Neurohormonal regulation of histamine synthesis in isolated rabbit fundic mucosal cells

1994 ◽  
Vol 266 (3) ◽  
pp. G395-G402
Author(s):  
F. Hollande ◽  
J. P. Bali ◽  
R. Magous
Keyword(s):  
Endocrine Cells ◽  
Inhibitory Concentration ◽  
Histidine Decarboxylase ◽  
Effective Concentration ◽  
Maximal Velocity ◽  
Michaelis Constant ◽  
High Potency ◽  
Cholecystokinin Octapeptide ◽  
Muscarinic Receptor Antagonists ◽  
Mucosal Cells

In a population of rabbit fundic mucosal cells enriched in mucous and endocrine cells, gastrin and cholecystokinin octapeptide (CCK-8) were shown to increase dose-dependently histidine decarboxylase (HDC) activity with the same efficacy and high potencies [50% effective concentration (EC50) 0.389 +/- 0.041 and 0.275 +/- 0.011 nM, respectively], whereas pentagastrin was less potent (EC50 2.90 +/- 0.13 nM). L-365,260 and PD-135,666 inhibited gastrin- and CCK-8-stimulated HDC activity with a high potency [50% inhibitory concentration (IC50) 1.00 +/- 0.08 and 4.2 +/- 0.7 nM for gastrin-stimulated and 1.95 +/- 0.21 and 1.78 +/- 0.12 nM for CCK-8-stimulated HDC activity, respectively], whereas L-364,718 was 50 to 100 times less potent (EC50 100 +/- 2.5 and 91.2 +/- 3.1 nM, respectively on gastrin- and CCK-8-stimulated HDC activity). Carbachol also dose-dependently increased HDC activity (EC50 7.08 +/- 0.32 nM), and its effect was reversed by selective muscarinic-receptor antagonists with the following order of potency: pirenzepine (IC50 15.1 +/- 1.2 nM) > para-fluoro-hexahydro-siladifenidol (IC50 0.316 +/- 0.02 microM) > 11-2[(2-[(diethyl-amino)-methyl]-1-piperidinyl)acetyl]-5,11-dihydro-6H- pyrido[2,3-b][1,4]benzodiazepine-6-one (IC50 28.5 +/- 1.1 microM). Moreover, gastrin and carbachol were able to modify slightly but significantly both the Michaelis constant (Km) and the maximal velocity (Vmax) of HDC in the same way (18-20% reduction of the Km and 25-30% increase of the Vmax).(ABSTRACT TRUNCATED AT 250 WORDS)

Autoregulation of histamine synthesis through H3 receptors in isolated fundic mucosal cells

1993 ◽  
Vol 265 (6) ◽  
pp. G1039-G1044 ◽  
Author(s):  
F. Hollande ◽  
J. P. Bali ◽  
R. Magous
Keyword(s):  
Gastric Mucosa ◽  
Endocrine Cells ◽  
Histidine Decarboxylase ◽  
Receptor Subtypes ◽  
Histamine Synthesis ◽  
H3 Receptors ◽  
Mucosal Cells ◽  
High Concentrations ◽  
The Relationship ◽  
Stimulation Of

Histamine plays an important role in the control of gastric acid secretion by activating H2 receptors located on parietal cells. In gastric mucosa, histamine is stored both in mast cells and in enterochromaffin-like cells, especially in rodents. It has been proposed that histamine may regulate its own synthesis and/or release through receptors pharmacologically distinct from H1- and H2-receptor subtypes. In this article, we studied the regulation by histamine of histidine decarboxylase (HDC) activity (enzyme responsible for the formation of histamine by decarboxylation of L-histidine) in a fraction of isolated rabbit gastric mucosal cells enriched in mucous and endocrine cells. Histamine and (R)-alpha-methylhistamine (H3 receptor agonist) dose dependently inhibited HDC activity with the same potency (mean effective concn: 32.2 +/- 0.7 and 50.5 +/- 3.1 pM, respectively) and efficacy (35 and 36% inhibition, respectively). In contrast, the H2 agonist dimaprit was devoid of effect. The H3 antagonist thioperamide was found to decrease the histamine- or (R)-alpha-methylhistamine-induced inhibition of HDC activity (mean ineffective concn = 28.3 +/- 1.8 and 9.87 +/- 0.8 nM, respectively), whereas H1 (promethazine) and H2 (ranitidine) antagonists were unable to affect HDC activity. Moreover, high concentrations of thioperamide (1-10 microns) increased histamine release from these cells. All these results allowed us to conclude that, in gastric mucosa, histamine downregulates its own synthesis (and perhaps release) through the stimulation of autoreceptors with pharmacological characteristics of H3 receptors. However, the relationship between histamine synthesis and release remains unclear and needs further investigation.

Gastrin stimulation of histamine synthesis in enterochromaffin-like cells from rabbit fundic mucosa

1996 ◽  
Vol 270 (3) ◽  
pp. G463-G469 ◽  
Author(s):  
F. Hollande ◽  
S. Combettes ◽  
J. P. Bali ◽  
R. Magous
Keyword(s):  
Histidine Decarboxylase ◽  
Polymyxin B ◽  
Maximal Velocity ◽  
Dependent Manner ◽  
Ecl Cells ◽  
Histamine Synthesis ◽  
Mucosal Cells ◽  
Enzyme Molecules ◽  
Stimulation Of

This work aimed to investigate the molecular role of gastrin in histamine synthesis in isolated rabbit fundic mucosal cells enriched in enterochromaffin-like (ECL) cells (37%). Gastrin stimulated histidine decarboxylase (HDC) activity by increasing the maximal velocity (Vmax) from 0.240 +/- 0.017 (basal value) to 0.332 +/- 0.012 pmol/mg protein/h and by decreasing the Michaelis-Menten constant value -Km; 73.90 +/- 2.2 vs. 93.42 +/- 4.32 microM (basal value)]. Pertussis toxin (PTX) (200 ng/ml) reduced the stimulation of HDC induced by 10 nM gastrin from 41.8 to 15.9%, whereas cholera toxin (CTX) (100 ng/ml) was without effect. Staurosporine and polymyxin B inhibited in a dose dependent manner the HDC activity stimulated by 10 nM gastrin. Phorbol 12-myristate 13-acetate (PMA; 100 nM) decreased Vmax (0.558 +/- 0.021 pmol/ mg protein/h) but did not change the Km. Furthermore, cycloheximide (0.1-10 microM) inhibited the gastrin-induced stimulation of HDC activity, whereas actinomycin D (up to 10 microM) was without effect. Finally, incubation of cells with gastrin (10 microM) left the expression of HDC mRNA unchanged. We concluded that gastrin, acting through "gastrin/CCK-B type" receptors coupled to PTX-sensitive G protein, exerts a short-term regulation of histamine synthesis in gastric ECL cells by increasing both the affinity of HDC for L-histidine and the number of active enzyme molecules. This last event, related to protein kinase C activation, could be due to a translational or posttranslational mechanism.

Peroxidases from Tulip Bulbs (Tulipa fosteriana, L.) Oxidize Xenobiotics NNitrosodimethylamine and N-Nitroso-N-methylaniline in vitro

1997 ◽  
Vol 62 (11) ◽  
pp. 1804-1814 ◽  
Author(s):  
Marie Stiborová ◽  
Hana Hansíková
Keyword(s):  
Cytochromes P450 ◽  
Kinetic Studies ◽  
The Other ◽  
Maximal Velocity ◽  
Michaelis Constant ◽  
Other Hand ◽  
Plant Peroxidases ◽  
Tulip Bulbs

Tulip bulbs (Tulipa fosteriana, L.) contain peroxidases catalyzing the oxidation of the xenobiotics N-nitrosodimethylamine (NDMA) and N-nitroso-N-methylaniline (NMA). Three anionic (A1, A2, A3) and four cationic (B, C, D, E) peroxidases were purified from this tissue, partially characterized and used for kinetic studies. Demethylation of NDMA and NMA producing formaldehyde is catalyzed by one anionic (A1) and three cationic (C, D, E) peroxidases. The oxidation of NDMA by tulip peroxidases exhibits the Michaelis-Menten kinetics. The apparent Michaelis constant and the maximal velocity values for this substrate were determined. On the other hand, non-Michaelian kinetics for the NMA oxidation were observed with tulip peroxidases. The most abundant cationic peroxidase (peroxidase C) was used for detailed enzymatic studies. In addition to formation of formaldehyde, methylaniline, aniline, 4-aminophenol and phenol were found to be metabolites formed from NMA. Phenol was formed presumably by N-demethylation via a benzenediazonium ion, while methylaniline, aniline and 4-aminophenol were products of denitrosation of the substrate. The efficiencies of plant peroxidases to oxidize NDMA and NMA in vitro are compared with those of cytochromes P450 and discussed.

Heat shock increases cytosolic free Ca2+ concentration via Na(+)-Ca2+ exchange in human epidermoid A 431 cells

1992 ◽  
Vol 263 (1) ◽  
pp. C30-C38 ◽  
Author(s):  
J. G. Kiang ◽  
M. L. Koenig ◽  
R. C. Smallridge
Keyword(s):  
Heat Treatment ◽  
Heat Shock ◽  
Time Dependent ◽  
Maximal Velocity ◽  
Dependent Manner ◽  
Michaelis Constant ◽  
Exchange System ◽  
Maximal Increase ◽  
Normal Cells ◽  
Dimethyl Amiloride

This study characterized cytosolic free Ca2+ concentration ([Ca2+]i) in normal and thermally injured human epidermoid A 431 cells. The resting [Ca2+]i in normal cells at 37 degrees C was 87 +/- 5 nM (n = 105). When cells were subjected to hyperthermia (40-50 degrees C), [Ca2+]i increased in a temperature- and time-dependent manner. The maximal increase in cells exposed to 45 degrees C was observed at 20 min; [Ca2+]i returned to normal within 1 h. The heat-induced [Ca2+]i increase depended on the presence of external Ca2+. La3+ and Cd2+ but not Co2+, verapamil, or nifedipine attenuated the heat-induced [Ca2+]i increase. TMB-8 partially blocked the increase in [Ca2+]i but pertussis toxin and cholera toxin pretreatment did not. The magnitude of the heat-induced [Ca2+]i increase or 45Ca2+ uptake depended on the presence of extracellular Na+. Heat treatment reduced the apparent Michaelis constant for external Ca2+ from 490 +/- 91 to 210 +/- 60 microM, whereas the maximal velocity remained the same. The intracellular Na+ concentration decreased 62.5% after heating. The heat-induced [Ca2+]i increase was completely blocked by amiloride (5 microM) and 5'-(N,N-dimethyl)-amiloride (1 microM). These results suggest heat activates the Na(+)-Ca2+ exchange system so as to increase [Ca2+]i and reduce [Na+]i.

Exponential function for calculating saturable enzyme kinetics.

1988 ◽  
Vol 34 (12) ◽  
pp. 2486-2489 ◽  
Author(s):  
F Keller ◽  
C Emde ◽  
A Schwarz
Keyword(s):  
Steady State ◽  
Enzyme Kinetics ◽  
Exponential Function ◽  
Association Constant ◽  
Time Dependent ◽  
Mathematical Representation ◽  
Hyperbolic Function ◽  
Maximal Velocity ◽  
Michaelis Constant ◽  
General Meaning

Abstract Enzyme kinetics are usually described by the Michaelis-Menten equation, where the time-dependent decrease of substrate (-dS/dt) is a hyperbolic function of maximal velocity (Vmax), Michaelis constant (Km), and amount of substrate (S). Because the Michaelis-Menten function in its most general meaning requires an assumption of steady-state, it is less curvilinear than true enzyme kinetics. A saturation-type exponential function is more curvilinear than the hyperbolic function and more closely approximates enzyme kinetics: -dS/dt = Vmax [1 - exp(-S/Km)]. The mathematical representation of enzyme kinetics can be further improved by introducing a deceleration term (Vdec), to make the assumption of a steady state unnecessary. For the action of chymotrypsin on N-acetyltyrosylethylester, the Michaelis-Menten equation yields the following: Vmax = 3.74 mumol/min and Km = 833 mumol. According to decelerated enzyme kinetics, the values Vmax = 4.80 mumol/min, Vdec = 0.0118 mumol/min, and the association constant (Ka) = 0.00111/mumol are more nearly accurate for this reaction (where 1/Ka = 901 mumol approximately Km).

The Inactivation by Carbenoxolone of Individual Human Pepsinogens and Pepsins

1973 ◽  
Vol 45 (2) ◽  
pp. 213-224 ◽  
Author(s):  
N. B. Roberts ◽  
W. H. Taylor
Keyword(s):  
Enzyme Inhibitor ◽  
Effective Concentration ◽  
Peptic Activity ◽  
Temperature Dependent ◽  
Mucosal Cells ◽  
Gastric Lumen ◽  
The Individual ◽  
Active Centres ◽  
Individual Human

1. Carbenoxolone, in suspension at pH 4·0, inhibits swine pepsin A, and human pepsins 1, 3 and 5. Human pepsin 5 is the most readily inhibited, and human pepsin 1 the least. 2. Inhibition occurs by a process which is time-dependent, temperature-dependent and proportional to the quantity of carbenoxolone suspended. 3. Carbenoxolone, in solution at pH 7·4 and pH 8·0, inhibits the activation of the total pepsinogens of human gastric mucosal extracts and of the individual pepsinogens 1, 3 and 5. Pepsinogen 1 was the most readily inhibited, pepsinogen 5 the least. 4. Chymotrypsin was readily inhibited by carbenoxolone at pH 7·4 and 8·0. Trypsin was not inhibited at pH 7·4 but was inhibited, relatively weakly, at pH 8·0. Pronase was weakly inhibited at pH 7·4 and 8·0 but papain was weakly activated. 5. Carbenoxolone is therefore not a general enzyme inhibitor but shows specificity for enzymes (pepsins and chymotrypsin) which split proteins at the same bonds, rather than for enzymes with similar active centres (chymotrypsin and trypsin). 6. The results suggest that, in vivo, carbenoxolone might diminish peptic activity in three ways: by inactivating pepsinogens irreversibly in the mucosal cells or at some point before their activation to pepsins; by inhibiting pepsins irreversibly in the gastric lumen; and by binding pepsins in the lumen without destroying their activity but decreasing their effective concentration. 7. These results are compatible with the hypothesis that pepsins, and pepsin 1 particularly, are factors in the aetiology of peptic ulcer.

INHIBITION AND ACTIVATION OF ADENYLOSUCCINIC SYNTHETASE BY 8-AZAGUANOSINE TRIPHOSPHATE

1963 ◽  
Vol 41 (1) ◽  
pp. 1495-1501 ◽  
Author(s):  
L. H. Cohen ◽  
R. E. Parks Jr.
Keyword(s):  
Reaction Rate ◽  
Maximal Velocity ◽  
Michaelis Constant ◽  
High Concentrations

8-Azaguanosine triphosphate can function in place of GTP in the reaction catalyzed by adenylosuccinic synthetase. The Michaelis constant and maximal velocity for the reaction obtained with the analogue are lower than those obtained with GTP. As a result, 8-azaGTP increases the reaction rate at low GTP concentrations and inhibits competitively at high GTP concentrations. It is suggested that this type of inhibition might be exploited in the development of chemo-therapeutic analogues specific for tissues containing high concentrations of a coenzyme or metabolite.

Hormone-induced rise in cytosolic Ca2+ in axolotl hepatocytes: properties of the Ca2+ influx channel

1997 ◽  
Vol 273 (5) ◽  
pp. C1526-C1532 ◽  
Author(s):  
Thomas Lenz ◽  
Jochen W. Kleineke
Keyword(s):  
Inhibitory Concentration ◽  
Cation Channels ◽  
Strong Inhibition ◽  
Michaelis Constant ◽  
Cyclic Monophosphate ◽  
First Order ◽  
First Order Kinetics ◽  
Nonselective Cation Channels ◽  
Store Depletion ◽  
Voltage Dependent

Calcium entry in nonexcitable cells occurs through Ca2+-selective channels activated secondarily to store depletion and/or through receptor- or second messenger-operated channels. In amphibian liver, hormones that stimulate the production of adenosine 3′,5′-cyclic monophosphate (cAMP) also regulate the opening of an ion gate in the plasma membrane, which allows a noncapacitative inflow of Ca2+. To characterize this Ca2+ channel, we studied the effects of inhibitors of voltage-dependent Ca2+ channels and of nonselective cation channels on 8-bromoadenosine 3′,5′-cyclic monophosphate (8-BrcAMP)-dependent Ca2+ entry in single axolotl hepatocytes. Ca2+ entry provoked by 8-BrcAMP in the presence of physiological Ca2+ followed first-order kinetics (apparent Michaelis constant = 43 μM at the cell surface). Maximal values of cytosolic Ca2+ (increment ∼300%) were reached within 15 s, and the effect was transient (half time of 56 s). We report a strong inhibition of cAMP-dependent Ca2+ entry by nifedipine [half-maximal inhibitory concentration (IC50) = 0.8 μM], by verapamil (IC50 = 22 μM), and by SK&F-96365 (IC50 = 1.8 μM). Depolarizing concentrations of K+were without effect. Gadolinium and the anti-inflammatory compound niflumate, both inhibitors of nonselective cation channels, suppressed Ca2+ influx. This “profile” indicates a novel mechanism of Ca2+ entry in nonexcitable cells.

scholarly journals Hydroxylated Tropolones Inhibit Hepatitis B Virus Replication by Blocking Viral Ribonuclease H Activity

2014 ◽  
Vol 59 (2) ◽  
pp. 1070-1079 ◽  
Author(s):  
Gaofeng Lu ◽  
Elena Lomonosova ◽  
Xiaohong Cheng ◽  
Eileen A. Moran ◽  
Marvin J. Meyers ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Inhibitory Concentration ◽  
Effective Concentration ◽  
Ribonuclease H ◽  
Human Pathogen ◽  
Hbv Genotype ◽  
Hbv Replication ◽  
B Virus ◽  
Current Therapies

ABSTRACTHepatitis B virus (HBV) remains a major human pathogen despite the development of both antiviral drugs and a vaccine, in part because the current therapies do not suppress HBV replication far enough to eradicate the virus. Here, we screened 51 troponoid compounds for their ability to suppress HBV RNaseH activity and HBV replication based on the activities of α-hydroxytropolones against HIV RNaseH, with the goal of determining whether the tropolone pharmacophore may be a promising scaffold for anti-HBV drug development. Thirteen compounds inhibited HBV RNaseH, with the best 50% inhibitory concentration (IC50) being 2.3 μM. Similar inhibition patterns were observed against HBV genotype D and C RNaseHs, implying limited genotype specificity. Six of 10 compounds tested against HBV replication in culture suppressed replication via blocking of viral RNaseH activity, with the best 50% effective concentration (EC50) being 0.34 μM. Eighteen compounds inhibited recombinant human RNaseH1, and moderate cytotoxicity was observed for all compounds (50% cytotoxic concentration [CC50] = 25 to 79 μM). Therapeutic indexes ranged from 3.8 to 94. Efficient inhibition required an intact α-hydroxytropolone moiety plus one or more short appendages on the tropolone ring, but a wide variety of constituents were permissible. These data indicate that troponoids and specifically α-hydroxytropolones are promising lead candidates for development as anti-HBV drugs, providing that toxicity can be minimized. Potential anti-RNaseH drugs are envisioned to be employed in combination with the existing nucleos(t)ide analogs to suppress HBV replication far enough to block genomic maintenance, with the goal of eradicating infection.

scholarly journals A rapid and efficient screening system for neutralizing antibodies and its application for the discovery of potent neutralizing antibodies to SARS-CoV-2 S-RBD

2020 ◽  
Author(s):  
Xiaojian Han ◽  
Yingming Wang ◽  
Shenglong Li ◽  
Chao Hu ◽  
Tingting Li ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Inhibitory Concentration ◽  
Emerging Infectious Diseases ◽  
Receptor Binding Domain ◽  
Screening System ◽  
High Potency ◽  
Preliminary Screening ◽  
Neutralization Activity ◽  
Prevention And Treatment ◽  
Efficient Screening

AbstractNeutralizing antibodies (Abs) have been considered as promising therapeutics for the prevention and treatment of pathogens. After the outbreak of COVID-19, potent neutralizing Abs to SARS-CoV-2 were promptly developed, and a few of those neutralizing Abs are being tested in clinical studies. However, there were few methodologies detailly reported on how to rapidly and efficiently generate neutralizing Abs of interest. Here, we present a strategically optimized method for precisive screening of neutralizing monoclonal antibodies (mAbs), which enabled us to identify SARS-CoV-2 receptor-binding domain (RBD) specific Abs within 4 days, followed by another 2 days for neutralization activity evaluation. By applying the screening system, we obtained 198 Abs against the RBD of SARS-CoV-2. Excitingly, we found that approximately 50% (96/198) of them were candidate neutralizing Abs in a preliminary screening of SARS-CoV-2 pseudovirus and 20 of these 96 neutralizing Abs were confirmed with high potency. Furthermore, 2 mAbs with the highest neutralizing potency were identified to block authentic SARS-CoV-2 with the half-maximal inhibitory concentration (IC50) at concentrations of 9.88 ng/ml and 11.13 ng/ml. In this report, we demonstrated that the optimized neutralizing Abs screening system is useful for the rapid and efficient discovery of potent neutralizing Abs against SARS-CoV-2. Our study provides a methodology for the generation of preventive and therapeutic antibody drugs for emerging infectious diseases.

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