Mepyramine Binding Assay Research Articles

Cell-based, animal and H1 receptor binding studies relative to the sedative effects of ketotifen and norketotifen atropisomers.

Ketotifen (K) and its active metabolite norketotifen (N) exist as optically active atropisomers. They both have antihistaminic and anti-inflammatory properties but the S-atropisomer of N (SN) causes less sedation than K and RN in rodents. This study investigated whether this could be related to a lower concentration of SN in brain or a lower affinity of SN for rat brain H1 receptors. Ketotifen and norketotifen atropisomers were quantified using a validated chiral HPLC assay. RBE4 and Caco-2 cell monolayers were used in uptake and permeability studies, respectively. Free and total brain-to-plasma (B/P) ratios were determined after injecting racemic K and N into rat tail veins. Affinity for rat brain H1 receptors (KI ) was determined using the [3 H]mepyramine binding assay. Uptake and permeation studies indicate no stereoselective transport for K or N. B/P ratios reveal the brain concentration of N is lower than K with no stereoselective transport into brain. Finally, the [3 H]mepyramine binding assay shows SN has the lowest affinity for rat brain H1 receptors. The lower sedative effect of SN in rodents is probably due to a combination of a lower uptake of N than K into the brain and less affinity of SN for CNS H1 receptors.

Expression and characterization of the bovine histamine H1 receptor in cDNA-transfected C6 astroglioma cells.

Rat C6 astroglioma cells (C6-bH1R cells) expressing cloned bovine histamine H1 receptors were established by transfection with a vector (pEF-BOS-bH1R) which carried a 2.7-kbp EcoRI fragment of the bovine H1 receptor cDNA [Yamashita, M. et al. (1991) Proc. Natl. Acad. Sci. USA 88, 11515-11519]. The cloned bovine H1 receptor in C6-bH1R cells was characterized by three established criteria: the [3H]mepyramine binding assay, the accumulation of inositol phosphates induced by histamine, and histamine-induced elevation of intracellular Ca2+ concentration ([Ca2+]i). The accumulation of inositol phosphates induced by histamine was time- and dose-dependent. The accumulation of inositol trisphosphate was biphasic with a prompt increase to the maximal level, followed by a sustained submaximal level. The histamine-induced accumulation of inositol phosphates was suppressed by phorbol ester, but not by pertussis toxin. Results from the [3H]-mepyramine binding assay and histamine-induced elevation of [Ca2+]i were characteristic of H1 receptors. Several compounds among tricyclic antidepressants, neuroleptics, and serotonin antagonists showed affinities to the cloned bovine H1 receptor with Ki values similar to reported values. Histamine neither induced cAMP accumulation nor attenuated forskolin-induced cAMP accumulation in C6-bH1R cells. C6-bH1R cells are particularly useful for studying the H1 receptor-mediated astroglial cell functions.

P-440 - Distribution of histamine H1 receptors in rat tissues by improved [3H]mepyramine binding assay.

P-440 - Distribution of histamine H1 receptors in rat tissues by improved [3H]mepyramine binding assay.

Re-examination of [ 3H]mepyramine binding assay for histamine H 1 receptor using quinine

[ 3H]Mepyramine, a potent antagonist of the histamine H 1 receptor, has been widely used as a radioligand binding assay for the H 1 receptor. Previously, we purified a mepyramine binding protein (MBP) from rat liver, but found that its partial amino acid sequences were very similar to those of debrisoquine 4-hydroxylase isozymes (P450 db1 and db2), which are members of the superfamily of cytochrome P450. Using cloned histamine H 1 receptor cDNA, we found that [ 3H]mepyramine could bind only the H 1 receptor did not bind MBP in the presence of 10 −5 M quinine, an inhibitor of debrisquine 4-hydroxylase isozymes. We developed a method to determine the contents of the H 1 receptor and MBP separately using [ 3H]mepyramine and quinine and found that MBP is abundant in certain areas of bovine brain.

The effect of histamine on cultured endothelial cells A study of the mechanism of increased vascular permeability

The role of endothclial cellS in the histamine-induced vascular response was investigated using cultured endothelial cells obtained from human umbilical veins. A single population of histamine H1 receptors was detected in in these cells by means of a [3H]mepyramine binding assay. Its Kd was 0.74 ± 0.07 nM and Bmax was 41.4 ±8.68 fmol/mg protein. Actin filaments were distributed as dense bands at the margin of the cells and as sparse microfilament bundles traversing the center of the cells. Histamine caused a decrease in peripheral bands and an increase in longitudinal bands. The changes evoked by histamine were dose-dependent, related to the duration of incubation of the cells with histamine, and blocked by mepyramine, a histamine H1 receptor antagonist. The results suggest that the endothelial cells respond to histamine through the histamine H1 receptor. This may explain one of the mechanisms of histamine-induced vascular response including vascular permeability increase.

3H]Mepyramine binding sites, histamine H1-receptors, in bovine retinal blood vessels

The presence of histamine H1-receptors in the bovine retinal blood vessels was studied with a [3H]mepyramine binding assay. The membranes of purified vessels obtained from bovine retinas showed specific [3H]mepyramine binding sites with a dissociation constant (KD) of 2.78 +/- 0.32 nM. This was similar to values obtained from the retinal neuronal fractions. The binding capacity (Bmax) was 53.8 +/- 1.7 fmol/mg protein, which was about a half that of the retinal neuronal fractions (108.9 +/- 3.1 fmol/mg protein). Some H1-antagonists proved to be potent competitors for [3H]mepyramine binding sites in bovine retinal blood vessels. These results indicate that histamine H1-receptors exist in the retinal blood vessels which may be involved in the physiological and the pathological responses of blood circulation in retinas.

Characterization of histamine H 1-receptors on astrocytes in primary culture: [ 3H]mepyramine binding studies

The characteristics of histamine H 1-receptors on astrocytes from the cerebral cortex of newborn rats in primary culture were analyzed with a [ 3H]mepyramine binding assay, and compared with those in the cerebral cortex. The apparent dissociation constant (K D) of [ 3H]mepyramine binding, the apparent inhibition constants (K i) of various H 1-ligands for [ 3H]mepyramine binding and the stereoselectivity of d- and l-chlorpheniramine for the inhibition of [ 3H]mepyramine binding to receptors on cultured astrocytes and to receptors in the brain tissue were very similar, indicating that these receptors are identical. The apparent density of H 1-receptors (B max) on astrocytes was 262 ± 60 fmol/mg protein, which was comparable to that in the brain tissue (194 ± 24 fmol/mg protein). The development of H 1-receptors on cultured astrocytes resembled the postnatal development of the receptors in the rat brain. These results suggest that astrocytes could be one of the main targets of the central histaminergic system.

Histamine H 1-receptor in endothelial and smooth muscle cells of guinea-pig aorta

The location of histamine H 1-receptors in the thoracic aorta of guinea-pigs was studied with a [ 3H]mepyramine binding assay. [ 3H]Mepyramine binding studies of whole and rubbed aortas, and of cultured endothelial and smooth muscle cells showed that the K d values were all in the range 0.53–0.76 nM, but that the B max values were 19.1, 10.1, 63.3 and 11.6 fmol/mg protein, respectively. Thus, the whole aorta contained more H 1-receptors than the rubbed one (free of endothelium), and cultured endothelial cells contained more H 1-receptors than smooth muscle cells. These results indicate that more histamine H 1-receptors were concentrated in the endothelial cells than in the smooth muscle cells in guinea-pig aorta.

Histamine H 1-receptor in the retina: Species differences

Histamine H 1-receptors in membranes of the various mamalian retinas were studied by [ 3H]mepyramine binding assay. Specific [ 3H]mepyramine bindings to bovine, pig, dog and human retinas were observed with the dissociation constants (K D), 3.8±1.2 nM, 1.8±0.6 nM, 2.6±0.6 nM and 3.0±0.9 nM, respectively, which were similar to those found in brains. But there was no detectable specific binding in the guinea-pig and rabbit retinas. The number of binding sites (Bmax) ranged from negligible value to 290.7±51.7 fmole/ mg protein (human retina). Some H 1-antagonists acted as potent agents in competing with [ 3H]mepyramine binding to bovine and pig retinas. These results indicated that histamine H 1-receptors exist in some mammalian retina and have similar characteristics to those in brain membranes, but they distributes in the wide difference of the binding capacities among the species, while in brain variations were smaller.

Effect of thyroid state on histamine H 1 receptors in adult and developing rat brain

The effect of thyroid status on histamine H 1 receptors in adult and developing rat brain was investigated using the ( 3H) mepyramine binding assay. Hypothyroidism induced by treatment with 6- n-propyl-2-thiouracil resulted in a 31% decrease in the density and total content of adult rat brain ( 3H) mepyramine binding sites and a significant retardation of the developmental increase in H 1 receptor binding in neonates. At 30 days of age, when euthyroid rats reached binding levels of the adult, hypothyroid animals presented reductions of 22 and 39% in ( 3H) mepyramine bound per unit weight and per brain respectively. In contrast, hyperthyroidism induced by treatment with l-thyroxine did not alter H 1 receptor numbers in the adult rat brain but accelerated the developmental increase in ( 3H) mepyramine bound per unit weight that reached normal adult levels by 21 days of age. The results suggest that thyroid dysfunction during early life and adulthood may cause derangements of the histaminergic system in the brain.