Isolation and localization of neurophysin-like proteins in rat uterus.

Neurophysins are part of the prohormones for vasopressin and oxytocin, and are localized with these hormones in the magnocellular cells of the neurohypophysis. New techniques have identified neurophysins in other areas within and outside the central nervous system, and we report here the isolation of neurophysins from the uterus of the rat. Using immunohistology the neurophysin immunoreactivity was localized to the epithelial lining cells of the uterus, and using radioimmunoassay was also present in uterine fluid suggesting secretion into the uterine cavity. The amount of uterine neurophysin increased in response to administered estrogen and was especially elevated in the pregnant uterus. The neurophysin-like material isolated from the uterus was similar to neurophysins from the neurohypophysis by radioimmunoassay, molecular sieve chromatography, isoelectric focusing and SDS gel electrophoresis. Both neurohypophyseal hormones, vasopressin and oxytocin, were also extracted from uterine endothelium and identified by radioimmunoassay and high pressure liquid chromatography.

Familial central diabetes insipidus: vasopressin and nicotine stimulated neurophysin deficiency with subnormal oxytocin and estrogen stimulated neurophysin.

Five members of a family with dominantly inherited diabetes insipidus were diagnosed and treated with deamino-d-arginine vasopressin (DDAVP), a vasopressin analogue given intranasally. All subjects demonstrated subnormal levels of arginine vasopressin (AVP) by radioimmunoassay in response to cigarette smoke inhalation, a standardized nicotine stimulation test. Levels of oxytocin (OT) were found to be normal and unstimulated after cigarette inhalation in two subjects, but when two affected male siblings ingested Ovulen, OT and ESN were stimulated to subnormal levels. After twelve months of DDAVP treatment, the low AVP response to nicotine was preserved whereas the carrier protein, nicotine stimulated neurophysin (NSN) remained undetectable.

Quantitation of changes in the content of neurohypophyseal peptides in hypothalamic nuclei after adrenalectomy.

In adrenal insufficiency there is an increase in the content of vasopressin in the external zone of the median eminence as determined by immunohistology. We studied rats after bilateral adrenalectomy and obtained punched samples of the supraoptic, the paraventricular, and the suprachiasmatic nuclei, and from the median eminence and the posterior pituitary. Vasopressin, neurophysin and oxytocin were all decreased in content in the posterior pituitary but in the suprachiasmatic nucleus and in the median eminence there was a significant increase in the content of vasopressin. The increased levels of vasopressin in the median eminence are interpreted as representative of the content in axons and demonstrate a large increase of transport of vasopressin. In the SON and PVN there was no increase in the content of vasopressin during this state of augmented synthesis and transport, indicating that vasopressin moves rapidly from the site of synthesis into the transport system. The increased content of vasopressin in the suprachiasmatic nucleus shows that vasopressin at this level also varies with changes in adrenal function. Levels of oxytocin throughout the neurohypophysis did not change in parallel with vasopressin demonstrating relatively selective responses of the neurohypophyseal hormones.

Vasopressin-dependent and -independent control of the release of adrenocorticotropin.

We tested the possibility that vasopressin mediates the responses of adrenocorticotropin (ACTH) to electrical stimulation of various areas of the hypothalamus. Thirty-three cats were anesthetized with chloralose-urethane, immobilized with gallamine, and respired artificially. Plasma ACTH was measured by RIA. Intraventricular administration of antiserum to vasopressin blocked the release of ACTH induced by electrical stimulation of the paraventricular nucleus (PVN), suggesting a role for the vasopressinergic projection from PVN to the external zone of the median eminence. In contrast, the release of ACTH induced by stimulation of areas ventral to PVN was unaffected by the antiserum. Thus, there is at least one corticotropin releasing factor released from nuclei other than PVN that is distinct from vasopressin.

Elevation of oxytocin and the oxytocin-associated neurophysin in the plasma of normal women during midcycle.

A positive correlation was observed between the midcycle elevation of estrogen (E) and the level of oxytocin- and estrogen-stimulated neurophysin (ESN), the protein carrier of oxytocin, in the plasma of five of six women. The time of the maximal level of E was associated with a level of oxytocin significantly greater than that in either the early follicular or late luteal phase (P less than 0.025). Likewise, the level of ESN at midcycle was greater than the level of ESN in the early follicular or late luteal phase (P less than 0.01). Other than states of lactation or pregnancy, this is the only described cyclic secretion of oxytocin in humans. Since oxytocin chronologically correlates with a rise in the level of E at midcycle, a role for oxytocin in ovulation may be considered.

Hyponatremia in intracranial disease: perhaps not the syndrome of inappropriate secretion of antidiuretic hormone (SIADH).

Patients with intracranial disorders are prone to develop hyponatremia with inability to prevent the loss of sodium in their urine. This was originally referred to as "cerebral salt wasting," but more recently is thought to be secondary to the syndrome of inappropriate secretion of antidiuretic hormone (SIADH). Blood volume determinations were made in 12 unselected neurosurgical patients with intracranial disease who fulfilled the laboratory criteria for SIADH. Ten of the 12 patients had significant decreases in their red blood cell mass, plasma volume, and total blood volume. The finding of a decreased blood volume in patients who fulfill the laboratory criteria for SIADH is better explained by the original concepts of cerebral salt wasting than by SIADH. The primary defect may be the inability of the kidney to conserve sodium.

Oxytocin in human plasma: correlation with neurophysin and stimulation with estrogen.

RIA for the measurement of oxytocin in human plasma is described. Extraction of oxytocin from larger peptides in plasma used acetone precipitation with a 75% +/- 2 SEM recovery of oxytocin. Nonspecific binding of the assay was less than 4%, and the minimum level of detection was 0.2 microunits/tube. No cross-reactivity was noted with neurophysins, arginine, or lysine vasopressin. The mean basal level (+/- SEM) of oxytocin in men was 1.80 +/- 0.07 microunits/ml and was not different in normal women (1.71 +/- 0.07 microunits/ml). Changes in posture had no effect on the levels of oxytocin. Samples obtained every 15 min over 4 h showed no pulsatile secretion of oxytocin. In women chronically receiving estrogen as an oral contraceptive, oxytocin was greater than normal, (4.59 +/- 0.51 microunits/ml; P less than 0.01). Estrogen-stimulated neurophysin was also elevated (8.45 +/- 1.99 ng/ml; P less than 0.005). Acute ingestion of estrogen caused an increase in the level of oxytocin in plasma by 12 h and a concomitant elevation of estrogen-stimulated neurophysin. When the neurophysin was isolated from plasma obtained from a subject after ingestion of estrogen, the neurophysin from plasma comigrated on a polyacrylamide gel with a human pituitary standard of estrogen-stimulated neurophysin. In the studies in which neurophysin was elevated, the correlation between the level of oxytocin and the level of estrogen-stimulated neurophysin in plasma was significant (P less than 0.01). The observation that estrogen administration stimulates the release of oxytocin and estrogen-stimulated neurophysin provides additional evidence that this neurophysin is the oxytocin-neurophysin of man.

PIP: RIA for the measurement of oxytocin in human plasma is described. Extraction of oxytocin from larger peptides in plasma used acetone precipitation with a 75% +or- 2 SEM recovery of oxytocin. Nonspecific binding of the assay was less than 4% and the minimum level of detection was 0.2 mcU/tube. No cross-reactivity was noted with neurophysins, arginine, or lysine vasopressin. The mean basal level (+or- SEM) of oxytocin in men was 1.80 +or- 0.07 mcU/ml and was not different in normal women (1.71 +or- 0.07 mcU/ml). Changes in posture had no effect on the levels of oxytocin. Samples obtained every 15 minutes over 4 hours showed no pulsatile secretion of oxytocin. In women chronically receiving estrogen as an oral contraceptive, oxytocin was greater than normal (4.59 +or- 0.51 mcU/ml; P0.01). Estrogen-stimulated neurophysin was also elevated *8.45 +or- 1.99 ng/ml; P0.005). Acute ingestion of estrogen caused an increase in the level of oxytocin in plasma by 12 hours and a concomitant elevation of estrogen-stimulated neurophysin. When the neurophysin was isolated from plasma obtained from a subject after ingestion of estrogen, the neurophysin from plasma comigrated on a polyacrylamide gel with a human pituitary standard of estrogen-stimulated neurophsin. In the studies in which neurophysin was elevated, the correlation between the level of oxytocin and the level of estrogen-stimulated neurophysin in plasma was significant (P0.01). The observation that estrogen administration stimulates the release of oxytocin and estrogen-stimulated neurophysin provides additional evidence that this neurophysin is the oxytocin-neurophysin of man.

Control of release of adrenocorticotropin and vasopressin by the supraoptic and paraventricular nuclei.

To determine the relative roles of the hypothalamic supraoptic (SON) and paraventricular (PVN) nuclei in the control of the release of vasopressin and of ACTH, we have examined the hormonal responses to electrical stimulation (200 microA, 0.2 msec, 100 Hz, 20 sec) of these regions. Cats were anesthetized with chloralose-urethane. Blood samples were taken 30 sec before stimulation and 1.5 min poststimulation. ACTH and vasopressin were measured by RIA. Electrical stimulation of the caudal pole of the SON increased vasopressin in plasma (1.82 +/- 0.41 microU/ml, n = 17, P less than 0.01) and decreased ACTH (-26 +/- 4 pg/ml, n = 13, P less than 0.01). In contrast, stimulation of the PVN increased vasopressin (2.01 +/- 0.60 microU/ml, n = 7, P less than 0.001) and increased ACTH (107 +/- 20 pg/ml, n = 32, P less than 0.01). Previous work has shown that vasopressinergic neurons of PVN, but not of SON, project to the zona externa of the median eminence. Other have suggested that the retrograde flow of blood from the neural lobe to the median eminence and thence to the anterior lobe would allow vasopressin to influence the release of ACTH. The present results indicate that both SON and PVN facilitate the release of vasopressin. However, PVN facilitates, but SON inhibits the release of ACTH. These findings suggest that the projection from PVN to the zona externa of the median eminence mediates the release of ACTH and that retrograde flow from the neural lobe is not important in the control of ACTH release during modest and transient increases in the release of vasopressin.

Chlorpropamide effect: measurement of neurophysin and vasopressin in humans and rats.

The effect of chlorpropamide on neurohypophyseal function was studied by measurement of neurophysin and vasopressin in humans and in rats. Administration of chlorpropamide was shown to inhibit the ability of the human subjects and of the rats to maximally dilute their urine after administration of water. Comparison of the levels of vasopressin and neurophysin before and after the administration of chlorpropamide in the basal state and after water loading in both the human subjects and in rats showed no lack of suppression due to the chlorpropamide. The levels of both peptides were measured at the limits of detection. In other studies where neurophysin and vasopressin were readily detected, administration of chlorpropamide did not result in any augmented release of neurophysin or vasopressin in response to stimulation of the neurohypophysis in humans nor in rats. Levels of neurophysin in the pituitaries of rats showed no change in rats given chlorpropamide, while the content of vasopressin was increased, possibly indicating a chronic decreased secretion of vasopressin in rats given chlorpropamide.

Levels of neurohypophyseal peptides in the rat during the first month of life. I. Basal levels in plasma, pituitary, and hypothalamus.

Vasopressin, oxytocin, and neurophysin were measured by RIA in the pituitary, hypothalamus, and plasma (except oxytocin) of the rat during the first month of life. In plasma, vasopressin was less than 1.7 microU/ml in most animals. Neurophysin was elevated above adult levles on day 2 and decreased with age. The three peptides were present in the pituitary at birth, but in amounts less than 1% of the adult level. The vasopressin content of the pituitary increased rapidly in the first days after birth, while the levels of oxytocin and neurophysin remained low until 8 days and then increased between 8-21 days. The ratio of vasopressin to oxytocin in the pituitary was 4.4 at birth and reached unity (the ratio in the adult) at 30 days. At birth, the moles of neurophysin in the pituitary relative to the moles of hormone (oxytocin plus vasopressin) was low (0.15), largely due to a molar excess of vasopressin. The ratio of neurophysin to hormone reached unity at 21-30 days. Assays to detect vasotocin gave negative results. It is postulated that a precursor neurophysin which was related to vasopressin was present in the fetal rat but was not measured in our study.

Levels of neurohypophyseal peptides in the rat during the first month of life. II. Response to physiological stimuli.

Levels of vasopressin, oxytocin, and neurophysin were measured by RIA in the hypothalamus, pituitary, and plasma of infant rats (2-30 days old). At all ages, ip injection of a hypertonic solution of 5 g/100 ml NaCl produced a marked increase in levels of vasopressin and neurophpysin in plasma, up to 21 microU/ml and 51 ng/ml, respectively. After dehydration, there was a decrease of 26-38% in the levels of neurohypophyseal peptides in the pituitary. Depletion of neurohypophyseal peptides from the pituitary was greater after 24 h of dehydration in younger rats (26%) than in older rats (7%). Levels of vasopressin in plasma were less than 1.7 microU/ml after dehydration in younger rats but were greater in older rats. Immaturity of the neurohypophysis may contribute to the inability of newborn rats to withstand prolonged dehydration.

Neurohypophyseal peptides in the developing rat fetus.

The quantitative changes in the content of the neurohypophyseal peptides, neurophysins, oxytocin, and vasopressin, were determined in the developing rat fetus. No neurohypophyseal peptides were found in 12-day fetuses. Neurophysins were first detected at day 13 and increased dramatically on day 14. The hormones vasopressin and oxytocin were not detected at day 13 and were measured at low levels at day 14. A 350-fold molar excess of neurophysin to hormones existed at day 14. From day 14 to day 19 the total content of neurophysin decreased while the content of vasopressin and oxytocin slowly increased. At day 19 there was a near molar equivalency between the content of neurophysin and that of the neurohypophyseal hormones. From day 18 to day 22 there was a sharp increase in the content of vasopressin while the content of neurophysin and oxytocin increased less dramatically. At term there was a molar excess of vasopressin, and the molar ratio of neurophysin to hormone at the time of delivery was 0.12. Measurement of vasopressin by different radioimmunoassays and by bioassay indicated no contribution of arginine-vasotocin to the measured vasopressin.

Studies on the antidiuretic effect of cyclophosphamide: vasopressin release and sodium excretion.

To elucidate the mechanism of cyclophosphamide (CTX)-induced antidiuresis, plasma and urine volume as well as serum electrolytes, creatinine, osmolality, and appropriate hormones were monitored serially during 19 courses of chemotherapy. In spite of plasma hypotonicity and urinary hypertonicity, the plasma vasopressin concentrations were unaltered. Intravenous isotonic hydration did not prevent water retention, but did not lead to plasma hypotonicity, and compensated for modest urinary sodium losses. Furosemide diuresis did not prevent the development of hyponatremia in patients receiving hypotonic hydration. The results indicate that the origin of this self-limited syndrome is a direct effect of CTX on the renal tubule, permitting increased water reabsorption and sodium loss. The likelihood that water and salt imbalance will develop after CTX administration can be reduced by vigorous isotonic hydration, and pharmacological diuresis.

Plasma neurophysin and vasopressin in the rat: response to adrenalectomy and steroid replacement.

Impaired excretion of a water load is known to occur in adrenal insufficiency and to be corrected by administration of glucocorticoid. Such impairment has been related to either a loss of a permissive effect of glucocorticoids on the diluting segments of the nephron or to an alteration of release, turnover, or action of antidiuretic hormone. Specific and sensitive RIAs for arginine vasopressin and neurophysin were utilized to measure plasma and pituitary levels of neurohypophyseal peptides at baseline and after an intragastrically administered water load. Conscious, unanesthetized, and nonstressed sham-operated, adrenalectomized, and adrenalectomized prednisone-treated rats were studied. The results demonstrate a significant elevation in vasopressin and neurophysin in plasma in adrenalectomized rats maintained in a normal state of hydration. After water loading, the adrenalectomized rats diluted their plasma osmolality but had a decreased urinary volume, increased urinary osmolality, and elevated vasopressin and neurophysin in their plasma. In the pituitary, vasopressin and neurophysin were depleted in adrenalectomized rats, indicating increased secretion of these peptides. It is concluded that elevated vasopressin in plasma may be an important factor in the incomplete water diuresis in adrenal insufficiency.

DDAVP (1-desamino-8-D-arginine-vasopressin) treatment of central diabetes insipidus--mechanism of prolonged antidiuresis.

DDAVP, 1-desamino-8-D-arginine-vasopressin, is a synthetic analog of arginine vasopressin which produces prolonged antidiuresis after intranasal administration to patients with complete central diabetes insipidus. We have studied the mechanism of the prolonged antidiuretic effect by specific radioimmunossay of DDAVP in plasma of patients and by in vitro studies on the adenylate cyclase-cylic AMP system of the rat outer renal medulla. When DDAVP was administredd to patients, all responded, but the duration of response among patients varied from 5-21 h. The peak level of DDAVP in plasma was achieved up to 4 h after administration indicating a slow absorption from the nasal mucosa. The disappearance time of DDAVP from plasma correlated significantly with the duration of antidiuresis, P less than 0.001. On a molar basis DDAVP was 3-fold greater than AVP in its stimulation of outer medullary adenylate cyclase activity and 10-fold greater than AVP in its stimulation of cyclic AMP content. The prolonged antidiuresis of intranasally administered DDAVP is due to slow absorption, presistence in plasma, and enchanced effect on the kidney.

Impaired inner medullary production of prostaglandin E2 in the kidney of the myxedematous rat.

Renal cAMP concentration and renal prostaglandin (PG) E2 and PGF2alpha production were determined in control rats and rats with surgically induced myxedema. Slices from the kidneys of each group were incubated in vitro. cAMP concentration of inner medullary slices from the kidneys of myxedematous rats was decreased in comparison to slices from control rats. There was no difference between the two groups when slices from outer medulla and cortex were compared for cAMP level. In myxedema, there was decreased inner medullary production of PGE2, but not PGF2alpha. The difference in PGE2 production between myxedema and control rat kidneys was not corrected by addition of either arachidonic acid (170 micronM) or phospholipase A2 (5 microgram/ml). Arachidonic acid, however, increased inner medullary cAMP concentration in myxedema kidneys to a value not measurably different from control. Thus, there appears to be a decreased PGE2 production in the renal inner medulla of rats with myxedema. In view of the fact that PGE2 has previously been shown to increase inner medullary cAMP content, the reduced inner medullary content of cAMP would appear to be attributable to reduced PGE2 production.