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Biomedical subjects

A Goll

Publications and source records attributed to A Goll.

At least 19 recordsLinked to original sources
PubMed2005

Optical coherence tomography guided retreatment of photodynamic therapy.

AIM: To evaluate the results of a retreatment modality of photodynamic therapy (PDT) based on optical coherence tomography (OCT) and fluorescein angiography (FA). To quantify the effect of PDT with the help of measurement of the retinal thickness. METHODS: Eyes with predominantly classic subfoveal choroidal neovascularisation (CNV) due to age related macular degeneration were included. PDT was performed every three months, when needed. OCT, FA, and measures of distance acuity were performed at baseline, after 6 weeks, 3 months, and from then on every 3 months. A control group of a consecutive series of eyes that had been retreated based only on FA results was installed. RESULTS: Forty eyes of 38 patients were included. The average age was 73 years. The maximum retinal thickness decreased from 404 mum at baseline to 281.6 mum at month 12. Furthermore there was a significant decrease of retinal thickness in both subgroups. The number of retreatments was reduced, when activity was diagnosed using OCT and FA. (2.4 v 4.0). The distance acuity correlated significantly with the maximum retinal thickness (p=0.0042). CONCLUSION: Information about the activity of a neovascular lesion can be obtained with the help of OCT. The retreatment modalities can be optimised by using OCT and FA and the number of retreatments can be reduced.

Aged↗
PubMed2005

Fetal and juvenile animal hemorheology.

The study provides information on the blood fluidity in healthy, juvenile sheep and rabbits during growth (n = 18), and shows also data from fetal rabbits and cats. In the fetal rabbit (n = 3) and cat (n = 2), whole blood viscosity (WBV; LS30, Contraves, Switzerland) and plasma viscosity (PV; OCR-D, Paar, Austria) was low (WBV (0.7 s(-1)): rabbit: 3.28/3.00/2.44; cat: 7.87/10.88; WBV (94 s(-1)): rabbit: 2.57/2.48/2.39; cat: 2.75/3.73 mPa s) (PV: rabbit: 1.10/1.10/1.05; cat: 1.27/1.39 mPa s), which was associated with a low plasma protein concentration and a low erythrocyte count despite a high erythrocyte volume. After parturition, blood viscosity increased in rabbits in parallel with hematocrit, while MCV decreased (WBV (0.7 s(-1)): 9.28 (8.07/10.88); WBV (94 s(-1)): 3.67 (3.62/3.82); PV: 1.15 (1.15/1.25) mPa s). In contrast, in the sheep, whole blood and plasma viscosity decreased after delivery (WBV (0.7 s(-1)): 1.31 (0.94/1.88); WBV (94 s(-1)): 2.45 (2.43/2.85) PV: 1.24 (1.23/1.29) mPa s). Hematocrit and MCV decreased, while erythrocyte count increased under these circumstances. In summary, whole blood viscosity was similar among fetal sheep, rabbits, and cats and is diminished compared to adult individuals to guarantee an optimal oxygen supply during a period of life in which the oxygen maintainance of the child depends on the health and the environment of the mother. However, during growth, blood viscosity rose in rabbits, while it continuously decreased in the sheep. At an unknown time point this fall in blood viscosity in lambs must reverse, since adult sheep again show a higher blood viscosity than juvenile lambs at the age of 2 months.

Analysis of Variance↗
PubMed1999

Hepatitis C virus infection risk factors in patients admitted in hospital emergency departments in Picardy. Value of oriented screening based on recommendations of the 'Direction Générale de la Santé'.

OBJECTIVE AND DESIGN: Oriented hepatitis C virus (HCV) screening on the basis of transfusion, previous or current parenteral drug addiction, invasive procedures, and in family members of patients with hepatitis C, was recommended in France by the 'Direction Générale de la Santé' (DGS). The aim of this study was to estimate the frequency of these risk factors in patients admitted in hospital emergency departments in Picardy. METHODS: Between 1 June and 31 July 1996, physicians of the emergency units of seven hospitals in Picardy were asked to question admitted patients about risk factors mentioned in the DGS recommendations, and to suggest a screening test when at least one of these risk factors was present. RESULTS: Among 1648 patients, 68.7% had at least one of these risk factors. Screening was accepted by 723 patients, 58.7% of those with at least one risk factor, and more than 70% of those with history of transfusion and/or drug addiction. It was immediately performed in 451, and 2.4% had anti-HCV antibodies. The prevalence of anti-HCV antibodies was 1.5% in patients without history of transfusion or drug addiction and 7.9% in those with at least one of these two risk factors. CONCLUSION: Oriented screening based on transfusion or drug addiction history seems to have better efficiency than the screening policy recommended by the DGS. Poor reliability of answers about medical history was observed probably because of stress related to emergency circumstances. A screening test proposed to patients with these major risk factors by their usual physician would be probably more efficient.

Aged↗
PubMed1995

Comprehensive allelotyping of human renal cell carcinomas using microsatellite DNA probes.

The von Hippel-Lindau locus on chromosome 3p is a tumor suppressor gene known to be involved in nonpapillary renal cell carcinoma. A previous loss of heterozygosity (LOH) study aimed at determining the allelotype of kidney tumors has indicated that in addition to 3p, chromosome arms 5q, 6q, 10q, 11q, 17p, and 19p may also harbor tumor suppressor genes. However, cytogenetic studies reveal that chromosomes 3p, 6q, 8p, 9pq, and 14q most frequently undergo karyotypic changes in renal tumors. To resolve these differences, a collection of microsatellite DNA probes has been used to scan for LOH so that 90% of individual tumor genomes were rendered informative for allele loss. The assay is capable of detecting quantitative genomic alterations in tumor cells as well. We find that LOH is most frequent for chromosome arm 3p. However, in no tumor is 3p exclusively affected. LOH for 6q, 8p, 9pq, and 14q is also distinctly elevated for both nonpapillary as well as papillary tumors and suggest that many of the tumor suppressor loci involved may be common to the etiology of both forms of kidney cancer.

Alleles↗
PubMed1994

[Pancreatic-portal fistula and subcutaneous fat necrosis].

The pancreaticoportal fistula is a very uncommon complication of pancreatic diseases, mostly occurring in the course of chronic pancreatitis. From 2 findings of pancreaticoportal fistula and a literature review, we emphasize in this study the frequency of subcutaneous fat necrosis during pancreaticoportal fistula, the pathogenic role of the pancreaticoportal fistula to induce a subcutaneous fat necrosis by means of a massive enzyme release in the systemic circulation, the protective role of portal thrombosis, and finally the diagnostic and therapeutic value of endoscopic retrograde cholangio-pancreatography in pancreaticoportal fistula.

Adult↗
PubMed1988

Characteristics of the binding of the antihypertensive agent moxonidine to alpha 2-adrenoceptors in rat brain membranes.

The characteristics of the alpha 2-adrenoceptor binding property of moxonidine (4-chloro-N-(4,5-dihydro-1H-imidazol-2-yl)-6-methoxy-2-methyl-5-pyrim idinamine, BDF 5895), a newly developed centrally acting antihypertensive agent, was investigated in receptor binding assays using [3H]-rauwolscine bound to rat cortex membranes. The results are as follows: 1. [3H]-Rauwolscine binds in a saturable manner to rat cerebral cortex membranes with a Bmax of 320 +/- 50 fmol/mg of protein and KD of 1.7 +/- 0.5 nmol/l. 2. The alpha 2-adrenoceptor antagonist yohimbine competes with a Ki value of 8.0 nmol/l and slope factor close to unity. (--)-Norepinephrine competes with an overall Ki-value of 50 nmol/l and slope factor of 0.61. Mg2+ concentrations of 10 mmol/l in the presence of 0.1 mmol/l 3'-guanylimidodiphosphate (Gpp(NH)p) (Mg2+/Gpp(NH)p) do not affect the yohimbine inhibition curve, but increase the (--)-norepinephrine Ki value to 2.8 mumol and the slope factor to unity. 3. Moxonidine competes with [3H]-rauwolscine binding with an overall Ki value of 460 nmol/l and slope factor of 0.72. Moxonidine binding inhibition was potentiated by Mg2+, but shifted to lower potency by Mg2+/Gpp(NH)p and 150 mmol/l NaCl. 4. Binding inhibition curves for (--)-norepinephrine and moxonidine fitted better to a 2-site model and could be interpreted in terms of high affinity site (with Ki value KH) and low affinity site (with Ki value KL). The effect of Mg2+/Gpp(NH)p and NaCl was to convert the high affinity sites to low affinity sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

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.

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.

Affinity Labels↗
PubMed1987

Interaction between calcium channel ligands and calcium channels.

Distinct drug receptors for 1,4-dihydropyridines, phenylalkylamines, and the benzothiazepine d-cis-diltiazem exist on voltage-dependent calcium channels. The drug receptors show reciprocal allosteric communication and are linked to calcium binding sites. The 1,4-dihydropyridine-selective receptor (probed with [3H]nimodipine) has a size (measured by radiation inactivation) identical in heart, skeletal muscle, and brain (180 kDa). To compare the sizes of 1,4-dihydropyridine receptors in different tissues, pure tritiated enantiomers of the arylazido photoaffinity probe [3H]azidopine were used to irreversibly label the purified 1,4-dihydropyridine receptor (155, 65, and 32 kDa) from guinea pig skeletal muscle transverse tubules and the membrane-bound cardiac receptor. The 155 kDa polypeptide region, but not the 65 or 32 kDa bands, was specifically labelled by (-)-[3H]azidopine. (+)-[3H]Azidopine did not label any of the polypeptides in the purified receptor preparation. In contrast with the results from other investigators, a 155 kDa polypeptide was also specifically labelled in cardiac membranes by (-)-[3H]azidopine. A 34 kDa photolabelled band carries a low-affinity 1,4-dihydropyridine binding site that has no obvious relation to the channel but is abundant in heart membranes and has apparently led to erroneous results in previous affinity or photoaffinity labelling experiments. Antibodies raised against the purified skeletal muscle channel precipitate the 125I-labelled 155 kDa channel polypeptide from skeletal muscle and precipitate the three drug receptor sites from both crude and purified channel preparations. We conclude that all three drug receptor sites are localized on this polypeptide.

Affinity Labels↗
PubMed1986

Stereoselective photoaffinity labelling of the purified 1,4-dihydropyridine receptor of the voltage-dependent calcium channel.

The voltage-dependent calcium channel from guinea-pig skeletal muscle T-tubules has been isolated with a rapid, two-step purification procedure. Reversible postlabelling of the channel-linked 1,4-dihydropyridine receptor and stereoselective photolabelling as a novel approach were employed to assess purity. A 135-fold purification to a specific activity of 1311 +/- 194 pmol/mg protein (determined by reversible equilibrium binding with (+)-[3H]PN200-110) was achieved. Three polypeptides of 155 kDa, 65 kDa and 32 kDa were identified in the purified preparation. The 155-kDa band is a glycoprotein. The arylazide photoaffinity probe (-)-[3H]azidopine bound with high affinity to solubilized membranes (Kd = 0.7 +/- 0.2 nM) and highly purified fractions (Kd = 3.1 +/- 2 nM), whereas the optical antipode (+)-azidopine was of much lower affinity. Irradiation of (-)-[3H]azidopine and (+)-[3H]azidopine receptor complexes with ultraviolet light led to preferential incorporation of the (-) enantiomer into the 155-kDa polypeptide in crude solubilized and purified preparations. The pharmacological profile of irreversible labelling of the 155-kDa glycoprotein by (-)-[3H]azidopine is identical to that found in reversible binding experiments. Specific photolabelling of the 155-kDa band by (-)-[3H]azidopine per milligram of protein increases 150-fold upon purification, whereas incorporation into non-specific bands in the crude solubilized material is identical for both, (-) and (+)-[3H]azidopine.

Affinity Labels↗
PubMed1986

Molecular structure of rat brain apamin receptor: differential photoaffinity labeling of putative K+ channel subunits and target size analysis.

Two photoreactive apamin derivatives were prepared with an aryl azide [[(azidonitrophenyl)amino]acetate (ANPAA)] group coupled at different positions on the neurotoxin molecule. These ligands were used to identify membrane components in the environment of the neuronal binding site that is associated with a Ca2+-activated K+ channel. 125I-[alpha-ANPAA-Cys1] apamin labeled a single Mr 86 000 chain in cultured neurons whereas two bands corresponding to Mr 86 000 and 59,000 were detected in synaptic membrane preparations, suggesting that the Mr 59,000 polypeptide may be a degradation product. 125I-[epsilon-ANPAA-Lys4]apamin however incorporated uniquely into two smaller components with Mr 33,000 and 22,000 in both cultured neurons and synaptic membranes. Randomly modified 125I-ANPAA-apamin gave a cross-linking profile equivalent to the sum of those obtained with the two defined derivatives. The apamin binding site seems to be located at the frontier between three or more putative K+ channel subunits which are only accessible from limited regions of the receptor-associated photoprobe. Irradiation of frozen rat brain membranes with high-energy electrons led to a reduction in 125I-apamin receptor capacity, yielding a target size for the functional binding unit of Mr 84,000-115,000, which could be constituted by the Mr 86,000 subunit alone or by the Mr 86,000 subunit in conjuction with one of the two smaller subunits.

Affinity Labels↗
PubMed1986

Purified calcium channels have three allosterically coupled drug receptors.

(-)-[3H]Desmethoxyverapamil and (+)-[3H]PN 200-110 were employed to characterize phenylalkylamine-selective and 1,4-dihydropyridine-selective receptors on purified Ca2+ channels from guinea-pig skeletal muscle t-tubules. In contrast to the membrane-bound Ca2+ channel, d-cis-diltiazem (EC50 = 4.5 +/- 1.7 microM) markedly stimulated the binding of (+)-[3H]PN 200-110 to the purified ionic pore. In the presence of 100 microM d-cis-diltiazem (which binds to the benzothiazepine-selective receptors) the Bmax for (+)-[3H]PN 200-110 increased from 497 +/- 81 to 1557 +/- 43 pmol per mg protein, whereas the Kd decreased from 8.8 +/- 1.7 to 4.7 +/- 1.8 nM at 25 degrees C. P-cis-Diltiazem was inactive. (-)-Desmethoxyverapamil, which is a negative heterotropic allosteric inhibitor of (+)-[3H]IN 200-110 binding to membrane-bound channels, stimulated 1,4-dihydropyridine binding to the isolated channel. (-)-[3H]Desmethoxyverapamil binding was stimulated by antagonistic 1,4-dihydropyridines [(+)-PN 200-110 greater than (-)(R)-202-791 greater than (+)(4R)-Bay K 8644] whereas the agonistic enantiomers (+)(S)-202-791 and (-)(4S)-Bay K 8644 were inhibitory and (-)-PN 200-110 was inactive. The results indicate that three distinct drug-receptor sites exist on the purified Ca2+ channel, two of which are shown by direct labelling to be reciprocally allosterically coupled.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1986

Correlation between the negative inotropic potency and binding parameters of 1,4-dihydropyridine and phenylalkylamine calcium channel blockers in cat heart.

Partially purified plasma membranes were prepared from cat ventricle. The purification factors for the calcium channel ligands (+)-3H-PN 200-110, 3H-nimodipine (1,4-dihydropyridines) and (-)-3-H-desmethoxyverapamil (a phenylalkylamine) were 3.1-, 3.4- and 2.9-fold, respectively, whilst beta-adrenoceptors labelled with (-)-3H-dihydroalprenolol were purified 3.0-fold. (+)-3H-PN 200-110 bound to 930 +/- 140 fmol/mg of membrane protein with a dissociation constant of 70 pmol/l at 25 degrees C. Under the same conditions 3H-nimodipine bound to 490 +/- 24 fmol/mg of sites with a KD of 120 pmol/l. (-)-3-H-desmethoxyverapamil bound to 530 +/- 55 fmol/mg of sites with a KD of 2.47 nmol/l. Twelve 1,4-dihydropyridines were evaluated for binding inhibition constants (Ki) with (+)-3H-PN 200-110 and 13 phenylalkylamines with (-)-3-H-desmethoxyverapamil in radioligand binding assays. Of the twelve 1,4-dihydropyridines evaluated (+/-)-nitrendipine was the most potent with a Ki-value of 280 pmol/l, nifedipine had a Ki-value of 500 pmol/l and the weakest drug tested, (+/-)-Bay b 4328, had a Ki-value of 14.3 nmol/l. The EC50-values of the same 1,4-dihydropyridines to inhibit the electrically driven cat papillary muscle were 77- to 3,450-fold higher and little correlation existed between Ki and EC50-values. Thirteen phenylalkylamines were tested for their potency to inhibit (-)-3-H-desmethoxyverapamil binding. The most potent phenylalkylamine with respect to negative inotropy was (+/-)-D 595 with an EC50-value of 794 nmol/l, the least potent substance was (+/-)-Sz 45 with an EC50-value of 39.8 mumol/l. The binding inhibition constants for the phenylalkylamines were 13- to 322-fold lower than the values for negative inotropy, but a significant positive correlation between the Ki and EC50-values (n = 12, r = 0.84) was observed. The absolute differences may reflect the state-dependent binding of phenylalkylamines to the channel. QSAR analysis revealed nearly identical correlations between physicochemical substituent properties on the one hand and binding affinities or functional potency on the other hand. In both cases the electronic properties (F-constant) of ring substituents mainly determine the variance in potency.

Animals↗
PubMed1985

Subunit composition of skeletal muscle transverse tubule calcium channels evaluated with the 1,4-dihydropyridine photoaffinity probe, [3H]azidopine.

The arylazide 1,4-dihydropyridine, [3H]azidopine, binds with high affinity to calcium channels in partially purified guinea-pig skeletal muscle transverse tubule membranes. Upon brief exposure to u.v. light, [3H]azidopine incorporates covalently into transverse tubule membrane proteins, as judged by SDS-PAGE. After alkylation of sulfhydryl groups with N-ethylmaleimide three specifically labelled bands of mol wts. 240 kd, 158 kd and 99 kd are always observed with fluorography after one-dimensional SDS-PAGE. Two other specific bands with mol. wts. of 52 kd and 55 kd, respectively, were sometimes observed. Two-dimensional SDS-PAGE (non-reduced but alkylated in the first dimension and reduced in the second dimension) revealed that the 240-kd band after reduction migrates with a mol. wt. of 99 kd. The 158-kd and 99-kd bands do not change in mobility. It is suggested that [3H]azidopine binds in such a way that the arylazide moiety of the ligand comes into contact with at least three calcium channel components: the A component of mol. wt. 240 kd, the B component of mol. wt. 158 kd and a C component of mol. wt. 99 kd. B and C are non-covalently bonded subunits of the channel, whereas A could be a heterodimer consisting of B and C, linked by disulfide bonds. Subunits of smaller mol. wt. may be also part of the ionic pore. Photolabelling of transverse tubule membranes after high energy irradiation with 10 MeV electrons supports this interpretation.

Affinity Labels↗
PubMed1985

Molecular approach to the calcium channel.

Tritiated 1,4-dihydropyridines (nimodipine, nitrendipine, nifedipine, PN 200-110) and [3H]D-cis-diltiazem as well as [3H]verapamil were employed to directly identify calcium channels in membranes for excitable tissues. The channels, when probed with 1,4-dihydropyridines, exhibit the following properties: 1,4-Dihydropyridine calcium channel blockers bind in a temperature-dependent, reversible manner and with high affinity (dissociation constants 0.2-2 nM at 37 degrees C) to a finite number of sites. For chiral 1,4-dihydropyridines, the binding is stereoselective. Hill slopes of approximately 1.0 are observed. In brain, heart, and solubilized skeletal-muscle membranes, an absolute requirement for certain divalent cations exists in order to bind the ligands with high affinity. Cooperativity (negative and positive) between Me2+ and 1,4-dihydropyridine binding sites is observed. 1,4-Dihydropyridine binding sites are down-regulated in a complex manner by the optically pure enantiomers of D-600 and verapamil. These channel blockers induce, to a different extent, a low-affinity state of the 1,4-dihydropyridine binding site. It is postulated that this allosteric site, at which these blockers act, is closely coupled to the 1,4-dihydropyridine binding site and that a spectrum of compounds exists that differ in their affinity as well as their intrinsic activity to induce the down-regulation. The 1,4-dihydropyridine binding sites are up-regulated by D-cis-diltiazem and KB-944. The up-regulation is temperature-dependent and induces a high-affinity state for 1,4-dihydropyridine channel blockers, accompanied by distinct alterations of the kinetics as well as the pharmacological profile of the 1,4-dihydropyridine binding sites. Complex interactions exist between the channel blockers that induce up-regulation and those that induce down-regulation of the binding. For a given radiolabeled 1,4-dihydropyridine, a tissue-specific (but not species-specific) equilibrium binding dissociation constant is observed. Thus, all heart (human, rat, guinea pig, frog, bovine) have the same KD (0.25 nM at 37 degrees C) for, [3H]nimodipine. The same is observed for brain (KD = 0.5 nM) and for skeletal muscle (KD = 1-2 nM). Three subtypes of channels can be distinguished on the basis of the KD and the tissue-specific up-regulation by D-cis-diltiazem. Subtype-selective drugs exist; e.g., AQA 39 is an inhibitor of [3H]nimodipine binding at skeletal-muscle calcium channels, but not at brain channels. Despite their different pharmacological and kinetic profiles, calcium channels in skeletal muscle and brain have the same molecular size (Mr) when probed by radiation inactivation.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1985

Calcium channels: basic properties as revealed by radioligand binding studies.

The structural features and molecular properties of calcium channels have been explored with 1,4-dihydropyridines, d-cis diltiazem and verapamil in radiolabeled form. The concept of multiple (at least three) drug receptor sites of the channel (sites 1, 2, and 3) has been proposed. Site 1 is labeled by 1,4 dihydropyridines, site 2 by (+/-) verapamil (in skeletal muscle) and by (-) desmethoxyverapamil (in brain, skeletal muscle, and heart), site 3 by d-cis diltiazem (in skeletal muscle). The three distinct drug receptor sites communicate with each other via reciprocal allosterism and are linked to divalent cation binding sites. The concept of the reciprocal allosteric communication between the receptor sites has been proven by individual labeling of each of the drug receptor sites in the skeletal muscle t-tubule membrane where the density of calcium channels is extremely high. In brain and heart, labeling of the receptor sites 1 and 2 has also been successful. Three novel ligands for the calcium channel are presented: 125I-iodipine, a 1,4 dihydropyridine ligand, which has an exceptionally high specific activity (2,200 Ci/mmol), 3H-azidopine, a high-affinity, photoaffinity ligand and finally (-) 3H-desmethoxyverapamil, a high-affinity radioligand for receptor site 2. These ligands will aid in the isolation and final characterisation of the calcium channel. 125I-iodipine, 3H-azidopine and (-) 3H-desmethoxyverapamil bind to detergent-solubilized channels from skeletal muscle t-tubules. Whereas the 1,4-dihydropyridines label only a 12.9 S macromolecule, (-) 3H-desmethoxyverapamil additionally labels a approximately 5.0 S peak, where almost no high-affinity 1,4 dihydropyridine binding is found. Taken together with the results from target-size analysis experiments and photoaffinity labeling, an oligomeric structure of the channel is most likely. One polypeptide chain (Mr = 145,000) in reduced form has been unambiguously identified and is currently further characterized. It remains to be seen if the all of the earlier postulated four elements of the channel (alpha, beta, delta, gamma) are on different polypeptide chains or if the 145,000 peptide corresponds to the (beta, delta) or the (delta, gamma) element.

Affinity Labels↗
PubMed1985

Calcium channels and calcium channel drugs: recent biochemical and biophysical findings.

The biochemical and biophysical features of the voltage-dependent calcium channels, as discovered in vitro by means of radiolabelled drugs, are presented. The concept of distinct but reciprocally allosterically coupled drug receptor domains linked to calcium binding sites is explained. The evidence for the existence of isochannels (and isoreceptors) is reviewed and the voltage-dependence of 1,4-dihydropyridine binding and action is discussed. The structure of the channel is investigated by radiation-inactivation and by photoaffinity labelling. Low affinity binding sites for calcium channel drugs are shown to reside on the nucleoside carrier.

Adenosine↗
PubMed1984

(-)-[3H]Desmethoxyverapamil, a novel Ca2+ channel probe. Binding characteristics and target size analysis of its receptor in skeletal muscle.

(-)-[3H]Desmethoxyverapamil (2,7-dimethyl-3-(3,4-dimethoxyphenyl)-3-cyan- 7-aza-9-(3-methoxyphenyl)-nonanhydrochloride) was used to label putative Ca2+ channels in guinea pig skeletal muscle. The binding sites for (-)-[3H]desmethoxyverapamil co-purified with t-tubule membrane markers in an established subcellular fractionation procedure. (-)-[3H]Desmethoxyverapamil bound to partially purified t-tubule membranes with a KD of 2.2 +/- 0.1 nM and a Bmax of 18 +/- 4 pmol/mg membrane protein at 25 degrees C. Binding was stereoselectively inhibited by phenylalkylamine Ca2+ antagonists and in a mixed, non-competitive fashion by the benzothiazepine Ca2+ antagonist d-cis-diltiazem and the 1,4-dihydropyridine Ca2+ antagonist (+)-PN 200-110. Target size analysis of the (-)-[3H]desmethoxyverapamil drug receptor site revealed a molecular mass of 107 +/- 2 kDa. In contrast, the target size of the allosterically coupled benzothiazepine drug receptor site, labelled by d-cis-[3H]diltiazem, was 130.5 +/- 4 kDa (p less than 0.01) and of the 1,4-dihydropyridine binding site 179 kDa, when labelled with [3H]nimodipine. It is concluded that (-)-[3H]desmethoxyverapamil is an extremely useful radioligand for the phenylalkylamine-selective receptor site of the t-tubule localized Ca2+ channel which is allosterically linked to two other distinct drug receptor sites.

Animals↗