Search

γ-Aminobutyric acid (GABA), one of the most important neurotransmitters in the brain, is also found in the periphery. GABAA receptors are chloride channels opened by GABA whose presence in the rat superior cervical ganglion has been indicated by functional and binding measurements. We describe the first molecular data on the possible subunit composition of these receptors, detecting mRNAs for 12 subunits (α1–5, β1–3, γ1–3, δ) by reverse-transcriptase polymerase chain reaction. Preliminary quantitation gave highest levels (in descending order) for the γ2 (both short and long forms), β3, γ3, and α1 subunits.

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.

The density of [3H]quinuclidinyl benzilate ([3H]QNB) binding sites in dissociated cell cultures of mouse spinal cord increased almost 4-fold between the 5th and 21st days of maturation in vitro. The mean specific binding of [3H]QNB in 21-day cultures was 340 pmol/g protein. Up to 79% of [3H]QNB binding sites were lost within a few days after exposure of the cultures to 1 mM glutamate. This neurotoxic action of glutamate showed a steep concentration dependence in the range 0.1-1 mM and did not vary with the duration of exposure of the cultures from 10 min to 24 h. Kainic acid was only moderately more potent than glutamate in reducing [3H]QNB binding. The susceptibility of 8-day cultures to glutamate-induced loss of [3H]QNB binding was less than that of 12- and 19-day cultures. These findings suggest that 80% or more of muscarinic binding sites in the spinal cord cell cultures are on neurons and that the sensitivity of these neurons to the toxic action of glutamate increases with their maturation in vitro.

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.

[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.