Richard L. Taylor

Species differences in the brain regional distribution of receptor binding for thyrotropin-releasing hormone.

Abstract: A survey of the regional distribution of binding of 1 nM [3H](3‐MeHis2)thyrotropin‐releasing hormone ([3H]MeTRH) to TRH receptors in the brains of eight mammalian species revealed major species differences in both the absolute and relative values of TRH receptor binding in different brain regions. Several brain regions exhibited binding equal to or exceeding that in the anterior pituitary gland of the same species, including the amygdaia in the guinea pig and rat, the hypothalamus in the guinea pig, the nucleus accumbens in the rabbit, and all these and other regions in the cat and dog, for which pituitary binding was exceptionally low. Species could be divided into two groups according to which brain region appeared highest in binding: rabbits, sheep, and cattle had highest binding in the nucleus accumbens/septal area, whereas guinea pigs, rats, dogs, cats, and pigs had highest binding in the amygdala/temporal cortex area. The nucleus accumbens consistently exceeded the caudate‐putamen in receptor binding. For most brain regions, rabbits, rodents, and sheep tended to be higher than carnivores, cattle, or pigs. Further regions that exhibited appreciable binding in most species included the olfactory bulb and tubercle, hippocampus, and various cortical and brain stem areas. In fact, essentially all brain regions appeared to have detectable levels of TRH receptors in at least some species, but no rat peripheral tissues have yet shown detectable receptor binding. The species differences appeared to reflect largely if not entirely differences in receptor density, although this was not tested in every species.

Properties of [3H](3-Me-His2)TRH binding to apparent TRH receptors in the sheep central nervous system

[3H](3-methyl-His2)thyrotropin releasing hormone ([3H]MeTRH) binds to sites in the sheep central nervous system (CNS) whose properties closely resemble both those of CNS binding sites for [3H]TRH and those of pituitary binding sites for [3H]MeTRH. Detailed studies for binding of [3H]MeTRH in the sheep nucleus accumbens and retina have yielded equilibrium dissociation constants of about 4 nM and densities of binding sites of about 3 and 2 pmol/g wet weight, respectively. The binding affinity of [3H]MeTRH was 8- to 10-fold higher than that of [3H]TRH, resulting in much lower non-specific binding with the new ligand. The association reaction had a rate constant of about 2-3 x 10(7) M-1 min-1, while the biphasic dissociation reaction had rate constants of 8-9 x 10(-2) min-1 for the fast phase and 1-2 x 10(-2) min-1 for the slow phase. The regional distribution of binding in the sheep CNS was similar to that observed previously with [3H]TRH. Highest binding outside the pituitary was in the nucleus accumbens area and retina, with another peak in the amygdala-temporal cortex area. Binding was widely distributed, so that no CNS region appeared totally devoid of binding. Nineteen TRH analogs, ranging in potency over 6 orders of magnitude, showed nearly identical abilities to complete for binding of [3H]MeTRH in the CNS areas and in the sheep anterior pituitary gland in side-by-side experiments. These findings argue strongly for identification of [3H]MeTRH binding sites in the CNS as TRH receptors.

Preparation of 3H-[3-Me-His2]TRH as an Improved Ligand for TRH Receptors

[3H]-3-methyl-His2]thyrotropin releasing hormone ([3H]MeTRH) binds to sites in the sheep anterior pituitary gland which appear to be the same as those occupied by [3H]TRH and which can therefore be identified as TRH receptors. In competition experiments performed in parallel, both ligands gave the same number of binding sites, 15 pmol/g wet weight, and showed the same pharmacology for 19 TRH analogs ranging over more than 5 orders of magnitude in potency. The apparent dissociation constant of binding of [3H]MeTRH was about 3.5 nM compared to 29 nM for [3H]TRH. Kinetic experiments with [3H]MeTRH yielded a rate constant for association of 1.4 x 10(7) M-1 min-1 and rate constants for the biphasic dissociation of 5 x 10(-2) min-1 (fast phase) and 7 x 10(-3) min-1 (slow phase). Detailed methods are described for preparation of [3H]MeTRH by reduction of the dehydroproline precursor and its purification by ion exchange and antibody affinity chromatography. The major advantage of the new ligand is that its higher affinity of binding gives relatively less non-specific binding than is obtained with [3H]TRH, particularly in central nervous tissue.

Muscarinic Receptor Binding in Sheep Anterior Pituitary

The existence of typical muscarinic receptors in membranes of sheep anterior pituitary was detected by binding of [3H]quinuclidinyl benzilate ([3H]QNB), a potent and specific muscarinic antagonist. [3H]QNB binding sites in anterior pituitary had an equilibrium dissociation constant of about 20-40 pM, a rate constant for association at 37 degrees C of about 2 x 10(8) M-1 min-1, a rate constant for dissociation at 37 degrees C of about 4 x 10(-3) min-1, and the expected specificities for a variety of cholinergic and other drugs. The concentration of [3H]QNB binding sites in anterior pituitary, about 4-6 pmol/g tissue, was at least twice that in posterior pituitary, but less than a quarter that in the hypothalamus. Since there is no established cholinergic or other innervation of the anterior pituitary, the presence there of typical muscarinic receptors, if we assume that they are functional, suggests that acetylcholine reaches the tissue through the hypophyseal portal circulation, and that it may thus have a role in the regulation of pituitary function.

Muscarinic receptors in pineal

Abstract The presence of muscarinic receptors in sheep and rat pineals was detected by binding of [ 3 H]quinuclidinyl benzilate ([ 3 H]QNB), a potent and specific muscarinic antagonist. [ 3 H]QNB binding to sheep pineal membrane resuspensions was saturable and reversible, with a rate constant for association at 37°C of 6×10 8 M −1 min −1 and a rate constant for dissociation of 1×10 −2 min −1 . Kinetic and saturation experiments yielded an equilibrium dissociation constant of 13–18 pM and a concentration of binding sites equivalent to 1.1 pmol/g of original wet weight. This is only about 5% of the level of β-adrenergic receptors. Competition by a variety of cholinergic drugs confirmed the muscarinic nature of the binding sites. Experiments in rats failed to detect a significant decrease in pineal [ 3 H]QNB binding following bilateral superior cervical ganglionectomy, suggesting that the binding sites are not localized exclusively on sympathetic terminals.

TRH receptor binding in avian pituitary and brain

Abstract The pituitary gland of the domestic fowl binds [ 3 H]-[3-methyl-His 2 ]thyrotropin-releasing hormone ([ 3 H]MeTRH) with properties very similar to those exhibited by mammalian TRH receptors, including affinity ( K D = 4.9 n M ), density of binding sites ( B max = 5.1 pmol/g), and pharmacology for eight TRH analogs. Chicken brain appears to contain similar binding sites, but the level of binding was too low for detailed characterization.

Pituitary cell cultures contain muscarinic receptors

The presence of muscarinic receptors in 5-day dissociated cell cultures of rat anterior pituitary glands was detected by atropine-sensitive binding of 3H-quinuclidinyl benzilate (3H-QNB). Muscarinic receptor levels in cultures were compared to levels of receptors for dopamine and thyrotropin releasing hormone.

TRH receptor binding in retina and pituitary: major species variation.

Retinas and anterior pituitary glands of nine readily available mammalian and one avian species have been examined for their TRH-sensitive binding of [3H]-[3-Me-His2]TRH. Among mammals, major species variations in TRH receptor binding have been detected in both tissues, amounting to about 100-fold in the retina and 20-fold in the pituitary. In the retina, TRH receptor binding was very high in the rat, quite high in sheep and guinea pig, intermediate in rabbit, pig, and mouse, and low but detectable in chicken, cat, calf and dog. In the pituitary gland, binding was very high in sheep, quite high in rabbit, rat, pig, calf and guinea pig, intermediate in chicken, and fairly low in mouse, cat, and dog. A number of possible interfering variables, including affinity differences, albino vs. non-albino strains, barbiturate anesthesia, time after death, sex and estrous cycle, age, and history of light exposure, were considered and/or tested directly, with generally negative results.

Muscarinic Receptors in the Posterior Pituitary Gland

The characteristics of atropine-sensitive binding of l-[3H]-quinuclidinyl benzilate ([3H]-QNB) to membrane suspensions of sheep posterior pituitary indicate that the binding sites represent muscarinic cholinergic receptors. Scatchard plots of 6 saturation experiments showed a single class of binding sites, with an equilibrium dissociation constant of 16 +/- 2 pM and a density equivalent to 1.8 +/- 0.2 pmol/g wet weight of tissue. Kinetic analysis of 2 association and 3 dissociation curves yielded mean association and dissociation rate constants of 3.9 x 10(8) M-1 min-1 and 4.3 x 10(-3) min-1, respectively. The binding had a detailed pharmacology for 12 drugs consistent with muscarinic receptor identification. In rat neurointermediate lobes, superior cervical ganglionectomy had no demonstrable effect on [3H]-QNB binding. The location(s) and functional role(s) of neurohypophyseal muscarinic receptors remain to be elucidated.