Binding of [3H]quinuclidinyl benzilate to intestinal mucus: An artifact in identification of epithelial cell muscarinic receptors

The widely used muscarinic receptor ligand [³H]quinuclidinyl benzilate ([³H]QNB) was found to bind in a site-specific but artifactual manner to rat intestinal mucus, obscuring specific binding to muscarinic receptors on intestinal epithelial cells. Atropine inhibited [³H]QNB binding to mucus with an apparent IC₅₀ of 2.1 × 10^?7 M, compared to an IC₅₀ of 1.4 × 10^?8 M obtained with a homogenate of intestinal epithelial cells. Unlabeled QNB also inhibited binding of [³H]QNB to mucus but the apparent IC₅₀(4 × 10^?7 M) was about 300-fold greater than the IC₅₀ determined with a control tissue, heart muscle (IC₅₀, 1.2 × 10^?9M). [³H]QNB binding was saturable over the concentration range of 1–7 nM in the heart, with an apparent k_D of 0.76 nM. As expected from the high IC₅₀ for QNB in the mucus binding experiments, binding to mucus was not saturable over the 1–15 nM concentration range. Based on pH profiles and temperature dependency of binding, it seems unlikely that mucin, the primary component of mucus, was responsible for [³H]QNB binding to the mucus. The findings have implications for studies which involve binding of [³H]QNB in particular and other ligands in general to mucus-secreting epithelial tissues.     

A mechanism of resistance to methotrexate NADPH but not NADH stimulation of methotrexate binding to dihydrofolate reductase

Characteristics of methotrexate (MTX) inhibition of dihydrofolic acid reductase (DHFR) enzyme activity and the effects of NADPH and NADH on enzyme-drug interaction were studied. Two highly sensitive assay procedures were used. The first utilized tritium-labeled MTX to measure direct binding properties of the enzyme and the second utilized tritium-labeled dihydrofolate (H₂PteGlu) and folate (PteGlu) to analyze kinetics of reduction of these substrates. NADPH was found to enhance DHFR binding of MTX (K_d = 2.6 × 1O^?11 M), whereas NADH was found to have no effect (K_d = 3.7 × 10^?9 M). However, NADH proved to be a good substrate for folate reduction compared to NADPH, especially in low salt buffer. The observation that NADH supports the reduction of folate and dihydrofolate but not MTX binding suggests that natural resistance to MTX could exist if NADH replaces NADPH as the main cofactor for DHFR.     

Inhibition of human lens aldose reductase by flavonoids,sulindac and indomethacin

The inhibition of human lens aldose reductase by flavonoids has been studied. Quercetin, the major pentahydroxyflavone, was observed to inhibit human lens aldose reductase by 50% at a concentration of 5 × 10^?6 M. The inhibitory activity of its 3-O-glucoside was similar to that of the parent aglycon. Glycosidation with l-sugar (quercitrin and guaijaverin), however, inproved the inhibitory activity (the IC₅₀ values being 1 × 10^?6 M and 2.5 × 10^?6 M respectively). The improvement in inhibitory activity with glycosidation with l-sugar was also apparent from the high inhibitory activity of myricitrin as compared to myricetin, although the improvement in this case of hexahydroxy flavone glycosidation was significantly less than in the case of penthahydroxy flavone glycosidation. The structure-activity relationship observed for human lens enzyme was similar to that reported previously for rat lens enzyme. Inhibitory activity on the whole however, was lower with human lens enzyme. Some known inhibitors of cyclo-oxygenase such as indomethacin, aspirin and sulindac also inhibited human lens aldose reductase. Thus, an inhibitor of one of the enzymes may actually inhibit both and. when administered, may exert mixed physiological effects.     

Anticholinesterase activity of the unsymmetric bisquaternary 6-aminoquinoline salt NSC-176319

The anticholinesterase activity of the unsymmetric bisquaternary 6-aminoquinoline salt NSC-176319 (QB) was studied in vitro. QB proved to be a noncompetitive inhibitor of both acetylcholinesterase (or true cholinesterase) and butyrylcholinesterase (or pseudocholinesterase) having a K₁ = 0.5 × 10^?6M for acetylcholinesterase and 1.5 × 10^6?M for butyrylcholinesterase. Further, QB inhibited esterase activity of murine plasma in a noncompetitive manner (K_i = 4.2 × 10^?6M). The inhibition was instantaneous in onset and did not diminish with prolonged incubation of the drug and enzyme. All mice treated intravenously with 2 mg QB/kg died within 5 min. Prior to death, mice developed severe parasympathomimetic effects and convulsions. Although the parasympathomimetic effects were diminished by atropine sulfate pretreatment, death could only be prevented by barbiturate anesthesia.     

1-methyl substituent and stereochemical effects of 2-phenylcyclopropylamines on the inhibition of rat brain mitochondrial monoamine oxidase A and B

(E)-2-Phenylcyclopropylamine ((E)-TCP), (Z)-2-phenylcyclopropylamine ((Z)-TCP), (E)-1-methyl-2-phenylcyclopropylamine ((E)-MTCP), and (Z)-1-methyl-2-phenylcyclopropylamine ((Z)-MTCP) were synthesized and used to determine to what extent 1-methyl substitution and stereochemistry of 2-phenylcyclopropylamines affect inhibition of monoamine oxidase (MAO). Inhibition of rat brain mitochondrial MAO-A and B by the compounds were measured using serotonin and benzylamine as the substrate, respectively and IC₅₀ values obtained with 95% confidence limits by the method of computation. For the inhibition of MAO-A, (E)-MTCP (IC₅₀=6.2×10^?8M) was found to be 37 times more potent than (Z)-MTCP (IC₅₀=2.3×10^?6 M), whereas the activity of (E)-TCP (IC₅₀=2.9×10^?7 M) was slightly less than that of (Z)-TCP (IC₅₀=2.3×10^?7 M). Similarly, for the inhibition of MAO-B, (E)-MTCP (IC₅₀=6.3×10^?8 M) was 7 times more potent than (Z)-MTCP (IC₅₀=4.7×10^?7 M) and (E)-TCP (IC₅₀=7.8×10^?8 M), 0.6 times as potent as (Z)-TCP (IC₅₀=4.4×10^?8 M). The results suggested that while without 1-methyl group, potency of a (Z)-isomer was comparable to that of (E)-isomer, the methyl group in its (Z)-position was very unfavorable to the inhibition of MAO and that in its (E)-position, the methyl group contributed positively to the potency as found by the fact that (E)-MTCP was 1–5 times more potent than (E)-TCP. In view of the selective inhibition of MAO-A or B, all compounds elicited 4–10 times higher preference for the inhibition of MAO-B over MAO-A and 1-methyl substitution as well as the stereochemical factors did not significantly influence the selectivity.     

Inhibitory activities of baicalin against renin and angiotensin-converting enzyme

Context: Baicalin has been characterized as the active compound and quality control marker in Scutellaria baicalensis Georgi, traditionally used as a hypotensive herb.

Objectives: To investigate the inhibitory activities of baicalin against renin and angiotensin-I converting enzyme (ACE) and their molecule mechanism of interactions.

Methods: The fluorescence method using renin substrate 1(R-2932) and the spectroscopy method by Cushman were used to determine renin and ACE activities, respectively. The fluorescence quench techniques were used to characterize their interactions.

Results: The results showed that baicalin inhibited renin activity with an IC₅₀ value of 120.36 µM and inhibited ACE activity with an IC₅₀ value of 2.24?mM in vitro. The fluorescence emission of both renin and ACE were efficiently quenched by baicalin and a complete quenching was achieved at a high concentration of baicalin. Furthermore, baicalin was more effective in quenching the fluorescence of renin (K_SV?=?60?×?10³ M^?1) than ACE (K_SV?=?17.1?×?10³ M^?1). The quenching of fluorescence of renin and ACE involved static interactions, which was characterized by the formation of quencher–enzyme complex. The baicalin–renin complex formed through three-sites binding including the active site with a binding constant of 796.15?×?10¹³ M^?1, but there was only one binding site for the baicalin–ACE complex with a much smaller binding constant of 6.8?×?10⁵ M^?1.

Conclusion: The inhibition activity of baicalin against renin was a result of the formation of stable complex through multisites binding including the active site, which could explain the higher inhibitory efficiency.     

Vascular and cardiac effects of HP 406 and nifedipine in vitro and in conscious spontaneously hypertensive rats and renal hypertensive dogs

The cardiovascular effects of the calcium antagonists HP 406 and nifedipine were assessed in a series of in vitro and in vivo studies. HP 406 was found to display moderate affinity for dihydropyridine binding sites, as determined by the inhibition of [³H]-nitrendipine binding on rat ventricular membranes. HP 406 was found to have an IC₅₀ of 1.3 × 10^?7 M as compared to nifedipine with an IC₅₀ of 7.0 × 10^?9 M. Additional in vitro studies were conducted with isolated spontaneously contracting and electrically paced guinea pig atria and rabbit aortic rings. HP 406 was found to have minimal effects on cardiac chronotropic and inotropic activity in isolated guinea pig atria. In contrast, nifedipine caused significant dose-related reductions in heart rate and myocardial contractile force. Rabbit aortic rings were precontracted with potassium chloride (KCI) or norepinephrine (NE), and HP 406 or nifedipine was added to tissue baths in a cumulative dose range. HP 406 and nifedipine both caused dose-related relaxation of KCI- and NE-contracted rings; however, the IC₅₀'s against KCI were found to be significantly lower than those against NE for both compounds. The IC₅₀'s against NE were found to be 4.8 × 10^?8 M and 5.9 × 10^?8 M for HP 406 and nifedipine, respectively. The IC₅₀'s against NE were 1.9 × 10^?5 M for HP 406 and 4.4 × 10^?5M for nifedipine. Finally, the antihypertensive activity of HP 406 and nifedipine were compared in the spontaneously hypertensive rat (SHR) and renal hypertensive dog. Both compounds produced significant reductions in mean arterial pressure at 1 mgikg p.o. in the SHR and at 3 mgikg p.o. in the renal hypertensive dog. Nifedipine was also found to cause a significantly greater tachycardia compared to HP 406. The results of this study show that HP 406 is a potent antihypertensive agent with minimal direct or reflex-mediated chronotropic effects. The mechanism of this antihypertensive activity appears to result from calcium-channel antagonism and vascular dilation.     

Interactions of D600 (methoxyverapamil) and local anesthetics with rat brain α-adrenergic and muscarinic receptors

D600 (methoxyverapamil) was found to inhibit the specific binding assayed in rat brain homogenates of the antagonist agents [³H]WB 4101 and [³H]QNB to the α-adrenergic and muscarinic receptors respectively. The IC₅₀ concentrations of D600 in standard binding experiments were 1.7 × 10_?6 M and 1.4 × 10 _?5M, with calculated k₁ values of 0.98 × 10_?6 M and 8.83 × 10_?6M.Scatchard analyses showed these inhibitions to be competitive. Lidocaine and tetracaine also inhibited radioligand binding to these receptors, with K₁ values of 5.25 × 10^?4M and 4.85 × 10^?5 M for the α-receptor and 8.2 × 10^t-5 M and 6.94 × 10^?6 M for the muscarinic receptor; these inhibitions also appeared to be competitive. Increasing the Ca²⁺ concentration in the assays to 10 mM did not influence the effects of D600 or the anesthetics. Analyses of inhibitions of muscarinic receptor binding produced by D600 and lidocaine over a range of pH indicated that the inhibitory species of D600 is the uncharged form, whereas the charged form of lidocaine is inhibitory. Interactions of D600 and lidocaine with the agonist site on the muscarinic receptor were studied by measuring the effects of these agents on the displacement of [³H]QNB by the muscarinic agonist carbachol. Comparison of these results with a theoretical model indicates that carbachol, [³H]QNB, and D600 or lidocaine competitively displace one another at the same agonist site. The binding of labeled naloxone to the opiate receptor was also inhibited by D600, the IC₅₀g being 4 × 10^t?6 M. These inhibitory effects of D600 and the local anesthetics on different receptors suggest that these agents may act by a common mechanism, namely by perturbing membrane structures. These results suggest caution in interpreting experiments in which D600 and verapamil are used analytically as Ca antagonists to assess the involvement of Ca in a biological system.     

Paralytic activity of (des-Glu1)conotoxin GI analogs in the mouse diaphragm

A series of 20 peptide analogs of (des-Glu¹) conotoxin GI were prepared by solid phase synthesis. The peptides were tested for their abilities to inhibit contractions in the mouse-diaphragm-with-phrenic-nerve assay. (Des-Glu¹) conotoxin has an IC₅₀, of 2.7 × 10^?7 M in this assay. Results from this assay show that total loss of paralytic activity occurs when Pro is replaced by Gly, Tyr by D-Tyr, or Gly by D-Phe. In most cases loss or change in length of one of the disulfide rings eliminates paralytic activity except with compound 17, which is weakly active, IC₅₀= 7.0 × 10^?5 M. Replacement of the Cys¹-Cys⁶ disulfide bond with an amide bond (compound 9) greatly lowers paralytic activity, IC₅, = 3.7 × 10^?5 M.     

Biochemical pharmacology and toxicology of 8-azaadenosine alone and in combination with 2′-deoxycoformycin(pentostatin)

The toxicology and metabolism of 8-azaadenosine (8-azaAdo) were examined both as a single agent and in combination with the adenosine deaminase inhibitor, 2′-deoxycoformycin (dCF). The LD₁₀ (mice) for 8-azaAdo alone on a once daily for 5 days (q.d. × 5) schedule was 30mg·kg^?1·day^?1. When the animals were pretreated with 0.1 mg·kg^?1·day^?1 of dCF, the ld₁₀ dose was reduced to 10 mg·kg^?1·day^?1× 5. The major organ toxicity seen was hepatic. Bone marrow cellularity was only slightly altered at the ld₁₀ dose. 8-AzaAdo nucleotides were detected in the livers of treated mice as determined by high performance liquid chromatography. Further, after 2 hr of incubation, isolated rat hepatocytes accumulated 8-azaATP to levels of 2.2 μmoles/g of cells with 8-azaAdo (1 mM) alone and to 4.3 μmoles/g of cells when 8-azaAdo was used in combination with dCF (1 μg/ml). ATP levels decreased to below the limits of detection after 2 hr in cells treated with the combination. The replacement of cellular ATP by 8-azaATP may provide an explanation for the hepatotoxicity observed in the murine toxicology studies.     

The anti-tumor effect of folate-targeted liposome microbubbles loaded with oridonin as ultrasound-triggered tumor-targeted therapeutic carrier system

In this study, folate receptor (FR) targeted liposome microbubbles loaded with oridonin (ORI) (F-LMB-ORI), liposome loaded with ORI (L-ORI) and liposome microbubbles loaded with ORI (LMB-ORI) were prepared. In vitro release properties, cellular uptake and cytotoxicity in HepG-2 cells as well as in vivo antitumor effects in HepG-2 cells tumor-bearing mice of F-LMB-ORI, L-ORI and LMB-ORI were evaluated upon ultrasound exposure. Results showed cytotoxicity assay on F-LMB-ORI gave IC₅₀ of 0.508?±?0.018?µmol/mL on HepG-2 cells and LMB-ORI; L-ORI gave IC₅₀ of 2.424?±?0.116?µmol/mL, 3.031?±?0.122?µmol/mL in vitro, respectively. These drug delivery carriers were able to control the release of ORI. F-LMB-ORI exhibited higher binding to HepG-2 cells in comparison to LMB-ORI and L-ORI. F-LMB-ORI improved antitumor activity of ORI obviously in comparison to L-ORI, LMB-ORI under in vivo ultrasound. After the treatment for 14 d, the tumor inhibition ratio for F-LMB-ORI (the dose of ORI: 1.5?×?10^?2?g·kg^?1, once a day) was 87.6%, obviously higher than that of LMB-ORI group, L-ORI group and free ORI (the dose of ORI: 1.5?×?10^?2?g·kg^?1, once a day) which were 71.5%, 64.3% and 43.4%, respectively.     

No evidence for reverse trans-synaptic regulation of neuronal uptake by beta-adrenoceptors in kitten atria

Kitten atria incubated with [³H]noradrenaline, 1.18 × 10^?7 M for 10 min or 3 × 10^?9 M for 30 min, actively accumulated the amine. Final tissue tritium concentrations were 2–4-fold and 8–12-fold higher, respectively, than those of the incubation fluid. Uptake was consistently greater in right than in left atria. The β₁-adrenoceptor antagonist, practolol, 10^?6 or 10^?5 M, and the β₁- and β₂-adrenoceptor antagonist, propranolol, 5 × 10^?8, 5 × 10^?7or 5 × 10^?6 M, did not affect noradrenaline uptake. Reverse trans-synaptic regulation of neuronal noradrenaline uptake by β-adrenoceptors therefore does not appear to operate in kitten atria as has been reported for rat atria and other tissues.     

Paracetamol does not potentiate the acetylsalicylate inhibition of prostaglandin release from macrophages

Prostaglandin (PGE₂ and PGI₂) synthesis can be stimulated in macrophages by the tumour promotor 12-O-tetradecanoylphorbol-13-acetate (TPA) and inhibited by acetylsalicyclic acid (IC₅₀ = 8.2 × 10^?6 M) and paracetamol (IC₅₀ = 1.9 × 10^?4 M). When mixtures of both drugs were tested only an additive effect was observed. This result is in contrast to recent reports describing both potentiation of acetylsalicylate-induced PG synthesis inhibtion and enhancement of PG production by paracetamol.     

Electrophysiologic effects of encainide on acutely ischemic rabbit myocardial cells

The electrophysiologic effects of encainide were determined in normal and acutely ischemic (30 min) rabbit ventricular muscle cells. Encainide (10^?6, 5 × 10^?5 M) had no effect on resting potential (RP); 10^?6 encainide reduced overshoot and action potential (AP) amplitude of cells in normal left ventricles and cells in normal areas of ischemic ventricles. Encainide, 5 × 10^?6 M and 10^?5M, depressed V?_max and prolonged AP duration of normalcells. Surviving cells within ischemic areas displayed AP with reduced RP, overshoot, AP amplitude, V?_max and shorterned AP duration. All encainide concentrations reduced overshoot, AP amplitude and V?_max of depressed AP. Encainide's lengthening of AP duration was greater in cells within ischemic areas than in surrounding normal cells. Encainide (10^?6 M) prolonged effective refractory period and often AP in ischematic cells. Encanides also caused depression in membrane responsiveness. Encainide's differential effect upon AP may significantly contribute to its antiarrhythmic activity in ischemic heart disease.     

Absorption mechanism of oxymatrine in cultured Madin–Darby canine kidney cell monolayers

Context Oxymatrine (OMT) is beneficial to human health by exerting various biological effects. Objective To investigate the absorption mechanism of OMT and discover absorption enhancers using Madin–Darby canine kidney (MDCK) cell monolayers.

Materials and methods Concentration effects on the transport of OMT were measured in the range of 1.0?×?10^?5–1.0?×?10^?3 M in 2?h. Then, the effect of time, direction, temperature and pH on the transport of OMT at 10^?4 M was studied. Moreover, P_app of OMT was determined in the absence/presence of cyclosporine and surfactants at 100?μM to further confirm the relative transport mechanism.

Results The P_{app AP→BL} ranged from (3.040?±?0.23)?×?10^?6 to (3.697?±?0.19)?×?10^?6?cm/s as the concentration varied from 10^?5 to 10^?3 M. OMT showed similar P_app at 4 and 37?°C (p?>?0.05). Increasing the apical pH 7.4 and 8.0 resulted in P_app versus pH 5.0 (p?<?0.01). Furthermore, in the presence of cyclosporine and surfactants including sodium citrate, sodium dodecyl sulphate (SDS) and deoxysodium cholate, P_app was (0.318?±?0.033)?×?10^?5, (0.464?±?0.048)?×?10^?5, (0.897?±?0.115)?×?10^?5 and (1.341?±?0.122)?×?10^?5?cm/s, respectively. In the presence of surfactants, P_app significantly increased up to 1.5–4.3-fold (p?<?0.05).

Discussion and conclusion OMT transport across MDCK cell monolayers was by passive diffusion. Sodium citrate, SDS and deoxysodium cholate serve as excellent absorption enhancers which are useful for the related research improving the oral bioavailability of OMT.     

Mouse neuroblastoma clone N1E-115: A suitable model for studying the action of dopamine agonists on tyrosine hydroxylase activity

The DOPA-content in neuroblastoma clone N1E-115 is higher than the dopamine or noradrenaline content. Blockade of tyrosine hydroxylase by ifα-methyl-p-tyrosine (1 × 10 su?3 M) resulted in a decrease of cellular DOPA-content to 24.9% after 4 hr. The accumulation of DOPA in these cells which is probably due to limited activity of l-aromatic amino acid decarboxylase led us to use DOPA-content as a measure of tyrosine hydroxylase (TH) activity. Dopamine and especially apomorphine were effective at low concentrations (dopamine ic₅₀1 × 10^?5 M, apomorphine 2 × 10^?7 M); lisuride had no effect on TH-activity. The low effective dose of apomorphine and the failure of lisuride to influence TH-activity are comparable to the observations in striatal synaptosomal preparations and make the N1E-115 clone a suitable model for studying the mechanism of TH-regulation. However, since haloperidol (1 × 10^?5 M) did not reverse the apomorphine-induced blockade of TH, a receptor-mediated blockade of TH seems to be improbable.     

Interactions between tetraethylammonium and methohexitone at the chick neuromuscular junction: Experiments with the moving fluid electrode technique

The effect of methohexitone on the depolarization and contracture responses produced by tetraethylammonium (TEA), acetylcholine (ACh) and repetitive indirect stimulation were investigated, using the moving fluid electrode technique, in the chick biventer cervicis (BVC) nerve muscle preparation. TEA (4.8 × 10^?4?4.8 × 10^?2 M) produced contracture and depolarization responses which were concn-dependent. These responses were potentiated by methohexitone (8.8 × 10^?5 M). The mean ED₅₀S for the contracture responses in the control Krebs solution and with methohexitone were (mean ± S.E.M.) 6.5 ± 0.03 × 10^?3 M and 1.3 ± 0.04 × 10^?3 M (N = 6) respectively. The mean ED₅₀S for the depolarizations were (mean ± S.E.M.) 5.9 ± 0.1 × 10^?3 and 1.5 ± 0.06 × 10^?3 M (N = 6) respectively. ACh (5.5 × 10^?6?1.1 × 10^?2 M) produced contracture and depolarization responses which were concn-dependent. These responses were reduced by methohexitone (8.8 × 10^?5 M). The mean ED₅₀S for the contracture responses in the control Krebs solution and with methohexitone were (mean ± S.E.M. 2.4 ± 0.21 × 10^?4 and 2.3 ± 0.1 × 10^?3 M (N = 6) respectively. The mean (± S.E.M.) ED₅₀S for the depolarizations were 8.4 ± 0.33 × 10^?4 and 3.7 ± 0.14 × 10^?3 M (N = 6), respectively. Repetitive indirect stimulation, at 1–20 Hz, produced contraction and depolarization responses which were frequency-dependent. These responses were slightly potentiated by methohexitone (8.8 × 10^?5 M). The mean (± S.E.M.) frequency₅₀S for the contractions produced in the control Krebs solution and with methohexitone were 9.2 ± 0.1 and 8.5 ± 0.2 Hz (N = 6) respectively. The mean frequency₅₀S for the depolarizations were (mean ± S.E.M.) 7.2 ± 0.1 and 5.8 ± 0.19 Hz (N = 6) respectively. It is concluded that TEA may have a direct post-synaptic action, in addition to releasing ACh from the presynaptic nerve terminals. TEA produces more contracture tension than does ACh for a given level of membrane depolarization. Methohexitone, non-competitively, reduces the responses produced by applied ACh whereas it potentiates those produced by TEA and repetitive nerve stimulation.     

Inhibition of serine proteases by steroids

Proteolysis of ¹⁴C-labeled globin, as well as the hydrolysis of the specific substrate benzoyl tyrosine ethyl ester, by purified bovine chymotrypsin was found to be inhibited by several steroid hormones. The inhibition of chymotrypsin by the steroids was of a competitive nature, with K_i values of 9.9 × 10^?5 M for triamcinolone (9-fluoro-11β, 16α,17,21-tetrahydroxy-1,4-pregnadiene-3,20-dione), 1.6 × 10^?4 M for cortisol (11β,17α,21-trihydroxypregn-4-ene-3,20-dione), 3.7 × 10^?4 M for testosterone (17β-hydroxy-4-androsten-3-one), 5.0 × 10^?4 M for dexamethasone (9-fluoro-11β,17,21-trihydroxy-16α-methyl-1,4-pregnadiene-3,20-dione), and 1.0 × 10^?4 M for epicortisol (11α,17,21-trihydroxy-4-pregnene-3,20-dione). The activity of purified bovine trypsin on its specific substrate, TAME (tosyl arginine methyl ester), also showed a similar pattern of inhibition by steroids. Both chymotrypsin and trypsin were found to bind ³H-labeled dexamethasone and cortisol. This binding was markedly inhibited by the general protease inhibitor, PMSF (phenylmethanesulfonyl fluoride), whereas the chymotrypsin-specific inhibitor, TPCK (l-[1-tosyl-amido-2-phenyl]ethylchloromethyl ketone), inhibited only the steroid binding to chymotrypsin but not to trypsin. These observations indicate that serine proteases recognize steroid hormones in a fashion similar to the recognition of their specific substrates and that the steroids inhibit activity of these enzymes at their binding sites.     

Effects of Calcium Blockers on the Cytosolic Calcium,H2O2 Production and Elastase Release in Human Neutrophils

Abstract: Activated neutrophils are assumed to be one plausible cause of tissue injury in the ischaemic and reperfused myocardium. We studied the inhibitory effects of the calcium antagonists felodipine, nimodipine and verapamil on human neutrophil activation in order to elucidate the mechanisms underlying their myocardioprotective effects and to determine whether calcium antagonists with different chemical structures vary in their effect on neutrophil activation. Neutrophils were stimulated with formyl-Met-Leu-Phe (0.1 μM) or by phorbol myristate acetate (0.16 μM), and the rise in cytosolic calcium and the H₂O₂ production were determined. For felodipine, the inhibitory effect on granulocyte elastase release was also studied. The calcium antagonists reduced formyl-Met-Leu-Phe and phorbol myristate acetate-induced neutrophil activation in a concentration-dependent manner, the order of potency being: felodipine >nimodipine >verapamil. For felodipine, the IC₅₀ (concentration causing 50% reduction) values were 3×10^?6 and 2×10^?6 M for the formyl-Met-Leu-Phe-induced cytosolic calcium increase and H₂O₂ production, respectively. The IC₅₀ -value for the phorbol myristate acetate-induced cytosolic calcium increase was 6×10^?6 and for H₂O₂ production 4×10^?6 M. For formyl-Met-Leu-Phe-induced granulocyte elastase release, the IC₅₀-value was 5×10^?6 M. The inhibitory effect of felodipine on the phorbol myristate acetate-induced granulocyte elastase release did not exceed 50%. Nimodipine was a less potent inhibitor than felodipine for both formyl-Met-Leu-Phe- and phorbol myristate acetate-induced cell activities. Verapamil was even less potent than the other two agents. The present study demonstrates that felodipine potentially suppresses neutrophil activation at micromolar concentrations. However, this observation should not be directly extrapolated to explain the tissue protection by the compounds without evidence of profound local accumulation.     

A new antiinflammatory compound, timegadine (N-cyclohexyl-N"-4-[2-methylquinolyl]-N'-2-thiazolylguanidine), which inhibits both prostaglandin and 12-hydroxyeicosatetraenoic acid (12-HETE) formation

Timegadine (N-cyclohexyl-N′′-4-[2-methylquinolyl]-N′-2-thiazolylguanidine), a new antiinflammatory agent, was found to be a potent, competitive inhibitor of prostaglandin synthetase derived from a variety of tissues, including bovine seminal vesicles, rabbit renal papillae, rabbit lung, rabbit platelets and rat brain. The concentration of timegadine required to obtain 50% inhibition (IC₅₀) varied from 5 × 10^?9M (washed rabbit platelets) to 2 × 10^?5M (rat brain). Timegadine was also found to be an inhibitor of lipoxygenase activity in the cytosol fraction of horse platelet homogenates, and in washed rabbit platelets. IC₅₀ was 1 × 10^?4 M in both cases. These effects are discussed in the view of recent evidence, suggesting that dual inhibitors of arachidonate cyclo-oxygenase and lipoxygenase may have an improved profile of antiinflammatory activity.