scholarly journals A novel 1,4-dihydropyridine-binding site on mitochondrial membranes from guinea-pig heart, liver and kidney

1988 ◽  
Vol 253 (1) ◽  
pp. 49-58 ◽  
Author(s):  
G Zernig ◽  
H Glossmann
Keyword(s):  
Guinea Pig ◽  
Binding Site ◽  
Binding Sites ◽  
Ph Dependence ◽  
High Capacity ◽  
Mitochondrial Membranes ◽  
Guinea Pig Heart ◽  
Heat Stable ◽  
Liver And Kidney ◽  
Dihydropyridine Binding Site

The 1,4-dihydropyridine (+/-)-[3H]nitrendipine reversibly binds to mitochondrial preparations from guinea-pig heart with a dissociation constant (Kd) of 593 +/- 77 nM and a maximum density of binding sites (Bmax.) of 1.75 +/- 0.27 nmol/mg of protein. This low-affinity high-capacity 1,4-dihydropyridine-binding site does not discriminate between the enantiomers of nitrendipine and is also found in mitochondrial membranes from guinea-pig liver (Kd 586 +/- 91 nM; Bmax. 0.36 +/- 0.04 nmol/mg of protein) and kidney (Kd 657 +/- 149 nM; Bmax. 0.56 +/- 0.12 nmol/mg of protein). Phenylalkylamines (e.g. verapamil) inhibit (+/-)-[3H]nitrendipine binding with micromolar inhibition constants, but the benzothiazepine D-cis-diltiazem, a potent Ca2+-channel blocker, is without effect. The binding is heat-stable, shows a V-shaped pH-dependence with a minimum around pH 7.0, and is strongly dependent on ionic strength in the incubation medium. The cations La3+ greater than Cd2+ much greater than Co2+ greater than Ca2+ much greater than Ba2+ greater than Mg2+ greater than Li+ greater than Na+ and the anions NO3- greater than C1- greater than or equal to F- stimulate the binding, whereas PO4(3-) greater than SO4(2-) slightly inhibit it. The low-affinity (+/-)-[3H]nitrendipine-binding site located on the mitochondrial inner membrane is biochemically and pharmacologically different from the 1,4-dihydropyridine-receptor domain of the L-type Ca2+ channel. Furthermore, it is not identical with any of the low-affinity 1,4-dihydropyridine-binding sites described so far.

Mitochondrial Oxidation of p-N, N-Dimethylamino-β-Phenethylamine (DAPA)

1975 ◽  
Vol 33 ◽  
pp. 458-459
Author(s):  
W. Allen Shannon ◽  
Hannah L. Wasserkrug ◽  
andArnold M. Seligman
Keyword(s):  
Guinea Pig ◽  
Oxidase Activity ◽  
Amine Oxidase ◽  
Histochemical Demonstration ◽  
Guinea Pig Heart ◽  
Pig Kidney ◽  
Cytoplasmic Vacuoles ◽  
Liver And Kidney ◽  
Mitochondrial Oxidation ◽  
Amine Oxidase Activity

The synthesis of a new substrate, p-N,N-dimethylamino-β-phenethylamine (DAPA)3 (Fig. 1) (1,2), and the testing of it as a possible substrate for tissue amine oxidase activity have resulted in the ultracytochemical localization of enzyme oxidase activity referred to as DAPA oxidase (DAPAO). DAPA was designed with the goal of providing an amine that would yield on oxidation a stronger reducing aldehyde than does tryptamine in the histochemical demonstration of monoamine oxidase (MAO) with tetrazolium salts.Ultracytochemical preparations of guinea pig heart, liver and kidney and rat heart and liver were studied. Guinea pig kidney, known to exhibit high levels of MAO, appeared the most reactive of the tissues studied. DAPAO reaction product appears primarily in mitochondrial outer compartments and cristae (Figs. 2-4). Reaction product is also localized in endoplasmic reticulum, cytoplasmic vacuoles and nuclear envelopes (Figs. 2 and 3) and in the sarcoplasmic reticulum of heart.

scholarly journals Photoaffinity labelling of the cardiac calcium channel. (−)-[3H]azidopine labels a 165 kDa polypeptide, and evidence against a [3H]-1,4-dihydropyridine-isothiocyanate being a calcium-channel-specific affinity ligand

1987 ◽  
Vol 243 (1) ◽  
pp. 127-135 ◽  
Author(s):  
D R Ferry ◽  
A Goll ◽  
H Glossmann
Keyword(s):  
Guinea Pig ◽  
Calcium Channel ◽  
Polyacrylamide Gel Electrophoresis ◽  
Dimethyl Ester ◽  
High Capacity ◽  
Bay K 8644 ◽  
Channel Blockers ◽  
Guinea Pig Heart ◽  
Affinity Ligand ◽  
Heart Membranes

The arylazide 1,4-dihydropyridine (-)-[3H]azidopine binds to a saturable population of sites in guinea-pig heart membranes with a dissociation constant (KD) of 30 +/- 7 pM and a density (Bmax.) of 670 +/- 97 fmol/mg of protein. This high-affinity binding site is assumed to reside on voltage-operated calcium channels because reversible binding is blocked stereoselectively by 1,4-dihydropyridine channel blockers and by the enantiomers of Bay K 8644. A low-affinity (KD 25 +/- 7 nM) high-capacity (Bmax. 21.6 +/- 9 pmol/mg of protein) site does not bind (-)- or (+)-Bay K 8644, but is blocked by high concentrations (greater than 500 nM) of dihydro-2,6-dimethyl-4-(2-isothiocyanatophenyl)-3,5-pyridinedicarboxy lic acid dimethyl ester (1,4-DHP-isothiocyanate) or, e.g., (+/-)-nicardipine. (-)-[3H]Azidopine was photoincorporated covalently into bands of 165 +/- 8, 39 +/- 2 and 35 +/- 3 kDa, as determined by SDS/polyacrylamide-gel electrophoresis. Labelling of the 165 kDa band is protected stereoselectively by 1,4-dihydropyridine enantiomers at low (nM) concentrations and by (-)- and (+)-Bay K 8644, whereas the lower-Mr bands are not. Thus, only the 165 kDa band is the calcium-channel-linked 1,4-dihydropyridine receptor. Photolabelling of the 39 or 35 kDa bands was only blocked by 10 microM-1,4-DHP-isothiocyanate or 50 microM-(+/-)-nicardipine but not by 10 microM-(-)-Bay K 8644. [3H]-1,4-DHP-isothiocyanate binds to guinea-pig heart membranes with a KD of 0.35 nM and dissociates with a k-1 of 0.2 min-1 at 30 degrees C. [3H]-1,4 DHP-isothiocyanate irreversibly labels bands of 39 and 35 kDa which are protected by greater than 10 microM-(+/-)-nicardipine or unlabelled ligand but not by 10 microM-(-)-Bay K 8644. Thus, [3H]-1,4-DHP-isothiocyanate is not an affinity probe for the calcium channel.

Hypoxia- and normoxia-induced reversibility of autonomic control in Andean guinea pig heart

1996 ◽  
Vol 81 (5) ◽  
pp. 2229-2234 ◽  
Author(s):  
Fabiola León-Velarde ◽  
Jean-Paul Richalet ◽  
Juan-Carlos Chavez ◽  
Rachid Kacimi ◽  
Maria Rivera-Chira ◽  
...  
Keyword(s):  
Guinea Pig ◽  
High Altitude ◽  
Binding Sites ◽  
Heart Rate Response ◽  
Differential Regulation ◽  
Autonomic Control ◽  
Cavia Porcellus ◽  
Guinea Pig Heart ◽  
Jean Paul ◽  
Cardiac Receptors

León-Velarde, Fabiola, Jean-Paul Richalet, Juan-Carlos Chavez, Rachid Kacimi, Maria Rivera-Chira, José-Antonio Palacios, and Daniel Clark. Hypoxia- and normoxia-induced reversibility of autonomic control in Andean guinea pig heart. J. Appl. Physiol. 81(5): 2229–2234, 1996.—We herein describe the regulation of cardiac receptors in a typical high-altitude native animal. Heart rate response to isoproterenol (HRIso) (beats ⋅ min−1 ⋅ mg Iso ⋅ kg−1) and atropine, the density of β-adrenergic (βAR) and muscarinic (M2) receptors, and the ventricular content of norepinephrine (NE) and dopamine (DA) were studied in guinea pigs ( Cavia porcellus). Animals native to Lima, Peru (150 m) were studied at sea level (SL) and after 5 wk at 4,300-m altitude (SL-HA). Animals native to Rancas [Pasco, Peru (4,300 m)] were studied at high altitude (HA) and after 5 wk at SL (HA-SL). HA animals had a lower HRIso, maximum number of βAR binding sites (Bmax), βAR dissociation constant ( K d), NE, and DA ( P < 0.05) and a higher M2Bmax( P < 0.001) when compared with the SL group. HA-SL showed an increase of the HRIso, βAR K d, and NE ( P < 0.05) and a decrease of the M2Bmax and K d( P < 0.0001) when compared with the HA group. The present study demonstrates the differential regulation and reversibility of the autonomic control in the guinea pig heart.

Oxidation of citrate by mitochondria from guinea pig heart, liver and kidney

1961 ◽  
Vol 200 (2) ◽  
pp. 355-358 ◽  
Author(s):  
Menard M. Gertler ◽  
Dino Mancini
Keyword(s):  
Guinea Pig ◽  
Heart Muscle ◽  
Tissue Preparation ◽  
Guinea Pig Heart ◽  
Atp Level ◽  
Liver And Kidney ◽  
Choice Of Species

There is no unanimity concerning the ability of mitochondrial systems from heart, liver and kidney to oxidize citrate. This disparity stems partly from lack of uniformity in the choice of species and partly from the mode of tissue preparation. The present results have been obtained with mitochondria prepared from guinea pig heart, liver and kidney. The QNOO2 for citrate at 2 mm ATP level is highest in kidney, 590, followed by liver, 417 and heart, 255. Kidney and liver oxidations are accompanied by phosphorylations while oxidation of citrate in the cardiac preparations are unaccompanied by any phosphorylation. Further differences are manifested by lowering ATP level in the medium to 0.33 mm. There is a decrease in the QNOO2 in liver and kidney with no change in phosphorylation ability, while the QNOO2 in the heart is increased by 25%. Greatest changes are those observed by addition of DPN and TPN which inordinately increase QNOO2 in the heart muscle, particularly at lower levels of ATP. Addition of CoA has virtually no effect on the QNOO2 or P/O ratios in any of the tissue preparations.

scholarly journals Interactions between inhibitors of dihydrofolate reductase

1989 ◽  
Vol 258 (2) ◽  
pp. 335-342 ◽  
Author(s):  
K Bowden ◽  
A D Hall ◽  
B Birdsall ◽  
J Feeney ◽  
G C K Roberts
Keyword(s):  
Steady State ◽  
Binding Site ◽  
Dihydrofolate Reductase ◽  
Binding Sites ◽  
High Field ◽  
Ph Dependence ◽  
Histidine Residue ◽  
Adenine Ring ◽  
Steady State Kinetics ◽  
Pka Value

The binding of substrates and inhibitors to dihydrofolate reductase was studied by steady-state kinetics and high-field 1H-n.m.r. spectroscopy. A series of 5-substituted 2,4-diaminopyrimidines were examined and were found to be ‘tightly binding’ inhibitors of the enzyme (Ki less than 10(-9) M). Studies on the binding of 4-substituted benzenesulphonamides and benzenesulphonic acids also established the existence of a ‘sulphonamide-binding site’ on the enzyme. Subsequent n.m.r. experiments showed that there are two binding sites for the sulphonamides on the enzyme, one of which overlaps the coenzyme (NADPH) adenine-ring-binding site. An examination of the pH-dependence of the binding of sulphonamides to the enzyme indicated the influence of an ionizable group on the enzyme that was not directly involved in the sulphonamide binding. The change in pKa value from 6.7 to 7.2 observed on sulphonamide binding suggests the involvement of a histidine residue, which could be histidine-28.

Effect of doxorubicin on calcium binding sites in guinea-pig heart

1986 ◽  
Vol 35 (7) ◽  
pp. 1203-1204 ◽  
Author(s):  
Fabrizio Villani ◽  
Elena Monti ◽  
Francesco Piccinini ◽  
Luigia Favall ◽  
Annalinda Rozza Dionigi ◽  
...  
Keyword(s):  
Guinea Pig ◽  
Binding Sites ◽  
Calcium Binding ◽  
Guinea Pig Heart ◽  
Calcium Binding Sites
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