Is blockade of alpha 1-adrenoceptors favourable in hypotension induced by stimulation of serotonin1A receptors in conscious dogs?
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8-Hydroxy-2-(di-n-propylamino)tetralin↗
Biomedical subjects
R Boer
Publications and source records attributed to R Boer.
Hydrophobic calcium channel ligands: methodical problems and their solution.
Niguldipine is a 1,4-dihydropyridine derivative that combines L-type Ca2+ channel-blocking effects and alpha 1-adrenolytic activity within a single molecule, exemplifying a novel approach in the treatment of hypertension. As niguldipine is a very hydrophobic compound, it (1) readily adsorbs to surfaces of the plastic-ware often used in radioligand binding assays and (2) partitions into the hydrophobic membrane compartments. Both phenomena decrease the actual free drug concentration in radioligand-binding assays and lead to gross underestimation of the affinity of niguldipine (and other hydrophobic ligands) for the 1,4-dihydropyridine binding domain of the L-type Ca2+ channel or for alpha 1A adrenoceptors, respectively. Partitioning of the hydrophobic molecules into the membrane phase leads to a dependence of the Ki value on "total receptor" concentration despite mathematic corrections of the experimentally determined IC50 values. The Ki dependence was mimicked by adding denatured membranes (devoid of high-affinity receptor-binding activity) to native membrane preparations. Loss to pipet tips and tubes was avoided by a special dilution protocol. Partitioning into the hydrophobic membrane compartments needed more elaborate correction procedures.
Adsorption↗
Affinity profiles of BTM-1086 and BTM-1041 at muscarinic receptor subtypes and at H1- and alpha 1-receptors.
The affinities of the (+) and (-) enantiomers of the antimuscarinic benzothiazepinone derivative, cis-2,3-dihydro-3-(4-methyl-1-piperazinylmethyl)-2-phenyl-1,5-benzoth iazepin-4 (5H)-one (BTM-1041 and BTM-1086), for muscarinic receptor subtypes, histamine H1-receptors and alpha 1-adrenoceptors were determined in vitro using isolated organs: field-stimulated rabbit vas deferens (M1-receptors), guinea-pig left atrium (M2-receptors), guinea-pig ileum (M3- and histamine H1-receptors) and rat vas deferens (alpha 1-adrenoceptors). We also assessed the binding profile of BTM-1041 and BTM-1086 at muscarinic receptor subtypes in guinea-pig cortex (M1), heart (M2) and salivary glands (M3) as well as at alpha 1-adrenoceptors in rat cerebral cortex. Functional and binding experiments showed that the (-) enantiomer (BTM-1086) had a high affinity (pA2 = 7.98-8.81; pKi = 8.31-9.15) for the three muscarinic receptor subtypes, whereas the (+) enantiomer (BTM-1041) showed a low antimuscarinic potency (pA2 = 4.87-5.31; pKi = 4.85-5.55). This results in an extremely high stereoselectivity for these optical isomers [-)/(+) ratios = 1023 to 6918). The affinity of the (-) enantiomer BTM-1086 was lower for both histamine H1- and alpha 1-receptors than for muscarinic receptors, whereas the reverse was true for the (+) enantiomer, BTM-1041. Thus, the stereochemical demands for the two optical isomers were most stringent at muscarinic receptors but were inverse and less pronounced at histamine H1- and alpha 1-receptors (stereoselectivity ratios = 0.16-0.22).
Adrenergic alpha-Antagonists↗
Stereoselective binding of niguldipine enantiomers to alpha 1A-adrenoceptors labeled with [3H]5-methyl-urapidil.
[3H]5-Methyl-urapidil, a potent antihypertensive derivative of urapidil, binds to alpha 1A-adrenoceptors in rat brain cortex membranes with a dissociation constant (KD) of 0.89 nM and a Bmax of 116 fmol/mg protein. The ligand does not bind to purified liver cell membranes (alpha 1B-adrenoceptors). [3H]5-Methyl-urapidil also labels 5-HT1A receptors in brain membranes (KD: 0.84 nM and Bmax: 235 fmol/mg protein). (+/-)-Niguldipine, a novel 1,4-dihydropyridine with Ca2+-antagonistic as well as alpha 1A-adrenoceptor blocking properties, is a competitive inhibitor of [3H]5-methyl-urapidil binding to alpha 1A-adrenoceptors. In contrast to those for prazosin, the Ki values for niguldipine were highly dependent on the membrane protein concentration, indicating partitioning of niguldipine into hydrophobic compartments unavailable for alpha-adrenoceptor interaction. The extrapolated, 'true' Ki values were as follows: (+/-)-niguldipine: 0.298 nM, (-)-niguldipine: 3.12 nM, (+)-niguldipine: 0.145 nM.
Animals↗
Interaction of telenzepine with muscarinic receptors in mammalian sympathetic ganglia.
The interaction of the antimuscarinic drug telenzepine with muscarinic receptors was studied in rabbit and rat isolated superior cervical sympathetic ganglia. Radioligand binding demonstrated two muscarinic receptor sites in rabbit ganglia, with the characteristics of M1- and M2-receptors. Telenzepine bound to the M1 sites with a KI of 0.94 nmol/l and to the M2 sites with a KI of 17.8 nmol/l; the corresponding values for pirenzepine were 18.6 and 588 nmol/l; for AF-DX 116 the values were 891 and 33 nmol/l respectively. [3H]Telenzepine dissociated from the M1-receptors with a half time of 46 min at 37 degrees C. Electrophysiological experiments demonstrated that telenzepine reduced the amplitude of the extracellularly recorded slow excitatory postsynaptic potential and the slow inhibitory postsynaptic potential (ED50: 38 and 253 nmol/l respectively). In rat ganglia, application of muscarine or the M1-receptor agonist McN-A-343 increased the amplitude of submaximal population action potentials. This facilitation of synaptic transmission was potently blocked by telenzepine and pirenzepine but only weakly by AF-DX 116 (ED50: ca. 30, 150 and 20 mumol/l, respectively). It is concluded that telenzepine blocks the generation of the slow excitatory postsynaptic potential and the excitatory action of muscarine and McN-A-343 via an action on muscarinic M1-receptors.
Animals↗
The affinity, selectivity and biological activity of telenzepine enantiomers.
The binding of the enantiomers of telenzepine to muscarinic receptor subtypes present in guinea-pig cerebral cortex, myocardium and salivary glands has been examined. The (+) enantiomer is more potent in all assays and exhibits a greater selectivity than the (-) enantiomer for the different receptor subtypes. As a consequence, the enantiomeric potency ratio varies from ca. 400 (cortical 'M1' receptors) to ca. 50 (cardiac receptors). In functional assays in vitro in the rabbit vas deferens and rat atria, the affinity constants and enantiomeric potency ratios for the two isomers agree with those found for the appropriate muscarinic receptor subtype in binding assays. A high enantiomeric potency ratio, 180, is found in vivo for the ability of the telenzepine enantiomers to inhibit the production of lesions in the modified Shay rat preparation. The data are compatible with the blockade of M1 receptors by (+)-telenzepine being responsible for this action of telenzepine and would tend to exclude the possibility that the anti-ulcer action of telenzepine is mediated via a muscarinic or non-muscarinic action of the (-) enantiomer.
(4-(m-Chlorophenylcarbamoyloxy)-2-butynyl)trimethy↗
(+)-Niguldipine binds with very high affinity to Ca2+ channels and to a subtype of alpha 1-adrenoceptors.
The enantiomers of the 1,4-dihydropyridine (DHP) niguldipine (3-methyl-5-[3-(4,4-diphenyl-1-piperidinyl)-propyl]- 1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-pyridine-3,5-dicarboxylate- hydrochloride) were investigated with respect to their interaction with 1,4-DHP receptors on L-type Ca2+ channels and alpha-adrenoceptors. The Ki values for niguldipine were dependent on the membrane protein concentrations in the radioligand binding assay. 'True' Ki values (at extrapolated 'zero' membrane protein) were determined with guinea-pig membranes for (+)-niguldipine and were found to be 85 pmol/l for the 1,4-DHP receptor of skeletal muscle, 140 pmol/l for that of brain and 45 pmol/l for that of heart. (-)-Niguldipine was approximately 40 times less potent. (+)-Niguldipine (Ki: 78 nmol/l) and (-)-niguldipine (Ki: 58 nmol/l) bound with approximately equal affinity to the alpha 1-adrenoceptors ('alpha 1B') in liver cell membranes. The (+)-niguldipine alpha 1-adrenoceptor inhibition data for rat brain cortex membranes were better fitted by a two-site model. The high-affinity component ('alpha 1A') had a Ki value of 52 pmol/l in competition experiments with [3H]prazosin. The low-affinity site (alpha 1B) had 200- to 600-fold less affinity. (-)-Niguldipine was greater than 40-fold less potent at alpha 1A- but was nearly equipotent to the (+)enantiomer at alpha 1B-sites. (+)-Niguldipine was the most selective compound for discriminating alpha 1A- from alpha 1B-adrenoceptors and is a novel prototype for 1,4-DHPs which bind with nearly equal affinity to skeletal muscle and brain or heart 1,4-DHP receptors.
Animals↗
Does the prolonged occupancy of M1 receptors by telenzepine protect them against the action of vagally released acetylcholine?
The affinities of telenzepine, pirenzepine and atropine for muscarinic acetylcholine receptors have been determined by receptor binding studies using brain cortex and heart membranes of calf and rat. The ratios of affinities of telenzepine and pirenzepine for the cortical M1 receptors, cortical 'non-M1' receptors and cardiac M2 receptors are 50:5:1 and 80:5:1, respectively. The time course of association of telenzepine and pirenzepine with M1 receptors is similar, whereas the half-times for dissociation are 35 and 2.3 min at 37 degrees C, respectively. In further experiments this slow dissociation rate of telenzepine is shown to be rate-limiting for the occupation of M1 receptors by other ligands. The prolonged occupation of M1 receptors by telenzepine is discussed as a possible protective mechanism against acetylcholine released from vagal nerve fibers.
Acetylcholine↗
Iodinated photoreactive vasopressin antagonists: labelling of hepatic vasopressin receptor subunits.
To identify and characterize V1 vasopressin receptors, photoreactive antagonists of the glycogenolytic and vasoconstrictor activity of vasopressin have been synthesized. The following analogues with 3-mercapto-3,3-cyclopentamethylene-propionic acid (Mca) and N-methylalanine (MeAla) in position 1 and 7 of vasopressin (VP) were effective V1 antagonists: [Mca1, D-Tyr2, MeAla7, Lys8]VP (1), [Mca1, MeAla7, Arg8, Lys9]VP (2), [Mca1, MeAla7, Arg8, D-Lys9]VP (3). Introduction of the photoreactive 4-azidophenylamidino group into the side-chain of Lys8 in analogue 1 or into Lys9 in analogues 2 and 3 increased the potency (for analogue 1 a tenfold increase in the antiglycogenolytic effect and a fivefold increase in the antivasopressor effect) and binding affinity for the rat hepatic V1 receptor. Mono-iodination at Tyr2 with 125I resulted in photoreactive antagonists of high specific radioactivity, which had roughly the same binding affinity as vasopressin for the rat hepatic V1 receptor (Kd = 0.9-1.8 nM). In photoaffinity labelling experiments with purified rat liver membranes, containing 2--3 pmol V1 receptor/mg protein, the analogues labelled specifically two proteins with the relative molecular masses (Mr) of 30,000 and 38,000. These results and the results of a recent study using 3H-labelled photoreactive vasopressin agonists [Boer, R. and Fahrenholz, F. (1985) J. Biol. Chem. 260, 15051-15054] provide evidence that both vasopressin agonists and antagonists can interact with the same two subunits of the heterodimeric hepatic V1 receptor. Furthermore the radioiodinated photoreactive V1 antagonists should be helpful to identify V1 receptor proteins in membranes of other cell types.
Affinity Labels↗
Arginine-vasopressin analogues with high antidiuretic/vasopressor selectivity. Synthesis, biological activity, and receptor binding affinity of arginine-vasopressin analogues with substitutions in positions 1, 2, 4, 7, and 8.
In a search for more selective agonists of arginine-vasopressin (AVP), 10 analogues of [Sar7]- and [MeLa7]AVP with additional substitutions in positions 1 (beta-mercaptopropionic acid), 2 (phenylalanine), 4 (valine), or 8 (D-arginine) were synthesized and tested for antidiuretic and vasopressor activities. All analogues are characterized by a relatively high antidiuretic activity and by a sharp decrease in pressor activity. Their antidiuretic/vasopressor (A/P) selectivities were 2-3 orders higher (except for peptides 2 and 3) than that of the parent hormone. The additivity of the effects of changes in positions 1, 2, 4, and 8 combined with the sarcosine or N-methylalanine substitutions in position 7 on the biological activity is observed. Binding affinities of AVP analogues to plasma membranes from bovine kidney inner medulla and from rat liver containing specific vasopressin receptors were also determined. Generally, these analogues retained high binding affinities to renal vasopressin receptors, and on the other hand they are characterized by a large decrease in binding affinities to hepatic vasopressin receptors, which share characteristics with vasopressor receptors.
Alanine↗
Photoaffinity labeling of the V1 vasopressin receptor in plasma membranes from rat liver.
Photoaffinity labeling experiments were performed with membranes from rat liver containing V1 vasopressin receptors. Photoreactive analogues of [1-beta-mercaptopropionic acid]vasopressin [( Mpa1], vasopressin, or deamino-vasopressin) retaining a high binding affinity (apparent dissociation constants: 5 X 10(-9) M-3 X 10(-8) M) and agonistic properties were used. The tritium-labeled analogue [Mpa1,Lys(N epsilon-4-azidobenzoyl)8]vasopressin preferentially and specifically labels a 30-kDa polypeptide and with lower efficiency a 38-kDa polypeptide. The analogue [Mpa1,Dab4(N gamma-(N-4-azido-2-nitrophenyl-beta-Ala4]arginine-vasopressin specifically labels the 38-kDa polypeptide. The labeling of these two membrane proteins is completely suppressed by an excess of arginine-vasopressin; bradykinin or angiotensin II do not inhibit the incorporation of the reactive vasopressin analogues into these proteins. The results suggest that the rat hepatic V1 receptor exists in the plasma membrane in an oligomeric form composed of two subunits with a molecular mass of 30 and 38 kDa.
Affinity Labels↗
Photoaffinity labelling of the renal V2 vasopressin receptor. Identification and enrichment of a vasopressin-binding subunit.
To identify renal vasopressin receptor proteins, analogues of 1-deamino-vasopressin i.e. ([1-(2-mercapto)propionic acid]vasopressin, [Mpa1]VP) with photoreactive aryl-azido groups in position 4 and 8 of the vasopressin sequence were prepared. In the absence of ultraviolet light, these ligands exhibit a high binding affinity for the V2 vasopressin receptor in plasma membranes from bovine and rat kidney medulla (apparent dissociation constants 1.8 X 10(-9) M to 1.7 X 10(-8)M); the photoreactive analogues stimulate the renal vasopressin-sensitive adenylate cyclase. In photoaffinity labelling experiments with tritium-labelled ligands (34-50 Ci/mmol), a membrane protein from bovine kidney or rat kidney medulla with an apparent relative molecular mass (Mr) of 30 000 was preferentially and specifically labelled. The labelling of the 30 000-Mr protein was completely inhibited by a 10-100-fold molar excess of vasopressin; in contrast, angiotensin II, bradykinin or low-affinity analogues of vasopressin did not suppress the incorporation of the reactive ligands into this protein. The highest specific labelling yield and only a low amount of unspecific labelling was obtained with the analogue [Mpa1,Lys(N6-4-azidobenzoyl)8]VP. Preparative sodium dodecyl sulfate gel electrophoresis of bovine kidney membranes photolabelled with this analogue resulted in a 20-30-fold enrichment of the 30 000-Mr vasopressin-binding protein. Our results suggest that this photoreactive analogue of [1-deamino, 8-lysine]vasopressin is a suitable tool for further purification of the renal V2 vasopressin receptor binding subunit.
Adenylyl Cyclases↗
Determination of the functional molecular size of vasopressin isoreceptors.
The molecular size of vasopressin receptors in the intact membrane-bound state was determined by radiation inactivation (target size analysis). For the V1 receptor in rat liver a molecular size of (76 +/- 8) kDa was determined. For the V2 receptor in rat kidney and bovine kidney molecular sizes of (95 +/- 4) and (108 +/- 11) kDa were found. Statistical analysis gave evidence for size differences between rat liver and rat kidney receptors or differences between rat liver and bovine kidney receptors, but not between kidney receptors from different species. The results suggest that V1 and V2 receptors can be distinguished by functional properties as well as by their size.
Animals↗
Interactions of vasopressin agonists and antagonists with membrane receptors.
Plasma membranes containing one class of non-cooperative binding sites for tritium-labelled [8-arginine]vasopressin were isolated from bovine kidney inner medulla and from rat liver. By using a weighted, non-linear least squares fit to logistic curves, the binding parameters of eight vasopressin agonists and antagonists were determined in competition experiments. Vasopressin analogues with sarcosine or N-methyl-L-alanine in position 7 instead of proline showed a high ratio of antidiuretic to vasopressor activity. These analogues retained a high binding affinity to the renal vasopressin receptor with apparent dissociation constants KD in the order proline less than sarcosine less than methylalanine . In contrast, the affinity to the hepatic vasopressin receptor, which shares characteristics with vasopressor receptors, was drastically reduced with KD values being in the order proline much less than N- methylalanine less than sarcosine. By combining the substitutions at position 7 with substitutions of cysteine in position 1 by either deaminopenicillamine or beta-mercapto-beta, beta-cyclopentamethylenepropionic acid, inhibitors of the oxytocoic and vasopressor responses were obtained. These additional substitutions at position 1 led to a drastic decrease in the binding affinity to the vasopressin receptor in bovine kidney. The intrinsic activity of these analogues to stimulate the renal vasopressin sensitive adenylate cyclase was strongly reduced or completely lost. In the rat liver system, however, these vasopressin antagonists showed a remarkably increased affinity to vasopressin receptors as compared to analogues substituted only at position 7. GTP reduced the binding affinity of all analogues to the hepatic receptor. The results show that these structural modifications which influence both the conformational properties of the vasopressin molecule and the biological activities of the hormone had strikingly different effects on the interactions of the resulting analogues with physiologically important receptors in the kidney and the liver. These studies may lead to the development of more specific vasopressin agonists and antagonists.
Adenylyl Cyclases↗
Solubilization of ligand-stabilized vasopressin receptors from plasma membranes of bovine kidney and rat liver.
The solubilization of vasopressin receptors from plasma membranes of bovine kidney and rat liver by different detergents was investigated. A prerequisite for the extraction of vasopressin receptors retaining binding affinity for their ligand was the stabilization of the receptors by the prior formation of the membrane-bound hormone-receptor complexes. The vasopressin-receptor complexes from both kidney and liver membranes were solubilized in a high yield with dodecyl-beta-D-maltoside and 3-laurylamido-N,N'-dimethylpropylaminoxide. Several other nonionic detergents including octyl-beta-D-glucopyranoside effectively extracted the hepatic vasopressin receptor. For the hormone-receptor complex solubilized from bovine kidney with dodecyl-beta-D-maltoside, a Stokes' radius of 5.8 nm was determined.
Animals↗
[Epidural analgesia for painless delivery?].
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Anesthesia, Epidural↗
Rescreen effect in conventional and PAPNET screening: observed in a study using material enriched with positive smears.
OBJECTIVE: To study the rescreen and PAPNET effects on enriched material derived from smears screened routinely using PAPNET and conventional microscopy. STUDY DESIGN: A series of 432 smears (containing 122 atypical squamous cells of undetermined significance [ASCUS] plus 44 at least dysplastic squamous intraepithelial lesion-positive [SIL+] ones), screened routinely with the conventional method, were rescreened using the PAPNET system. Another series of 461 smears (containing 140 ASCUS + 52 SIL+ ones) screened routinely with PAPNET were rescreened conventionally. The rescreen effect, defined as the effect of differences between the rescreen and routine screening situation, was investigated by comparing the rescreen results in both series of smears with the routine results in both series. The effect of using either method of screening was studied by comparing the PAPNET results in both series with the results of conventional screening in both series. RESULTS: The rescreen effect was statistically significant both for a higher number of smears classified as negative (less than ASCUS) and a higher number of smears classified as high grade SIL or more. PAPNET-assisted screening resulted in a significantly higher number of smears classified as high grade SIL+, although for this latter finding there is an unexplained significant difference between conventional and PAPNET-screened cases in the changes made by the cytopathologist in the cytotechnologists' diagnoses. CONCLUSION: The rescreen effect should not be ignored when enriched material is used.
Carcinoma, Squamous Cell↗