Biomedical subjects

C Romanin

Publications and source records attributed to C Romanin.

At least 37 records · Page 2Linked to original sources

PubMed1997

Estimating the number of channels in patch-clamp recordings: application to kinetic analysis of multichannel data from voltage-operated channels.

Important kinetic information of voltage-operated ion channels can be obtained by estimating the open probability, the availability, and the first latency, and by applying run analysis. In the case of multichannel patches, estimation of the number of available channels is a prerequisite for the above analysis. Here we describe a method for calculation of the a posteriori probability of the number of available channels in each sweep by using the Bayes formula. This probability serves as a measure for the number of channels and allows for first latency determination and run analysis. The methods described were applied to simulated and experimental data obtained from L-type Ca2+ channel recordings.

Bayes Theorem↗

PubMed1997

Intracellular Ca2+ inhibits smooth muscle L-type Ca2+ channels by activation of protein phosphatase type 2B and by direct interaction with the channel.

Modulation of L-type Ca2+ channels by tonic elevation of cytoplasmic Ca2+ was investigated in intact cells and inside-out patches from human umbilical vein smooth muscle. Ba2+ was used as charge carrier, and run down of Ca2+ channel activity in inside-out patches was prevented with calpastatin plus ATP. Increasing cytoplasmic Ca2+ in intact cells by elevation of extracellular Ca2+ in the presence of the ionophore A23187 inhibited the activity of L-type Ca2+ channels in cell-attached patches. Measurement of the actual level of intracellular free Ca2+ with fura-2 revealed a 50% inhibitory concentration (IC50) of 260 nM and a Hill coefficient close to 4 for Ca2+- dependent inhibition. Ca2+-induced inhibition of Ca2+ channel activity in intact cells was due to a reduction of channel open probability and availability. Ca2+-induced inhibition was not affected by the protein kinase inhibitor H-7 (10 microM) or the cytoskeleton disruptive agent cytochalasin B (20 microM), but prevented by cyclosporin A (1 microg/ ml), an inhibitor of protein phosphatase 2B (calcineurin). Elevation of Ca2+ at the cytoplasmic side of inside-out patches inhibited Ca2+ channels with an IC50 of 2 microM and a Hill coefficient close to unity. Direct Ca2+-dependent inhibition in cell-free patches was due to a reduction of open probability, whereas availability was barely affected. Application of purified protein phosphatase 2B (12 U/ml) to the cytoplasmic side of inside-out patches at a free Ca2+ concentration of 1 microM inhibited Ca2+ channel open probability and availability. Elevation of cytoplasmic Ca2+ in the presence of PP2B, suppressed channel activity in inside-out patches with an IC50 of approximately 380 nM and a Hill coefficient of approximately 3; i.e., characteristics reminiscent of the Ca2+ sensitivity of Ca2+ channels in intact cells. Our results suggest that L-type Ca2+ channels of smooth muscle are controlled by two Ca2+-dependent negative feedback mechanisms. These mechanisms are based on (a) a protein phosphatase 2B-mediated dephosphorylation process, and (b) the interaction of intracellular Ca2+ with a single membrane-associated site that may reside on the channel protein itself.

Calcineurin↗

PubMed1996

A type 2A phosphatase-sensitive phosphorylation site controls modal gating of L-type Ca2+ channels in human vascular smooth-muscle cells.

The patch-clamp technique was employed to investigate phosphorylation/dephosphorylation-dependent modulation of L-type Ca2+ channels in smooth-muscle cells isolated from human umbilical vein. Okadaic acid, an inhibitor of phosphoprotein phosphatases type 1 (PP1) and 2A (PP2A), increased the probability of channels being in the open state (Po) in intact cells. This increase in Po was due mainly to promotion of long-lasting channel openings, i.e. promotion of 'mode 2' gating behaviour. Exposure of the cytoplasmic side of excised patches of membrane to the purified catalytic subunit of PP2A (PP2Ac) resulted in the opposite modulation of channel function. PP2Ac (0.2 unit/ml) reduced the Po of Ca2+ channels mainly via suppression of 'mode 2' gating. This effect of PP2Ac was completely prevented by 1 microM okadaic acid. The catalytic subunit of PPI (0.2 unit/ml), however, barely affected channel activity. Our results provide evidence for a PP2A-sensitive regulatory site that controls modal gating of L-type Ca2+ channels in smooth muscle.

Calcium Channels↗

PubMed1996

Inhibition of a store-operated Ca2+ entry pathway in human endothelial cells by the isoquinoline derivative LOE 908.

1. The novel cation channel blocker, LOE 908, was tested for its effects on Ca2+ entry and membrane currents activated by depletion of intracellular Ca2+ stores in human endothelial cells. 2. LOE 908 inhibited store-operated Ca2+ entry induced by direct depletion of Ca2+ stores with 100 nM thapsigargin or 100 nM ionomycin with an EC50 of 2 microM and 4 microM, respectively. 3. LOE 908 did not affect thapsigargin- or ionomycin-induced Ca2+ release from intracellular stores up to concentrations of 3 microM. 4. LOE 908 reversibly suppressed thapsigargin- as well as ionomycin-induced whole-cell membrane currents. 5. The LOE 908-sensitive membrane conductance corresponded to a cation permeability of 5.5 and 6.9 fold selectivity for Ca2+ over K+ in the presence of thapsigargin and ionomycin, respectively. 6. Our results suggest that the isoquinoline, LOE 908 is a novel, potent inhibitor of the store-operated (capacitive) Ca2+ entry pathway in endothelial cells.

Acetamides↗

PubMed1995

Basal dephosphorylation controls slow gating of L-type Ca2+ channels in human vascular smooth muscle.

The role of cellular phosphatase activity in regulation of smooth muscle L-type Ca2+ channels was investigated using tautomycin, a potent and specific inhibitor of serin/threonin phosphatases type 1 and 2A. Tautomycin (1-100 nM) inhibited Ca2+ channel activity in smooth muscle cells isolated from human umbilical vein. Tautomycin-induced inhibition of Ca2+ channel activity was due to a reduction of channel availability which originated mainly from prolongation of the lifetime of unavailable states of the channel. Pretreatment of smooth muscle cells with the protein kinase inhibitor H-7 (10 microM) prevented the inhibitory effect of tautomycin. Our results suggest modulation of slow gating between available and unavailable states as a mechanism of phosphorylation-dependent down-regulation of Ca2+ channels in vascular smooth muscle.

Antifungal Agents↗

PubMed1995

Action of calpastatin in prevention of cardiac L-type Ca2+ channel run-down cannot be mimicked by synthetic calpain inhibitors.

Activity of L-type Ca2+ channels in a membrane patch disappears rapidly when the patch is excised from the cell into an artificial solution. This channel run-down observed in isolated membrane patches can however, be prevented by application of calpastatin, an endogenous protease inhibitor, and ATP. The high specificity of calpastatin for the protease calpain would clearly point to a participation of calpain activity in the run-down of Ca2+ channels. In an attempt to examine a possible involvement of calpain, three synthetic and rather specific calpain inhibitors were substituted for calpastatin. One of these inhibitors chosen for its membrane permeability in addition allowed calpain activity to be inhibited even before patch excision. The potency of these compounds in inhibiting calpain, specifically mu- and m-calpain, was first determined in a biochemical assay and then compared with their efficacy in preventing Ca2+ channel run-down. Surprisingly, calpastatin was least effective in calpain inhibition but by far the most potent in prevention of Ca2+ channel run-down. In addition run-down of Ca2+ channel activity was examined for its reversibility, which would not be expected upon involvement of a proteolytic process. However, Ca2+ channel activity clearly recovered after run-down by application of calpastatin. In contrast, synthetic calpain inhibitors were unable to reverse Ca2+ channel run-down. These results indicate that proteolysis might only be partially responsible for channel run-down and suggest an as yet unidentified function for calpastatin beyond its inhibitory action on calpain in the regulation of Ca2+ channel activity.

Adenosine Triphosphate↗

PubMed1995

Trypsin increases availability and open probability of cardiac L-type Ca2+ channels without affecting inactivation induced by Ca2+.

The patch-clamp technique was employed to investigate the response of single L-type Ca2+ channels to the protease trypsin applied to the intracellular face of excised membrane patches from guinea pig ventricular myocytes. Calpastatin and ATP were used to prevent run-down of Ca2+ channel activity monitored with 96 mM Ba2+ as charge carrier in the presence of 2.5 microM (-)-BAYK 8644. Upon application of trypsin (100 micrograms/ml) channel activity was enhanced fourfold and remained elevated upon removal of trypsin, as expected of a proteolytic, irreversible modification. The trypsin effect was not mediated by a proteolytic activation of protein kinases, as evidenced by the insensitivity of this effect to protein kinase inhibitors. Trypsin-modified Ca2+ channels exhibited the usual run-down phanomenon upon removal of calpastatin and ATP. In ensemble average currents trypsin-induced changes of channel function are apparent as a threefold increase in peak current and a reduction in current inactivation. At the single channel level these effects were based on about a twofold increase in both Ca2+ channels' availability and open probability. Neither the actual number of channels in the patch nor their unitary conductance as well as reversal potential was changed by trypsin. The Ca(2+)-induced inactivation was not impaired, as judged by a comparable sensitivity of trypsin-modified Ca2+ channels to intracellular Ca2+. Similarly, trypsin treatment did not affect the sensitivity of Ca2+ channels to phenylalkylmine inhibition. The observed alterations in channel function are discussed in terms of possible structural correlates.

Adenosine Triphosphate↗

PubMed1995

Blockade of capacitive Ca2+ influx by Cl- channel blockers inhibits secretion from rat mucosal-type mast cells.

Whole-cell patch-clamp recordings of membrane currents were performed in combination with measurements of mediator secretion from mucosal-type mast cells (rat basophilic leukemia cells, subline 2H3), to determine the involvement of membrane conductances induced upon depletion of intracellular Ca2+ stores. In patch-clamp experiments, ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid-induced depletion of internal Ca2+ stores led to activation of two distinct membrane conductances, a Ca2+ current and a Cl- current. The Ca2+ current was blocked by 100 microM La3+, which did not affect the Cl- current. In contrast, 500 microM 4,4'-diisothiocyanato-2,2'-disulfonic acid produced selective blocked of the Cl- current. Remarkably, the Cl- channel blockers 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), niflumic acid, and N-phenylanthranilic acid (NPAA) inhibited not only the Cl- current but also the Ca2+ current. IC50 values for the blockade of the Ca2+ inward current by NPPB, niflumic acid, and NPAA were determined to be 23, 150, and 190 microM, respectively. In secretion experiments, thapsigargin-induced depletion of internal Ca2+ stores stimulated serotonin release, which was found to be strictly dependent on extracellular Ca2+. In the presence of 100 microM La3+ secretion was almost completely inhibited. In contrast, only 50% of secretion was suppressed by 500 microM 4,4'-diisothiocyanato-2,2'-disulfonic acid, which fully blocked the Cl- current without affecting Ca2+ influx, as monitored by electrophysiological experiments. The other Cl- channel blockers produced a very different pattern for the inhibitory dose dependence of secretion, with IC50 values for NPPB, niflumic acid, and NPAA of 23, 60, and 180 microM, respectively. Taken together, these findings suggest that Ca2+ store depletion leads to concomitant activation of Cl- and Ca2+ currents. Blockade of the latter is apparently an additional mode of action for diarylaminocarboxylate-type Cl- channel blockers inhibiting mast cell secretory responses.

Animals↗

PubMed1993

The benzazepine/benzothiazepine binding domain of the cardiac L-type Ca2+ channel is accessible only from the extracellular side.

The whole-cell tight seal recording technique was used to investigate location of benzothiazepine binding site of the cardiac L-type Ca2+ channel. For this we utilized a permanently charged compound, SQ 32.428, out of a series of benzazepine drugs which have been characterized as competitive inhibitors of diltiazem binding. The non-permanently charged derivative SQ 32.910 was initially tested to electrophysiologically establish Ca2+ antagonistic properties of benzazepines. Upon extracellular application, either compound was able to completely block Ca2+ currents. At a stimulating frequency of 0.2 Hz IC50 concentrations of SQ 32.910 and SQ 32.428 were determined as 35 nM and 15 microM, respectively. Intracellular application of SQ 32.428 was then compared to control experiments in the absence of drug. Initially, adequate drug dialysis was confirmed with 100 microM D890, which produced a progressive inhibition of Ca2+ currents within 10 min after whole-cell access. In contrast, internal application of 100 microM SQ 32.428 did not change time-course of Ca2+ currents compared to control run-down. These results show that the benzazepine/benzothiazepine binding domain of the cardiac L-type Ca2+ channel is accessible only from the extracellular side and therefore suggest an extracellular location on the alpha 1-subunit of the Ca2+ channel protein.

Animals↗

PubMed1993

Heterogeneity of L-type calcium channel alpha 1 subunits: stereoselective discrimination of different populations by the novel 1,4-dihydropyridine B 874-67.

The basic (pKa = 8.49) 1,4-dihydropyridine B 874-67 [[3-(C1R,2S)- 2-methylamino-1-phenylpropyl]-5-methyl-1,4-dihydro-2,6-dimethyl-(4R)-4-( 3-nitrophenyl)pyridine-3,5-dicarboxylate hydrochloride] has unique properties; it can discriminate two populations of alpha 1 subunits in 1,4-dihydropyridine-sensitive calcium channels labeled with the neutral 1,4-dihydropyridine (+)-[3H]PN 200-110. The two populations, which occur in proportions of approximately 2:1 in rabbit skeletal muscle membranes and highly purified calcium channel preparations, differ approximately 20-fold in their affinity. The corresponding diastereomer, B 874-66, and other 1,4-dihydropyridines (neutral, basic, or permanently charged) do not share this property. The two populations were observed at 2 degrees, 22 degrees, and 37 degrees in similar proportions. Heterogeneity was also observed for guinea pig heart membrane calcium channels labeled with (+)-[3H]PN 200-110. Heterotropic allosteric regulators, Ca2+, and Mg2+, but not Ba2+ and Ni2+, abolished the discriminatory activity of B 874-67 at 2 degrees and 22 degrees, regardless of whether binding of the neutral 1,4-dihydropyridine was stimulated or inhibited. It is proposed that the two alpha 1 subunit populations differ with respect to their 1,4-dihydropyridine binding domain. The structural basis for the two populations is unclear but may relate to the functional heterogeneity of membrane-bound and highly purified calcium channel preparations previously observed by others.

Allosteric Regulation↗

PubMed1992

Potent block of Cl- channels by antiallergic drugs.

Two antiallergic drugs, cromolyn and RU 31156, were examined for their potency to block intermediate-conductance Cl- channels. For this, single Cl- channel activities of mucosal-type mast cells (RBL-2H3) and alternatively, of colonic carcinoma cells (HT29) were monitored employing the patch-clamp technique in the inside-out patch configuration. Here we show that intermediate-conductance Cl- channels of either cell type were blocked by both compounds. Cl- channel inhibition occurred by a slow rather than a flickering block and with a Hill coefficient around 1. RU 31156 was about one order of magnitude more potent (IC50 about 1 microM) than cromolyn. The IC50 values of each compound were not significantly different (p < 0.01) in the two cell types. Our data provide evidence for two novel Cl- channel blockers, which may be of further use in Cl- channel characterization as well as purification.

Animals↗

PubMed1992

Activity of cardiac L-type Ca2+ channels is sensitive to cytoplasmic calcium.

Ca(2+)-induced inactivation of L-type Ca2+ channels is proposed as an important negative feedback mechanism regulating Ca2+ entry. Here, for the first time, evidence for modification of heart L-type Ca2+ channel activity by cytoplasmic calcium is provided from excised inside-out membrane patches. Ba2+ currents through cardiac L-type Ca2+ channels exhibited only modest inactivation in the absence of cytoplasmic Ca2+. Elevation of cytoplasmic Ca2+ to micromolar concentrations strikingly affected L-type Ca2+ channel activity as evaluated from ensemble average Ba2+ currents. Inactivation was markedly increased concomitant with a reduction of peak inward current, which was almost completely eliminated at about 15 microM cytoplasmic Ca2+ concentration. Half maximal suppression of Ba2+ currents was observed at 2.3 microM Ca2+. The observed modifications of L-type Ca2+ channel activity show that cytoplasmic Ca2+ induces channel closure. Below 4 microM Ca2+, channels can be reversibly reactivated during repetitive depolarizations, while at high Ca2+ concentrations (approximately 15 microM) most Ca2+ channels reside in a closed state. This may allow for a delicate regulation of Ca2+ entry, and consequently of heart contraction.

Animals↗

PubMed1992

The dihydropyridine niguldipine inhibits T-type Ca2+ currents in atrial myocytes.

The whole-cell tight seal recording technique was used to investigate the effects of niguldipine, a novel dihydropyridine, on Ca2+ currents in guinea pig atrial cells. Ca2+ currents were separated into T-type and L-type components by an appropriate voltage protocol. Extracellular application of 1 microM (+/-)-niguldipine (NIG) resulted in a pronounced blockade of both T-type (to 20 +/- 10% of control, n = 5) and L-type Ca2+ currents (to 28 +/- 12% of control, n = 5). Current to voltage relationships clearly showed that both Ca2+ currents were blocked over the whole voltage range examined (-60 to +40 mV). The inhibitory effect of niguldipine on T-type Ca2+ currents was found to be voltage-dependent, i.e. prolonged hyperpolarization to -90 mV led to a partial and transient removal of NIG block. The IC50 for T-type Ca2+ current inhibition by (+/-)-NIG was determined as 0.18 microM. NIG action is stereospecific. (+)-niguldipine was found to be more potent than (-)-niguldipine in blocking both Ca2+ currents. This study demonstrates the Ca2+ antagonistic action of the dihydropyridine NIG, which may not discriminate between T- and L-type Ca2+ channels.

Animals↗

PubMed1992

Membrane potential modulates release of tumor necrosis factor in lipopolysaccharide-stimulated mouse macrophages.

Lipopolysaccharide (LPS)-mediated synthesis of macrophage gene products such as tumor necrosis factor (TNF) is controlled by different signaling pathways. We investigated intracellular free Ca2+ (Ca2+ic) and the membrane potential as early cellular responses to LPS and their role in the synthesis and release of TNF. In peritoneal macrophages and in the RAW 269 mouse macrophage cell line, LPS and its biologically active moiety lipid A stimulated TNF synthesis but exerted no significant effects on these early cellular responses using Fura-2/Indo-1 to measure Ca2+ic and bis-oxonol, as well as the patch-clamp technique to monitor membrane potential. In contrast, the platelet-activating factor transiently induced both an increase in Ca2+ic and cell membrane depolarization but no significant TNF release. Increased extracellular K+ concentrations or K(+)-channel blockers, such as quinine, tetraethylammonium, or barium chloride, inhibited the LPS-stimulated release of TNF alpha, as well as the accumulation of cell-associated TNF alpha as found by enzyme-linked immunosorbent assay analysis, but did not inhibit TNF alpha mRNA accumulation. Concentrations of quinine (greater than 125 microM) or of enhanced extracellular K+ (25-85 mM) required to inhibit TNF production both significantly depolarized macrophages. These results indicate a lack of ion transport activities as early cellular responses of macrophages to LPS but suggest an important regulatory role of the membrane potential on the posttranscriptional synthesis and release of TNF in macrophages.

Animals↗

PubMed1991

Immunologically activated chloride channels involved in degranulation of rat mucosal mast cells.

Crosslinking of type I Fc epsilon receptors (Fc epsilon RI) on the surface of basophils or mast cells initiates a cascade of processes leading to the secretion of inflammatory mediators. We report here a correlation between mediator secretion and the activation of Cl- channels in rat mucosal-type mast cells (line RBL-2H3). Stimulation of RBL cells by either IgE and antigen or by a monoclonal antibody specific for the Fc epsilon RI, resulted in the activation of Cl- ion channels as detected by the patch-clamp technique. Channel activation occurred slowly, within minutes after stimulation. The channel has a slope conductance of 32 pS at potentials between 0 and -100 mV, and an increasing open-state probability with increasing depolarization. Activation of apparently the same Cl- channels could be mimicked without stimulation by isolating inside-out membrane patches in tyrode solution. Parallel inhibition of both Cl- channel activity and mediator secretion, as monitored by serotonin release, was observed by two compounds, the Cl- channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and the anti-allergic drug cromolyn. NPPB inhibited both the antigen-induced Cl- current and the serotonin release, where half-maximal inhibition occurred at similar doses, at 52 microM and 77 microM, respectively. The drug cromolyn, recently found to inhibit immunologically induced mediator secretion from RBL cells upon intracellular application, also blocks Cl- channels (IC50 = 15 microM) when applied to the cytoplasmic side of an inside-out membrane patch. The observed Cl- channel activation upon immunological stimulation and the parallel inhibition of channel current and of serotonin release suggests a functional role for this Cl- channel in mediator secretion from the mast cells studied.

Animals↗

PubMed1991

Calpastatin and nucleotides stabilize cardiac calcium channel activity in excised patches.

The activity of single L-type Ca2+ channels is rapidly lost (run-down) when contact between the membrane and cytosol is interrupted. We have now achieved the stabilization of cardiac Ca2+ channel activity of guinea-pig ventricular myocytes by using either cytosol or defined components added to excised patches. The endogenous protease inhibitor, calpastatin, together with nucleotides, ATP + GTP, was found to prevent run-down as effectively as cardiac cytosolic solution. These results suggest the involvement of proteolysis by calpain in run-down of channel activity and enable the study of cardiac Ca2+ channel regulation with free access to both sides of the membrane.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1990

Heparin binds with high affinity to voltage-dependent L-type Ca2+ channels. Evidence for an agonistic action.

Heparin and related polyanions are a new class of compounds interacting with 1,4-dihydropyridine-sensitive L-type Ca2+ channels in a tissue-specific manner. Labeling of membrane-bound Ca2+ channels in rabbit skeletal muscle transverse tubules at the phenylalkylamine, benzothiazepine, and 1,4-dihydropyridine-selective domains was inhibited reversibly by a noncompetitive mechanism as shown by equilibrium saturation analysis and kinetic studies. (+)-cis-diltiazem but not (-)-cis-diltiazem reduced the inhibitory potency of heparin for 1,4-dihydropyridines. Antagonistic but not agonistic 1,4-dihydropyridines reversed heparin inhibition at the benzothiazepine site. Heparin forms a tight complex with the purified Ca2+ channel which is highly sensitive with respect to heparin inhibition (IC50 value: 0.05 microgram/ml) of 1,4-dihydropyridine binding. Reconstituted channel complexes have completely lost 1,4-dihydropyridine binding-inhibition by heparin and are not retained by lectin or heparin affinity columns. In whole cell patch clamp experiments with guinea-pig cardiac myocytes heparin increased the current through L-type Ca2+ channels when applied extracellulary. Synthetic peptides (representing putative heparin binding domains) which were derived from the rabbit skeletal muscle alpha 1-subunit reversed the inhibitory effects of heparin on 1,4-dihydropyridine receptors. Reversal for a peptide representing an extracellular domain occurred by an apparently competitive mechanism. It is suggested that heparin and related polyanions may interact with an evolutionary conserved cluster of basic amino acids in the large putative extracellular domain connecting the fifth and sixth putative transmembrane segment in the first motif of the ionic pore-forming alpha 1-subunit from skeletal muscle.

Allosteric Regulation↗

PubMed1989

Tolerance to nitroglycerin is caused by reduced guanylate cyclase activation.

Since the diminished vasodilatation characterizing tolerance to organic nitrates is associated with lower rises in 3', 5'-cyclic guanosine monophosphate (cGMP) levels, the possibility that nitrovasodilators desensitized guanylate cyclase (GC) when pre-incubated with coronary supernatants was studied. In the absence of cysteine, pre-incubation with nitroglycerin (NG) decreased GC-activity during subsequent incubation to 24 +/- 7% of control values, whereas six other nitrovasodilators had much smaller effects. When cysteine was present during pre-incubation, NG-stimulation of GC remained significantly higher (59 +/- 3%; P less than 0.05), whereas the effects of other nitrovasodilators were not significantly changed. We also found that GC-activity, when reduced by pre-incubation with NG could only be restored by readdition of native coronary supernatant, suggesting that the enzyme became inactivated. NG pre-incubation of GC (in contrast to coronary strips) almost completely abolished the direct and thiol-independent stimulatory effect of 3-morpholinosydnonimine (SIN-1) down to 4.5 +/- 0.2%, whereas pre-incubation with other nitrovasodilators reduced the stimulatory response to SIN-1 to only 59 to 98%. Increasing concentrations of NG during pre-incubation dose-dependently (IC50 = 0.13 mM) reduced the activating effect of SIN-1 during incubation. There was also a time dependence in NG-induced inactivation of GC which followed first order kinetics with a calculated half life of 2.5 min in the absence of a thiol. The latter was increased to 4.0 or 19.2 min, respectively, when glutathione or cysteine-methylester were present during pre-incubation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗