Gonadotrophin-releasing activity of neurohypophysial hormones: II. The pituitary oxytocin receptor mediating gonadotrophin release differs from that of corticotrophs

Neurohypophysial hormones stimulate gonadotrophin release from dispersed rat anterior pituitary cells in vitro, acting through receptors distinct from those which mediate the secretory response to gonadotrophin-releasing hormone (GnRH). The LH response to oxytocin was not affected by the presence of the phosphodiesterase inhibitor, methyl isobutylxanthine, but was diminished in the absence of extracellular calcium and was progressively increased as the calcium concentration in the medium was raised to normal. In addition, the calcium channel antagonist, nifedipine, suppressed oxytocin-stimulated secretion of LH. It is likely that the mechanisms of LH release induced by GnRH and neurohypophysial hormones are similar, although stimulation of gonadotrophin secretion is mediated by separate receptor systems. Oxytocin was more active than vasopressin in releasing LH, but less active in releasing ACTH. The highly selective oxytocin agonist, [Thr4,Gly7]oxytocin, elicited concentration-dependent secretion of LH but had little effect on corticotrophin secretion. The neurohypophysial hormone antagonist analogues, [d(CH2)5Tyr(Me)2]vasopressin, [d(CH2)5Tyr(Me)2,Orn8]vasotocin and [d(CH2)5D-Tyr(Et)2Val4,Cit8]vasopressin, inhibited the LH response to both oxytocin and vasopressin. However, [d(CH2)5Tyr(Me)2]vasopressin was much less effective in inhibiting the ACTH response to the neurohypophysial hormones, and [d(CH2)5Tyr-(Me)2,Orn8]vasotocin and [d(CH2)5D-Tyr(Et)2,Val4,Cit8]vasopressin exhibited no inhibitory activity against ACTH release. Thus, agonist and antagonist analogues of neurohypophysial hormones display divergent activities with regard to LH and ACTH responses, and the neuropeptide receptor mediating gonadotroph activation is clearly different from that on the corticotroph. Whereas the corticotroph receptor is a vasopressin-type receptor an oxytocin-type receptor is responsible for gonadotrophin release by neurohypophysial hormones.     

Contribution of the peptide backbone to the action of oxytocin analogs.

This investigation was undertaken to evaluate the functional contribution of the peptide backbone of oxytocin in its interaction with receptor. Corey-Pauling-Koltun models of (Gly-7) deaminooxytocin, deaminotocinamide, and their respective retro-D-analogs built in any specific conformation (e.g., the Walter-Urry model for oxytocin) have a quai-equivalent topochemical arrangement of amino-acid side chains; however, the CO and NH elements of the peptide backbone of the retro-D-analog are reversed. The retro-D-analogs of deaminotocinamide and (Gly-7) deaminooxytocin (prepared using D-alle for L-Ile) and their respective N-formyl derivatives were assayed for uterotonic activity relative to related L-peptides. All retro-D-analogs (tested at concentrations ranging from 10-10 to 10-5 M) were devoid of angonistic (or antagonistic) activity in the isolated rat uterus, except for the retro-D-(D-alle-3, Gly-7) deaminooxytocinamine, which retains a terminal NH-2 group on the tail; the latter is a partial agonist with very low affinity. The results obtained with retro-D-analogs indicate that one or more of the elements of the peptide backbone of the tocinamide ring are essential for "occupation" and "activation" of uterine receptors. Oxytocin action may be the resultant of multiple hydrogen-bonding interactions between CO, NH, NH-2, and OH groups of the hormone with complementary groups on receptor, made possible by appropriate hydrophobic bonding.     

Oxytocin and vasopressin are present in human and rat pancreas

Immunoreactive oxytocin and vasopressin were found in human and rat pancreatic extracts. The pancreatic oxytocin and vasopressin eluted on Sephadex G-75 gel filtration chromatography and on reverse phase high pressure liquid chromatography in the same positions as their respective reference preparations. The immunoreactive oxytocin was biologically active in the rat milk ejection assay. The presence of oxytocin and vasopressin in human and rat pancreatic extracts suggests the possibility of local synthesis of both hormones. The neurohypophysial hormones are known to be endocrine mediators of insulin and glucagon release. The finding of oxytocin and vasopressin in the pancreas raises the possibility, although yet unproven, of local synthesis and perhaps a paracrine function for the neurohypophysial peptides upon pancreatic hormone release or for a local function upon the liver.     

Special evolution of neurohypophysial hormones in cartilaginous fishes: asvatocin and phasvatocin, two oxytocin-like peptides isolated from the spotted dogfish (Scyliorhinus caniculus).

In contrast to most vertebrate species that possess one oxytocin-like hormone and one vasopressin-like hormone, a few groups, such as marsupials or cartilaginous fishes, are endowed with two peptides of either or both types, suggesting possible gene duplications. We have now isolated two oxytocin-like hormones from the pituitary of the spotted dogfish Scyliorhinus caniculus (suborder Galeoidei). Microsequencing as well as chromatographic and pharmacological comparisons with synthetic peptides show that these peptides are [Asn4,Val8]oxytocin (asvatocin) and [Phe3,Asn4,Val8]-oxytocin (phasvatocin). Asvatocin and phasvatocin display oxytocic activity on rat uterus, about 80 and 5 milliunits per nmol, respectively, and virtually no pressor activity on anesthetized rats. They occur in roughly equal molar amounts in the gland; vasotocin is also present in a proportional amount that is lower by about a factor of 20. In addition to the duality, conservative amino acid substitutions are observed in the two oxytocic peptides in positions 4 (Gln-4-->Asn) and 8 (Leu-8-->Val), when compared with oxytocin. Furthermore, replacement of the isoleucine residue found in position 3 of all other oxytocin-like hormones by phenylalanine in phasvatocin is exceptional; it determines a dramatic decrease of the oxytocic activity. Preservation of the C-terminal-amidated nonapeptide pattern in the 12 vertebrate neurohypophysial hormones known to date suggests that both precursors and processing enzymes have coevolved tightly. On the other hand, whereas the great evolutionary stability of the mature hormones (generally observed in vertebrates) suggests a strict messenger-receptor coevolution, the exceptional diversity found in cartilaginous fishes (six oxytocin-like peptides identified out of eight known) might be due to a looseness of selective constraints, perhaps in relationship with their specific urea osmoregulation.     

Relatively greater increase of oxytocin than vasopressin in the supraoptic nucleus of rats after hypophysectomy.

The vasopressor and milk ejection activities were estimated in the supraoptic region of the hypothalamus of rats which had been hypophysectomized 30 min, 4 and 8 days earlier. Both pressor and milk ejection activities were significantly greater 4 days after hypophysectomy than those in sham-operated control rats. Eight days after hypophysectomy, pressor activity decreased below control values but milk ejection activity was still significantly raised. Increased biological activity after hypophysectomy is thus associated with the previously observed increase in neurosecretory material within the supraoptic nucleus so these results provide additional evidence that neurosecretory material represents stored hormone. Milk ejection activity increased relatively more than pressor activity which supports the suggestion that the final stage in the formation of neurohypophysial hormones occurs as they pass from the hypothalamus to the neural lobe. If this final maturation normally occurs more slowly for oxytocin than for vasopressin, the obstruction to the flow of hormones down the neural stalk caused by hypophysectomy would result in a greater increase in milk ejection than pressor activity.     

Biofunctional evaluation of a hydrogen bond linking the ring and tail beta-turns of oxytocin.

Deamino-[8-N-methylleucine]oxytocin and deamino-[8-alpha-hydroxyisocaproic acid]oxytocin were synthesized to study the importance of hydrogen bonding between the carboxamide carbonyl of asparagine and the peptide N-H of leucine in stabilizing the biologically active conformation of oxytocin. The analogs were synthesized by coupling deaminotocinoic acid with Pro-Leu(Me)-Gly-NH2 and Pro-HyIc-Gly-NH2, respectively. (HyIc is alpha-hydroxyisocaproic acid). Deamino-[8-N-methylleucine]oxytocin was found to possess 48 +/- 7 units of uterotonic activity, 33 +/- 5 units of avian vasodepressor activity, and 3.15 +/- 1.5 units of antidiuretic activity per mg; deamino-[8-alpha-hydroxyisocaproic acid]oxytocin possessed 134 +/- 12 units of uterotonic activity, 31 +/- 3 units of avian vasodepressor activity, 9.6 +/- 3.0 units of antidiuretic activity, and 0.26 +/- 0.02 unit of pressor activity per mg. Neither of the analogs possesses the peptide N-H at residue 8 required for the formation of a hydrogen bond with the asparagine carboxamide; however, both can assume the conformation needed to evoke the characteristic biological activities of oxytocin although in lower potency. It is concluded that such a hydrogen bond does not constitute a conformational constraint that is essential for hormone action.     

Vasotocin biosynthesis by neurohypophysial cells from human fetuses. Evidence for its ependymal origin.

Cultured neurohypophysial cells from human fetuses aged 130-155 days release into the culture medium an active principle which has antidiuretic, hydroosmotic and rat uterine activities. The chromatographic mobility of the active principle, as well as the susceptibility of all of the above activities to tryptic digestion, indicates the presence of a basic peptide identical to arginine vasotocin (AVT). The ability of these cultured cells, which are probably ependymal cells, to release the above activities during 43 days of incubation, suggests that AVT is synthesized and secreted by specialized ependymal cells in the developing human neurohypophysis. Neither in the culture media from anterior pituitaries of the same fetuses nor in control culture media could antidiuretic, rat uterine or hydroosmotic activities be detected. Although the nonincubated neurohypophyses of the same age contain pharmacological activities suggesting the presence of vasopressin, oxytocin and AVT, the neurohypophysial cells cultured in vitro apparently release only AVT into the medium. This supports the suggestion that whereas vasopressin and oxytocin are synthesized in the hypothalamic neurosecretory cells, AVT, on the contrary, appears to be synthesized by ependymal neurohypophysial cells in the developing human neurohypophysis. Consequently, the fetal human neurohypophysis, and presumably the fetal mammalian neurohypophysis, appears not only as a storage site for neurohypophysial hormones but also as an endocrine structure which synthesizes and secretes AVT by ependymosecretion.     

Inactivation of oxytocin by the hen oviduct during oviposition.

All parts of the hen's oviduct were able to inactivate oxytocin in the following decreasing order: uterus>magnum>vagina>isthmus. A decrease in oxytocin-inactivating activity of the oviduct tissues was observed 4 hr before oviposition and during oviposition. It is probable that a decrease in oxytocin-inactivating activity of the hen's oviduct during oviposition intensifies the oxytocic effect of neurohypophysial hormones on the uterine muscle.     

Cloning and sequence analysis of cDNAs for neurohypophysial hormones vasotocin and mesotocin for the hypothalamus of toad, Bufo japonicus.

The primary structures of the precursors of neurohypophysial hormones vasotocin (VT) and mesotocin (MT) in the hypothalamus of the toad Bufo japonicus were determined by analyzing the nucleotide sequences of the cloned cDNAs encoding them. The MT precursor consists of 125 amino acid residues containing a signal peptide followed directly by MT, which in turn is connected to the MT neurophysin by Gly-Lys-Arg, a processing and carboxyl-terminal amidation signal. In contrast, the VT precursor includes a glycoprotein of 36 amino acids following the VT neurophysin. Except for glycoprotein, the structures of MT and VT precursors are quite similar. RNA transfer blotting analysis showed that both MT and VT mRNAs are present in the brain but not in the liver, ovaries, and testes of the toad. The sequences and the structural organizations of the MT and VT precursors are highly homologous to those of their mammalian counterparts, oxytocin and arginine vasopressin precursors, respectively. This fact suggests that, in the evolutionary pathway of neurohypophysial hormones, VT is the ancestor molecule of vasopressin, while MT is that of oxytocin.     

Identification and characterization of arginine vasopressin receptors in the rat testis

We have previously shown that arginine vasopressin (AVP) directly inhibits testicular steroidogenesis in vitro. In the present study, binding of neurohypophysial peptides to interstitial cells of the rat testis was studied using [3H]AVP as the ligand. Interstitial cells were obtained from adult rat testis after collagenase dispersion and were incubated with [3H]AVP in the presence or absence of unlabeled AVP. Binding equilibrium was reached by 60 min at 4 C, while incubation at higher temperatures (23 and 37 C) resulted in an apparent decrease in binding. Scatchard plot analysis of equilibrium binding data revealed the existence of one class of high affinity, low capacity binding sites (Kd = 1.0 +/- 0.3 nM; maximal binding = 8.5 fmol/10(6) cells). In addition, the rate constants of association and dissociation were calculated to be 0.024 nM-1 min-1 and 0.009 min-1, respectively. Addition of naturally occurring neurohypophysial hormones as well as their synthetic analogs inhibited [3H]AVP binding to testis cells, resulting in parallel displacement curves. The order of potencies for the native peptides was: AVP = lysine vasopressin = arginine vasotocin (IC50, 5 X 10(-10) M) greater than oxytocin = mesotocin (IC50, 4 X 10(-7) M) greater than isotocin = glumitocin (IC50 greater than 10(-6) M). Furthermore, two potent vasopressor antagonists, d(CH2)5Tyr(Me)AVP ([1-(beta-mercapto-beta, beta-cyclopentamethylenepropionic acid), 2-(O-methyl)tyrosine]AVP) and dPTyr(Me)AVP ([1-deaminopenicillamine-2-(O-methyl)tyrosine]AVP) competed for [3H]AVP binding with a higher affinity (IC50, approximately 10(-11) M) than native AVP. In contrast, a selective antidiuretic agonist, dDAVP (1-deamino-8-D-AVP), only competed weakly for receptor binding, while a specific oxytocic agonist, (Thr4,Gly7)oxytocin, did not affect AVP binding. These results suggested that the testis may contain the V1 receptor subtype. Studies on the intratesticular distribution of AVP receptors indicated minimal binding to cells derived from the seminiferous tubule, while most of the AVP-binding sites sediment with enriched fractions of Leydig cells after Metrizamide density gradient centrifugation. AVP-binding sites were also found in rat liver, kidney, and anterior pituitary (10.7, 2.6, and 1.7 fmol/mg protein), whereas adrenal, cerebellum, prostate, and hypothalamus were devoid of AVP-binding sites. Thus, we have demonstrated the presence of high affinity, stereospecific receptors for AVP in the interstitial cell compartment of the rat testis. These V1 receptors may mediate the direct inhibitory action of neurohypophysial hormones on testicular Leydig cell steroidogenesis.     

"Antigonadal" activity of the neurohypophysial hormones in cultured rat testicular cells: abolition by pertussis toxin

The intermediary role of the putative inhibitory regulatory membrane protein "Ni" in the "antigonadal" activity of the neurohypophysial hormones was investigated in vitro with the use of a primary culture of rat testicular cells. To this end, use was made of the pertussis toxin (PT) probe, an exotoxin of Bordetella pertussis presumed to modify and inactivate a membrane protein related to or identical with Ni. Testicular cells were pretreated with PT (70 ng/ml) for 24 h, a duration of exposure known to result in a maximal PT effect. Thereafter, the cells were cultured for an additional 48 h in the absence or presence of hCG (10 ng/ml), with or without a maximal inhibitory dose (10(-6) M) of the neurohypophysial principle arginine vasotocin (AVT). Although concomitant treatment of control cells with AVT produced a profound (95%) inhibition of hCG-stimulated testosterone accumulation, PT pretreatment resulted in a long-lasting (greater than 4 days) abolition of this "antigonadal" effect. Furthermore, pretreatment with PT resulted in significant (P less than 0.05) augmentation of the hCG-stimulated accumulation of extracellular cAMP (1.5- to 2.0-fold) and testosterone (1.7- to 3.8-fold), presumably as a result of abolition of the "basal" tonic inhibition of adenylate cyclase activity. Taken together, these findings constitute the first demonstration of the involvement of Ni in the "antigonadal" activity of the neurohypophysial hormones and in the regulation of testicular function, thereby raising the intriguing possibility that testicular Ni may serve as the coupling unit of inhibitory receptors other than those for the neurohypophysial hormones, integrating varied incoming signals of endocrine, paracrine or autocrine nature.     

Gonadotrophin-releasing activity of neurohypophysial hormones: I. Potential for modulation of pituitary hormone secretion in rats

Neurohypophysial hormones have been implicated in the control of anterior pituitary function, and oxytocin has been shown to stimulate gonadotrophin excretion and ovarian follicular development in certain species. To determine the role of neurohypophysial peptides in the control of gonadotrophin release, their actions on LH and FSH secretion were analysed in rats in vivo and in vitro. In adult female rats, administration of oxytocin during early pro-oestrus advanced the spontaneous LH surge and markedly increased peripheral LH levels at 15.00 h compared with control animals. In cultured pituitary cells from adult female rats, oxytocin and vasopressin elicited dose-related increases in LH and FSH release. Such responses were not affected by a potent gonadotrophin-releasing hormone (GnRH) antagonist that abolished GnRH agonist-induced release of LH and FSH. Oxytocin did not enhance GnRH agonist-stimulated gonadotrophin release to the same extent as it increased basal secretion, but at low concentrations of GnRH agonist the effects were additive. The gonadotrophin responses to oxytocin and vasopressin were inhibited by the specific neurohypophysial hormone antagonists, [d(CH2)5D-Ile2,Ile4,Arg8]vasopressin and [d(CH2)5Tyr (Me),Arg8]vasopressin. These results provide direct evidence that neurohypophysial hormones can stimulate gonadotrophin secretion through a receptor system distinct from the GnRH receptor. Such a mechanism could represent a complementary hypothalamic control system for long-term modulation of LH and FSH secretion by exerting a basal or tonic influence on gonadotrophin production.     

Neurohypophysial hormones in fetal life and pregnancy II. Chromatographic studies in the sheep (Ovis aries)

The neurohypophysial principles of the pituitary of the pregnant sheep were purified by a single-step gel-filtration system. Amino acid analysis, and other evidence, indicated the presence of oxytocin, AVP, and no other neurohypophysial principles.The principles of late-term fetal sheep (138–141 days of gestation) were purified by the same system. Amino acid analysis, with chromatographic and pharmacological evidence, demonstrated oxytocin, with larger amounts of AVP. In addition, there were small quantities of a principle with strong frog bladder activity—probably AVT. The original glands were estimated to contain FB:V:O activities of 233:1133:147 mU/mg dry gland, where FB is frog bladder activity due to AVT, V is vasopressor and O is oxytocic activity.Paper chromatography of the principles from midterm fetuses (89–91 days of gestation) showed evidence for small quantities of oxytocin, and large amounts of AVP. However, the predominant activity present was a powerful frog bladder effect, which indicated substantial quantities of AVT. The original glands were estimated to contain FB:V:O activities of 12,259:616:30 mU/mg dry tissue.The fetal pituitary appears to contain AVT, AVP, and oxytocin. At midterm, AVT and AVP are the predominant neurohypophysial peptides present. At that time, there are almost 5 molecules of AVT for every 1 molecule of oxytocin, and 1 molecule of AVT for every 5 of AVP. The ratio of AVT to AVP molecules falls to 1:400 close to birth, and AVT appears to be lost in the adult.     

Effects of glucagon-like peptide-1(7-36) amide on neurohypophysial and cardiovascular functions under hypo- or normotensive hypovolaemia in the rat

To date, glucagon-like peptide 1(7-36) amide (tGLP-1) has been found to affect the neurohypophysial and cardiovascular functions in normotensive and normovolaemic rats. The aim of the present study was to investigate possible effects of tGLP-1 on the mean arterial blood pressure and the release of vasopressin and oxytocin under conditions of blood volume depletion in the rat. In the first series of experiments, the animals were injected i.p. with either 0.15 M saline or 30% polyethylene glycol (PEG). PEG caused an 18% reduction of blood volume 1 h after injection. No significant changes in the mean arterial blood pressure were found in either normo- or hypovolaemic rats during the experiment. tGLP-1 injected i.c.v. at a dose of 1 microg/5 microl 1 h after the i.p. injection increased similarly the arterial blood pressure in normo- and hypovolaemic rats. The plasma vasopressin/oxytocin concentrations were markedly elevated in hypovolaemic animals and tGLP-1 further augmented the release of both hormones. In the second study, hypovolaemia was induced by double blood withdrawal. The haemorrhage resulted in a marked decrease of the mean arterial blood pressure and in the elevated plasma vasopressin/oxytocin concentrations. tGLP-1 injected immediately after the second blood withdrawal increased the arterial blood pressure. In parallel, tGLP-1 enhanced significantly vasopressin and oxytocin secretion when compared with haemorrhaged, saline-injected rats. The results of this study indicate that tGLP-1 may affect the arterial blood pressure and the secretion of neurohypophysial hormones under pathological conditions brought about by blood volume depletion.     

The effect of opioid antagonism and environmental restriction on plasma oxytocin and vasopressin concentrations in parturient gilts

Oxytocin plays an important role at parturition due to its involvement in uterine contractions, foetal expulsion and the onset of maternal behaviour. The role of the related neurohypophysial hormone, vasopressin, is less clear; however, there is some evidence that it is also involved in maternal behaviour and its role in osmotic regulation is well established. The aim of this study was to investigate the inhibitory effects of endogenous opioids on these hormones during the expulsive phase of parturition in the pig, and to examine how opioid restraint interacts with environmental restriction. The subjects of this study were 31 Large Whitex Landrace primiparous sows (gilts). An indwelling jugular catheter was implanted under general anaesthesia at 12 days before the expected parturition day (EPD). From 5 days before the EPD 15 of the gilts were individually housed in a restrictive parturition crate without straw and 16 were individually housed in a straw-bedded pen. Blood samples were taken with increasing frequency towards and during parturition through a catheter extension to reduce disturbance. At 7.5 min after the birth of the first piglet half of the gilts in each environment received a dose of the opioid receptor antagonist naloxone (1 mg/kg, i.v.) with the remaining gilts receiving saline as a control. Overall, there was no effect of environment on either circulating oxytocin or vasopressin. However, both oxytocin and vasopressin were inhibited by endogenous opioids during the expulsive phase. The inhibitory effects of opioids on these hormones did not appear to have any adverse effects on the progress of parturition as judged by cumulative piglet birth intervals. The regulation of the opioid inhibition of oxytocin and vasopressin during parturition is discussed in relation to other neurotransmitters and whether opioid inhibition of these neurohypophysial hormones is part of the 'normal' physiological response to parturition or whether it is stress-induced.     

Oxytocin mediates atrial natriuretic peptide release and natriuresis after volume expansion in the rat.

Our previous studies have shown that stimulation of the anterior ventral third ventricular region increases atrial natriuretic peptide (ANP) release, whereas lesions of this structure, the median eminence, or removal of the neural lobe of the pituitary block ANP release induced by blood volume expansion (BVE). These results indicate that participation of the central nervous system is crucial in these responses, possibly through mediation by neurohypophysial hormones. In the present research we investigated the possible role of oxytocin, one of the two principal neurohypophysial hormones, in the mediation of ANP release. Oxytocin (1-10 nmol) injected i.p. caused significant, dose-dependent increases in urinary osmolality, natriuresis, and kaliuresis. A delayed antidiuretic effect was also observed. Plasma ANP concentrations increased nearly 4-fold (P < 0.01) 20 min after i.p. oxytocin (10 nmol), but there was no change in plasma ANP values in control rats. When oxytocin (1 or 10 nmol) was injected i.v., it also induced a dose-related increase in plasma ANP at 5 min (P < 0.001). BVE by intra-atrial injection of isotonic saline induced a rapid (5 min postinjection) increase in plasma oxytocin and ANP concentrations and a concomitant decrease in plasma arginine vasopressin concentration. Results were similar with hypertonic volume expansion, except that this induced a transient (5 min) increase in plasma arginine vasopressin. The findings are consistent with the hypothesis that baroreceptor activation of the central nervous system by BVE stimulates the release of oxytocin from the neurohypophysis. This oxytocin then circulates to the right atrium to induce release of ANP, which circulates to the kidney and induces natriuresis and diuresis, which restore body fluid volume to normal levels.     

Extraction of small amounts of oxytocin from biological fluids by means of agarose-bound neurophysin.

The use of agarose-bound neurophysin for the extraction of oxytocin from biological fluids is described. Oxytocin can be extracted from plasma, urine and cerebrospinal fluid with a high rate of recovery and samples varying widely in volume and oxytocin concentration can be tested by the method. Columns can be used to extract and concentrate dilute samples, or to help identify small amounts of neurohypophysial hormones by affinity chromatography. The oxytocin can be eluted from the column directly into the buffer used for subsequent bioassay. The composition of the final extract is constant and independent of the composition of the sample. The specificity of the binding is high. It is suggested that the method has many advantages over others in current use.     

The Influence of Melatonin on the Content of Vasopressin and Oxytocin in the Hypothalamus and Neurohypophysis in Euhydrated and Dehydrated Male Rats

Melatonin injected in a single intraperitoneal dose of 100 micrograms/100 g b.w. to euhydrated rats resulted in a decrease of neurohypophysial oxytocin content but the hypothalamic oxytocin storage as well as the hypothalamo-neurohypophysial storage of vasopressin were not changed. Following 8 d of once-daily melatonin treatment the hypothalamic and neurohypophysial oxytocin and vasopressin content was decreased. It might be therefore suggested that melatonin increases the release of neurohypophysial hormones and/or decreases their synthesis. Melatonin did not significantly modify the neurohypophysial vasopressin depletion rate in animals deprived of water up to 8 days. No consistent effects of melatonin on the decrease of hypothalamo-neurohypophysial content of oxytocin were noted under conditions of dehydration and simultaneous administration of melatonin up to 8 d.     

Developmental changes in arginine vasopressin receptors and testosterone stimulation in Leydig cells

The steroidogenic response of purified Leydig cells from mice at various ages (from 10-95 days old) was investigated after exposure of cells to arginine vasopressin (AVP) or oxytocin (OT). A 24-h pretreatment by the neurohypophysial hormones significantly increased the acute (3-h) testosterone production by 1.3- to 3-fold at all ages studied, with the exception of pubertal Leydig cells which responded with a 5- to 7-fold increment of testosterone production (P less than 0.05). The higher responsiveness of pubertal Leydig cells to AVP does not result from an increased sensitivity, since the half-maximal effective doses (ED50) of AVP needed to stimulate testosterone production were quite similar and were in nanomolar range for both pubertal and adult Leydig cells. In addition, the ED50 for OT was 10 times higher than that for AVP. Cycloheximide totally abolished the AVP stimulation, suggesting that the AVP effect is dependent upon new protein synthesis. No modification of hCG-stimulated testosterone production occurred after a 24-h pretreatment of Leydig cells by AVP or OT for all ages studied. A 72-h AVP pretreatment resulted in a marked decrease (50-60%) in acute hCG-stimulated testosterone production in both pubertal and adult Leydig cells. Binding studies of [3H]AVP to purified Leydig cells from prepubertal, pubertal, and adult mice showed the presence of a single set of high affinity V1 sites. The Kd values, in the nanomolar ranges, remained unchanged regardless of age. Maximal capacities were of the same order in the prepubertal and adult Leydig cells (9,460 vs. 10,314 sites/cell), while a 50% decrease (P less than 0.01) in the number of AVP receptors occurred in pubertal Leydig cells (5,048 sites/cell). These data indicate developmental changes in the steroidogenic responsiveness of Leydig cells to neurohypophysial hormones and suggest that AVP receptors might be under a regulatory mechanism(s) during puberty. They provide additional evidence for participation of neurohypophysial hormones in autocrine/paracrine regulation of testicular steroidogenesis.     

The neurohypophysial principles of the western brook lamprey, Lampetra richardsoni: Studies in the adult

Pharmacological and purification studies were carried out on lyophilised neurohypophysial tissues from Lampetra richardsoni and on control brain tissues. Crude neurohypophysial extracts contained 5.45 (4.41–6.73) mU/mg dry tissue of oxytocic activity; 3.27 mU/mg dry tissue appeared to be due to a neurohypophysial peptide which showed frog-bladder and other activities typical of arginine vasotocin (AVT). The use of trichloroacetic acid (TCA) and a carboxymethyl cellulose column resulted in a 190-fold purification of the peptide. Assay of the purified principle directly against synthetic AVT by different methods of assay suggested that it was AVT. Partition chromatography supported this finding. Control spinal cord tissues contained only low oxytocic activity (0.27 mU/mg dry tissue), but hindbrain materials showed the surprisingly high potency of 1.39 (1.19–1.62) mU/mg dry tissue. Purification of the hindbrain extract with TCA and carboxymethyl cellulose resolved an oxytocic agent with the thioglycollate lability and chromatographic and pharmacological properties of AVT. It amounted to approximately 0.18 mU/mg dry tissue, or only 12% of the total oxytocic activity present. The purification resolved small amounts of a number of oxytocic agents other than AVT. Some showed chromatographic similarities to oxytocin. However, none was destroyed by sodium thioglycollate. Any true neutral neurohypophysial principle could not have amounted to more than 1% of the AVT present (in terms of oxytocic activity), and there was no positive evidence for its existence in the neurohypophysis. The results suggest that AVT is the only neurohypophysial principle of L. richardsoni.