NEUROPHYSINS OF THE RAT
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Abstract:
Three neurophysins have been isolated and biochemically characterized from the ox (Rauch, Hollenberg & Hope, 1969), pig (Uttenthal & Hope, 1970) and sheep (Watkins, 1972) while the presence of a neurophysin has been demonstrated in cod pituitary glands (Pickering, 1968). By means of immunological and biosynthesis techniques, Norstrom, Sjöstrand, Livett, Uttenthal & Hope, (1971) have tentatively identified one neurophysin in the rat posterior pituitary gland. In a recent extension of this work, Burford, Jones & Pickering (1971) using polyacrylamide electrophoresis have been able to resolve the major rat posterior pituitary gland protein into two major components one of which was tentatively described as an oxytocin-neurophysin and the other as a vasopressin—neurophysin. In this present investigation, the neurophysins of the rat were fractionated using procedures previously established for the purification of the ox, pig and sheep neurophysins. The protein-hormone complex was prepared in a yield of 26·9 mg from anKeywords:
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Neurophysins
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Vasotocin
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Previous studies have suggested that oxytocin may have an intragranular association with the posterior pituitary binding protein neurophysin I (NPI), and vasopressin with neurophysin II (NPII) in the ox. We have studied the possible relationship between oxytocin and NPI, and vasopressin and NPII at their presumed sites of synthesis in the bovine hypothalamus. Separate regions of the supraoptic (SON) and paraventrieular (PVN) nuclei from both sides of the hypothalamus and the posterior lobes of the pituitary glands (PL) of 4 steers were homogenized and extracted. The oxytocin and vasopressin content of these homogenates was determined by bioassay. Vasopressin was also measured by radioimmunoassay, as were NPI and NPII. Both hormones and both neurophysins were found in SON, PVN and PL. The concentrations of NPI and oxytocin in SON and PVN were the same. In the PVN the concentrations of oxytocin and vasopressin and those of NPI and NPII were similar. In the SON, however, NPII and vasopressin concentrations were higher than those of oxytocin and NPI. The SON contained 2–3 times as much NPII and vasopressin as did the PVN. A significant correlation between the vasopressin/oxytocin ratio and the NPII/PI ratio was found in the hypothalamic regions. This correlation was not found in PL, however, where the vasopressin/oxytocin ratio was 0.46 and the NPII content was greater than NPI. These data demonstrate a relationship between NPI and oxytocin, and NPII and vasopressin in the bovine hypothalamus and suggest that a specific neurophysin may be synthesized with each neurohypophysial hormone. (Endocrinology95: 931, 1974)
Neurophysins
Supraoptic nucleus
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Posterior pituitary
Neurophysins
Osmoreceptor
Desmopressin
Plasma osmolality
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Neurophysin and the octapeptide hormones oxytocin and vasopressin are synthesized in the hypothalamus and stored in the posterior lobe of the pituitary gland. It has recently been shown that the release of both oxytocin and vasopressin or of vasopressin alone, in response to potent stimuli, is accompanied by a simultaneous release of neurophysin into the circulation (Burton, Forsling & Martin, 1971; McNeilly, Legros & Forsling, 1972). However, it has yet to be shown that neurophysin can be released at the same time as a specific release of oxytocin. This situation occurs in animals during both parturition (Folley & Knaggs, 1965) and lactation (Folley & Knaggs, 1966; McNeilly, 1972). The present report describes the simultaneous release of oxytocin and neurophysin during parturition in the goat. Serial blood samples (approx. 10 ml each) were taken from an indwelling jugular cannula during the whole of labour in two pedigree British Saanen goats. Samples
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A pituitary glycopeptide whose amino acid sequence was previously identified has now been recognized as the final portion of the precursor to arginine vasopressin and its associated neurophysin. Immunocytochemical techniques with antiserums against this 39 amino acid peptide and vasopressin were used to study their distribution in the rat central nervous system. The peptide is located in vasopressin-synthesizing cells in the neurosecretory magnocellular nuclei. Positively stained fibers project from the magnocellular nuclei through the median eminence to the posterior pituitary. Studies of the homozygous Brattleboro rat, which is known to be deficient in the production of vasopressin and its related neurophysin, also show the absence of immunoreactivity to this peptide. These immunocytochemical data strongly indicate that the peptide is synthesized with vasopressin.
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Median eminence
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Neurophysins
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Some evidence suggests that oxytocin, which is a neuropeptide conventionally thought to be synthesized in the hypothalamus and released by the posterior pituitary, is generated in peripheral keratinocytes, but the details are lacking and the mRNA analysis is further required. Oxytocin and neurophysin I are generated together as cleavage products after splitting the precursor molecule, preprooxyphysin. To confirm that oxytocin and neurophysin I are also generated in the peripheral keratinocytes, it must first be clarified that these molecules contained in peripheral keratinocytes did not originate in the posterior pituitary gland and then the expression of oxytocin and neurophysin I mRNAs must be established in keratinocytes. Therefore, we attempted to quantify preprooxyphysin mRNA in keratinocytes using various primers. Using real-time PCR, we observed that the mRNAs of both oxytocin and neurophysin I were located in keratinocytes. However, the mRNA amounts of oxytocin, neurophysin I, and preprooxyphysin were too small to confirm their co-existence in keratinocytes. Thus, we had to further determine whether the PCR-amplified sequence was identical to preprooxyphysin. The PCR products analyzed by DNA sequencing were identical to preprooxyphysin, finally determining the co-existence of both oxytocin and neurophysin I mRNAs in keratinocytes. In addition, the immunocytochemical experiments showed that oxytocin and neurophysin I proteins were located in keratinocytes. These results of the present study provided further support indicating that oxytocin and neurophysin I are generated in peripheral keratinocytes.
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Individual hypothalamic nuclei were microdissected from brain tissue of ten human subjects who had died suddenly while in apparent good health. Appreciable amounts of vasopressin and oxytocin immunoreactivity were found by specific radioimmunoassay in six hypothalamic nuclei including supraoptic and paraventricular nuclei. Vasopressin and oxytocin are presumed to be synthesized in supraoptic and paraventricular nuclei for axonal transport to the posterior pituitary for storage and release. Vasopressin and oxytocin in other hypothalamic nuclei may be a part of this system of neurosecretion or may serve some other function.
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Oxytocin and vasopressin content of the neurohypophysis was determined by means of bioassays in pregnant rats before, during, and after spontaneous as well as oxytocin- induced parturition. Before parturition the levels of oxytocin and vasopressin in the neural lobe were equal on Days 21 and 22 of gestation. Immediately after spontaneous parturition the content of both hormones was about 60% of the prelabor values. At the delivery of the first fetus the oxytocin level had decreased by only about 10%, which is not a significant difference, while the vasopressin content of the posterior lobe was almost as low as at the end of parturition. When parturition was induced by oxytocin or combined oxytocin-arginine-vasopressin infusions, the pituitary oxytocin level was unchanged but the level of vasopressin was as low as after spontaneous delivery. Infusions of exogenous hormone to pregnant rats before term did not alter the endogenous hormone levels, indicating that no neurophypophysial accumulation of oxytocin occurs in spite of high circulating blood levels. It is concluded that significant amounts of both oxytocin and vasopressin are released during spontaneous parturition but that these hormones are secreted independently. The release of oxytocin is not a consequence of labor since it did not occur in induced deliveries, whereas vasopressin release is associated with spontaneous as well as induced delivery, probably in response to the pain and stress of labor. No negative feedback effect occurred with respect to vasopressin. (Endocrinology88: 574, 1971)
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SummaryThe oxytocic and pressor activities of the human posterior pituitary gland were separated chromatographically after preliminary purification. The behavior of purified components on ion exchange chromatography, paper chromatography and paper electrophoresis indicated that they possessed the properties of oxytocin and arginine vasopressin. The amino acid composition of the oxytocin and vasopressin isolated from the human glands was determined. It is evident that the oxytocin and vasopressin found in the human pituitary are identified as oxytocin and arginine vasopressin.
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