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Studies of the oxytocic activity of the neurointermediate lobe of Squalus acanthias (Pacific variety) Swiatkiewicz, Victor Joseph

Abstract

The oxytocic activity of the neurointermediate lobe of the pituitary of the elasraobranch, Squalus acanthias (Pacific variety), was studied with chromatographic and pharmacological techniques, in an effort to determine whether the activity was due to a single or to a number of similar neurohypophysial principles. Neurointermediate lobes (NIL's) were dissected from live specimens of Squalus acanthias caught on the Pacific coast of Canada during 1966-67. The tissues were acetone dried and extracted in 0.25% acetic acid in saline. The extracts were assayed on the isolated rat uterus for their oxytocic activity, both in the presence and absence of magnesium ions, and in one case for rabbit milk ejection activity and antidiuretic activity in the anaesthetized rat. The ratio of milk ejection:rat uterus: rat antidiuretic activity was 2.6:1:0.046 which was in excellent agreement with the observations of previous investigators. However, this ratio contrasts with the 1:1:1 ratio of International standard ox posterior pituitary powder and indicates the presence of an elasmobranch oxytocic principle or principles similar to but not identical with oxytocin. This result was confirmed by the potentiation of the elasmobranch oxytocic activity in the presence of magnesium ions, a property typical of oxytocin analogues but not of oxytocin itself. The elasmobranch extracts showed a 1.2 – 3.2 fold range of magnesium potentiations and agreed with the values published by Sawyer (1965b). However, no extract in the 16 samples assayed showed sufficient potentiation to indicate the presence of significant amounts of a highly potentiated peptide (6 - 10 fold potentiation) such as postulated by Heller and Roy (1964) or Acher, Chauvet, Chauvet and Crepy (1965). Paper chromatography of the oxytocic activity using butanol/ acetic acid (glacial)/water (4:1:5) solvent system, was extended from the low oxytocic activity load levels utilized by Perks (i960) to loads beyond those used by Heller and Roy (196k) in which they detected two separable oxytocic activity peaks. Paper chromatography of our crude extracts at loads of 30 - 500 milli-units (without Mg⁺⁺), showed only one chromatographic peak, with an Rf approximating 0.4 - 0.5, which ran slightly slower than synthetic oxytocin Rf 0.5 - 0.6. No magnesium potentiation greater than a 3-fold potentiation could be detected in any regions of these chromatograms. Crude extracts ranging from 1148 to 7356 milliunits oxytocic activity were partially purified by the mild procedure of gel filtration on Sephadex G-15 in order that higher loads of oxytocic activity could be chrornatogrammed without artifacts due to overloading with protein. Despite the chromatography of levels of oxytocic activity higher than any previously.reported, the oxytocic activity never resolved into more than one chromatographic peak, and no region of the chromatograms potentiated beyond 3.4 when assayed with magnesium. The study of 16 different extracts from the neurointermediate lobes of Squalus acanthias (Pacific variety) gave no evidence for any large content of an oxytocic neurohypophysial principle potentiated 6-10 fold by magnesium ions. The use of paper chromatography techniques identical to those used by Heller and Roy (1964) failed to resolve more than one oxytocic principle, despite the use of oxytocic activity loads beyond those used by these authors. It is concluded that in the case of certain extracts of the oxytocic moiety of the Squalus acanthias (Pacific variety) neurointermediate lobe, there is no evidence for the presence of more than one oxytocic neurohypophysial principle.

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