Vasopressin and Its Analogues: From Natural Hormones to Multitasking Peptides.

Human neurohormone vasopressin (AVP) is synthesized in overlapping regions in the hypothalamus. It is mainly known for its vasoconstricting abilities, and it is responsible for the regulation of plasma osmolality by maintaining fluid homeostasis. Over years, many attempts have been made to modify this hormone and find AVP analogues with different pharmacological profiles that could overcome its limitations. Non-peptide AVP analogues with low molecular weight presented good affinity to AVP receptors. Natural peptide counterparts, found in animals, are successfully applied as therapeutics; for instance, lypressin used in treatment of diabetes insipidus. Synthetic peptide analogues compensate for the shortcomings of AVP. Desmopressin is more resistant to proteolysis and presents mainly antidiuretic effects, while terlipressin is a long-acting AVP analogue and a drug recommended in the treatment of varicose bleeding in patients with liver cirrhosis. Recently published results on diverse applications of AVP analogues in medicinal practice, including potential lypressin, terlipressin and ornipressin in the treatment of SARS-CoV-2, are discussed.

Two Lys-vasopressin-like peptides, EFLamide, and other phasmid neuropeptides.

Phasmid neuropeptide genes were identified in the genomes of two phasmids, Timema cristinae and Clitarchus hookeri. The two species belong to two sisters groups, the Timematodea and Euphasmatodea respectively. Neuropeptide genes were identified using the BLAST+ program on the genome assemblies and the absence of some neuropeptides was confirmed by the concomitant absence of their G-protein coupled receptors. Both genomes were assembled using short reads and the average coverage of the genome is more than 166 times for both species. This makes it virtually impossible that there would not be a single short read for at least one of the conserved transmembrane regions of a GPCR coded by such a genome. Hence, when not a single read can be found for a specific GPCR, it can be concluded that the particular gene is absent from that species. Most previously identified insect neuropeptides are used by these two species. Of the three arthropod allatostatin C related peptides, only allatostatins CC and CCC are present. Both species lack leucokinin, while sulfakinin and dilp8 signaling is absent from Clitarchus, but present in Timema. Interestingly, whereas Timema has lost a vasopressin-related peptide, the gene coding such a peptide is amplified in the Clitarchus genome. Furthermore, while Clitarchus has a specific tryptopyrokinin gene, Timema does not and in this species tryptopyrokinin is coded only by the pyrokinin and periviscerokinin genes. Finally, both species have genes coding EFLamide and its GPCR; in phasmids these genes codes for one (Clitarchus) or two (Timema) EFLamide paracopies.

Effects of terlipressin and naloxone compared with epinephrine in a rat model of asphyxia-induced cardiac arrest.

OBJECTIVE: To evaluate the hemodynamic and metabolic effects of terlipressin and naloxone in cardiac arrest. METHODS: Cardiac arrest in rats was induced by asphyxia and maintained for 3.5 minutes. Animals were then resuscitated and randomized into one of six groups: placebo (n = 7), epinephrine (0.02 mg/kg; n = 7), naloxone (1 mg/kg; n = 7) or terlipressin, of which three different doses were tested: 50 µg/kg (TP50; n = 7), 100 µg/kg (TP100; n = 7) and 150 µg/kg (TP150; n = 7). Hemodynamic variables were measured at baseline and at 10 (T10), 20 (T20), 30 (T30), 45 (T45) and 60 (T60) minutes after cardiac arrest. Arterial blood samples were collected at T10, T30 and T60. RESULTS: The mean arterial pressure values in the TP50 group were higher than those in the epinephrine group at T10 (165 vs. 112 mmHg), T20 (160 vs. 82 mmHg), T30 (143 vs. 66 mmHg), T45 (119 vs. 67 mmHg) and T60 (96 vs. 66.8 mmHg). The blood lactate level was lower in the naloxone group than in the epinephrine group at T10 (5.15 vs. 10.5 mmol/L), T30 (2.57 vs. 5.24 mmol/L) and T60 (2.1 vs. 4.1 mmol/L). CONCLUSIONS: In this rat model of asphyxia-induced cardiac arrest, terlipressin and naloxone were effective vasopressors in cardiopulmonary resuscitation and presented better metabolic profiles than epinephrine. Terlipressin provided better hemodynamic stability than epinephrine.

Effects of terlipressin and naloxone compared with epinephrine in a rat model of asphyxia-induced cardiac arrest

OBJECTIVE: To evaluate the hemodynamic and metabolic effects of terlipressin and naloxone in cardiac arrest. METHODS: Cardiac arrest in rats was induced by asphyxia and maintained for 3.5 minutes. Animals were then resuscitated and randomized into one of six groups: placebo (n = 7), epinephrine (0.02 mg/kg; n = 7), naloxone (1 mg/kg; n = 7) or terlipressin, of which three different doses were tested: 50 µg/kg (TP50; n = 7), 100 µg/kg (TP100; n = 7) and 150 µg/kg (TP150; n = 7). Hemodynamic variables were measured at baseline and at 10 (T10), 20 (T20), 30 (T30), 45 (T45) and 60 (T60) minutes after cardiac arrest. Arterial blood samples were collected at T10, T30 and T60. RESULTS: The mean arterial pressure values in the TP50 group were higher than those in the epinephrine group at T10 (165 vs. 112 mmHg), T20 (160 vs. 82 mmHg), T30 (143 vs. 66 mmHg), T45 (119 vs. 67 mmHg) and T60 (96 vs. 66.8 mmHg). The blood lactate level was lower in the naloxone group than in the epinephrine group at T10 (5.15 vs. 10.5 mmol/L), T30 (2.57 vs. 5.24 mmol/L) and T60 (2.1 vs. 4.1 mmol/L). CONCLUSIONS: In this rat model of asphyxia-induced cardiac arrest, terlipressin and naloxone were effective vasopressors in cardiopulmonary resuscitation and presented better metabolic profiles than epinephrine. Terlipressin provided better hemodynamic stability than epinephrine. .

Stress in an Island kangaroo? The Barrow Island euro, Macropus robustus isabellinus.

Selected physiological parameters were monitored over a 4-year period in the Barrow Island euro, Macropus robustus isabellinus, in Western Australia in a study of this species' homeostatic capabilities in an extremely arid habitat where individuals are exposed to high environmental temperatures and a lack of free water for much of the year. Evidence was found of a significant change in the animal's milieu intérieur on only one occasion on Barrow Island: in November 1994, following a protracted 8-month drought. Euros had significantly elevated levels of plasma osmolality, cortisol, anti-diuretic hormone (lysine vasopressin - LVP), and a reduced eosinophil count. This suggests that these animals may have been dehydrated, despite the operation of appropriate physiological responses to water deprivation. Lower eosinophil counts also suggest that immune function may have been suppressed as a result of the elevated corticosteroid levels. Comparisons with the mainland sub-species of the euro revealed the presence of a non-generative normocytic hypochromic anaemia in Barrow Island euros that potentially compromises their aerobic capacity. Barrow Island is Australia's most important A Class Reserve, harbouring 8 species of marsupials, 4 of which are now extinct, or virtually so, on the adjacent mainland. This study reveals the remarkable effectiveness of the euro's homeostatic capacities, however, its future conservation depends on ensuring that potential stress due to declining water availability and environmental change is avoided.

Resident-intruder trait aggression is associated with differences in lysine vasopressin and serotonin receptor 1A (5-HT1A) mRNA expression in the brain of pre-pubertal female domestic pigs (Sus scrofa).

Aggressive behaviour exhibited by domestic pigs following encounters with unfamiliar individuals is a serious welfare and economical problem. Aggression resulting in skin lesions is similarly prevalent in prepubertal pigs of either sex. Little is known about the neural circuits and neuropeptides that control aggression in the pig. Because there is evidence for the involvement of the vasopressin and serotonergic systems in the regulation of aggressive behaviour in male mammals, we sought differences using quantitative in situ hybridisation of vasopressin and serotonin 1A receptor (5-HT1A) mRNA expression within specific brain regions of aggressive and nonaggressive prepubertal female pigs. The number of cells expressing vasopressin mRNA was significantly higher in aggressive pigs in the medial amygdala, lateral septum (LS) and showed a similar trend in the bed nucleus of the stria terminalis (BnST) but not the paraventricular nucleus (PVN) or supraoptic nucleus. The 5-HT1A receptor was widely expressed through the porcine brain and a significantly lower intensity (silver grain density) of 5-HT1A mRNA expression was observed in the BnST. In the medial amygdala and LS fewer cells expressed 5-HT1A mRNA in aggressive pigs but no differences were found in the PVN. In the absence of inbred strains or selection lines, these findings have shown that prior identification of phenotypic behavioural extremes in a population in advance of neural studies is a useful technique. Moreover, these findings support a central role for vasopressin and serotonin in the mediation of high trait aggression in prepubertal female pigs.

Phospholipid mediators and MgATPase modulation causes changes in the cardiovascular effects of vasopressin in lithium carbonate-induced polyuric rats.

The effect of phospholipid and MgATPase modulation was evaluated on the cardiovascular actions of vasopressin in normal and lithium carbonate- (Li2CO3) induced polyuric rats. We examined the effects of the phospholipase inhibitor neomycin, the diacylglycerol kinase II inhibitor R59949 and the MgATPase activator sphingosine on heart rate (HR) and blood pressure (BP) responses to vasopressin analogues lysine vasopressin (LVP) and arginine vasopressin (AVP). R59949 (20 microg/kg) produced an increase while sphingosine (30 microg/kg) caused a decrease in HR responses in both control and polyuric rats. Pretreatment with sphingosine caused significant enhancement of LVP- (10 microg/kg) induced bradycardia in polyuria rats compared with control animals (p < 0.01). R59949 induced a potentiation of vasopressin-induced bradycardia in control animals compared with polyuria rats. Pretreatment with sphingosine and R59949 produced a significant increase in BP per se and potentiated the actions of LVP in control animals, while the response in the lithium-treated animals was attenuated. Neomycin caused a reduction in HR and BP in control and lithium-treated animals. To evaluate the central role of the MgATPase enzyme we used sphingosine, which significantly increased the locomotor activity of lithium-treated animals, suggesting a possible central interaction of lithium and MgATPase (p < 0.05). These results strongly suggest that phospholipid mediators and MgATPase modulation contribute to the alteration of the cardiovascular effects of vasopressin in lithium carbonate-induced polyuric rats.

Structures of an unliganded neurophysin and its vasopressin complex: implications for binding and allosteric mechanisms.

The structures of des 1-6 bovine neurophysin-II in the unliganded state and as its complex with lysine vasopressin were determined crystallographically at resolutions of 2.4 A and 2.3 A, respectively. The structure of the protein component of the vasopressin complex was, with some local differences, similar to that determined earlier of the full-length protein complexed with oxytocin, but relatively large differences, probably intrinsic to the hormones, were observed between the structures of bound oxytocin and bound vasopressin at Gln 4. The structure of the unliganded protein is the first structure of an unliganded neurophysin. Comparison with the liganded state indicated significant binding-induced conformational changes that were the largest in the loop region comprising residues 50-58 and in the 7-10 region. A subtle binding-induced tightening of the subunit interface of the dimer also was shown, consistent with a role for interface changes in neurophysin allosteric mechanism, but one that is probably not predominant. Interface changes are suggested to be communicated from the binding site through the strands of beta-sheet that connect these two regions, in part with mediation by Gly 23. Comparison of unliganded and liganded states additionally reveals that the binding site for the hormone alpha-amino group is largely preformed and accessible in the unliganded state, suggesting that it represents the initial site of hormone protein recognition. The potential molecular basis for its thermodynamic contribution to binding is discussed.

Characterization of vasopressin receptor in rat lung.

This study characterized rat lung membrane arginine vasopressin (AVP) receptors in detail. Specific binding of [3H]AVP to rat lung membranes was dependent upon time, temperature and membrane protein concentration. Scatchard plot analysis of equilibrium binding data revealed the existence of a single class of high-affinity binding sites with a Kd of 0.45 nM and a Bmax of 76.6 fmol/mg protein. Competitive inhibition of [3H]AVP binding showed that neurohypophysial hormones as well as their synthetic analogues displaced [3H]AVP in a concentration-dependent manner. The order of potencies for the native peptides was: AVP > lysine vasopressin = arginine vasotocin > oxytocin. Furthermore, potent V1A receptor antagonists, d(CH2)5Tyr(Me)AVP and dPTyr(Me)AVP, showed high affinity for lung membranes. In contrast, the V2 receptor agonist, dDAVP, and the specific oxytocin receptor agonist, [Thr4,Gly7]oxytocin, did not affect AVP binding. These results suggest that the lung contains the V1A receptor subtype. The lung membrane AVP receptor characterized in this study may play an important role in mediating the physiological effects of AVP in the lung.

Interrelated adrenocortical and neurohypophysial responses associated with fever in endotoxin-treated pigs.

Low intravenous doses of endotoxin [lipopolysaccharide (LPS), 0.7 microgram/kg] induce monophasic fever, increase anterior and posterior pituitary hormone release, and enhance hypothalamic c-Fos expression in pigs, all of which can be prevented by indomethacin (Ind). The present study shows that the synthetic corticosteroid dexamethasone (Dex, 5 mg/kg) has a similar action to Ind and, when given alone, lowers core temperature. In addition, the corticosteroid synthesis inhibitor metyrapone (Met, 3.3 mg/kg, every one-half hour) reduces LPS fever and amplifies the effect of LPS on vasopressin, but not on oxytocin, release. The similar actions of Dex and Ind suggest that phospholipase A2 pathways controlling prostaglandin synthesis mediate the responses of prepubertal pigs to immunological challenge with LPS. The increased vasopressin release induced when animals receiving Met are also given LPS supports findings in other nonrodent species indicating an inverse relationship between cortisol and vasopressin. The attenuation of LPS fever by Met is suggestive of an endogenous antipyretic mechanism associated with enhanced neurohypophysial vasopressin secretion.

Effects of corticotropin-releasing hormone, lysine vasopressin, oxytocin, and angiotensin II on adrenocorticotropin secretion from porcine anterior pituitary cells.

The aim of this study was to determine the ability of corticotropin-releasing hormone (CRH), lysine vasopressin (LVP), oxytocin (OT), and angiotensin II (AII) to stimulate adrenocorticotropin (ACTH) secretion from porcine anterior pituitary (AP) cells in vitro and to evaluate the role of protein kinase C (PKC) in the interaction between CRH and LVP. In this study, porcine AP cells were enzymatically and mechanically dispersed, cultured (150,000 cells/well) for 4 d, and then challenged with doses of various neuropeptides for 3 hr. CRH (10(-7)-10(-10) M) was the most potent of the peptides tested in stimulating ACTH release from porcine AP cells. In fact, none of the other peptides consistently affected ACTH concentrations relative to basal levels. However, LVP potentiated CRH action, even though by itself, it failed to stimulate ACTH production. Neither OT or AII potentiated CRH-stimulated ACTH release from porcine AP cells. To determine whether the inter-action between CRH and LVP was regulated partially by the protein Kinase C (PKC) pathway, we challenged AP cells in a 30-min incubation with 10(-7) M staurosporine (ST), a treatment predicted to decrease PKC activity. Then, cells were washed and challenged with 10(-9) M LVP, 10(-9) M CRH, and 10(-9) M CRH + LVP. Treatment with ST decreased (P < 0.05) CRH + LVP-stimulated ACTH release. To further demonstrate an interaction between protein kinase A (PKA) and PKC transduction pathways in the observed synergism between CRH and LVP to enhance ACTH secretion, we also challenged AP cells with 10(-7) M phorbol 12, 13-myristate acetate (PMA) and 5 microM forskolin (FOR) for 3 hr. This treatment was predicted to enhance PKA and PKC activities, respectively, and thereby enhance ACTH concentrations. Challenging cells with FOR + PMA enhanced (P < 0.001) ACTH release above basal concentrations, but more important, it increased (P < 0.001) ACTH concentration above that elicited by either drug given alone. Taken together, our in vitro studies support the conclusion that CRH is the principal regulator of ACTH secretion in the pig. In contrast to the results in most other species evaluated, vasopressin alone did not affect ACTH release. However, LVP can enhance the effectiveness of CRH in releasing ACTH, and this enhancement appears to rely, at least in part, on the activation of the PKC signal transduction pathway.

A proposed role for oxytocin in regulation of endometrial prostaglandin F2 alpha secretion during luteolysis in swine.

Pulsatile secretion of endometrial prostaglandin (PG)F2 alpha is stimulated by oxytocin (OT) during late diestrus in domestic ruminants (i.e., cattle, sheep and goats) and results in corpus luteum (CL) regression leading to the onset of a new estrous cycle. Pulsatile PGF2 alpha release is also responsible for CL regression in swine, but the stimulus for its secretion from the uterine endometrium is not known. We propose that OT binds to specific OT receptors (OTR) on the endometrium to stimulate phosphoinositide (PI) hydrolysis, thereby activating the inositol trisphosphate (IP3)-diacylglycerol (DAG) second-messenger system to promote pulsatile PGF2 alpha secretion. Exogenous OT administered to cyclic gilts during late diestrus (days 10-16) decreased interestrous interval in three of four experiments. However, OT did not promote CL regression in hysterectomized gilts indicating that the effect of OT was uterine-dependent. Circulating concentrations of 13,14-dihydro-15-keto PGF2 alpha (the major stable metabolite of PGF2 alpha) were increased (p < 0.01) 10 min after i.v. injection of OT on days 14 and 16 in cyclic gilts and on days 10-16 in pregnant gilts, but the magnitude of the response to OT on all days in pregnant gilts was markedly reduced compared to the response in cyclic gilts on days 14 and 16. Mean density and Kd of OTR detected on endometrium of cyclic pigs 15 days post-estrus were 29.2 +/- 5.5 fmol/mg protein and 1.59 +/- 0.23 nM, respectively. Density of OTR was correlated with OT-stimulated PI hydrolysis (r = 0.83, p < 0.05) and PGF2 alpha secretion (r = 0.87, p < 0.10). Endometrial IP3 was increased within 30 seconds after OT treatment and preceded the increase in PGF2 alpha release stimulated by OT. Endometrial PI hydrolysis and PGF2 alpha secretion were similarly increased by AIF4-(phospholipase C activator), but not by cholera toxin (adenylyl cyclase activator). Although OT binding to OTR could be displaced by lysine-vasopressin and lysine-vasopressin stimulated PI hydrolysis, lysine-vasopressin did not stimulate PGF2 alpha release. Distinct receptors for OT and lysine-vasopressin on pig endometrium were confirmed by treatment with 100 nM OT + 100 nM lysine-vasopressin which stimulated PI hydrolysis more than 100-200 nM OT or lysine-vasopressin alone. These results support the hypothesis that OT stimulates phospholipase C to hydrolyze PI, yielding IP3 and DAG second-messengers which promote endometrial PGF2 alpha release during CL regression in pigs.

Cellular colocalization of diuretic peptides in locusts: a potent control mechanism.

Locust abdominal ganglia are shown to colocalize Locusta-diuretic peptide-, leucokinin I-, and lysine vasopressin-like immunoreactivity in posterior lateral neurosecretory cells. Extracts of abdominal ganglia were partially purified by RP-HPLC then dot immunoassay screened with the same antisera used for immunocytochemistry. Locusta-diuretic peptide-like immunoreactive material coeluted with synthetic Locusta-diuretic peptide, and leucokinin-like immunoreactive material coeluted with locustakinin. Lysine vasopressin-like material eluted in fractions that also showed Locusta-diuretic peptide and leucokinin I immunoreactivity. The diuretic activity of synthetic Locusta-diuretic peptide and locustakinin is demonstrated, and they are shown to act at least additively to promote Malpighian tubule fluid secretion. The immunoreactive neurosecretory cells are assumed to express at least these two peptides, and a model for promoting fluid secretion is proposed.

The CCKA receptor antagonist devazepide inhibits the effect of apomorphine on vasopressin release in pigs.

1. A transient increase in plasma vasopressin concentrations represents a physiological correlate of nausea in animals that vomit. 2. The CCKA receptor antagonist devazepide has previously been shown to inhibit vasopressin release induced in pigs by intravenous (i.v.) CCK. 3. This study investigated whether devazepide (70 micrograms/kg i.v.) would affect vasopressin secretion induced in pigs (n = 6) by the emetic drug apomorphine (25 micrograms/kg i.v.). 4. Apomorphine stimulated vasopressin release in the 30 min period following injection; this effect was prevented by prior administration of devazepide. 5. The results suggest that CCKA receptor antagonists may have the ability to prevent nausea and/or emesis.

Vasopressin-binding sites in the pig pituitary gland: competition by novel vasopressin antagonists suggests the existence of an unusual receptor subtype in the anterior lobe.

Arginine vasopressin (AVP) acts in the pituitary gland, in synergy with corticotrophin-releasing factor, to induce ACTH release in response to stressful stimuli. Pituitary AVP receptors in the rat are coupled to phospholipase C, as are the so-called V1-type AVP receptors. The present study examined [3H]AVP binding in membranes prepared from the anterior lobe of the pituitary gland of the pig. [3H]AVP, alone or in competition with analogues, bound to sites in the pig anterior lobe which are pharmacologically similar to those described previously by others in the rat pituitary gland. For comparison, the same competition studies were performed on membrane preparations from the rat liver which contain the classic V1-type AVP receptor. Pituitary and liver AVP-binding sites were dissimilar; both cyclic and linear V1 antagonists had, in general, a much lower affinity for pituitary AVP-binding sites than for those in the liver. Thus, Phaa-D-Tyr(Et)-Phe-Gln-Asn-Lys-Pro-Arg-NH2 (Phaa = phenylacetyl) has a 2500-fold greater affinity for the latter (negative logarithm of inhibition constant (pKi) = 9.64) than for the former (pKi = 6.22). One linear antagonist, Pa-D-Tyr-Phe-Val-Asn-Arg-Pro-Arg-Arg-NH2 (Pa = propionyl) had about equal affinities for liver and pituitary membranes (pKi = 6.39 and 6.53 respectively). Another compound, Phaa-D-Tyr-Phe-Val-Asn-Arg-Pro-Arg-Arg-NH2 had the highest affinity found to date for binding to AVP sites in the pituitary (pKi = 7.43). These findings suggest some ideas for the design of more potent and/or selective AVP analogues acting in the pituitary gland.

Effect of bacitracin on the degradation of a vasopressin receptor ligand with high affinity for the V1 and V2 vasopressin isoreceptors.

We previously described a new iodinated vasopressin analogue (N epsilon-[125I]L-Tyr-[Lys8]-vasopressin) with high affinity for the vasopressin V1 and V2 isoreceptors. The aim of the present study was: i) to analyse the degradation pathway of N epsilon-[125I]L-Tyr-[Lys8]-vasopressin and (ii) to look for an effective inhibitor of radioligand degradation. N epsilon-[125I]L-Tyr-[Lys8]-vasopressin was processed in a temperature-dependent manner by crude cell membranes from LLC-PK1 cells. Only one degradation product was seen using RP-HPLC. The degradation product co-eluted with monoiodotyrosine. The stereoisomer, N epsilon-[125I]D-Tyr-[Lys8]-vasopressin, underwent the same degradation process. Bacitracin prevented degradation at doses as low as 40 mg/l without alterating the binding affinity.

Determination of specific activity of labeled ligands by nonlinear regression analysis.

The specific radioactivity (SA) of 125I-lysine vasopressin (LVP) was determined by analyzing the binding B (cpm/tube) of variable amounts of tracer T (cpm/tube) to a constant amount of an LVP antibody, in the presence of known quantities L (mol/tube) of LVP standards. The parameters of the equations B = f(F) and B/F = g(T), describing B as a function of free F (cpm/tube) tracer or the ratios B/F as a function of T, were first calculated by nonlinear regression analysis of the results obtained with tracer alone. Then the dependent variables B or B/F were measured in the presence of LVP and analyzed with the same equations by substituting the independent variables F or T with (F + alpha FL) and (T + alpha L), respectively, where alpha (cpm/mol) represents a measure of the SA and FL (FL = L.F/T), free LVP, respectively. The SA was thus treated as an unknown parameter to be calculated by nonlinear regression. This method was compared with the traditional interpolation of the SA from the self-displacement and standard curves. Tracer and ligand were found to have the same affinity for the binding sites, since the set of equations B = f(F + alpha FL) and B/F = g(T + alpha L), describing the binding of the tracer in the presence of LVP and equations B = f(F) and B/F = g(T) to which these equations reduce in the absence of LVP (L = 0), had identical binding parameters. To be valid, any method based on self-displacement requires that the tracer and standards have the same affinity for the binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular cloning and functional characterization of V2 [8-lysine] vasopressin and oxytocin receptors from a pig kidney cell line.

[Arg8]vasopressin and oxytocin are the two main members of the neurohypophysial hormone family found to be present in nearly all mammals. [Lys8]vasopressin ([Lys8]VP) has been identified as the antidiuretic hormone in pig and some marsupial families. The porcine-derived kidney epithelial cell line, LLC-PK1, expresses both [Lys8]VP receptors coupled to the activation of adenylate cyclase (V2 receptors) and oxytocin receptors. Here we report the molecular cloning of the V2 [Lys8]VP receptor and the oxytocin receptor from LLC-PK1 cells. The cloned V2 [Lys8]VP receptor differs from human and rat V2 [Arg8] receptors mainly in its N-terminal region, in residues located in the extracellular loops and in intracellular phosphorylation sites. When expressed in COS7 cells, the V2 [Lys8]VP receptor exhibits the relative order of ligand affinity [Lys8]VP = [Arg8]VP >> 1-deamino[D-Arg8]VP > or = oxytocin and adenylate-cyclase stimulation, expected for the porcine V2 [Lys8]VP receptor but different from V2 [Arg8]VP receptors. Adenylate-cyclase activation by [Lys8]VP was inhibited in COS7 cells by a V2 antagonist. The cloned oxytocin receptor exhibits in COS7 cells a ligand specificity typical of mammalian oxytocin receptors. mRNA-distribution analysis revealed a single 5.5-kb transcript in the uterus from pregnant guinea pig.

Expression of vasopressin receptors (V2-subtype) on LLC-PK1 cells during cell culture.

Vasopressin receptor expression on LLC-PK1-cells (a porcine renal tubular cell line) during cell culture is still not fully understood. We studied receptor expression using a novel vasopressin analogue with high specific radioactivity ([125I][8-p-hydroxy-phenylpropionyl]-lys8-vasopressin, 74EBq/mol (2000 Ci/mmol)). LLC-PK1 cells were grown in monolayers for 1 to 6 days. Scatchard analysis performed with membranes of LLC-PK1 cells revealed a single binding site with a binding constant (Kd) of 0.46 +/- 0.04 nmol/l. During cell culture, the binding constant (Kd) was not altered, but receptor density increased significantly (21,115 +/- 645 receptors per cell, day 2; 42,315 +/- 1512 receptors per cell, day 6). A receptor occupancy of about 30% was found to be associated with a cAMP stimulation of 50%. The receptor reserve might be even higher because, by using a highly specific oxytocin antagonist, we found that 20% of the occupied [125I][8-p-hydroxy-phenylpropionyl]-lys8-vasopressin-binding sites are oxytocin receptors. For lys8-vasopressin receptor studies, great care has to be taken to examine cells in identical culture phases.