James M. Schaeffer

Gonadotropin-releasing hormone as a paracrine hormone and neurotransmitter in extra-pituitary sites

Gonadotropin-releasing hormone (GnRH), in addition to its classical releasing action at the pituitary level, acts on multiple extrapituitary sites to regulate various reproductive functions. In the rat ovary, specific high affinity GnRH receptors have been identified in granulosa and theca cells. These binding sites mediate the inhibitory effects of GnRH and its agonists on gonadotropin-stimulated estrogen, progestin and androgen biosynthesis. At the granulose cell level, GnRH treatment decreases aromatase activity as well as the biosynthesis of pregnenolone and progesterone via inhibition of cholesterol side-chain cleavage and 3 beta-hydroxysteroid dehydrogenase enzymes. High concentrations of GnRH also stimulate low but significant levels of various steroids. In addition, treatment with high concentrations of GnRH induces ovulation and oocyte maturation in hypophysectomized rats. This is associated with the ability of GnRH to stimulate plasminogen activator activity in cultured granulosa cells. In the rat testis, GnRH receptors have been identified in Leydig but not Sertoli cells. Treatment with GnRH inhibits gonadotropin-stimulated androgen biosynthesis by the cultured Leydig cells. The inhibitory effect of GnRH on testicular androgen production occurs at sites distal to the formation of cyclic AMP and pregnenolone and may be due to decreases in the activity of the enzyme 17 alpha-hydroxylase and 17-20 desmolase. Since hypothalamic GnRH is unlikely to act at the gonadal level, several laboratories have attempted to isolate gonadal GnRH-like peptide which may serve as the ligand for specific gonadal GnRH receptors. Although the presence of ovarian GnRH-like substance still remains elusive, testicular GnRH-like substance has been identified. This gonadal peptide(s) may be an important local paracrine hormone. In addition to its action at the gonadal level, GnRH or GnRH-like peptides may play an important role as a neurotransmitter in the central nervous system. Exogenous administration of GnRH in selected brain areas has been shown to modulate sexual behavior in experimental animals, while neural pathways containing GnRH-like immunoreactive substances have been identified in several brain areas. We have recently synthesized a bioluminescent GnRH analog capable of serving as a specific GnRH ligand for a bioluminescent ligand receptor assay which is more sensitive than classical 125I-ligand assays. We have identified GnRH receptors in small, discrete brain regions. Thus, GnRH and GnRH-like peptides may play important paracrine and neurotransmitter roles in the regulation of various reproductive functions in extra-pituitary sites.

Identification of gamma-aminobutyric acid and its binding sites in the rat ovary

Gamma-aminobutyric acid (GABA), GABA synthesizing enzyme and GABA binding sites were measured in rat ovaries. The concentration of GABA in the ovary (0.56 microgram/mg protein) was less than that in the brain (1.2-3.4 microgram/mg protein), but was six-fold higher than any other non-neuronal tissue examined. Glutamate decarboxylase, the GABA synthesizing enzyme was also found in high concentrations in whole ovarian homogenate but not in enriched ovarian granulosa cells, testis, anterior pituitary or muscles. Furthermore, high affinity (Kd = 15-21 nM), specific GABA binding sites were identified in the ovaries by specific [3H]muscimol binding and the majority of GABA binding sites were associated with the granulosa cells. These data suggest a possible role of GABA in the regulation of ovarian functions.

Identification of dopamine receptors in the rat retina

Specific [3H]spiroperidol binding was used to measure dopamine receptors in membrane fractions isolated from rat retinae. Spiroperidol binding is saturable with an apparent dissociation constant, KD, of 3·5 × 10−10m and approximately 155 fmol bound/mg protein. Specific binding is proportional to protein concentration, and time-dependent. The ability of various dopamine agonists and antagonists to compete with [3H]spiroperidol binding is compatible with the relative in vivo dopaminergic effect of these drugs.

Nucleoside uptake by rat retina cells

Abstract Adenosine and guanosine uptake have been studied in the rat retina. Both nucleosides are taken up in a time- and temperature-dependent manner by dispersed rat retinal cells. The uptake of both nucleosides is Na + -dependent and Ca ++ -independent. Initial rate studies of guanosine and adenosine uptake demonstrate a single uptake process for each nucleoside with K D values of 2.1 and 2.9 uM, and maximal rates of 24 and 17 pmol/mg protein/min, respectively. Guanosine uptake was inhibited by adenosine with a K I of 12.1 uM whereas guanosine inhibited adenosine uptake with a K I value greater than 10 −3 M. LN 6 -phenylisopropyladenosine, a nucleoside analog, was the most potent inhibitor of adenosine and guanosine uptake with K I values of 25 and 8 uM, respectively. Phosphodiesterase inhibitors (isobutylmethylxanthine and theophylline) and biogenic amines (dopamine, norepinephrine, and histamine) had no significant effect on the uptake of guanosine or adenosine at concentrations up to 100 uM.

[3H]muscimol binding in the rat retina

Abstract Crude membrane fractions were prepared from rat retinae and used to study the specific binding of [ 3 H]muscimol, a potent GABA agonist. Specific [ 3 H]muscimol binding was enhanced 2–3 fold by pretreatment of the membranes with 0.025% Triton X-100. Two muscimol binding sites were demonstrated with K D values of 4.4 and 12.3 nM. GABA, muscimol, and 3-aminopropanesulfonic acid were the most potent inhibitors of specific [ 3 H]muscimol binding with K I values of 15, 10, and 50 nM, respectively. These data are consistent with binding to the synaptic GABA receptor.

A simple and rapid method to isolate rat retinal cells for biochemical analysis

Abstract We have developed a rapid method to enzymatically disperse rat retinae and to separate large quantities of various cell types by centrifugation through a metrizamide density gradient. Seven distinct bands of cells were detected in the gradient (I–VII). Cells from layers III and V were morphologically identified as red blood cells and photoreceptor cells, respectively. [ 125 I]Horseradish peroxidase injected into the lateral geniculate bodies labels the ganglion cells via retrograde transport. This technique allowed us to demonstrate the presence of ganglion cells in layer I of the metrizamide gradient. This method will be useful for biochemical and pharmacological characterization of several types of retinal cells.

Biochemical characterization of isolated rat retinal cells: The γ-aminobutyric acid system

Rat retinae were chemically dissociated with collagenase and eight populations of cells (layers I–VIII, from bottom to top, respectively) were separated by centrifugation through a linear metrizamide density gradient. Each cell type was examined to determine its content of γ-aminobutyric acid (GABA), glutamate decarboxylase, GABA uptake capacity and GABA binding sites. The cells in layers I and II had the highest concentration of GABA (a six-fold increase in GABA concentration as compared to the whole retina). Glutamate decarboxylase, the enzyme which catalyzes the decarboxylation of glutamate to form GABA, was also found in highest concentration in the cells of layers I and II. Only the cells in layer VII had the ability to take up exogenous GABA. Initial rate studies demonstrated a high and low affinity site for GABA uptake with dissociation constants of 4·7 and 50 μ m , respectively. GABA uptake was Na + -dependent and Ca 2+ -independent. Diaminobutyric acid and GABA were the two most potent inhibitors of [ 3 H]GABA uptake with K I values of 3 and 5 μ m , respectively. Muscimol and 3-aminopropanesulfonic acid, inhibitors of GABA binding, did not significantly effect GABA uptake at concentrations up to 10 m m . Specific [ 3 H]muscimol binding, used to quantitate GABA binding to GABA receptor sites, was present only in layer VI. Specific [ 3 H]muscimol binding to the membranes isolated from layer VI was saturable with a dissociation constant of 6·5 n m and was competitively inhibited by muscimol and GABA with K I values of 3 n m and 10 n m , respectively. [ 3 H]Muscimol binding was Ca 2+ - and Na + -independent and was not inhibited by diaminobutyric acid, nipecotic acid or β -alanine at concentrations up to 100 μ m .

2-Hydroxyestradiol Interaction with Dopamine Receptor Binding in Rat Anterior Pituitary

Dopamine receptors in the rat anterior pituitary were quantitated by specific binding of [3H]spiroperidol, a potent dopamine antagonist, to a crude membrane fiattion. [3H]Spiroperidol binding is saturable with high affinity (apparent KD = 1.01 -C 0.15 X 10-l’ M) and low capacity (215 fmol bound/mg of protein). The specificity of the dopamine receptor was demonstrated by the ability of various dopamine agonists and antagonists to inhibit [3H]spiroperidol binding. Dopamine is lo-fold more potent than serotonin, 50-fold more potent than norepinephrine and 2500-fold more potent than epinephrine in displacing bound [3H]spiroperidol. In addition, the physiologically active (+)-isomer of butaclamol is approximately lO,OOO-fold more potent than the inactive (-)-butaclamol. In order to evaluate the ability of estrogen and catecholestrogens to interact with the dopamine receptor, estrone, estradiol, estriol, and the P-hydroxyand 2-methoxyestrogen derivatives were used for competition experiments. 2-Hydroxyestradiol was the only steroid tested found to compete effectively for the [3111spiroperidol binding site (KI = 9 x 10m6 M), whereas the parent estrogens and related estrogen derivatives were ineffective at concentrations up to lo+ M. These results present the first evidence that 2-hydroxyestradiol, a naturally occurring estrogen metabolite, interacts with anterior pituitary receptors for dopamine, and provide a clue for understanding one mechanism by which estrogens may regulate pituitary prolactin release.