UBC Theses and Dissertations
A pharmacological, chromatographic, and chemical study of the neurohypophysial principles of two elasmobranch species: Raia rhina and Squalus acanthias (Pacific variety) Heinicke, Elizabeth Anne
A wide and confusing array of neurohypophysial principles has been attributed to the elasmobranch fish; several of these principles are notably similar, either in their distinguishing characteristics or in the species in which they have been reported to occur. Here, an attempt was made to resolve some of the confusion regarding the exact number and nature of the elasmobranch principles, by a study of the pharmacological, chromatographic, and chemical characteristics of the agents produced by two species: the skate Raia rhina and the dogfish Squalus acanthias (Pacific variety) = Squalus suckleyi. The neurointermediate lobe extracts of both species contained a major neutral oxytocic fraction and small amounts of a basic antidiuretic principle which appeared to be the widespread agent, arginine vasotocin. This identification was based on direct pharmacological comparison of the purified Squalus principle against synthetic arginine vasotocin. It was the first such characterization of an elasmobranch principle, and possibly of any naturally occurring oxytocin analogue, against its possible synthetic equivalent; earlier comparisons had been made against the customary oxytocin or vasopressin standards. Neurointermediate lobe extracts of Raia rhina showed an unexpected variation in the extent to which their activity on the isolated rat uterus was potentiated by the presence of magnesium ions in the bathing solution (1.8 fold to 17.5 fold). This could not be correlated with factors such as season or location of the catch; it was shown to result from anomalies in the distinction which different uterine preparations made between the unknown peptide and the oxytocin standard. The potency of Raia extract also varied on the milk-ejection assay, with a corresponding variation in the ratio of milk-ejection activity to rat uterus activity of from 4.4 to 17.5. Many of the oxytocic principles ascribed to the elasmobranchs have been distinguished by their degree of magnesium potentiation or by the ratio of their milk-ejection activity to their oxytocic activity on the rat uterus. The variability described here for these two values may invalidate such distinctions; in conjunction with other available data, these observations raised the possibility that the many elasmobranch oxytocin-like principles might be reduced to two -- the agent designated as E₁, which did not occur in the two species studied here, and glumitocin, a principle of known structure. Later studies on the neutral fraction of Raia rhina indicated that the agent was glumitocin, but the studies on Squalus suggested the introduction of several new principles to the elasmobranch spectrum. The identification of the Raia principle as glumitocin was based on direct pharmacological comparison of the agent against synthetic glumitocin, and on chemical analysis of the principle. Direct pharmacological comparison of the Squalus neutral fraction-against synthetic glumitocin showed clear differences between the two principles, notably in their degree of magnesium potentiation and in their milk-ejection activities. The Squalus neutral fraction was purified by gel filtration through G-15 Sephadex, by chromatography on Sephadex ion exchangers, and by chromatography on paper. The activity was separated into two distinct fractions on CM Sephadex. These, however, behaved identically throughout numerous comparative procedures; they were subsequently shown to be experimental artifacts, and were recombined. The purified neutral fraction showed an activity of 109 IU/mg, the most active preparation of the Squalus oxytocic agent so far obtained. Amino acid analyses of the purified material suggested that it consists of an equimolar mixture of two peptides, neither of which has been encountered previously in nature. The work presented here lays a basis for a better understanding of the elasmobranch neurohypophysial principles, but the details of the distribution and structure of these peptides must await yet more extensive studies.
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