William G. North

A new extraction of arginine vasopressin from blood: The use of octadecasilyl-silica

SummarySmall columns packed with octadecasilylsilica were used to extract arginine-vasopressin in femtomole amounts from biological fluids for radioimmunoassay. This method is effective in isolating the peptide from substances in plasma and serum which interfere with its quantitation. Consistent and reproducible results and recoveries >80% were obtained with this procedure. High pressure liquid chromatography and serial dilution of extracted samples confirm the identity of the extracted product.

The role of central oxytocin in obsessive compulsive disorder and related normal behavior

Oxytocin (OT) is a neurosecretory nonapeptide synthesized in hypothalamic cells, which project to widely distributed sites in the CNS as well as the neurohypophysis. Central OT affects a variety of cognitive, grooming, affiliative, sexual, and reproductive behaviors in animals. Obsessive Compulsive Disorder (OCD) includes a range of cognitive and behavioral symptoms that bear some relationship to dimensions of behavior associated with OT. Anecdotal data and a recently completed cerebrospinal fluid (CSF) study provide evidence that some forms of OCD are related to OT dysfunction. Based on these findings, we hypothesize: 1) that some forms of OCD are at the extreme end of a range of normal behaviors that are mediated by OT and related systems; and that 2) some normal cognitive, affiliative, and sexual behaviors contain elements that are similar to features of OCD. Alternative hypotheses are considered, and a series of predictions are presented concerning the relationship between central OT and the onset, course, treatment response, and response to challenge procedures seen in this form of OCD.

Influence of oxytocin on renal hemodynamics and sodium excretion.

Acute administration of physiological doses of synthetic OT to conscious Long-Evans and Brattleboro homozygous diabetes insipidus rats produced a modest increase in GFR and effective filtration fraction. Chronic administration of OT to DI rats for 9 days in dosages that were antidiuretic (plasma OT ca. 100 pg/ml) increased both GFR and ERPF by 40%. Table 1 summarizes these renal hemodynamic changes and compares them to the renal effects of VP. Further investigation is needed to define the mechanisms responsible for the changes in GFR and/or ERPF produced by acute and chronic administration of OT to conscious rats. Acute administration of physiological doses of synthetic OT to conscious LE and DI rats also produced a brisk natriuresis with a marked increase in the fractional excretion of sodium. A natriuresis was also observed in conscious Sprague-Dawley rats administered physiological amounts of OT by subcutaneous osmotic minipump. The natriuretic effect of the hormone was short lived, however, being observed only during the first 24-hr period of treatment. The nephron site where OT exerts its natriuretic action, either directly or indirectly, is unknown. Renal prostaglandins may contribute to OT-induced natriuresis, but other mechanisms such as increased renal production of nitric oxide and cGMP have not been tested. Although the natriuretic response to OT has also been described for conscious dogs, it probably does not occur in humans and nonhuman primates. Precise localization of specific renal OT receptors has recently been reported for the rat. OT receptors were identified in the macula densa cells of the adult, rat kidney. This location suggests a possible role for OT in the regulation of tubuloglomerular feedback and solute transport. The signal transduction of the renal OT receptor has been recently evaluated in various kidney epithelial cells in culture. OT stimulates phosphoinositide hydrolysis and increases cytosolic calcium concentrations. In fact, VP produces similar cellular responses in renal epithelia, possibly through the OT receptor. Also, OT stimulates soluble guanylate cyclase and increases intracellular cGMP. Whether OT activates soluble guanylate cyclase secondarily through the production of nitric oxide has not been tested. An important role for OT in renal sodium homeostasis under basal conditions is likely, at least for the rat. Moreover, OT possibly mediates dehydration natriuresis in lower animal species. The contribution of OT to renal physiology in humans and in nonhuman primates, if any, remains uncertain.

Cerebrospinal fluid levels of oxytocin in Prader–Willi syndrome: a preliminary report

BACKGROUND Prader-Willi syndrome (PWS) is a genetic disorder characterized by mental retardation, appetite dysregulation, and a high risk for obsessive-compulsive disorder (OCD). Microscopic abnormalities of the hypothalamus have been described in PWS, and oxytocin has been implicated in both appetite regulation and OCD. METHODS Oxytocin and arginine vasopressin (AVP) were measured in the cerebrospinal fluid of 5 subjects with PWS (2 male, 3 female) and in 6 normal control subjects (all female). RESULTS CSF oxytocin was elevated in PWS (9.2 +/- 3.9 pmol/L) as compared to normal control subjects (5.1 +/- 0.9 pmol/L, p = 0.045), a finding that was more significant when excluding male subjects from analysis (p = 0.02). AVP was not significantly different between the groups as a whole. CONCLUSIONS These data provide further evidence for hypothalamic and oxytocinergic dysfunction in PWS. The associations between oxytocin, appetite regulation, and obsessive compulsive symptomatology in PWS warrant further investigation.

Gene regulation of vasopressin and vasopressin receptors in cancer

It is proposed that neuropeptide production by tumours is an important part of a special process of oncogenic transformation rather than a pre‐existing condition of progenitor cells; this concept is called Selective Tumour gene Expression of Peptides essential for Survival (STEPS). All small‐cell lung cancers and breast cancers evidently express the vasopressin gene, and this gene seems to be structurally normal in all but exceptional cases. Vasopressin gene expression in cancer cells leads to the production of both normal and abnormal forms of tumour vasopressin mRNA and proteins. Although the necessary post‐translational processing enzymes are expressed in these cells, most processing seems to be extragranular, and most of the protein products become components of the plasma membrane. Small‐cell lung cancer and breast cancer cells also express normal genes for all vasopressin receptors and produce normal vasopressin receptor mRNAs and V1a and V1b receptor proteins, and the vasopressin‐activated calcium mobilising (VACM) protein; plus both normal and abnormal forms of the V2 receptor. Through these receptors, vasopressin exercises multifaceted effects on tumour growth and metabolism. A normal protein vasopressin gene promoter seems to be present in small‐cell lung cancer cells, and this promoter contains all of the transcriptional elements known to be involved in gene regulation within hypothalamic neurones. Since these elements largely account for regulation of tumour gene expression observed in vitro, it is likely that as yet unknown factors are selectively produced by tumours in vivo to account for the observed seemingly autonomous or unregulated production of hormone in tumour patients. Promoter elements thought to be responsible for selective vasopressin gene expression in small‐cell lung cancer probably include an E‐box and a neurone restrictive silencer element close to the transcription start site. It is possible that transcription factors acting at these same elements can explain selective vasopressin expression, not only in small‐cell tumours, but also in all other tumours such as breast cancer. By extrapolation, similar mechanisms might also be responsible for the expression of additional features that characterize the ‘neuroendocrine’ profile of these cancers.

Vasopressin gene related products are markers of human breast cancer

SummaryImmunohistochemical analysis for products of vasopressin and oxytocin gene expression was performed on acetone-fixed tissues from 19 breast cancers representing a variety of tumor sub-types. Studies employed the avidin-biotin complex (ABC) immunohistochemical procedure and utilized rabbit polyclonal antibodies to arginine vasopressin (VP), provasopressin (ProVP), vasopressin-associated human glycopeptide (VAG), oxytocin (OT), oxytocin-associated human neurophysin (OT-HNP), and a mouse monoclonal antibody to vasopressin-associated human neurophysin (VP-HNP). Western Blot analysis was performed on protein extracts of fresh-frozen tissues from 12 additional breast tumors. While VP gene related proteins were not detected in normal breast tissue, immunohistochemistry revealed the presence of VP, Pro VP, and VAG in all neoplastic cells for all of the tumor tissues examined. Vasopressin-associated human neurophysin was evident in only one of 19 acetone-fixed tumor preparations. However, Western blot analysis for all 12 fresh-frozen tumor samples showed the presence of two proteins, 42,000 and 20,000 daltons, that were immunoreactive with antibodies to VP, VP-HNP, and VAG. Oxytocin and OT-HNP, by immunohistochemistry, were found to be common to cells of normal breast tissues. For tumors, positive staining for OT was observed in 8 of 18 tumors, while OT-HNP was not detected in any of the tumors examined. These findings indicate that VP gene expression is a selective feature of all breast cancers, and that products of this expression might therefore be useful as markers for early detection of this disease and as possible targets for immunotherapy.

Biosynthesis of Vasopressin and Neurophysins

Vasopressin (VP) is a product of magnocellular neurons in the anterior hypothalamus, most of which have axons that terminate in the neural lobe. The VP gene is also probably expressed by neurons in other brain loci (Caffe and Van Leeuwen, 1983; Sofroniew, 1983; Caffe et al., 1985), and by cells outside the central nervous system (CNS) (Lim et al., 1984; Nussey et al., 1984), but it has yet to be established that VP itself is the final secreted product of this expression. In hypothalamic neurons, VP is translated as a preprohormone that loses an N-ter-minal signal peptide and undergoes final glycosylation in the Golgi apparatus to form a prohormone. This prohormone is packaged into neurosecretory vesicles (NSV); during transport within these vesicles to axonal terminals, it is acted on by intravesicular enzymes that generate the active nonapeptide amide, a 10,000-M r protein called neurophysin, and a 39-amino acid glycopeptide. All three products are released into the peripheral circulation and have been immunologically identified in plasma (North et al., 1983a; Groesbeck et al., 1983). The nomenclature preproVP and pro VP is used in this text to denote preprohormone and prohormone. Distinct neurons in the hypothalamus produce oxytocin (OX), a peptide that is chemically and structurally similar to VP. In fact, it is highly likely that the OX and the VP genes are derived from a common ancestral gene—that VP and OX neurons share a common ancestral neuron (Sawyer, 1977). Therefore, not surprisingly, OX was found to be first translated as a preprohormone (prepro-oxytocin); this converted to a prohormone (pro-oxytocin). Pro-oxytocin undergoes enzymatic modification in the NSV of OX neurons to form oxytocin and a neurophysin (NP). However, through what is currently believed to have arisen as a base deletion or a base insertion in the OX gene, pro-oxytocin does not contain a moiety that will become a glycopeptide (Ivell and Richter, 1984). Since the neu-rophysins produced with each hormone are distinct molecules, they have been named vasopressin-associated neurophysin (VP-NP) and oxytocin-associated neurophysin (OX-NP) (North et al., 1911a).

Vasopressin reduces release from vasopressin-neurons and oxytocin-neurons by acting on V2-like receptors

The effects of arginine vasopressin (AVP), and of the V2-AVP receptor agonist 1-deamino[8-D-arginine] vasopressin (DDAVP) on release from the vasopressin-neurons and oxytocin-neurons of Long-Evans rats were evaluated using specific radioimmunoassays for rat neurophysins. AVP (1 microgram, 1 nmol) or DDAVP (25 ng, 25 pmol) was administered i.p. to animals 1 h before they received an i.v. infusion of 18% saline at 10 microliters/100 g b. wt./min for 60 min. Both AVP and DDAVP decreased the responsiveness (slope) but not the sensitivity threshold of vasopressin-neurons to acute changes in plasma osmolality. Since the amounts of the peptides giving comparable decreases in responsiveness were directly related to their antidiuretic potencies, it is most probable that this influence is mediated through V2-like receptors. However, while ruling out a significant contribution of V1-type receptors, the data do not exclude involvement of other vasopressin receptors (e.g. V3-type receptors). Both AVP and DDAVP also appeared to have an inhibitory effect on release from oxytocin-neurons, but in this case they significantly altered sensitivity threshold but not responsiveness to acute changes in plasma osmolality. Because AVP produced a shift in sensitivity threshold larger than that by DDAVP when the peptides were used in amounts related to their antidiuretic potencies, our results suggest that the feedback influence of AVP on oxytocin-neurons is largely, although not entirely, exercised through V2-like receptors.

Oxytocin and vasopressin hexapeptide fragments have opposing influences on conditioned freezing behavior

We investigated the influence of C-terminal fragments of oxytocin (OT) and arginine vasopressin (AVP) on conditioned freezing behavior. Subcutaneous injections of 0.3 microgram AVP(4-9) or OT(4-9) given to rats after shock training or before behavioral observation significantly altered fear-induced freezing behavior. Animals treated with OT hexapeptide froze less than controls, while animals treated with AVP hexapeptide froze more. These results support the concept that the hexapeptide metabolites of oxytocin and vasopressin can selectively modulate certain behavioral processes, and that these peptides have opposite effects on performance in behavioral tests designed to evaluate memory consolidation and retrieval.

Evidence for expression of vasopressin V2 receptor mRNA in human lung

Studies using fetal sheep, goats, and guinea pigs indicate that vasopressin may play a role in preparing the fetal lung for the transition from a uterine to an air-breathing environment by slowing lung liquid secretion. The mechanism of vasopressin action is believed to occur through V2 receptors with subsequent activation of amiloride-sensitive sodium channels. However, the presence of the V2 receptor in human lung has not yet been documented. In the present study, expression of the vasopressin V2 receptor in fetal and adult human lung was examined using reverse transcription-polymerase chain reaction (RT-PCR), Northern blot analysis, and DNA sequencing. Using RT-PCR and primer pairs specific for the human V2 receptor, PCR products of the predicted sizes of 512 and 862 bp were obtained from adult human lung. DNA sequencing of the cloned PCR products revealed exact identity with the published sequence for the V2 receptor. Northern blot analysis revealed the expression of a approximately 1.9 kb mRNA in adult human lung as well as in kidney, but not in fetal human lung at 22-24 weeks of gestation. However, using the more sensitive RT-PCR assay the 862-bp product was successfully amplified from human fetal lung, although the data indicate the mRNA for this receptor is expressed in lower levels than in adult human lung or kidney. Using RT-PCR and primers specific for the rat V2 receptor, a PCR product of the predicted size of 461 bp was amplified from adult rat lung and kidney, despite an earlier report that this receptor mRNA is absent from the lung of this species. The role for the V2 receptor in adult human lung is unknown at this time, but, as in the human kidney and lungs of fetal sheep, goats, and guinea pigs, this receptor may play a role in fluid balance.