Reproductive stage- and sex-dependant effects of neurohypophyseal nonapeptides on gonadotropin subunit mRNA expression in the catfish Heteropneustes fossilis: An in vitro study.

In the present study, in vitro effects of synthetic vasotocin (VT), isotocin (4Ser, 8Ile- oxytocin; ITb) and the recently cloned IT gene paralog product (8Val-Isotocin, ITa) were studied on the expression of pituitary gonadotropin (GtH) subunit mRNA levels. In male pituitaries of early (preparatory phase) and late (prespawning phase) recrudescing catfish, Heteropneustes fossilis, VT (10 nM, 100 nM and 1000 nM) stimulated fshß expression dose-dependently. But in females, the dose-dependent effect was found only in the preparatory phase. In males, VT stimulated lhß expression only at higher doses. In females, VT produced a significant dose-dependent increase of the lhß expression only in the prespawning phase. VT stimulated the expression of gpα, dose-dependently in the preparatory phase in males and in the prespawning phase in females. The incubation of the pituitaries with ITb did not alter the fshß expression in either sex in both preparatory and prespawning phases. In males, ITb stimulated the expression of lhß and gpα only at the highest concentration (1000 nM) in both phases. In females, ITb stimulated both lhß and gpα expression only at 1000 nM in the preparatory phase and dose-dependently in the prespawning phase. The incubation of the pituitaries with ITa produced effects similar to ITb on the expression of fshß, lhß, and gpα. The results show that the basic peptide VT modulates both fshß and lhß expressions, which are influenced by the sex and reproductive stage. The neutral peptide ITA/ITb exerts an insignificant effect on the fshß expression regardless of sex or season. Both VT and ITa/ITb elicit a significant effect on the lhß expression in late recrudescent phase especially in females.

[Effects of desmopressin acetate and pituitrin on proliferation, contraction, and secretion of hepatic stellate cells].

Objective: To investigate the effects of desmopressin acetate and pituitrin on the proliferation, contraction, and secretion of hepatic stellate cells (HSCs). Methods: The human HSC cell line LX-2 was selected as the research model. And three groups were designed: blank control group, desmopressin acetate group (three subgroups: 1×10-10mol/L, 1×10-9mol/L, and 1×10-8mol/L desmopressin acetate), and pituitrin group (three subgroups: 0.1 U/L, 1.0 U/L, and 10.0 U/L pituitrin). Water-soluble tetrazolium salt (WST)-1 assay was used to evaluate cell proliferation; collagen gel contraction assay was used to assess cell contraction; enzyme-linked immunosorbent assay (ELISA) was used to identify cell secretion. The data was subjected to one-way analysis of variance. Results: (1) The results of WST-1 assay showed that the values of A450in three desmopressin acetate subgroups (1×10-10mol/L, 1×10-9mol/L, and 1×10-8mol/L) were 0.459±0.017, 0.467±0.024, and 0.436±0.015, respectively. And the values of A450 in three pituitrin subgroups (0.1 U/L, 1.0 U/L, and 10.0 U/L) were 0.495±0.011, 0.507±0.015, and 0.501±0.009, respectively. Compared with the control group, the desmopressin acetate at high concentration significantly inhibited the cell proliferation (P< 0.05), but the pituitrin at three different concentrations significantly promoted the cell proliferation (P< 0.05). (2) The collagen gel area ratios in three desmopressin acetate subgroups (1×10-10mol/L, 1×10-9mol/L, and 1×10-8mol/L) were 77.07±4.42, 75.85±3.70, and 72.74±3.92, respectively. And the collagen gel area ratios in three pituitrin subgroups (0.1 U/L, 1.0 U/L, and 10.0 U/L) were 57.83±3.96, 50.28±6.69, and 43.56±7.68, respectively. Compared with the control group, the pituitrin at three different concentrations significantly reduced the collagen gel area (P< 0.01). (3) The matrix metalloproteinase(MMP)-2 concentrations in three desmopressin acetate subgroups (1×10-10mol/L, 1×10-9mol/L, and 1×10-8mol/L) were 13.321±0.098, 12.230±0.153, and 12.061±0.126, respectively. And the MMP-2 concentrations in three pituitrin subgroups (0.1 U/L, 1.0 U/L, and 10.0 U/L) were 12.899±0.150, 13.662±0.152, and 13.698±0.119, respectively. Compared with the control group, the desmopressin acetate at low concentration significantly increased the secretion of MMP-2 (P< 0.01); the desmopressin acetate at high concentration significantly decreased the MMP-2 concentration (P< 0.05); the pituitrin at three different concentrations significantly increased the MMP-2 concentration (P< 0.01). The transforming growth factor-beta 1 (TGF-ß1) concentrations in three desmopressin acetate subgroups (1×10-10mol/L, 1×10-9mol/L, and 1×10-8mol/L) were 5.233±0.102, 17.749±0.188, and 36.060±0.227, respectively. And the TGF-ß1 concentrations in three pituitrin subgroups (0.1 U/L, 1.0 U/L, and 10.0 U/L) were 15.615±0.099, 38.460±0.209, and 49.053±0.115, respectively. Compared with the control group, desmopressin acetate and pituitrin significantly promoted the secretion of TGF-ß1 in a concentration-dependent manner (P< 0.01) and pituitrin had a stronger effect than desmopressin acetate (P< 0.01). Desmopressin acetate and pituitrin had no effect on the secretion of the collagenase type I and III (P> 0.05). Conclusion: Desmopressin acetate and pituitrin can induce the changes in the function and morphology of HSCs and may increase vascular resistance in the hepatic sinus. However, desmopressin acetate has less influence on HSCs than pituitrin.

[Protective effect and mechanisms of pituitrin on acute paraquat-induced lung injury in rats].

OBJECTIVE: To explore the protective effect of pituitrin on the development of paraquat-induced lung injury in rats. METHODS: Sixty healthy Sprague Dawley female rats were randomized into 3 groups of control, paraquat and treatment (80 mg/kg, intragastric) groups (n = 20 each) Each group was divided into 4, 6, 12 and 24 h subgroups (n = 5 each). The treatment group received pituitrin, injection via internal jugular vein 30 minutes after paraquat dosing. As controls, control and paraquat groups were injected with an equal volume of saline. The paraquat content in serum and lung tissue was measured by high-performance liquid chromatography-mass spectrometry (HPLC-MS). And the levels of tumor necrosis factor-alpha (TNF-α) in sera and nuclear factor-kappa B (NF-κB) in lung tissue and the content of protein in bronchoalveolar lavage (BAL) fluid were detected at various timepoints. Lung wet-to-dry weight ratio (W/D) was recorded after pituitrin dosing. In addition, pathological changes were also observed. RESULTS: The highest drug concentration of paraquat in lung tissue was far lower in the treatment group than that in the paraquat group ((7.67 ± 0.91) vs (13.27 ± 0.95) µg/g, P = 0.002). There were the same result in sera ((1 695 ± 274) vs (5 377 ± 576) ng/ml, P = 0.003). The area under the concentration-time curve in the treatment group was significantly lower than that in the paraquat group (10 482 vs 43 441, P = 0.000). The levels of NF-κB in lung tissue and TNF-α in sera in the treatment group were lower than those in the paraquat group (TNF-α: 24 h: (1.85 ± 0.22) vs (2.59 ± 0.13) ng/ml, P = 0.020; NF-κB: 24 h: (88.0 ± 2.7) vs (101.8 ± 2.8) ng/g, P = 0.003). And there was a decrease in the content of protein in BAL fluid in the treatment group versus the paraquat group (BALF protein: 24 h: (125.9 ± 4.2) vs (192.7 ± 6.5)µg/ml, P = 0.003), lung W/D significantly decreased in the treatment group versus the paraquat group (12 h: 3.50 ± 0.14 vs 3.73 ± 0.15, P = 0.043; 24 h: 3.41 ± 0.06 vs 3.61 ± 0.09, P = 0.047). In addition, when compared with the paraquat group, the pituitrin-treated rats showed a mitigation of inflammatory response in lungs and reduced pulmonary edema. CONCLUSION: Pituitrin treatment decreases the content of paraquat in sera and lung homogenate in intoxicated rats and alleviates lung injury so that it may become a useful adjuvant in the treatment of acute lung injury.

[Effect of hormonal compounds on embryogenesis of the pond snail Lymnaea stagnalis (L., 1758)].

Effect of preparations ofa peptide nature (pituitrin and oxytocin) and of a steroid nature (progesterone and hydrocortisone) on embryonic development of freshwater gastropod Lymnaea stagnalis (Mollusca, Gastropoda, Pulmonata) is described. The hormonal preparations used, which differed in chemical nature and physiological activity, may render diverse effects on embryogenesis of the studied mollusk. Of neurohormones, pituitrin rendered the most noticeable and principally stimulating effect. Oxytocin was incorporated in regulatory processes much later and its effect on the rate of realization of particular stages depended more on the quality of occurring changes. In final stages of development, this hormone principally inhibited growth and development of embryos. The female sex hormone progesterone rendered an expressed stimulatory effect, especially notable in later developmental stages of embryos. The hormone hydrocortisone stimulated initial stages of embryogenesis. Its effect was almost not expressed in the final stages. The discovered differences seem to be related both to the functional specificity of the investigated compounds and to specific traits of mechanisms of realization of their effects. A hypothesis is formulated: in gastropods, similarly to vertebrates, the hormones are systemic embryonic and postnatal inducers of differentiation processes.

[Influence of new neurohypophyseal hormone analogs on sodium and water reabsorption in rat kidney].

New analogs of some neurohypophyseal hormones (oxypressin, hydrin, glumitocin, vasotocin) have been synthesized. Experiments with injection of these peptides to rats showed that substitution of C-terminal glycinamide on beta-ethanolamine (glycinol) or ethylamine in 1-deamino-arginine vasotocin resulted in loss of natriuretic but not antidiuretic activity. Analogs of oxypressin and hydrin exhibited neither natriuretic activity nor ability to affect water reabsorption. Glumitocin analog induced renal sodium ion excretion and did not influence potassium ion excretion.

Metabolic and ultrastructural changes in the frog ovarian follicle in response to pituitary stimulation.

Frog ovarian fragments were prevented from ovulating in vitro by the addition of actinomycin D up to 3 hr following pituitary stimulation; but addition of Actinomycin D 6 hr after stimulation was far less effective. Puromycin, on the other hand, effectively inhibited ovulation when added as late as 6 hr after pituitary stimulation. Although actinomycin D reduced uptake of uridine-(3)H, and puromycin reduced uptake of leucine-(3)H and lysine-(14) by pituitary-stimulated ovarian tissue minus oocytes (OTMO) in vitro, it was found that pituitary stimulation did not significantly increase uptake of these compounds by OTMO. Radioautographs of ovarian follicles fixed 6 hr after the addition of pituitary extract and uridine-(3)H in vitro revealed increased RNA synthesis in the peritoneal surface epithelium, compared with unstimulated controls, while the ovarian sac epithelium showed no increase. Gross ultrastructural changes occurred in the peritoneal area of ovarian follicles following pituitary stimulation in vivo, including loss of collagen fibrils, and general disorganization of the connective tissue theca. Changes in the rough endoplasmic reticulum of the peritoneal epithelial cells, while frequently encountered, were less pronounced. None of these changes was observed in the ovarian sac area, or in the interfollicular region. The above data are consistent with the hypothesis that pituitary stimulation of the frog ovary results in increased synthesis of RNA and protein by the peritoneal epithelial cells, and that the protein may be collagenase.

The role of hypothalamo-hypophyseal-adrenocortical system hormones in controlling pain sensitivity.

The present review addresses analysis of data demonstrating the role of the hypothalamo-hypophyseal-adrenocortical axis (HHACA) in controlling pain sensitivity. Experiments on rats have demonstrated the analgesic effects of exogenous hormones of all components of the HHACA - corticotropin-releasing hormone (CRH), adrenocorticotropic hormone (ACTH), and glucocorticoids - in the same models, and have also shown that the opioid and non-opioid mechanisms contribute to the development of the analgesia induced by these hormones. Endogenous glucocorticoids are involved in the development of analgesia mediated by non-opioid mechanisms. Along with the non-opioid mechanisms associated with endogenous glucocorticoids, the analgesic effect of ACTH can be mediated by the opioid mechanism. Unlike the situation with ACTH, the analgesic effect of CRH is mediated exclusively by non-opioid mechanisms, one of which is associated with HHACA hormones, while the other, appearing only on systemic administration, is not associated with these hormones. The actions of glucocorticoids on pain are mediated by neurons in the central gray matter of the midbrain.

Development and clinical application of a new method for the radioimmunoassay of arginine vasopressin in human plasma.

A radioimmunoassay has been developed that permits reliable measurements of plasma arginine vasopressin (AVP) at concentrations as low as 0.5 pg/ml in sample volumes of 1 ml or less. Nonhormonal immunoreactivity associated with the plasma proteins is eliminated by acetone precipitation before assay, leaving unaltered a component that is immunologically and chromatographically indistinguishable from standard AVP. Storage of plasma results in a decline in AVP concentration and, thus, must be carefully regulated. The plasma AVP values obtained by our method approximate the anticipated levels and vary in accordance with physiologic expections. In recumbent normal subjects, plasma AVP ranged from (mean +/-SD) 5.4+/-3.4 pg/ml after fluid deprivation to 1.4+/-0.8 pg/ml after water loading, and correlated significantly with both plasma osmolality (r=0.52; P<0.001) and urine osmolality (r=0.77; P<0.001). After fluid restriction, plasma AVP was uniformly normal relative to plasma osmolality in patients with nephrogenic diabetes insipidus and primary polydipsia but was distinctly subnormal in all patients with pituitary diabetes insipidus. The infusion of physiologic amounts of posterior pituitary extract caused a dose-related rise in plasma vasopressin that afterwards declined at the expected rate (t(1/2)=22.5+/-4 min). We conclude that, when used appropriately, our radioimmunoassay method provides a useful way of assessing AVP function in man.

Laser Doppler flowmetry for assessment of myocardial microperfusion in the beating rat heart.

Although laser Doppler flowmetry (LDF) is widely used for measuring microperfusion, it is rarely used to measure coronary microcirculation. The present in vivo study investigated the use of LDF to measure myocardial microperfusion in beating rat hearts. Ascending aortic flow and other hemodynamic parameters were simultaneously recorded. A needle probe with a holder was adhered to the epicardium of the left ventricular myocardium close to the left anterior descending coronary artery in an anaesthetized open-chest rat. Myocardial microperfusion was measured in response to bolus intravenous administration of both two representative vasodilators (captopril and nifedipine) and a vasoconstrictor (pituitrin). Myocardial microperfusion was found to be predominately diastolic, and in an opposing phase to the ascending aortic flow. Captopril (5 or 10 mg/kg) increased the initial myocardial microperfusion phase. Nifedipine at 75 microg/kg caused a sustained myocardial microperfusion elevation with a peak increase of 7.1+/-1.1%, but this was not observed using 150 microg/kg nifedipine. Both drugs caused an increase in the cardiac index. In contrast, myocardial microperfusion decreased (28.7+/-0.1% maximum decrease) in response to 1 IU/kg pituitrin. In conclusion, LDF provided a means of assessing myocardial microperfusion in beating rat hearts, and can be applied to evaluate the coronary microcirculation response to drugs.

Threshold and receptor reserve in the action of neurohypophyseal peptides. A study of synergists and antagonists of the hydroosmotic response of the toad urinary bladder.

The interrelationship of several physiological receptors which influence the hydroosmotic response of the toad urinary bladder was studied employing neurohypophyseal peptides, prostaglandin E(1), theophylline, and cyclic nucleotides. The binding property of agonists (pD(2)), synergists (pS(2)), competitive antagonists (pA(2)), and noncompetitive antagonists (pD(2)') was determined after a suitable methodology had been developed. A series of neurohypophyseal peptides was examined in detail for their catalytic activity. It was found that the replacement of the hydroxy radical of the tyrosine residue in oxytocin by a methoxy and then by an ethoxy radical led to a progressive decline in the catalytic activity of the hormone-corresponding to a change from agonist to partial agonist to competitive antagonist. [4-Leucine]-mesotocin behaved as a competitive antagonist of oxytocin. Prostaglandin E(1) (PGE(1)) was found to be a noncompetitive inhibitor of neurohypophyseal peptides and theophylline; whereas the maximal hydroosmotic response of the bladder to [2-O-methyltyrosine]-oxytocin and theophylline was greatly depressed by PGE(1), the response to saturating concentrations of oxytocin was only slightly diminished-a finding which reveals a "receptor reserve" for oxytocin. Saturating concentrations of [2-O-ethyltyrosine]-oxytocin, inactive per se, potentiate theophylline-disclosing a "threshold phenomenon" for the mediation of neurohypophyseal hormone action. It is concluded that neurohypophyseal peptides are capable of producing graded effects on adenyl cyclase both below and above the range of enzyme activity which evokes graded changes in membrane permeability.

A sensitive hydroosmotic toad bladder assay. Affinity and intrinsic activity of neurohypophyseal peptides.

A sensitive and precise method for assaying the water permeability response evoked by neurohypophyseal hormones and their synthetic analogues on the isolated urinary bladder of the toad (Bufo marinus L.) is described. The method permits detection of 8-arginine-vasotocin at concentrations as low as 10(-12)M. This sensitivity, not achieved heretofore with this tissue, results largely from minimizing interference of inhibitory substances by means of an "in vitro circulation assembly." The precision of the method derives from a direct comparison between the cumulative dose-response curve of an agonist of unknown potency acting on one hemibladder and that of a reference compound acting on the contralateral hemibladder. Crystalline deamino-oxytocin is used as the reference standard in this assay. The intrinsic activity of 2-(O-methyltyrosine)-oxytocin, as defined by the maximal response, is 12% lower than that of deamino-oxytocin. All other hormonal peptides investigated have the same intrinsic activity as deamino-oxytocin, even 5-valine-oxytocin, in spite of its extremely low affinity. A comparison of the potencies of 8-arginine-vasotocin vs. 8-arginine-vasopressin, 8-ornithine-vasotocin vs. 8-ornithine-vasopressin, 8-alanine-oxytocin vs. 8-alanine-oxypressin, and deamino-8-alanine-oxytocin vs. deamino-8-alanine-oxypressin suggests that an isoleucine residue in position 3 imparts a higher specificity for binding of the hormonal peptide molecule to the bladder receptor than a phenylalanine residue in this locus.

Sites of behavioral and neurochemical action of ACTH-like peptides and neurohypophyseal hormones.

In order to localize the site of action of neuropeptides in relation to their effects on behavior and memory various approaches have been used. As a result of studies using rats bearing lesions in different areas of the limbic system as well as of studies in which neuropeptides were locally applied into various areas of the brain it appeared that the limbic system (amygdala, hippocampus, septum and some thalamic areas) plays an essential role in the effect of vasopressin and ACTH and their derivatives on behavior and memory. Neurochemical studies generally indicate that changes occur in catecholamine utilization in these various limbic regions upon administration of these neuropeptides. It can be concluded that the effects of vasopressin in the terminal regions of the coeruleo-telencephalic noradrenalin system correlate with its effects on consolidation of memory. It is likely that the effects of vasopressin on other transmitter systems (e.g. dopamine in the amygdala and serotonin in the hippocampus) correspond with the effect of this neuropeptide on retrieval processes. In addition, regional differences in biotransformation of the neurohypophyseal hormones suggest that different patterns of behaviorally active fragments of these peptides may be present locally in the brain.

Prolactin-releasing activity of neurohypophysial hormones: structure-function relationship.

Oxytocin (OT) and arginine vasopressin (AVP) have been reported to release PRL both in vivo and in vitro. The objectives of this study were 1) to compare the potencies of the PRL-releasing activities of OT and TRH using cultured anterior pituitary (AP) cells, and 2) to assess the PRL-releasing activity of naturally occurring neurohypophysial hormones and selected analogs. AP cells were incubated with peptides for 15 min, and medium PRL concentrations were determined by enzyme-linked immunosorbent assay. OT at 25, 100, and 400 nM increased PRL release by 110%, 175%, and 270%, respectively; higher concentrations (1600 and 6400 nM) did not cause a further increase in PRL release. TRH was 5-10 times more potent than OT on a molar basis. GH3 cells, a somatommamotroph tumor cell line, did not respond to OT and related compounds, but showed a similar responsiveness to TRH as AP cells. Twelve neurohypophysial peptides and selected analogs were incubated with AP cells, and their relative PRL-releasing activities were compared. OT and arginine vasotocin (AVT) showed the highest PRL-releasing activity. T4-G7-oxytocin, mesotocin, isotocin, lysine vasotocin, and AVP showed a moderate PRL-releasing activity, whereas, lysine vasopressin, desmopressin, tocinoic acid, pressinoic acid, and oxytocin free acid showed very low or no PRL-releasing activity. Coincubation of OT, AVT, or AVP with a specific OT receptor antagonist abolished their PRL-releasing activity. We conclude that 1) OT and related peptides are capable of stimulating PRL release in vitro, but their potencies are significantly lower than that of TRH; 2) unlike primary AP cells, GH3 cells are unresponsive to OT and related peptides; 3) AVT and AVP probably stimulate PRL release by acting via an OT receptor; and 4) the amino acid residues in positions 3 and 8 in the peptide chain and an amidated C-terminus are critical for the PRL-releasing activity of the neurohypophysial peptides.

An osmometric method for the bioassay of vasotocin and related peptides in the toad bladder.

This study describes a new method for quantitating the antidiuretic activity of 8-arginine vasotocin (AVT) and related peptides on the isolated toad urinary bladder. The method is based on measuring changes in the osmolality of the surface fluid film of bladders that have been filled with a dilute solution and suspended in humidified air. Eight microliters of Ringer's fluid containing a known concentration of hormone are applied to small paper discs (0.7 cm in diameter), and the discs are then placed with fine forceps onto the outer surface of the bladder to which they adhere. The hormone increases the permeability to water of the epithelium that is underneath the area of the disc, and as water moves from the interior of the bladder along its osmotic gradient to the outer surface of the bladder, the Ringer's fluid in the disc becomes diluted. The magnitude of this dilution is quantitated by removing the discs to a vapor pressure osmometer at timed intervals. A supramaximal dose of AVT reduced disc fluid osmolality by 188 mosmol/kg H2O within 15 min. Similar maximal responses were observed with 8-arginine vasopressin (AVP), oxytocin, and 8-lysine vasopressin, although the potencies of these hormones diminished in the order listed above. AVT was 112-fold more potent than AVP, and AVP, in turn, was 329-fold more potent than 8-lysine vasopressin. The lower limit at which AVT was detected in this assay was 0.25 pg/disc (3 X 10(-11) M). The intra- and interassay variabilities for AVT were 14% and 28% (+/- SD), respectively. This assay is suitable for measuring the biological activity of hormone analogs lacking vasopressor activity, such as desmopressin, which was found to have a hydroosmotic activity of 8.3 +/- 2.4 U/mg. After osmotic stimulation, AVT was detected in toad plasma at a concentration of 1.4 X 10(-10) M. Therefore, this method has the requisite sensitivity for measuring this hormone in biological fluids of amphibia, reptiles, fish, and birds.

Effects of synthetic ovine corticotropin-releasing factor, glucocorticoids, catecholamines, neurohypophysial peptides, and other substances on cultured corticotropic cells.

Synthetic ovine corticotropin-releasing factor (CRF) is a 41-residue peptide with high potency and intrinsic activity to stimulate the secretion of ACTH and beta-endorphin-like immunoactivity (beta-End-LI) by cultured adenohypophysial corticotropic cells. The action of CRF in vitro can be potentiated by the weaker secretagogues, vasopressin, oxytocin, epinephrine, norepinephrine, and angiotensin II. CRF-mediated secretion of ACTH and beta-End-LI is noncompetitively inhibited by pretreatment of cells with glucocorticoids. Long term exposure of adenohypophysial cells to CRF results in an increase in total medium plus cell ACTH in the cultures, suggesting that CRF can enhance rates of ACTH synthesis as well as release. CRF also stimulates the secretion of beta-End-LI by corticotropic cells cultured from the neurointermediate lobe. Higher concentrations of CRF are required to stimulate secretion by this cell type than by anterior lobe corticotropic cells. These in vitro results are consistent with CRF playing a major physiological role in the neuroregulation of secretion by anterior lobe corticotropic cells, where the peptide may interact with other modulators.

Short-term behavioural effects of neurohypophyseal hormones: pharmacological characteristics.

Neurohypophyseal hormones and related peptides cause behavioural alterations after intracerebroventricular injection in mice. In the present study, these effects, consisting of excessive grooming and scratching, and of escape-directed activity in stressful situations, could easily be distinguished from those of other centrally acting peptides and drugs by means of two different behavioural bioassays. The effects were not antagonized by drugs that block cholinergic or adrenergic receptors, but they were powerfully suppressed by some potent psychotropic agents. Some compounds with strong vasoconstrictor or vasodilatory actions did not mimick or antagonize the behavioural alterations, suggesting that vasoconstriction is not essential for the induction of these effects. A considerable degree of tolerance could be induced and cross-tolerance was observed between different neurohypophyseal hormones. In rats, behavioural alterations caused by oxytocin and vasopressin could be demonstrated as well, but they were by far less pronounced than those observed in mice. For comparison, some data on the behavioural effects of bombesin are included. This peptide caused behavioural alterations similar to those of the neurohypophyseal hormones, but these were apparently mediated by different mechanisms. It is suggested that centrally-released oxytocin and/or vasopressin might be physiologically involved in the regulation of animal behaviour.

Oxytocin analogues with combined high smooth muscle and negligible antidiuretic activities. Investigation of position 7 in neurohypophyseal hormones.

The proline residue in position 7 of oxytocin occupies one of the four corner positions in the two beta turns proposed for the preferred conformation of the pituitary hormone. It has been suggested that synthetic modifications of the residues in these corner positions will yield analogues in which one or more of the biological activities of the parent hormone is highly accentuated in terms of potency relative to other activities. In a continued effort to test this hypothesis the following analogues of oxytocin were prepared: [7-glycine]oxytocin, [1-beta-mercaptopropionic acid,7-glycine]oxytocin, [7-alanine]oxytocin, and [1-beta-mercaptopropionic acid,7-alanine]oxytocin. These peptides were found to possess the following specific activities, respectively: rat uterotonic, 65 +/- 2, 355 +/- 3, 22 +/- 1, 123 +/- 4; avian vasodepressor, 5.3 +/- 0.8, 17 +/- 0.4, 4.8 +/- 0.1, 9.8 +/- 0.5; rat antidiuretic, less than0.01, 0.062, 0.081 +/- 0.01, 0.17 +/- 0.01; rat pressor, 0.3, 0.5, 0.4, 0.5 unit/mg. Thus the analogues retain high uterotonic activity but exhibit strongly diminished renal and vascular activities relative to oxytocin. Especially noteworthy is [1-beta-mercaptopropionic acid,7-glycine]oxytocin with its high uterotonic activity but very low antidiuretic and pressor activities. The activity profile of this analogue combined with the fact that it is only slowly enzymatically degraded warrants further investigations of this peptide for clinical applications.

Inhibitory effect of neurohypophysial peptides on cyclic AMP accumulation by isolated hepatocytes of the trout.

Cyclic AMP levels were measured in freshly isolated hepatocytes of the rainbow trout. Compared with basal values, the average levels were increased up to 60 times in a dose-dependent manner either by mammalian glucagon (concentration range 1 nmol-1 mumol/l; dose giving half maximum response (EC50) 0.18 mumol/l) or by forskolin (concentration range 0.1-100 mumol/l; EC50 about 10 mumol/l). These stimulatory effects were partially inhibited by fish or mammalian neurohypophysial hormones used at relatively high concentrations (1-5 mumol/l). It is suggested that these results are evidence for the presence of V1-type receptors in fish hepatocytes. Together with previous results obtained with gills on the hormonal inhibition of adenylate cyclase activity, they suggest that teleost fish may possess only V1-type receptors (or two V1-related types), while the V2 receptors have evolved (or have become functional) in higher vertebrates.

A V1-type receptor for mediating the neurohypophysial hormone-induced ACTH release in trout pituitary.

We analysed the effects of specific neurohypophysial analogues for pharmacological characterization of the type of vasotocin receptor involved in the control of the adrenocorticotrophin hormone (ACTH) release from the perifused pituitary in the rainbow trout, Oncorhynchus mykiss. Mammalian corticotrophin releasing factor (CRF) and teleostean neurohypophysial peptides (arginine vasotocin (AVT) and isotocin (IT)) stimulated ACTH release. Analysis of concentrations giving half-maximal effects (D50) showed that these peptides affected ACTH release in the following order of potency: CRF (8 x 10(-13) M) > AVT (2 x 10(-10) M) > IT (10(-7) M). Maximal responses (Dmax) were obtained for hormonal concentrations of 10(-10) M, 10(-8) M and 10(-6) M respectively. This suggests that AVT and IT have different roles in the control of ACTH release. The values obtained for AVT and IT were in agreement with the circulating levels we previously found for these peptides. Specific V1 or V2 agonists or antagonists (with reference to vasopressin in mammals) were used to define the specificity of the neurohypophysial peptide receptor involved in this stimulation. The V1 agonist, [Phe2, Orn8]-oxytocin, stimulated ACTH release while the V2 agonist, [deamino1, Val4, D-Arg8]-vasopressin, had no such effect. Maximal and half-maximal responses were obtained in the presence of the V1 agonist with 10(-7) M and 7 x 10(-9) M respectively, and were in the range of values obtained with natural peptides. The V1 antagonist, [d(CH2)5(1), O-Me-Tyr2, Arg8]-vasopressin, and the V2 antagonist, [d(CH2)5(1), D-Ile2, Ile4, Arg8, Ala9]-vasopressin, maximally reversed the 10(-9) M AVT-stimulated ACTH release by 60% and 25% respectively, for a 5 x 10(-10) M concentration of the analogues and a D50 approximately 2 x 10(-11) M. These results demonstrated the presence of only one V1-type receptor in fish pituitary, with some of the structural and functional peculiarities typically displayed by the mammalian V1a-type receptor, but distinct from it. In this sense, the fish pituitary vasotocin receptor may represent a novel type of neurohypophysial hormone receptor, more closely related to the mammalian V1b-type.

Inter-relationships between nonapeptide hormones and cyclic nucleotides within cultured porcine granulosa cells.

The reciprocal control of nonapeptide hormone (oxytocin, vasopressin) and cyclic nucleotide (cAMP, cGMP) release by porcine granulosa cells was studied. In particular, the influence of vasopressin and oxytocin treatment (10-10 000 ng/ml) on basal and LH-induced cAMP and cGMP output, as well as the effects of dibutyryl cAMP (dbcAMP; cAMP analogue) and forskolin (a stimulator of cAMP formation; 0.1-1000 ng/ml) on vasopressin and oxytocin secretion by cultured porcine granulosa cells were examined. It was observed that the addition of arginine-8-vasopressin or oxytocin stimulated both cAMP and cGMP output from granulosa cells. Moreover, both vasopressin and oxytocin also increased LH-stimulated cAMP and cGMP release. On the other hand, both dbcAMP and forskolin decreased vasopressin secretion. Oxytocin release was stimulated under the influence of dbcAMP. The same stimulating effect occurred with forskolin given at a low dose (1 ng/ml), whilst higher doses of forskolin (10 or 1000 ng/ml) were inhibitory. The present observations demonstrate the reciprocal influence of nonapeptide hormones and cyclic nucleotides in porcine ovarian cells. Oxytocin and vasopressin, like LH, exert their action on the ovary via the activation of cAMP- and cGMP-dependent intracellular mechanisms. cAMP in turn inhibits vasopressin release through a negative feedback mechanism. On the other hand, a reciprocal stimulation of oxytocin and cAMP output in granulosa cells is suggested. Thus, cyclic nucleotides can be both regulators of nonapeptide hormone secretion and mediators of their action within porcine ovaries.