Thyrotropin-Releasing Hormone in the Blood of the Frog,Rana pipiens: Its Nature and Possible Derivation from Regional Locations in the Skin*

Abstract

Immunoreactive TRH (IR-TRH) circulates in the blood of the leopard frog (Rana pipiens) in high concentrations [227 ± 57 ng/ml (mean ± SEM of individual estimations in 35 animals]. Ethanol extracts of blood from this amphibian show parallelism with standard TRH in the RIA and elute from a Sephadex G-10 column identically with synthetic TRH. Further evidence of the authenticity of this immunoreactive substance is shown by its biological activity. Injection of an extract of blood containing 100 ng IR-TRH produced a serum TSH rise in the rat comparable to that after 100 ng synthetic TRH, whereas a similar quantity of blood, allowed to incubate at 37 C before extraction, contained only 12 pg IR-TRH and caused no change in rat TSH levels. The turnover of TRH in the circulation appears to be very fast, as endogenous blood TRH is degraded in vitro with a t_1/2 of 0.95 min at 37 C and 1.8 min at 26 C, while [³H]TRH rapidly disappears from the blood in vivo after injection. The rate of TRH secretion into the blood estimated from measurements of fractional disappearance (39%/min at 26 C), volume of distribution, and blood level is 0.8 μg/min. Since the frog brain weighs only 100–150 mg and contains around 0.1 μg TRH, it is improbable that brain tissue could be the source of circulating TRH. Of other tissues surveyed, the skin contains the largest quantities of IR-TRH, especially in the back area where mean levels are 109 ng/mg protein, a concentration over 4 times that found in the hypothalamus of the same animals. That skin IR-TRH is the authentic peptide is supported by the results of studies using high pressure liquid chromatography, column chromatography, and bioassay. Marked regional variation in the skin distribution of TRH is found, with concentrations in the abdomen being one sixteenth of those in the dorsal region, while skin from other areas contains intermediate levels. The total quantity of TRH in the skin from one frog is approximately 85 μg, so that the secretion of 1% of the content/min could account for TRH circulating in the blood. It appears likely that within the skin, TRH and other neural peptides are located in the cutaneous glands that are derived from specialized neuroectodermal tissue. We postulate that frog skin is an active peptide-secreting organ and that the physiological role of TRH extends outside the strict anatomical confines of the nervous system.