Comparison of the interaction of equine LH and human chorionic gonadotrophin to equine testicular receptors.

Human chorionic gonadotrophin (hCG) can be used to study horse luteinizing hormone (LH) receptors in stallion testicular tissue. hCG was more stable than horse LH during radioiodination when compared by their abilities to bind to testicular receptor sites. During incubation, neither hormone lost binding activity at 4 degrees C. Horse LH lost binding activity during incubation at 25 degrees C and both hormones lost binding activity at 37 degrees C. Both hormones bound to the same receptor sites which are specific for the hormones. The receptor sites were not degraded when incubated at 4 degrees C for up to 16 h. However, a rapid loss of binding ability occurred at 37 degrees C and a continuous but slower loss at 25 degrees C. Scatchard plots were similar for both hormones. Affinity constants (Ka) of 4 . 5-8 x 10(10) M-1 and receptor site numbers of 3 . 9-7 . 3 x 10(-11) M were calculated from the Scatchard plots, which were linear, indicating a single class of receptors. Similar association and dissociation rate constants were obtained for hCG and horse LH. Association rate constants (K1) between 1 and 32 x 10(5) M-1 sec-1 and dissociation rate constants (K-1) between 1 . 1 and 5 . 6 x 10(-6) sec-1 were observed.

Effect of PGF-2 alpha on LH receptors in the equine corpus luteum.

As quantified by Scatchard analysis, a 27 000 g crude luteal membrane fraction contained a single population of unoccupied LH receptors characterized by high affinity, ka = 0.647 +/- 0.158 X 10(11) M-1 and low binding capacity, Rt = 4.91 +/- 0.78 X 10(-11) M/mg membrane fraction. Acceptable hormonal specificity, reversibility, saturability, high affinity and tissue specificity indicated that the binding protein was a physiological receptor. To ensure that the methods used for Scatchard analysis were valid, hCG was characterized for specific activity and maximum bindability, non-specific binding was monitored, equilibrium binding assay conditions were optimized and the amount of hormone and receptor degradation was evaluated. Serum concentrations of LH and progesterone significantly increased within 1 h after PGF-2 alpha treatment (P less than 0.05). Serum and luteal progesterone concentrations were significantly reduced (P less than 0.05, P less than 0.01, respectively) by 3 h, long before a decline in luteal LH receptors was observed. A significant decline in receptor number (P less than 0.05) was detected by 36 h. This loss of receptors was associated with a decrease in luteal progesterone (P less than 0.05) and a significant increase in luteal cell degeneration, as judged by morphology. The affinity of the receptor for hCG did not differ at any of the times studied. Luteal weights remained unchanged. PGF-2 alpha treatment of the mare resulted in a rapid decline of serum and luteal progesterone before the loss of luteal LH receptors that presumably leads to the irreversible functional and structural demise of the equine CL.

The response of single melanophores to extracellular and intracellular iontophoretic injection of melanocyte-stimulating hormone.

The purpose of this study was to determine if MSH, a peptide hormone, injected within a frog skin melanophore could elicit a physiological response, melanosome dispersion. Multibarreled electrodes were used to iontophoretically inject alpha-MSH inside frog skin melanophores of Rana pipiens pipiens. In 46 cells, intracellular MSH was ineffective in producing melanosome dispersion as viewed through the microscope. Because the frog skin is a complex of closely spaced cells, at times the microelectrode may have impaled cells other than melanophores. Therefore, in order to verify that the electrode was in a melanophore and not some other cell type, cAMP, shown to produce melanosome dispersion, was iontophoretically injected to 17 cells, causing the melanosomes to disperse. In these 17 cells, prior injection of MSH caused no dispersion. The response was monitored by observing the target cell with surrounding cells serving as a control. As an additional control to determine if adequate amounts of MSH were released, the electrode was withdrawn from the cell and placed near a group of melanophores, and in all cases the cells close to the electrode tip showed melanosome dispersion after MSH injection. The results of this study remain consistent with the view that MSH receptors in frog skin melanophores are located on the external surface of the plasma membrane, and MSH injected into the cytoplasm of the cell has no short term effect.

Iontophoretic release of cyclic AMP and dispersion of melanosomes within a single melanophore.

Selective dispersion of melanosomes was often observed after iontophoretic injection of cyclic adenosine monophosphate (AMP) from a glass microelectrode positioned in a target melanophore in frog skin (as viewed from above through a microscope), with other melanophores in the field serving as controls. Because the skin has orderly arrays of several types of closely spaced cells, it is probable that at times the microelectrode also impales cells other than melanophores. When cyclic AMP injection inside a cell resulted in dispersion of melanosomes from a perinuclear position into dendritic processes, the onset of dispersion was relatively rapid, in many cases less than 4 min (mean time of onset, 5.3 +/- 2.9 [SD] min). A much slower dispersion (mean time of onset, 19.0 +/- 5.0 min) of melanosomes was observed when the microelectrode was positioned adjacent to a melanophore, and much larger quantities of cyclic AMP were released. In addition, no changes were observed for injections of 5'-AMP or cyclic guanosine monophosphate (GMP) through electrodes positioned inside or adjacent to melanophores. Potential measurements showed that after impaling a clell, a constant transmembrane potential could often be recorded over many minutes, indicating that the membrane tends to seal around the microelectrode. The results indicate that cyclic AMP acts more rapidly on the inside of a cell than when applied outside a cell and allowed to diffuse through the plasma membrane. This study introduces a model system whereby the properties of the plasma membrane and melanocyte-stimulating hormone (MSH) receptors can be studies within a single target cell.