Fas/CD95/Apo-I activates the acidic sphingomyelinase via caspases.
Fas/CD95/Apo-I has been shown to stimulate a variety of molecules including several members of the caspase family and the acidic sphingomyelinase (Martin and Green 1995; Gulbins et al, 1995). Here, we demonstrate that Fas receptor-triggered activation of the acidic sphingomyelinase, consumption of sphingomyelin, release of ceramide, and subsequent activation of JNK and p38-K are regulated by caspases. Inhibition of caspases by Ac-YVAD-chloromethylketone or transient CrmA transfection prevented stimulation of acidic sphingomyelinase, release of ceramide and activation of JNK and p38-K upon Fas-receptor crosslinking. Likewise, Fas triggered apoptosis was almost completely blocked by Ac-YVAD-chloromethylketone or CrmA mediated inhibition of caspases. The results suggest a new signalling cascade from the Fas receptor via caspases to acidic sphingomyelinase, ceramide and JNK/p38-K. (+info)
Effects of imipramine, an uptake inhibitor, on double-peaked constrictor responses to periarterial nerve stimulation in isolated, perfused canine splenic arteries.
Using a cannula insertion method, periarterial nerve electrical stimulations were performed at 1 and 10 Hz in the isolated, perfused canine splenic artery. Electrical nerve stimulation readily caused double-peaked vasoconstrictions. The 1st-peak response at 1 Hz was not influenced by treatment with imipramine but the 2nd one was significantly enhanced by it. The 2nd-peak response was markedly blocked by prazosin. An additional treatment with alpha,beta-methylene ATP, a P2X-purinoceptor desensitizer, abolished electrical stimulation-induced vascular responses that remained. At 10 Hz, the responses to electrical stimulation were not significantly influenced by imipramine. On the other hand, the imipramine treatment inhibited the tyramine-induced vasoconstriction but potentiated the noradrenaline-induced one. ATP-induced responses were not modified by imipramine. From these results, it is concluded that 1) the 1st-peaked constriction is mainly due to a P2X-purinoceptor-dependent mechanism, 2) the 2nd one is mainly due to an alpha1-adrenoceptor-dependent mechanism, and 3) presynaptic uptake mechanisms may perform an important role in the regulation of vascular reactivity, especially at a low frequency. (+info)
Transmembrane domain I contributes to the permeation pathway for serotonin and ions in the serotonin transporter.
Mutation of a conserved Asp (D98) in the rat serotonin (5HT) transporter (rSERT) to Glu (D98E) led to decreased 5HT transport capacity, diminished coupling to extracellular Na+ and Cl-, and a selective loss of antagonist potencies (cocaine, imipramine, and citalopram but not paroxetine or mazindol) with no change in 5HT Km value. D98E, which extends the acidic side chain by one carbon, affected the rank-order potency of substrate analogs for inhibition of 5HT transport, selectively increasing the potency of two analogs with shorter alkylamine side chains, gramine, and dihydroxybenzylamine. D98E also increased the efficacy of gramine relative to 5HT for inducing substrate-activated currents in Xenopus laevis oocytes, but these currents were noticeably dependent on extracellular medium acidification. I-V profiles for substrate-independent and -dependent currents indicated that the mutation selectively impacts ion permeation coupled to 5HT occupancy. The ability of the D98E mutant to modulate selective aspects of substrate recognition, to perturb ion dependence as well as modify substrate-induced currents, suggests that transmembrane domain I plays a critical role in defining the permeation pathway of biogenic amine transporters. (+info)
Diurnal variation in 5-HT1B autoreceptor function in the anterior hypothalamus in vivo: effect of chronic antidepressant drug treatment.
1. Intracerebral microdialysis was used to examine the function of the terminal 5-hydroxytryptamine (5-HT) autoreceptor in the anterior hypothalamus of anaesthetized rats at two points in the light phase of the light-dark cycle. 2. Infusion of the 5-HT1A/1B agonist 5-methoxy-3-(1,2,3,6-tetrahydro-4-pyridyl)-1H-indole (RU24969) 0.1, 1.0 and 10 microM through the microdialysis probe led to a concentration-dependent decrease (49, 56 and 65% respectively) in 5-HT output. The effect of RU24969 (1 and 5 microM) was prevented by concurrent infusion of methiothepin (1 and 10 microM) into the anterior hypothalamus via the microdialysis probe. Infusion of methiothepin alone (1.0 and 10 microM) increased (15 and 142% respectively) 5-HT output. 3. Infusion of RU24969 (5 microM) through the probe at mid-light and end-light resulted in a quantitatively greater decrease in 5-HT output at end-light compared with mid-light. 4. Following treatment with either paroxetine hydrochloride (10 mg kg(-1) i.p.) or desipramine hydrochloride (10 mg kg)(-1) i.p.) for 21 days the function of the terminal 5-HT1B autoreceptor was more markedly attenuated at end-light. 5. The data show that, as defined by the response to RU24969, the function of the 5-HT1B receptors that control 5-HT output in the anterior hypothalamus is attenuated following chronic desipramine or paroxetine treatment in a time-of-day-dependent manner. (+info)
Endothelium is required in the vascular spasm induced by tetraethylammonium and endothelin-1 in guinea-pig aorta.
1. To investigate the role of endothelium in vascular spasm, we studied the influence of endothelin-1 (ET-1) on the contracting and spasmogenic effect of the K+-channel blocker, tetraethylammonium (TEA), in aorta rings of reserpine-treated guinea-pigs, perfused with either control (5.5 mM) or elevated (50 mM) glucose concentration. 2. Endothelium-dependent relaxation induced by acetylcholine was lost in rings contracted by noradrenaline in the presence of elevated glucose. In control medium, TEA (1-20 mM) induced a sustained tonic contraction, followed by a phasic spasm, characterized by rhythmic contractions. Elevated glucose, ET-1 (3 nM), or both, reduced the EC50 of TEA-induced tonic contraction, without modifying the maximum contractile effect. 3. In control medium, ET-1 reduced the time before TEA-induced spasm and increased the rate of rhythmic contractions. TEA-induced spasm was abolished by elevated glucose, and restored by ET-1. The spasm induced by TEA and ET-1 was amplified by the ETA antagonist, EMD94246, and suppressed by the ET(A)-ET(B) antagonist, bosentan. In endothelium-denuded vessels incubated with high glucose and ET-1, TEA evoked only a tonic contraction. 4. In control medium, L-NAME (N(G)-nitro-L-arginine methyl ester) abolished TEA-induced rhythmic contractions. L-arginine, but not D-arginine, prevented the effect of L-NAME. In the presence of elevated glucose and ET-1, TEA-induced spasm was not affected by L-NAME, whereas verapamil, indomethacin, metyrapone, glybenclamide or apamin abolished the phasic spasm, unmasking the tonic contracture. 5. In conclusion, endothelium plays a regulatory role in the genesis and maintenance of TEA-induced rhythmic contractions, through the release endothelium derived relaxing factor and vasodilating eicosanoids. (+info)
Neuronal uptake affects dynamic characteristics of heart rate response to sympathetic stimulation.
Recently, studies in our laboratory involving the use of a Gaussian white noise technique demonstrated that the transfer function from sympathetic stimulation frequency to heart rate (HR) response showed dynamic characteristics of a second-order low-pass filter. However, determinants for the characteristics remain to be established. We examined the effect of an increase in mean sympathetic stimulation frequency and that of a blockade of the neuronal uptake mechanism on the transfer function in anesthetized rabbits. We found that increasing mean sympathetic stimulation frequency from 1 to 4 Hz significantly (P < 0.01) decreased the dynamic gain of the transfer function without affecting other parameters, such as the natural frequency, lag time, or damping coefficient. In contrast, the administration of desipramine (0.3 mg/kg iv), a neuronal uptake blocking agent, significantly (P < 0.01) decreased both the dynamic gain and the natural frequency and prolonged the lag time. These results suggest that the removal rate of norepinephrine at the neuroeffector junction, rather than the amount of available norepinephrine, plays an important role in determining the low-pass filter characteristics of the HR response to sympathetic stimulation. (+info)
Decrease in hepatic CYP2C11 mRNA and increase in heme oxygenase activity after intracerebroventricular injection of bacterial endotoxin.
We previously reported (Arch. Toxcol. 1998, 72, 492-498) that the differential decrease in the levels of hepatic cytochrome P450 (CYP) isozymes in rats was observed 24 hr after intracerebroventricular (i.c.v.) injection of bacterial lipopolysaccharide (LPS) at the dose ineffective (0.1 microgram) when injected intraperitoneally (i.p.). Among CYP isozymes we examined, the male specific CYP isozyme, CYP2C11 was most severely affected by i.c.v. injection of LPS. In this study, we examined the gene expression of CYP2C11, the total P450 contents, the CYP2C11-dependent activity of imipramine N-demethylase (IMND) and protein of CYP2C11 10 hr after i.c.v. or i.p. injections of LPS. Intracerebroventricular injection of LPS significantly decreased the level of CYP2C11 mRNA (to 63% of saline i.c.v. control), the total P450 contents (to 70% of saline i.c.v. control), the IMND activity (to 74% of saline i.c.v. control), but not protein of CYP2C11 in rat liver. In contrast, i.p. injection of LPS at the same dose as i.c.v. did not significantly affect these parameters. Since CYP is a heme protein, we also measured the activity of heme oxygenase (HO) using the same rat liver microsomes. The HO activity was increased to 166% by i.c.v. injection of LPS and 135% by i.p. injection of LPS compared to corresponding saline control. It is suggested that i.c.v. injection of LPS down-regulates the expression of CYP2C11 at transcriptional level and that both the decrease in CYP2C11 mRNA and the increase in heme degradation may be involved in the decreased level of protein and activity of CYP2C11 by i.c.v. injection of LPS in rat liver. (+info)
Activities of trovafloxacin compared with those of other fluoroquinolones against purified topoisomerases and gyrA and grlA mutants of Staphylococcus aureus.
Frequencies of mutation to resistance with trovafloxacin and four other quinolones were determined with quinolone-susceptible Staphylococcus aureus RN4220 by a direct plating method. First-step mutants were selected less frequently with trovafloxacin (1.1 x 10(-10) at 2 to 4x the MIC) than with levofloxacin or ciprofloxacin (3.0 x 10(-7) to 3.0 x 10(-8) at 2 to 4x the MIC). Mutants with a change in GrlA (Ser80-->Phe or Tyr) were most commonly selected with trovafloxacin, ciprofloxacin, levofloxacin, or pefloxacin. First-step mutants were difficult to select with sparfloxacin; however, second-step mutants with mutations in gyrA were easily selected when a preexisting mutation in grlA was present. Against 29 S. aureus clinical isolates with known mutations in gyrA and/or grlA, trovafloxacin was the most active quinolone tested (MIC at which 50% of isolates are inhibited [MIC(50)] and MIC(90), 1 and 4 microg/ml, respectively); in comparison, MIC(50)s and MIC(90)s were 32 and 128, 16 and 32, 8 and 32, and 128 and 256 microg/ml for ciprofloxacin, sparfloxacin, levofloxacin, and pefloxacin, respectively. Strains with a mutation in grlA only were generally susceptible to all of the quinolones tested. For mutants with changes in both grlA and gyrA MICs were higher and were generally above the susceptibility breakpoint for ciprofloxacin, sparfloxacin, levofloxacin, and pefloxacin. Addition of reserpine (20 microg/ml) lowered the MICs only of ciprofloxacin fourfold or more for 18 of 29 clinical strains. Topoisomerase IV and DNA gyrase genes were cloned from S. aureus RN4220 and from two mutants with changes in GrlA (Ser80-->Phe and Glu84-->Lys). The enzymes were overexpressed in Escherichia coli GI724, purified, and used in DNA catalytic and cleavage assays that measured the relative potency of each quinolone. Trovafloxacin was at least five times more potent than ciprofloxacin, sparfloxacin, levofloxacin, or pefloxacin in stimulating topoisomerase IV-mediated DNA cleavage. While all of the quinolones were less potent in cleavage assays with the altered topoisomerase IV, trovafloxacin retained its greater potency relative to those of the other quinolones tested. The greater intrinsic potency of trovafloxacin against the lethal topoisomerase IV target in S. aureus contributes to its improved potency against clinical strains of S. aureus that are resistant to other quinolones. (+info)