Evaluation of a novel aminopeptidase N inhibitor,in vitro,using two assay systems

The activities of the novel aminopeptidase N inhibitor (APNI), β-Amino-α-Hydroxyl-Phenyl butanic acid-Valine (AHPA-Val), were compared with APNI (amastatin). AHPA-Val and amastatin produced competitive inhibition of the hydrolysis of Tyr-Gly in the guinea-pig striatal membrane preparation, with K_i equal to 14.06 μM and 12.48 μM respectively. Met-enkephalin-induced twitch inhibition of the guinea-pig ileum preparation was enhanced by AHPA-Val and amastatin with pA_1/2 values (the negative logarithm concentration of APNI that decreased the IC₅₀ of Met-enkephalin by half), of 7.08 and 7.79 respectively. These results suggest that AHPA-Val has good activity as an APNI and that these two assay systems are useful for evaluating the potency of novel APNIs.     

A specific enzyme assay for aminopeptidase M in rat brain.

A specific enzyme assay for aminopeptidase M (APM) activity on rat brain membranes has been developed through selective use of enzyme inhibitors. Amastatin was the most potent inhibitor (amastatin > actinonin > MDL73347 > bestatin) for purified porcine kidney APM, giving 98% inhibition at a 6 microM concentration, while actinonin, yielded only 57% inhibition at this concentration. Puromycin (10 microM) was used to inhibit puromycin-sensitive aminopeptidase activity in the rat brain membrane preparation. Puromycin (10 microM) had only a slight effect on the Km of porcine kidney APM, and had negligible effect on APM velocity at the high substrate concentration (2 mM) used in the APM assay. The assay produced a linear accumulation of product for increasing amount of rat brain membranes used, and for increasing incubation time. The Km of APM on rat brain membranes for L-Leucine-p-nitroanilide (0.383 mM) was similar to the Km of purified porcine kidney APM (0.558 mM). APM-activity, involved in the metabolism of several biologically important neuropeptides in different brain regions, can be specifically measured with this enzyme assay.     

Proctolin degradation by membrane peptidases from nervous tissues of the desert locust (Schistocerca gregaria).

The hydrolysis of the insect neuropeptide proctolin (Arg-Tyr-Leu-Pro-Thr) by enzyme preparations from the nervous tissue of the desert locust (Schistocerca gregaria) was investigated. Neural homogenate degraded proctolin (100 microM) at neutral pH by cleavage of the Arg-Tyr and Tyr-Leu bonds to yield Tyr-Leu-Pro-Thr, Arg-Tyr and free tyrosine. Arg-Tyr was detected as a major metabolite when the aminopeptidase inhibitors amastatin and bestatin were present to prevent Arg-Tyr breakdown. Around 50% of the proctolin-degrading activity was isolated in a 30,000 g membrane fraction and was shown to be almost entirely due to aminopeptidase activity. The aminopeptidase had an apparent Km of 23 microM, a pH optimum of 7.0 and was inhibited by 1 mM-EDTA and amastatin [IC50 = 0.3 microM], but was relatively insensitive to bestatin, actinonin and puromycin. Phenylmethanesulphonyl fluoride (1 mM) and p-chloromercuriphenylsulphonic acid (1 mM) had no effect on this enzyme activity. Although the bulk of the Tyr-Leu hydrolytic activity was located in the 30,000 g supernatant, some weak activity was detected in a washed membrane preparation. This peptidase displayed a high affinity for proctolin (Km = 0.35 microM) and optimal activity at around pH 7.0. Synaptosome- and mitochondria-rich fractions were prepared from crude neural membranes. The aminopeptidase activity was concentrated in the synaptic-membrane preparation, whereas activity giving rise to Arg-Tyr was predominantly localized in the mitochondrial fraction. The subcellular localization of the membrane aminopeptidase is consistent with a possible physiological role for this enzyme in the inactivation of synaptically released proctolin.     

Calcitonin gene-related peptide (CGRP) inhibits contractions of the prostatic stroma of the rat but not the guinea-pig

This study investigated the presence and effects of calcitonin gene-related peptide (CGRP) within the rat and guinea-pig prostate glands. Immunohistochemical studies demonstrated that CGRP immunoreactive nerve fibres are sparsely distributed throughout the prostatic fibromuscular stroma in both species. These CGRP immunopositive nerve fibres shared a similar distribution profile but were not colocalized with tyrosine hydroxylase immunopositive nerve fibres which also innervate the prostatic stroma of these species. Nerve terminals within rat and guinea-pig prostatic tissues were electrically field stimulated (60 V, 0.5 ms, 10 Hz, 20 pulses every 60 s). In guinea-pig preparations, application of human alpha-CGRP, rat adrenomedullin or rat amylin (0.1 nM-1 microM) had no effect on responses to field stimulation. In contrast, both rat and human alpha-CGRP (10 pM-300 nM), rat adrenomedullin (0.3 nM-1 microM) and rat amylin (3 nM-1 microM) concentration-dependently inhibited electrically evoked contractile responses in the rat prostate. The relative order of potency was rat alpha-CGRP=human alpha-CGRP>rat adrenomedullin>rat amylin. The inhibition by rat alpha-CGRP of field stimulation-induced contractions in the rat prostate was competitively antagonized by human CGRP((8-37)) (1, 3 and 10 microM) with a pA(2) of 6.20+/-0.13. Rat alpha-CGRP (10 nM) attenuated contractile responses of the rat prostate to exogenously added noradrenaline (1-100 microM). Inhibitory concentration-response curves to rat alpha-CGRP in rat prostates were unaffected by preincubation in either glibenclamide (10-100 microM), N-nitro-L-arginine methyl ester (L-NAME) (10 microM), bestatin (10 microM), captopril (10 microM) or phosphoramidon (3 microM). Our results indicate that CGRP-induced inhibition of electrically evoked contractions in the rat prostate occurs through activation of postjunctional CGRP(2) receptors which act independently of a K(ATP) channel or nitrergic mechanisms. Degradation of rat alpha-CGRP via peptidases does not appear to occur in the rat prostate.     

Vasoactive intestinal polypeptide induces neurogenic contraction of guinea-pig ileum. Involvement of acetylcholine and substance P.

The effect and mode of action of vasoactive intestinal polypeptide (VIP), a peptidergic neuromodulator in the gastrointestinal nervous system, were investigated in isolated muscle strips of the guinea-pig ileum. VIP induced concentration-dependent (20 nM-1 microM) contractions of longitudinal ileal strips. TTX (1 microM), a mixture of atropine (3 microM) and spantide (30 microM), a mixture of atropine (3 microM) and omega-conotoxin GVIA (100 nM), somatostatin (60 nM) and dynorphin (100 nM) abolished the effect of VIP. In most cases a small relaxation became evident. Desensitization to substance P in the presence of atropine prevented VIP-induced contraction. A partial inhibition was observed in the presence of atropine (3 microM), spantide (30 microM), omega-conotoxin GVIA (100 nM), beta-endorphin (265 nM), met-enkephalin (1100 nM) and a mixture of spantide (30 microM) and omega-conotoxin GVIA (100 nM). The action of VIP was not significantly modified by guanethidine (3 microM) or hexamethonium (150 microM). In circular ileal strips VIP (10-300 nM) caused concentration-dependent relaxations through a direct myogenic effect. These results indicate that the VIP produced contractions of the guinea-pig ileum are exclusively neurally mediated and involve a cholinergic as well as a noncholinergic-nonadrenergic (NANC) pathway. It is concluded that besides acetylcholine (Ach) VIP releases the peptidergic transmitter substance P from postganglionic nerve fibers of myenteric plexus. Opioid peptides and somatostatin modulate the activity of cholinergic and peptidegic nerves in the guinea-pig ileum. The release of substance P appears to depend completely on N-type voltage sensitive calcium channels.     

The identification of a new cyclic nucleotide phosphodiesterase activity in human and guinea-pig cardiac ventricle. Implications for the mechanism of action of selective phosphodiesterase inhibitors.

Four cyclic nucleotide phosphodiesterase (PDE) activities were separated from low-speed supernatants of homogenates of human cardiac ventricle by DEAE-Sepharose chromatography, and designated PDE I-PDE IV in order of elution with an increasing salt gradient. PDE I was a Ca2+/calmodulin-stimulated activity, and PDE II was an activity with a high Km for cyclic AMP which was stimulated by low concentrations of cyclic GMP. Human ventricle PDE III had Km values of 0.14 microM (cyclic AMP) and 4 microM (cyclic GMP), and showed simple Michaelis-Menten kinetics with both substrates. PDE IV is a previously unrecognized activity in cardiac muscle, the human enzyme having Km values of 2 microM (cyclic AMP) and 50 microM (cyclic GMP). PDE III and PDE IV were not activated by cyclic nucleotides or calmodulin. Four PDE activities were also isolated from guinea-pig ventricle, and had very similar kinetic properties. By gel filtration, the Mr of PDE III was 60,000, and that of PDE IV 45,000. The drug SK&F 94120 selectively and competitively inhibited PDE III with a Ki value of 0.8 microM (human), showing simple hyperbolic inhibition kinetics. Rolipram (Schering ZK 62711) and Ro 20-1724 (Roche), which have previously been reported to inhibit PDE III-like activities strongly, were shown to be weak inhibitors of human and guinea-pig PDE III enzymes (Ki values greater than 25 microM), but potent inhibitors of PDE IV [Ki values 2.4 microM (Rolipram) and 3.1 microM (Ro 20-1724) with human PDE IV]. The inhibition in all cases demonstrated simple hyperbolic competition. These observations suggest that the previously reported complex inhibition of PDE III-type activities from cardiac muscle was caused by incomplete separation of the PDE III from other enzymes, particularly PDE IV.     

The effects of non-steroidal inhibitors of phospholipase A2 on leukotriene and histamine release from human and guinea-pig lung

The effects of chloroquine and mepacrine were determined on the release of slow reacting substances (leukotrienes) from lung fragments in vitro. These drugs have been shown in a variety of tissues to inhibit phospholipase A2, and thus to reduce the availability of arachidonate, which is a substrate for leukotriene biosynthesis. Leukotriene and histamine release from unsensitized human lung was stimulated by calcium ionophore A23187, and from actively sensitized guinea-pig lung, by ovalbumin. Chloroquine (10 microM and 100 microM) significantly inhibited leukotriene release in lung from both species, and at 100 microM also inhibited histamine release. Mepacrine (10 microM) inhibited leukotriene release in human lung and at 100 microM in guinea-pig lung. The effects of chloroquine (100 microM) on leukotriene release were counteracted by the presence of arachidonic acid (10 microM), which suggests that chloroquine had impaired the availability of arachidonate. It seems probable that chloroquine and mepacrine inhibit leukotriene release by inhibition of phospholipase A2 in lung.     

Characterization of the effects of AHN 086, an irreversible ligand of "peripheral" benzodiazepine receptors, on contraction in guinea-pig atrial and ileal longitudinal smooth muscle

The effects of AHN 086 and its reversibly acting structural analogue Ro 5-4864 were studied in the spontaneously beating guinea-pig atria and field-stimulated guinea-pig ileal longitudinal smooth muscle in the presence and absence of dihydropyridine calcium channel modulators. The treatment of guinea-pig atria with AHN 086 followed by extensive washing did not alter contraction. However, AHN 086 (0.5 microM) potentiated (88%) the positive inotropic responses by BAY K 8644, an effect that was not reversed by extensive washing of the tissue. Higher concentrations of AHN 086 (greater than 2 microM) irreversibly inhibited the intropic, but not the chronotropic responses to BAY K 8644, nifedipine, and isoproterenol. Ro 5-4864 (10 microM) produced a reversible enhancement of the inotropic responses and block of the chronotropic responses to BAY K 8644. In guinea-pig ileal longitudinal smooth muscle, both AHN 086 and Ro 5-4864 reversibly inhibited field-stimulated contractions. Neither Ro 5-4864 nor AHN 086 affected the ability of nifedipine to inhibit field-stimulated contractions of ileal longitudinal smooth muscle. Treatment of intact atrial with 5 microM AHN 086 followed by extensive washing resulted in a significant inhibition (30-50%) of [3H]Ro 5-4864 binding to peripheral benzodiazepine receptors and of [3H]nitrendipine binding to voltage-operated calcium channels, but did not affect [3H]dihydroalprenolol binding to beta-adrenergic receptors on atrial membranes. The same treatment applied to intact ileal longitudinal smooth muscle affected neither [3H] (-)-quinuclidinyl benzilate binding to muscarine receptors nor [3H]nitrendipine binding, but did result in a significant inhibition (30-50%) of [3H]Ro 5-4864 binding to ileal longitudinal smooth muscle membranes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacological profile of a new peptidic gastrin antagonist

Boc-Trp-Met-Asp-NH2 was described as the smallest peptidic fragment which presented gastric antisecretory activity. Some pharmacological aspects of a peptide analogue, Boc-Trp-Leu-Asp-NH2 (Boc-WLD-NH2), were studied on the main biological functions of gastrin. This compound was found to inhibit the binding of gastrin to isolated gastric fundic mucosal cells (IC50 50 microM). On pentagastrin-induced gastric acid secretion in the rat, a dose-dependent inhibition was observed with an ID50 of 55 mumol/kg when pentagastrin (1 microgram/kg per h) was continuously infused and with an ID50 of 7.8 mumol/kg when pentagastrin (1 microgram/kg) was bolus i.v. injected. Similar inhibition was observed on acid secretion induced by pentagastrin in the isolated rat gastric mucosa (IC50 100 microM), whereas the tripeptide had no effect when acid output was triggered by histamine. A dose-dependent inhibition with the tripeptide was shown on pentagastrin induced guinea-pig ileum contractions (IC50 31 microM). The compound had no activity on histamine-stimulated guinea-pig atria (histamine H2-receptor). These results suggest some evidence for a selective antigastrin activity.     

Synthesis, biological evaluation and molecular modelling studies of novel ACD- and ABD-ring steroidomimetics as inhibitors of CYP17

Two novel classes of non-steroidal substrate mimetics were synthesised and examined for their potency as inhibitors of human CYP17. Selected compounds were tested for inhibition of hepatic CYP enzymes 3A4, 1A2, 2C9 and 2C19. The most promising compound 15 showed a good inhibition of the target enzyme (31% and 66% at 0.2 and 2 microM, respectively), and little inhibition of the most important hepatic enzyme CYP3A4 (6% and 19% inhibition at 0.2 and 2 microM, respectively) and the key enzyme of glucocorticoid biosynthesis CYP11B1 (3% and 23% inhibition at 0.2 and 2 microM, respectively). Docking studies revealed that this compound does not assume the same binding mode as steroidal ligands.     

Modulation of Anopheles gambiae Epsilon glutathione transferase activity by plant natural products in vitro

Elevated glutathione transferase (GST) E2 activity is associated with DDT resistance in the mosquito Anopheles gambiae. The search for chemomodulators that inhibit the function of AgGSTE2 would enhance the insecticidal activity of DDT. Therefore, we examined the interaction of novel natural plant products with heterologously expressed An. gambiae GSTE 2 in vitro. Five of the ten compounds, epiphyllocoumarin (Tral-1), knipholone anthrone, isofuranonaphthoquinones (Mr 13/2, Mr13/4) and the polyprenylated benzophenone (GG1) were shown to be potent inhibitors of AgGSTE2 with IC(50) values of 1.5 microM, 3.5 microM, 4 microM, 4.3 microM and 4.8 microM respectively. Non-competitive inhibition was obtained for Tral 1 and GG1 with regards to GSH (K(i) of 0.24 microM and 0.14 microM respectively). Competitive inhibition for Tral1 was obtained with CDNB (K(i) = 0.4 microM) whilst GG1 produced mixed type of inhibition. The K(i) and K(i)' for GSH for Tral-1 and GG1 were 0.2 microM and 0.1 microM respectively. These results suggest that the novel natural plant products, particularly Tral-1, represent potent AgGSTE2 in vitro inhibitors.     

Effects of N-phthalamoyl-L-glutaminic acid, a selective agonist of NMDA receptor, on binding of 3H-L-glutamate with synaptic membranes of the hippocampus]

In the present investigation the interaction of a novel selective NMDA receptors agonist, N-phthalamoyl-L-glutamic acid (PhGA), with the synaptic membranes preparation of human hippocampus was examined against NMDA. It was established that there are two binding sites of 3H-L-Glu, Kd1 = 0.35 +/- 0.11 nM, Bmax1 = 6.5 +/- 2.3 pmol/mg and Kd2 = 51 +/- 12 nM, Bmax2 = 98 +/- 17 pmol/mg. The inhibition constants (Ki) were calculated for NMDA and PhGA and were equal: Ki(NMDA) = 19 microM, Ki (PhGA) = 13 microM, respectively. It was concluded that PhGA is the partial agonist of the NMDA receptors.     

Wy-48,252 (1,1,1-trifluoro-N-[3-(2-quinolinylmethoxy)phenyl]methane sulfonamide) an orally active leukotriene D4 antagonist: pharmacological characterization in vitro and in vivo in the guinea pig

The following communicates the pharmacology of Wy-48,252 (1,1,1-trifluoro-N-[3-(2-quinolinylmethoxy)phenyl]methanesulfonamide) a chemically novel and orally potent leukotriene (LT) D4 receptor antagonist. In the isolated guinea-pig trachea pretreated with indomethacin (5 microM) and L-cysteine (10 mM), Wy-48,252 antagonized TD4-induced contraction with a pKB = 7.6. Against LTC4 on tissues pretreated with IND and glutathione (10 mM), Wy-48,252 had a pKB greater than 5. Wy-48,252 (10 microM) did not antagonize pilocarpine-, histamine- or PGF2 alpha-induced tracheal contraction. Further, in the presence of indomethacin and chlorpheniramine (1 microM), Wy-48,252 dose-dependently inhibited the antigen-induced contraction of guinea-pig trachea in a manner consistent with antagonism at the LTD4 receptor and inhibition of LT synthesis. In the Konzett-Rossler model of i.v. LTD4-induced bronchoconstriction in indomethacin treated guinea pigs, intragastric Wy-48,252 (2 hr) had an ID50 of 100 micrograms/kg and a functional half-life of 5 hr. Against i.v. antigen-induced bronchoconstriction in guinea pigs treated with indomethacin and chlorpheniramine, intragastric Wy-48,252 (2 hr) had an ID50 of 0.6 mg/kg and a 5 hr half life. Intragastric Wy-48,252 also selectively blocked the cutaneous wheal reaction to intradermal LTD4 but not histamine. We conclude that Wy-48,252 is distinguished from other selective LTD4 receptor antagonists by its oral potency and should be useful in ascertaining the role of LTD4 mediated processes in asthma, allergy and animal models.     

Aminopeptidases in Caenorhabditis elegans and Panagrellus redivivus: detection using peptide and non-peptide substrates

Aminopeptidase activities were detected in extracts of the free-living nematodes Caenorhabditis elegans and Panagrellus redivivus using the aminoacyl substrate L-alanine-4-nitroanilide. The activities exhibited similarities in Km (C. elegans = 2.22 mM; P. redivivus = 2.09 mM) and specific activity (C. elegans = 1.38 +/- 0.43 mAU min(-1) x g(-1); P. redivivus, 1.23 +/- 0.18m AU min(-1) microg(-1). Each is inhibited competitively by amastatin (C. elegans IC50 = 0.46 microM; P. redivivus IC50 = 15.90 microM) and non-competitively by leuhistin (C. elegans IC50 = 3.00 microM; P. redivivus IC50 = 37.35 microM). The bioactive peptides adipokinetic hormone and substance P decrease the apparent aminopeptidase activities of each extract suggesting that the peptides compete with the Ala-pNA as substrates. With each extract, adipokinetic hormone appeared to be the more effective substrate. Digestion of adipokinetic hormone by C. elegans and P. redivivus extracts in the presence and absence of 1 mM amastatin produced distinct chromatographic profiles that suggest different digestion patterns for the two species. However, amastatin had clear effects on chromatographic profiles from each species indicating that an aminopeptidase is involved in the digestion of the peptide substrates. The data presented indicate that extracts of free-living nematodes are capable of metabolizing peptide hormones, and that this metabolism involves substrate-selective aminopeptidases.     

Effect of insulin, prostaglandin E1 and uptake inhibitors on glucose transport in the perfused guinea-pig placenta

The effects of insulin, prostaglandin E1 (PGE1) and uptake inhibitors on unidirectional D-glucose influx at brush border (maternal) and basal (fetal) sides of the guinea-pig syncytotrophoblast were investigated in the intact, perfused guinea-pig placenta by rapid, paired-tracer dilution. Experiments were performed in either an in situ preparation artificially perfused through the umbilical vessels (intact maternal circulation) or in the fully isolated dually-perfused placenta in which both interfaces were studied simultaneously. Kinetic characterization of unidirectional D-glucose influx gave apparent Km values (mean +/- SEM) at maternal and fetal sides of 70 +/- 6 and 87 +/- 16 mM respectively; corresponding Vmax values were 53 +/- 3 and 82 +/- 6 mumol min-1g-1. At the fetal side (singly-perfused placenta) cytochalasin B (50 microM), ethylidene-D-glucose (100 mM) and PGE1 (1 microM) partially inhibited D-glucose uptake whereas cortisol (50 microM) and progesterone (100 microM) had no effect. Abolition of the sodium gradient across the fetal interface did not modulate the kinetics of influx. In the presence of 150 mu units ml-1 insulin (dually-perfused placenta), unidirectional uptake into the trophoblast and transplacental D-[3H]glucose transfer were unaltered. In contrast, prostaglandin E1 (1 microM) markedly reduced the Km and Vmax for D-glucose at both interfaces and the inhibitory effect was reflected in a reduction in specific transplacental D-glucose transfer. Further experiments showed that the isolated placenta releases prostaglandins (PGE; PGF2 alpha) into both circulations. Bilateral insulin perfusion did not affect either lactate release by the placenta or rapid metabolism of D-[14C]glucose to [3H]lactate (usually less than 10% effluent [14C]lactate in 5 min). An asymmetric degradation of exogenous insulin was observed in the dually-perfused placenta: uterine venous samples contained 24 +/- 7 microunits ml-1 immunoreactive insulin when compared to the arterial concentration (151 +/- 3 microU ml-1 perfusate) while no change was measureable in the fetal circulation within the same time period (152 +/- 5 microU ml-1). This asymmetry was confirmed in experiments employing [125I]insulin. These results demonstrate that glucose transport in the intact guinea-pig placenta occurs by a sodium-independent, cytochalasin B-inhibitable system which is insulin-insensitive. Prostaglandin E1 appeared to be a potent transport inhibitor which suggests that prostaglandins may be involved in the 'down' regulation of placental glucose transport in vivo.     

Activation of superoxide production and differential exocytosis in polymorphonuclear leukocytes by cytochalasins A, B, C, D and E. Effects of various ions

All of the common cytochalasins activate superoxide anion release and exocytosis of beta-N-acetylglucosaminidase and lysozyme from guinea-pig polymorphonuclear leukocytes (neutrophils) incubated in a buffered sucrose medium. Half-maximal activation of both processes is produced by approx. 0.2 microM cytochalasin A, C greater than 2 microM cytochalasin B greater than or equal to 4-5 microM cytochalasin D, E. While maximal rates of O2- release and extents of exocytosis require extracellular calcium (1-2 mM), replacing sucrose with monovalent cation chlorides is inhibitory to neutrophil activation by cytochalasins. Na+, K+ or choline inhibit either cytochalasin B- or E-stimulated O2- production with IC50 values of 5-10 mM and inhibition occurs whether Cl-, NO3- or SCN- is the anion added with Na+ or K+. Release of beta-N-acetylglucosaminidase in control or cytochalasin B-stimulated cells is inhibited by NaCl(IC50 approximately 10 mM), while cytochalasin E-stimulated exocytosis is reduced less and K+ or choline chloride are ineffective in inhibiting either cytochalasin B- or E-stimulated exocytosis. Release of beta-glucuronidase, myeloperoxidase or acid phosphatase from neutrophils incubated in buffered sucrose is not stimulated by cytochalasin B. Stimulation of either O2- or beta-N-acetylglucosaminidase release by low concentrations of cytochalasin A is followed by inhibition of each at higher concentrations. It appears that all cytochalasins can activate both NAD(P)H oxidase and selective degranulation of neutrophils incubated in salt-restricted media and that differential inhibition of these two processes by monovalent cations and/or anions is produced at some step(s) subsequent to cytochalasin interaction with the cell.     

Effects of hirsutine and dihydrocorynantheine on the action potentials of sino-atrial node, atrium and ventricle

The effects of hirsutine, an indole alkaloid from Uncaria rhynchophylla MIQ. JACKSON with antihypertensive, negative chronotropic and antiarrhythmic activity, and its C3 structural epimer, dihydrocorynantheine, on membrane potentials of rabbit sino-atrial node and guinea-pig right ventricle and left atrium were studied with microelectrode techniques. In sino-atrial node preparations, hirsutine and dihydrocorynantheine (0.1 microM to 10 microM) concentration-dependently increased cycle length, decreased slope of the pacemaker depolarization (phase 4 depolarization), decreased maximum rate of rise and prolonged action potential duration. In atrial and ventricular preparations, both compounds (0.1 microM to 30 microM) concentration-dependently decreased maximum rate of rise and prolonged action potential duration. These results indicate that hirsutine and dihydrocorynantheine have direct effects on the action potential of cardiac muscle through inhibition of multiple ion channels, which may explain their negative chronotropic and antiarrhythmic activity.     

Photoaffinity labelling of nucleoside-transport proteins in plasma membranes isolated from rat and guinea-pig liver.

Nitrobenzylthioinosine (NBMPR) was employed as a probe of the nucleoside transporters from rat and guinea-pig liver. Purified liver plasma membranes prepared on self-generating Percoll density gradients exhibited 16-fold (rat) and 10-fold (guinea pig) higher [3H]NBMPR-binding activities than in crude liver homogenates (3.69 and 14.7 pmol/mg of protein for rat and guinea-pig liver membranes respectively, and 0.23 and 1.47 pmol/mg of protein for crude liver homogenates respectively). Binding to membranes from both species was saturable (apparent Kd 0.14 and 0.63 nM for rat and guinea-pig membranes respectively) and inhibited by uridine, adenosine, nitrobenzylthioguanosine (NBTGR) and dilazep. Uridine was an apparent competitive inhibitor of high-affinity NBMPR binding to rat membranes (apparent Ki 1.5 mM). There was a marked species difference with respect to dipyridamole inhibition of NBMPR binding (50% inhibition at 0.2 and greater than 100 microM for guinea-pig and rat respectively). These results are consistent with a role of NBMPR-binding proteins in liver nucleoside transport. Exposure of rat and guinea pig membranes to high-intensity u.v. light in the presence of [3H]NBMPR resulted in the selective radio-labelling of membrane proteins which migrated on sodium dodecyl sulphate/polyacrylamide gels with apparent Mr values in the same range as that of the human erythrocyte nucleoside transporter (45 000-66 000). Covalent labelling of these proteins was abolished when photolysis was performed in the presence of non-radio-active NBTGR as competing ligand.     

Surface aminopeptidase activity of human lymphocytes. I. Biochemical and biologic properties of intact cells

Surface aminopeptidase activity in intact lymphocytes was studied and was shown to have the following properties when alanine-p-nitroanilide was used as substrate: 1) The activity was surface associated and not secreted as determined by extracellular location of product and the effect of proteases and diazotized sulfanilic acid on enzyme activity. 2) The enzyme activity was shown to have a pH optimum of 7.4 to 8.0. 3) Enzyme activity was shown to be inhibited by amastatin, bestatin, and 1,10 phenanthroline. Inhibition by amastatin consisted of a high-affinity component (Ki = 3.5 x 10(-6) M) which accounted for approximately 20% of the total activity and a low-affinity component (Ki = 3.5 x 10(-5) M) which accounted for the remainder suggesting that two forms of aminopeptidase exist. Only a single component of inhibition was seen with bestatin (Ki = 3.5 x 10(-6) M) and 1,10 phenanthroline (Ki = 2.0 x 10(-4) M) which accounted for 80 and 90% of the total enzyme activity, respectively. Unlike the competitive inhibitors bestatin and amastatin, inhibition by 1,10 phenanthroline was shown to be non-competitive. Finally, surface aminopeptidase activity essentially doubled in the presence of PHA (10 micrograms/ml) or Con A (10 micrograms/ml), at 72 h. This enhancing effect was shown to be dose dependent, time dependent, and mitogen dependent and correlated with the cellular state of activation as determined by [3H]TdR incorporation.