Molecular and functional characterization of choline transporter in the human trophoblastic cell line JEG-3 cells.

INTRODUCTION: Choline is essential for the synthesis of the major membrane phospholipid phosphatidylcholine (PC), the methyl donor betaine and the neurotransmitter acetylcholine (ACh), which is involved in several vital biological functions that play key roles in fetal development. In this study, we examined the molecular and functional characteristics of choline uptake in the human trophoblastic cell line JEG-3. METHODS: We examined [(3)H]choline uptake in the human trophoblastic cell line JEG-3. The expression of CTL1 and CTL2 was evaluated by quantitative real-time PCR, western blotting and immunocytochemistry. RESULTS: We demonstrated that JEG-3 cells take up [(3)H] choline by a saturable process that is mediated by a Na(+)-independent and pH-dependent transport system. The cells have two different [(3)H] choline transport systems, high- and low-affinity, with Km values of 28.4 ± 5.0 µM and 210.6 ± 55.1 µM, respectively. Cationic compounds and hemicholinium-3 (HC-3) inhibited choline uptake. Choline transporter-like protein 1 (CTL1) and CTL2 mRNA and protein were highly expressed in JEG-3 cells and were localized to the plasma membrane. DISCUSSION: The present results suggest that choline is mainly transported via a high-affinity choline transport system (CTL1) and a low-affinity choline transport system (CTL2) in human trophoblastic JEG-3 cells. These transporters play an important role in the growth of the fetus.

Responses of trabecular and cortical bone turnover and bone mass and strength to bisphosphonate YH529 in ovariohysterectomized beagles with calcium restriction.

Thirty-six beagles, 18 months of age, underwent ovariohysterectomy (OHX) or a sham operation. Sham-operated animals were given a diet with standard calcium (1.4%) (group 1, n = 6) or a restricted calcium diet (0.14%) (group 2, n = 6). The OHX animals were given the restricted calcium diet and YH529 orally with respective daily doses of 0, 0.02, 0.1, and 0.5 mg/kg of body weight (groups 3-6, n = 6 each) for 12 months. At the end of this period, the lumbar bone mineral densities (BMDs) in groups 2 and 3 and the load values for group 3 were significantly smaller than those for group 1. The midfemur BMD did not differ among the groups. The urinary deoxypyridinoline (U-Dpy) and bone formation rates (BFR/BS, BFR/BV) in groups 2 and 3 and the osteonal BFR/BS and trabecular osteoclast number (Oc.N/BS) in group 3 were significantly larger than the respective values for group 1. However, these parameters did not significantly differ between groups 2 and 3. The serum osteocalcin (OC) level, wall thickness (W.Th), and mineral apposition rate values for group 3 were significantly larger than those for group 2. In group 2, the trabecular activation frequency (Ac.F) increased by 3.11 times, and the percent values of the number of labeled osteons (L-Ot.N/T-Ot.N, %) in the tibia by 3.28 times over those for group 1. In group 3, the Ac.F increased by 3.20 times and the number of labeled osteons by 3.77 times over those for group 1. In groups 4-6, the U-Dpy and Oc.N/BS values were smaller, but their OC levels did not significantly differ from the level for group 3. The lumbar BMD, the load, and W.Th were dose-dependently significantly larger than those for group 3. The Ac.F values were significantly smaller, and the respective value in groups 4-6 was 67.9, 25.5, and 10.2% of that in group 3. The BMDs of the midfemur in groups 4-6 were significantly larger than those in group 3, but the ultimate load values did not significantly differ. The L-Ot.N/T-Ot.N values were also significantly smaller, and the respective value in groups 4-6 was 82.0, 48.5, and 55.2% of that in group 3. The tibial endocortical and periosteal BFR/BSs did not differ significantly. These data demonstrate that the effects of OHX on bone mass and turnover were small in the beagles fed a restricted calcium diet. YH529 maintained the mass and strength of the lumbar bone by reducing the bone resorption. The cortical bone appeared to be less sensitive to the agent than the trabecular bone in this animal model.

Detection of eumelanogenic and pheomelanogenic melanosomes in the same normal human melanocyte.

Ellipsoid and spheroid melanosomes similar to those found in the hair matrix melanocytes of eumelanic C57BL mice and pheomelanic Ay mice, respectively, have been shown to coexist in the same human melanocyte. The difference in the three-dimensional ultrastructure of these melanosomes of the human hair matrix melanocyte has been determined by high-voltage transmission electron microscopy using a goniometer. By energy-dispersive X-ray spectroscopy, sulfur, one of the main characteristic chemical properties of pheomelanin, is detected in a significant amount in each spheroid melanosome, but is absent in ellipsoid melanosomes. Furthermore, the internal structure of the spheroid melanosomes is dissolved by treatment with 0.5 N NaOH solution, whereas the ellipsoid melanosomes are not affected. We proposed that in normal human melanocytes pheo- and eumelanogenesis occurs in spheroid and ellipsoid melanosomes, respectively.

Melanogenic regulatory factors in coated vesicles from melanoma cells.

Coated vesicles have been found to contain much higher tyrosinase and gamma-glutamyl transpeptidase activities than premelanosomes. This indicates that similar to tyrosinase, gamma-glutamyl transpeptidase, an enzyme responsible for pheomelanogenesis, is highly concentrated in coated vesicles after its maturation in Golgi associated endoplasmic reticulum (GERL). Furthermore, in the pre- and post-dopaquinone melanogenic pathway, coated vesicles convert dopachrome to colorless indole compounds more quickly than in premelanosomes because of their higher dopachrome conversion factor activity. Melanosomes have been found to exhibit indole conversion factor activity, while coated vesicles show indole blocking factor activity. In moderately tyrosinase-rich premelanosomes, the levels of dopachrome conversion factor and indole blocking factor are lower than in coated vesicles or melanosomes. High levels of indole blocking factor in coated vesicles may indicate why melanin polymer formation does not occur there in vivo despite their high tyrosinase activity.

Morphological study of recombinant human transforming growth factor beta 1-induced intramembranous ossification in neonatal rat parietal bone.

Recombinant human transforming growth factor beta 1 (rhu TGF beta 1) was injected singly or repeatedly for 3-12 days into the periosteum of the right side parietal bone of neonatal rats under the period of bone growth, and the time course of histological changes of the bone was observed by light and electron microscopy and by enzyme histochemistry. The repeated injections of rhu TGF beta 1 at 200 ng/day increased the thickness of the bone tissue on the treated side, which was about twice the nontreated side value after 12-day injections. On the dermal side, preosteoblasts in the periosteum increased in an early stage of treatment, and thereafter, differentiation into osteoblasts, increase of bone matrix, bone marrow cavity formation, and increase of osteoclasts within the bone marrow cavities were observed. Activation of osteoblasts on the dura mater side was also seen. The single injection of rhu TGF beta 1 at 200 ng resulted only in increased osteoprogenitor cell layers and bone matrix formation in an early stage, and the thickness of the osteoprogenitor cell layers and bone tissue at 12 days after single injection was comparable to the values on the nontreated side. At 1 microgram, however, the osteoblasts were activated, and the osteoprogenitor cell layers and bone matrix formation were markedly increased. At 12 days, the bone tissue thickness on the treated side was about twice the nontreated side value, as in the repeated treatment groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacological characterization and visualization of the glial serotonin transporter.

Astrocytes contain transport systems that are capable of removing various neurotransmitters from the synaptic cleft by transporters present in the plasma membrane. Glial serotonin transporter (SERT) plays an important role in the re-uptake of 5-hydroxytryptamine (5-HT). We examined the pharmacological characterization of 5-HT uptake into rat cortical synaptosomes and cultured rat astrocytes, and the immunodetection of glial SERT proteins using specific site-directed monoclonal antibodies (MoAb). Furthermore, using a reverse transcriptase-polymerase chain reaction (RT-PCR) method, we addressed the expression of SERT mRNA in cultured rat astrocytes. We investigated the inhibitory effects of various monoamine uptake inhibitors on the uptake of [3H]5-HT into cultured astrocytes and cortical synaptosomes. Tricyclic antidepressants (clomipramine and imipramine) as well as selective serotonin re-uptake inhibitors (fluvoxamine, fluoxetine and zimelidine) were very potent inhibitors of [3H]5-HT uptake in both preparations. In contrast, the inhibitory effects of NE uptake inhibitors (nisoxetine and desipramine) and cocaine were weaker than those of 5-HT uptake inhibitors. In addition, dopamine (DA) uptake inhibitors (nomifensine and GBR-12935) exhibited a Ki value in the low micromolar range. The inhibitory potencies were in the order 5-HT uptake inhibitors (clomipramine, fluvoxamine, fluoxetine, imipramine and zimelidine) > NE uptake inhibitors (nisoxetine and desipramine) = cocaine > DA uptake inhibitors (nomifensine and GBR-12935). There was no difference in the order of the inhibitory effects of various monoamine uptake inhibitors between the two preparations. A correlation analysis of the potencies of various monoamine uptake inhibitors in the inhibition of [3H]5-HT into cultured astrocytes and cortical synaptosomes produced a highly significant correlation coefficient of 0.9893 (P < 0.0001). Immunocytochemical staining using anti-SERT MoAb in cultured astrocytes revealed that the plasma membrane, as well as intracellular, perinuclear compartments, presumably endoplasmic reticulum or golgi membranes, showed a considerable level of immunoreactivity. Extracts of astrocytes and synaptosomes from the cortex were immunoblotted with anti-SERT MoAb. SDS-PAGE/Western blots indicate that anti-SERT MoAb recognized two bands of 120 and 73 kDa in both preparations. RT-PCR demonstrated that astrocytes in cultured expressed mRNA for the cloned SERT protein, which has been characterized as the neuronal SERT. These pharmacological experiments indicate that this uptake process takes place through glial SERT that is very similar to neuronal SERT. Furthermore, the present data also indicate that the presence of the mRNA and protein for the neuronal SERT were established in cultured rat astrocytes, and the polypeptide portion of SERT in astrocytes and frontal cortex could be the same gene product.

Methyl-4-phenylpyridinium (MPP(+))-evoked dopamine release from rat striatal slices: possible roles of voltage-dependent calcium channels and reverse dopamine transport.

We examined the properties of voltage-dependent Ca(2+) channels (VDCCs) mediating 1-methyl-4-phenylpyridinium (MPP(+))-evoked [3H]DA release from rat striatal slices. In some cases, the Ca(2+)-independent efflux of neurotransmitters is mediated by the high-affinity neurotransmitter-uptake systems. To determine whether such a mechanism might be involved in MPP(+)-evoked [3H]DA release. MPP(+) (1,10 and 100 microM) evoked the release of [3H]DA from rat striatal slices in a concentration-dependent manner. In the absence of Ca(2+), MPP(+) (10 and 100 microM)-evoked [3H]DA release was significantly decreased to approximately 50% of control (a physiological concentration of Ca(2+)). In the presence of Ca(2+), nomifensine (0.1,1 and 10 microM) dose-dependently and significantly inhibited the MPP(+)-evoked release of [3H]DA. Nomifensine (1 and 10 microM) also dose-dependently and significantly inhibited the MPP(+)-evoked release of [3H]DA under Ca(2+)-free conditions. MPP(+)-evoked [3H]DA release was partly inhibited by nicardipine (1 and 10 microM), an L-type Ca(2+) channel blocker. On the other hand, the N-type Ca(2+) channel blocker omega-conotoxin-GVIA (omega-CTx-GVIA) (1 and 3 microM) did not affect this release. omega-agatoxin-IVA (omega-Aga-IVA) at low concentrations (0.1 microM), which are sufficient to block P-type Ca(2+) channels alone, also had no effect. On the other hand, MPP(+)-evoked [3H]DA release was significantly decreased by high concentrations of omega-Aga-IVA (0.3 microM) that would inhibit Q-type Ca(2+) channels. In addition, application of the Q-type Ca(2+) channel blocker omega-conotoxin-MVIIC (omega-CTx-MVIIC) (0.3 and 1 microM) also significantly inhibited MPP(+)-evoked [3H]DA release. These results suggest that MPP(+)-evoked [3H]DA release from rat striatal slices is largely mediated by Q-type Ca(2+) channels, and the Ca(2+)-independent component is mediated by reversal of the DA transport system.

Regulation of dopamine uptake by basic fibroblast growth factor and epidermal growth factor in cultured rat astrocytes.

We examined the characteristics of dopamine (DA) uptake and its regulation by neurotrophic factors such as basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) in cultured rat astrocytes. In the presence of inhibitors of monoamine oxidase (MAO) and catechol-O-methyl-transferase (COMT), astrocytes took up DA by Na(+)-dependent and Na(+)-independent mechanisms that were sensitive to a reduction in temperature. The Na(+)-dependent and Na(+)-independent components increased linearly with increasing [3H]DA concentration (1-1000 microM), and showed no saturation. Na(+)-dependent DA uptake was significantly inhibited by ouabain, a Na(+)-K+ ATPase inhibitor. In bFGF-treated astrocytes, [3H]DA uptake increased in a time-dependent manner until 48 h, and declined after 72 h in both the presence and absence of Na+. In EGF-treated astrocytes, [3H]DA uptake increased in a time-dependent manner until 72 h in both the presence and absence of Na +. This enhancement of DA uptake induced by EGF or bFGF was significantly inhibited when the cells were cultured with actinomycin D, cycloheximide, or brefeldin A. Actinomycin D and brefeldin A also significantly inhibited the basal uptake of [3H]DA into astrocytes. These results suggest the existence of Na(+)-dependent and Na(+)-independent DA uptake in cultured rat astrocytes, and that EGF or bFGF might stimulate the expression and translocation of the extraneuronal DA transporter.

Sodium pump activity and contraction of renal artery from spontaneously hypertensive rats.

We investigated the hypertensive changes in renal arteries isolated from 21-week-old spontaneously hypertensive rats (SHR), and from age-matched normotensive Wistar-Kyoto rats (WKY). The maximum contraction of renal arteries from SHR in response to norepinephrine (NE), serotonin (5-HT) and KCl was greater than that of arteries from WKY. The threshold and EC50 concentrations of NE, 5-HT and KCl were not significantly different between SHR and WKY. Contraction induced by removal of K+ was inhibited by 10(-8) M prazosin. Less than 10(-7) M NE in K(+)-free solution did not cause contraction. Addition of 5.9 mM KCl to K(+)-free solution in the presence of 10(-5) M NE induced relaxation, which was followed by contraction to about the same level as that before KCl addition. The duration of the K(+)-induced relaxation in SHR (22.4 +/- 0.9 min) was slightly, but significantly shorter than that in WKY (26.6 +/- 0.8 min) arteries. In K(+)-free solution with reduced Na+, the duration of the relaxation induced by KCl was shorter than that in the normal solution, for both SHR (13.8 +/- 0.3 min) and WKY (14.1 +/- 0.5 min). Such differences could be caused by increased influx and decreased efflux of Ca2+, which depend on the Na+ concentration and are related to the Na(+)-Ca2+ exchange. The results suggest that enhanced renal vascular reactivity in hypertension may depend on structural changes and increased Na+ pump activity.

Contractile responses and calcium mobilization in renal arteries of diabetic rats.

The causes of diabetes-associated change of renal artery vasomotion have not been established. We investigated both contractile responses to KCl and norepinephrine (NE) in renal arteries of rats with streptozotocin-induced diabetes and age-matched controls and the effects on Ca2+ mobilization. Renal arteries from diabetics had greater maximum contractile responses to KCl and NE, but the threshold concentrations and EC50 values of KCl and NE were similar in controls and diabetics. The concentration-response for Bay K 8644, a dihydropyridine Ca2+ channel agonist in the presence of 20 mM KCl was significantly greater in diabetics than in controls. The maximum contractile responses to Ca2+ in the presence of 10(-6) M NE were significantly (P less than 0.05) greater in diabetics than in controls. The increased contractile response at low concentrations of Ca2+ (0.01-0.05 mM) was inhibited in both preparations by 10(-6) M nifedipine, but at high concentrations of Ca2+ (0.1-2.5 mM) the inhibition by nifedipine was significantly less in diabetics than in the controls. 45Ca2+ uptake had significantly greater resting levels in diabetics than in controls. The uptake of 45Ca2+ induced by 10(-5) M NE was significantly greater in diabetics than in controls, and 10(-7) M prazosin diminished both responses. The results suggest hyperreactivity of contractile responses to KCl or NE, and hyperpermeability of renal artery smooth muscle membrane to Ca2+ in streptozotocin-induced diabetics.

Calcium mobilization and phosphatidylinositol turnover in vas deferens smooth muscle of diabetic rats.

The study concerned Ca2+ channels that are receptor-operated by norepinephrine (NE) and mediate hyper-reactivity of vas deferens smooth muscle from rats with streptozotocin (STZ)-induced diabetes, and the mediatory responses of these channels, such as tension development, Ca2+ uptake and phosphatidylinositol (PI) turnover. The contractile responses induced by adrenoceptor agonists were significantly greater in diabetic rat vas deferens than in the controls. A greater Ca2+ uptake was induced by 10(-5) M NE in strips from diabetic rats than in the controls. The uptake of Ca2+ was completely inhibited by 10(-6) M prazosin but not by 10(-5) M verapamil. Enhancement of Ca2+ release by 10(-5) M NE was faster and greater in diabetic muscles than in the controls. The accumulation of [3H]inositol phosphates was increased 4-fold in the controls and 7-fold in diabetic muscles by 10(-5) M NE. This increase was completely inhibited by 10(-6) M prazosin but not by 10(-6) M yohimbine. The data suggest that vas deferens smooth muscle hyper-reactivity in diabetic rats is due to increased PI turnover mediated by alpha 1-adrenoceptors, to the release of intracellular bound Ca2+ and to an increase of Ca2+ uptake through receptor-operated Ca2+ channels.

Cyclopiazonic acid stimulates Ca2+ influx through non-specific cation channels in endothelial cells.

A non-specific cation channel in cultured human umbilical vein endothelial cells was obtained by cell-attached patch-clamp study. This channel showed a conductance of 28 pS when both pipette and bath contained 140 mM potassium chloride. when pipette solution was changed into 140 sodium chloride with 5 mM calcium chloride, the conductance was 26 pS. when 120 mM calcium chloride was used as the only cation in the pipette, a conductance of 6 pS was obtained. Bath application of cyclopiazonic acid, an inhibitor of the sarcoplasmic reticulum Ca2+ pump in smooth muscle and other tissues, dose dependently activates this non-specific cation channel. It is assumed that cyclopiazonic acid by blockade of the refilling of Ca2+ stores depletes the rapidly exchanging intracellular Ca2+ stores and this action stimulates Ca2+ influx through the non-specific cation channels in human umbilical vein endothelial cells.

Effects of various dopamine uptake inhibitors on striatal extracellular dopamine levels and behaviours in rats.

In vivo central effects of some dopamine uptake inhibitors were evaluated in both brain microdialysis and behavioural studies in rats, and compared with their in vitro affinities to dopamine uptake sites. IC50 values of GBR12909 (1-[2- bis(4-fluorophenyl)methoxy]ethyl]-4-(3- phenylpropyl)piperazine), diclofensine, mazindol, amfonelic acid and nomifensine for inhibiting 1 nM [3H]GBR12935 (1-[2-(diphenylmethoxy)ethyl]-4-(3-phenylpropyl)piperazine) binding to rat striatal membrane were 7.0, 36, 81, 187 and 290 nM, respectively. In the brain microdialysis study, dopamine levels in the striatal dialysates were increased to 16.3- (GBR12909), 14.1- (nomifensine), 4.8- (diclofensine) and 1.9-fold (amfonelic acid) the respective basal levels 40-60 min after i.p. administration (0.1 mmol/kg) and thereafter decreased slowly but remained at the elevated levels for a further 3 h, while mazindol gradually increased dopamine levels though less pronouncedly than others (1.7-fold 200 min after administration). Remarkable and comparable stereotyped behaviours (licking and forepaw treading) were continuously observed at least for 3 h after administration of GBR12909, nomifensine and amfonelic acid, while stereotypies induced by diclofensine and mazindol were moderate and marginal, respectively. In vivo potencies of dopamine uptake inhibitors to increase the extracellular dopamine levels in the striatum tended to correlate with their in vitro affinities to dopamine uptake sites except in the case of nomifensine, and correlated significantly with their potencies to induce stereotyped behaviours except in the case of amfonelic acid. Based on these findings, pharmacological characteristics of these dopamine uptake inhibitors are discussed.

Effect of LP-805, a releaser of endothelium-derived nitric oxide, on systemic vasodilatation in vivo.

We have investigated relations between hypotensive responses to LP-805, a newly synthesized vasodilator, and the production of nitric oxide (NO), in anesthetized rats. LP-805 (0.1-0.5 mg/kg, i.v.) or acetylcholine (ACh) (0.3-3.0 micrograms/kg, i.v.) caused a dose-dependent transient decrease in diastolic blood pressure. The decrease induced by 0.3 mg/kg LP-805 (i.v.) was partially inhibited by pretreatment with NG-nitro-L-arginine (L-NNA), a specific inhibitor of endothelial NO synthase, but the responses to lower or higher doses of LP-805 (0.1 or 0.5 mg/kg, i.v.) were not affected. The dose-dependent decrease in diastolic blood pressure, caused by LP-805, was not affected by pretreatment with L- or D-arginine. The dose-dependent decrease in diastolic blood pressure caused by ACh was not affected by pretreatment with L-NNA or with L- or D-arginine. The hypotensive response to 20-min infusions of LP-805 (100 micrograms/kg per min) was significantly inhibited by pretreatment with L-NNA (10 mg/kg, i.v.). The half-recovery times (T 1/2) of LP-805 or ACh-induced depressor responses were shortened by pretreatment with L-NNA. They were prolonged by L-arginine, but not by D-arginine. This shortening, by L-NNA, of the half-recovery time after LP-805 or ACh was reversed by L-arginine, but not by D-arginine. The T 1/2 of the LP-805-induced hypotensive response was not affected by pretreatment with indomethacin (1 mg/kg, i.v.). In the presence of L-NNA (10 mg/kg, i.v.), the T 1/2 of the LP-805-induced hypotensive response was not affected by pretreatment with indomethacin.(ABSTRACT TRUNCATED AT 250 WORDS)

Different mechanisms can activate Ca2+ entrance via cation currents in endothelial cells.

Effects of Endothelin-1 (ET-1) and cyclopiazonic acid (CPA) on non-specific cation channels in cultured bovine pulmonary artery endothelial cells (BPAECs) were investigated using the patch-clamp technique. In a bath solution containing Ca2+ as a permeant cation, 10 nM ET-1 increased inward and outward currents and this current reversed at -10 mV instead of -60 mV. Under similar conditions, 10 microM CPA, an inhibitor of Ca2+ pumps in the sarcoplasmic reticulum, also increased both currents which now reversed near -10 mV. An inorganic Ca2+ influx blocker, La3+ at 50 microM completely blocked ET-1 and CPA-evoked currents restoring the reversal potential to -60 mV. ET-1 and CPA evoked currents were partially blocked by 50 microM SK&F 96365 (a putative inhibitor of receptor-mediated Ca2+ entry). ET-1 and CPA increased Ca2+ influx by activation of the Ca(2+)-permeable non-specific cation channels, which are gated by the depletion of intracellular Ca2+ stores in endothelial cells. These results, together with a previous study demonstrating that this Ca2+ entrance pathway can be opened directly by one vasodilator (LP-805) reveal that different mechanisms exist to activate Ca2+ entrance into endothelial cells. All may allow sustained release of endothelium-derived relaxing factor (EDRF).

LP-805, a releaser of endothelium-derived nitric oxide, activates an endothelial calcium permeable non-specific cation channel.

The actions of LP-805, a releaser of endothelium-derived nitric oxide, on Ca2+ permeable non-specific cation channel in cultured bovine pulmonary artery endothelial cells (BPAECs) were investigated using patch-clamp technique. In Ca2+ saline bath solution, 10 microM LP-805 increased inward and outward currents. LP-805 evoked currents were reversibly blocked by 50 microM La3+, an inorganic Ca2+ influx blocker. In Ca2+ and Na+ free saline bath solution, 10 microM LP-805 did not evoke these currents, when 2 mM Ca2+ was then added to the bath the inward current increased. Furthermore, in the Na+ saline bath solution (Ca2+ free), 10 microM LP-805 increased similar inward and outward currents, which were blocked by 50 microM La3+. LP-805 evoked currents were carried by either Ca2+ or Na+. With the pipette solution containing 11 mM EGTA, 10 microM LP-805 activated the inward and outward currents. The increase of inward and outward currents by 10 microM LP-805 was inhibited by 1 mM Ni2+ but not by 1 microM nicardipine or 50 microM SKF 96365. In conclusion, LP-805 increases Ca2+ influx into vascular endothelial cells through Ca2+ permeable non-specific cation channels. This may explain the release of endothelium-derived nitric oxide in endothelial cells by LP-805.

Relations between contractile responses and beta-adrenoceptors in gastric fundus of diabetic rats.

Contractile responses to norepinephrine (NE), and the population of beta-adrenoceptors, were determined in gastric fundus smooth muscle from rats with diabetes induced by streptozotocin (STZ), and age-matched controls. Relaxation and/or contraction of fundus strips of controls and diabetics were induced by 10(-5)M NE. Responses to NE were mainly relaxation in gastric fundus isolated from controls, and contraction in fundus isolated from diabetics. Contraction was blocked by 10(-8) M prazosin and relaxation was blocked by 10(-6) M propranolol. Relaxation by isoproterenol of contraction induced by 10(-6) M acetylcholine was significantly less in fundus from diabetics than in that from controls. The number of beta-adrenoceptors, measured with [125I] iodocyanopindolol as a ligand, was significantly less in gastric fundus membrane isolated from diabetics than in that from controls, but affinity was no different. The level of plasma catecholamine was higher in diabetics than in controls. Results suggest that depression of gastric fundus relaxation and increase of contraction by NE in diabetics could be due to fewer beta-adrenoceptor binding sites caused by down-regulation by higher catecholamine level in diabetic rats.

Pharmacological characterization of dopamine transport in cultured rat astrocytes.

The effects of GBR-12909 (selective DA uptake inhibitor), zimelidine (selective 5-HT uptake inhibitor) and nisoxetine (selective NE uptake inhibitor) on the uptake of 30 nM [3H]DA into cultured rat astrocytes were examined. [3H]DA uptake was inhibited by approximately 50% by GBR-12909 or zimelidine in a concentration-dependent manner (100 nM to approximately 10 microM). Furthermore, the inhibition curves of GBR-12909 were biphasic, and uptake was completely inhibited by a high concentration of GBR-12909 (100 microM). [3H]DA uptake was also inhibited by approximately 50% by nisoxetine in a concentration-dependent manner (0.1 to approximately 100 nM), and nisoxetine was more potent than GBR-12909 or zimelidine. The inhibitory potencies were in the order nisoxetine > GBR-12909 > zimelidine. The uptake of [3H]DA under Na+-free conditions was approximately 50% of that under normal conditions. Thus, DA was taken up by both Na+-dependent and Na+-independent mechanisms. Nisoxetine (100 nM), zimelidine (100 microM) and GBR-12909 (10 microM) inhibited [3H]DA uptake into astrocytes only in the presence of Na+. On the other hand, this uptake was completely inhibited by a high concentration of GBR-12909 (100 microM) in the absence of Na+. The present data suggest that the Na+-dependent uptake of [3H]DA in cultured rat astrocytes may occur in the NE uptake system. Furthermore, astrocytes express the extraneuronal monoamine transporter (uptake2), which is an Na+-independent system, and this transporter is involved in the inactivation of centrally released DA.

Contractile hyperreactivity and alteration of PKC activity in gastric fundus smooth muscle of diabetic rats.

Contraction dose dependently induced in gastric smooth muscle of diabetic rats by Bay K 8644 in the presence of 20 mM KCl was about two times that induced in controls, and was inhibited more than 50% by 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7). Contraction was caused in diabetics but usually not in controls by 10(-5) M phorbol 12-myristate 13-acetate (PMA). In diabetics, this contraction was about 2.5 times that in controls. Protein kinase C (PKC) activity in the soluble fraction was depressed by H-7 or staurosporine, and depended on PMA concentration, but was greater in diabetics than in controls at any PMA concentration. PKC activity in the soluble fraction was inhibited by lower Ca2+ concentration, and was greater in diabetics than in controls. Affinity and density of binding sites of a Ca2+ channel antagonist ligand, [3H]PN200-110, were the same in plasma membrane-enriched fractions isolated from either controls or diabetic preparations. Thus, hyperreactivity in diabetic fundus may depend, in part, on alteration of PKC properties, but not on the density of Ca2+ channels.

[Effect of cefodizime sodium on the kidney function in male rabbits--single and 7-day repeated intravenous administration].

Renal effects of cefodizime sodium (THR-221) administered by the intravenous route singly and for 7 consecutive days to male rabbits, were compared with those of cefazolin sodium (CEZ) and cephalothin sodium (CET). Four animals were used in each group including control groups. In the single-dose study, THR-221 (600 and 1800 mg/kg) and CET (1800 mg/kg) caused no nephrotoxic effects. In the CEZ groups (600 and 1800 mg/kg), findings indicative of the decreased renal function were obtained: serum urea nitrogen and creatinine levels increased over the control values, and phenolsulfonphthalein (PSP) retention test showed a delay in PSP excretion from the blood. In addition, the white surface of the kidney was macroscopically observed, and microscopic examination revealed renal proximal tubular changes such as necrosis, hyaline cast and calcification, suggesting renal disorders. The repeated-dose study also showed similar results to those described above. Administration of THR-221 (200 and 600 mg/kg/day) and CET (600 mg/kg/day) caused no effects on the kidney. In the CEZ groups (200 and 600 mg/kg/day), serum chemical and PSP test results suggested the decreased renal function, and macroscopic and microscopic findings included organic changes in the kidney. These results suggest that under the conditions tested THR-221 dose not elicit signs of nephrotoxicity in contrast to CEZ, and behaves almost equally to CET.