyzing glutamatergic transmission and synapse formation.