Brain iron accumulation affects myelin-related molecular systems implicated in a rare neurogenetic disease family with neuropsychiatric features.

The 'neurodegeneration with brain iron accumulation' (NBIA) disease family entails movement or cognitive impairment, often with psychiatric features. To understand how iron loading affects the brain, we studied mice with disruption of two iron regulatory genes, hemochromatosis (Hfe) and transferrin receptor 2 (Tfr2). Inductively coupled plasma atomic emission spectroscopy demonstrated increased iron in the Hfe-/- × Tfr2mut brain (P=0.002, n ≥5/group), primarily localized by Perls' staining to myelinated structures. Western immunoblotting showed increases of the iron storage protein ferritin light polypeptide and microarray and real-time reverse transcription-PCR revealed decreased transcript levels (P<0.04, n ≥5/group) for five other NBIA genes, phospholipase A2 group VI, fatty acid 2-hydroxylase, ceruloplasmin, chromosome 19 open reading frame 12 and ATPase type 13A2. Apart from the ferroxidase ceruloplasmin, all are involved in myelin homeostasis; 16 other myelin-related genes also showed reduced expression (P<0.05), although gross myelin structure and integrity appear unaffected (P>0.05). Overlap (P<0.0001) of differentially expressed genes in Hfe-/- × Tfr2mut brain with human gene co-expression networks suggests iron loading influences expression of NBIA-related and myelin-related genes co-expressed in normal human basal ganglia. There was overlap (P<0.0001) of genes differentially expressed in Hfe-/- × Tfr2mut brain and post-mortem NBIA basal ganglia. Hfe-/- × Tfr2mut mice were hyperactive (P<0.0112) without apparent cognitive impairment by IntelliCage testing (P>0.05). These results implicate myelin-related systems involved in NBIA neuropathogenesis in early responses to iron loading. This may contribute to behavioral symptoms in NBIA and hemochromatosis and is relevant to patients with abnormal iron status and psychiatric disorders involving myelin abnormalities or resistant to conventional treatments.

Brain transcriptome perturbations in the transferrin receptor 2 mutant mouse support the case for brain changes in iron loading disorders, including effects relating to long-term depression and long-term potentiation.

Iron abnormalities within the brain are associated with several rare but severe neurodegenerative conditions. There is growing evidence that more common systemic iron loading disorders such as hemochromatosis can also have important effects on the brain. To identify features that are common across different forms of hemochromatosis, we used microarray and real-time reverse transcription polymerase chain reaction (RT-PCR) to assess brain transcriptome profiles of transferrin receptor 2 mutant mice (Tfr2(mut)), a model of a rare type of hereditary hemochromatosis, relative to wildtype control mice. The results were compared with our previous findings in dietary iron-supplemented wildtype mice and Hfe(-/-) mice, a model of a common type of hereditary hemochromatosis. For transcripts showing significant changes relative to controls across all three models, there was perfect (100%) directional concordance (i.e. transcripts were increased in all models or decreased in all models). Comparison of the two models of hereditary hemochromatosis, which showed more pronounced changes than the dietary iron-supplemented mice, revealed numerous common molecular effects. Pathway analyses highlighted changes for genes relating to long-term depression (6.8-fold enrichment, p=5.4×10(-7)) and, to a lesser extent, long-term potentiation (3.7-fold enrichment, p=0.01), with generalized reductions in transcription of key genes from these pathways, which are involved in modulating synaptic strength and efficacy and are essential for memory and learning. The agreement across the models suggests the findings are robust and strengthens previous evidence that iron loading disorders affect the brain. Perturbations of brain phenomena such as long-term depression and long-term potentiation might partly explain neurologic symptoms reported for some hemochromatosis patients.

Iron absorption and hepatic iron uptake are increased in a transferrin receptor 2 (Y245X) mutant mouse model of hemochromatosis type 3.

Hereditary hemochromatosis type 3 is an iron (Fe)-overload disorder caused by mutations in transferrin receptor 2 (TfR2). TfR2 is expressed highly in the liver and regulates Fe metabolism. The aim of this study was to investigate duodenal Fe absorption and hepatic Fe uptake in a TfR2 (Y245X) mutant mouse model of hereditary hemochromatosis type 3. Duodenal Fe absorption and hepatic Fe uptake were measured in vivo by 59Fe-labeled ascorbate in TfR2 mutant mice, wild-type mice, and Fe-loaded wild-type mice (2% dietary carbonyl Fe). Gene expression was measured by real-time RT-PCR. Liver nonheme Fe concentration increased progressively with age in TfR2 mutant mice compared with wild-type mice. Fe absorption (both duodenal Fe uptake and transfer) was increased in TfR2 mutant mice compared with wild-type mice. Likewise, expression of genes participating in duodenal Fe uptake (Dcytb, DMT1) and transfer (ferroportin) were increased in TfR2 mutant mice. Nearly all of the absorbed Fe was taken up rapidly by the liver. Despite hepatic Fe loading, hepcidin expression was decreased in TfR2 mutant mice compared with wild-type mice. Even when compared with Fe-loaded wild-type mice, TfR2 mutant mice had increased Fe absorption, increased duodenal Fe transport gene expression, increased liver Fe uptake, and decreased liver hepcidin expression. In conclusion, despite systemic Fe loading, Fe absorption and liver Fe uptake were increased in TfR2 mutant mice in association with decreased expression of hepcidin. These findings support a model in which TfR2 is a sensor of Fe status and regulates duodenal Fe absorption and liver Fe uptake.

Molecular pathogenesis of iron overload.

Our current understanding of iron absorption under normal conditions is presented, together with an overview of the clinical disorders of iron overload and the molecular processes that contribute to increased iron deposition in iron overload. Recently, a number of new genes involved in iron metabolism have been identified which is allowing the molecular mechanisms of iron absorption to be elucidated.

Cloning and gastrointestinal expression of rat hephaestin: relationship to other iron transport proteins.

The membrane-bound ceruloplasmin homolog hephaestin plays a critical role in intestinal iron absorption. The aims of this study were to clone the rat hephaestin gene and to examine its expression in the gastrointestinal tract in relation to other genes encoding iron transport proteins. The rat hephaestin gene was isolated from intestinal mRNA and was found to encode a protein 96% identical to mouse hephaestin. Analysis by ribonuclease protection assay and Western blotting showed that hephaestin was expressed at high levels throughout the small intestine and colon. Immunofluorescence localized the hephaestin protein to the mature villus enterocytes with little or no expression in the crypts. Variations in iron status had a small but nonsignificant effect on hephaestin expression in the duodenum. The high sequence conservation between rat and mouse hephaestin is consistent with this protein playing a central role in intestinal iron absorption, although its precise function remains to be determined.

The new iron age.

In the last four years there has been a major change in the approach to diagnosis of the iron overload disorder hereditary haemochromatosis (HH) following the discovery of the gene that is mutated in HH called HFE. In the first part of this review we will give a concise overview of the disease. Also the current literature on the role of HFE in iron absorption and transport at a molecular level and how mutations in HFE may lead to the break down in the regulation of iron homeostasis is reviewed. The second part of the review focuses on the molecular aspects of iron storage. Different chemical forms of storage iron deposits such as ferrihydrite and geotite are present in the iron storage proteins ferritin and haemosiderin. The type of iron storage deposits is thought to be an important factor in determining the degree of iron toxicity and tissue damage in patients with iron overload. Variations in the form of iron deposits in different types of iron overload disease e.g. HH or beta-thalessemia, the site of iron deposition and the clinical treatment used will be discussed.

Gastrointestinal function, divalent metal transporter-1 expression and intestinal iron absorption.

Iron absorption involves two carriers, one involved in the uptake of iron across the microvillus membrane of the enterocyte and the other in its transfer to the plasma at the basolateral surface. The uptake phase is thought to involve divalent metal transporter-1 (DMT1) which may move from the cytoplasm to the microvillus membrane under conditions of iron deficiency. To examine this possibility we used fasted animals previously fed an iron-deficient diet and then gavaged with iron. We measured the processes of iron absorption using in vivo gut sacs and correlated the changes observed with the intensity of DMT1 staining and gene expression in the duodenum. Fasting resulted in increased iron absorption, whereas gavage with iron decreased absorption. These changes were due to alterations in the uptake phase of absorption but not the transfer phase. There was also a highly significant correlation between the reduction in iron absorption, microvillus DMT1 staining and messenger ribonucleic acid (mRNA) expression. The loss of DMT1 from the microvillus membrane was not associated with an increase in cytoplasmic staining, suggesting that its loss was due to destruction of the carrier protein. It is concluded that DMT1 functional activity is determined by de novo synthesis and that the latter is regulated post-transcriptionally by enterocyte iron levels.

Cellular distribution of ferric iron, ferritin, transferrin and divalent metal transporter 1 (DMT1) in substantia nigra and basal ganglia of normal and beta2-microglobulin deficient mouse brain.

We examined whether high levels of circulatory iron may cause iron accumulation in the brain. In particular, we focussed on the substantia nigra and basal ganglia as several papers have indicated that iron may accumulate here and cause death of dopaminergic neurons. Normal mice and a mouse model of hereditary haemochromatosis, the beta2-microglobulin (beta2m) knock out [beta2m (-/-)] mouse, which has high levels of circulating iron due to increased iron absorption, were examined. The iron concentration in livers were: 170+/-15 microg/g (mean +/- SD) in controls and 1010+/-50 microg/g in beta2m (-/-) mice (p<0.001), whereas in the brain the respective values were 47 +/-1 microg/g and 53+/-2 microg/g (p<0.02). Hence, the difference between cerebral iron levels of normal and beta2m (-/-) mice was small. Histological examination of the brains revealed an unequivocal distribution of ferric iron, ferritin, transferrin and divalent metal transporter 1 (DMT1), which were indistinguishable when normal and beta2m (-/-) mice were compared. In the substantia nigra and basal ganglia, ferric iron and the iron-binding proteins were present in identical cell types, which mainly comprised oligodendrocytes and microglia. Neurons were lightly labelled with transferrin and DMT1. The virtual lack of an increase in cerebral iron in beta2m (-/-) mice clearly shows that the blood-brain barrier (BBB) is capable of restricting the transport of excess plasma iron into the brain.

Localisation of divalent metal transporter 1 (DMT1) to the microvillus membrane of rat duodenal enterocytes in iron deficiency, but to hepatocytes in iron overload.

BACKGROUND: The mechanism of iron absorption by the intestine and its transfer to the main iron storage site, the liver, is poorly understood. Recently an iron carrier was cloned and named DMT1 (divalent metal transporter 1). AIMS: To determine the level of DMT1 gene expression and protein distribution in duodenum and liver. METHODS: A DMT1 cRNA and antibody were produced and used in in situ hybridisation and immunohistochemistry, respectively, in rats in which the iron stores were altered by feeding diets with normal, low, and high iron content. RESULTS: Duodenal DMT1 mRNA was low in crypts and increased at the crypt-villus junction in iron deficient and control rats; it fell in the iron loaded state. Staining for DMT1 protein was not detected in crypts. In villus enterocytes, protein staining was localised to the microvillus membrane in iron deficiency, in the cytoplasm and to a lesser extent in the membrane in controls, and entirely in the cytoplasm of iron loaded animals. Liver DMT1 mRNA was distributed evenly across hepatocytes. DMT1 protein staining was observed on hepatocyte plasma membranes, with highest values in the iron loaded state, lower values in control animals, and none after iron depletion. CONCLUSIONS: Results are consistent with a role for DMT1 in the transmembrane transport of non-transferrin bound iron from the intestinal lumen and from the portal blood.

Mechanisms of ferric citrate uptake by human hepatoma cells.

The mechanisms of uptake of non-transferrin-bound iron by human hepatoma cells (HuH7) were investigated using 59Fe-citrate and [14C]citrate. The amount of iron associated with the cells increased linearly with time, whereas citrate uptake reached a plateau after 45 min, resulting in a cellular accumulation of iron over citrate. The cells displayed high-affinity membrane binding sites for citrate with maximum binding of 118 +/- 17 pmol citrate/mg protein and a dissociation constant of 21 +/- 2 microM (n = 3). Iron uptake was saturable with a maximum uptake rate of 1.95 +/- 0.43 pmol . mg protein-1 . min-1 and an apparent Michaelis constant of 1.1 +/- 0.1 microM. Nonradioactive ferric citrate and citrate inhibited 59Fe uptake to a similar degree. This suggests that the uptake of citrate-bound iron is dependent on either binding to specific citrate binding sites or the concentration of unbound iron. The uptake of iron was inhibited by ferricyanide (>100 microM) and ferrous iron chelators but stimulated by ferrocyanide and ascorbate, suggesting that the iron is reduced from Fe3+ to Fe2+ and transported into the cell by an iron carrier-mediated step.

Inhibition of uptake of transferrin-bound iron by human hepatoma cells by nontransferrin-bound iron.

The liver acquires iron from transferrin by transferrin receptor-mediated (TR) and transferrin receptor-independent pathways (NTR) and from nontransferrin-bound iron (NTB-Fe). Iron uptake by the NTR processes involves an iron-carrier mediated step. Experiments, using human hepatoma cells (HuH7) transfected with TR antisense (sense for control) RNA expression vectors to suppress TR expression, were performed to examine the effect of unlabeled NTB-Fe as iron citrate on the uptake of 59Fe-125I-transferrin. This was to determine if the uptake of transferrin-bound iron (Tf-Fe) and NTB-Fe uptake is mediated by a common iron-carrier. Iron citrate inhibited the uptake of 59Fe-transferrin (2.5 micromol/L Fe) in a concentration-dependent manner with a maximum effect when the citrate-iron:Tf-Fe molar ratio was 10:1. Transferrin uptake was not affected. At a lower Tf-Fe concentration of (0.125 micromol/L) when uptake of iron is TR-mediated, a 10-fold molar excess of iron citrate had no effect on Tf-Fe uptake by HuH7 TR antisense and sense cells. However, at a higher Tf-Fe concentration (2.5 micromol/L), when uptake occurs mainly by the NTR-mediated process, there was a 40% reduction in the membrane-bound and intracellular uptake of iron. Iron citrate did not affect the maximum rate (Vmax) of Tf-Fe uptake but the Michaelis-Menten constant (Km) for Tf-Fe uptake by the NTR-mediated process was increased, indicating there was competitive inhibition of Tf-Fe uptake by iron citrate. These results suggest that the uptake of NTB-Fe and Tf-Fe by the NTR- mediated process occurs by the same cellular pathway, using a common iron-carrier.

Transferrin receptor-independent uptake of differic transferrin by human hepatoma cells with antisense inhibition of receptor expression.

The hepatic uptake of transferrin-bound iron by a nontransferrin receptor (NTR)-mediated process was investigated using the human hepatoma cell line HuH7. Because HuH7 cells also acquire iron from transferrin by a receptor (TR)-mediated process, TR expression was inhibited by transfecting the cells with a plasmid containing human TR complementary DNA in antisense orientation relative to a human cytomegalovirus promoter/enhancer element. Cell clones were obtained that expressed a 50% to 60% reduction in cell surface TR, leading to a corresponding decrease in transferrin and iron uptake compared with wild-type cells. Uptake of transferrin by a second process was nonsaturable and not inhibited by a 100-fold excess of unlabeled transferrin. The amounts of transferrin taken up by the wild-type and antisense cells by this process were similar, showing that it did not involve TR. The proteolytic enzyme Pronase reduced the uptake of transferrin, suggesting that the NTR-mediated process entailed the nonsaturable binding of transferrin to plasma membrane proteins. This process, like the TR-mediated one, involved the internalization and recycling of transferrin, leading to accumulation of iron with time. Iron uptake mediated by NTR process was saturable and displaced by 100-fold excess unlabeled transferrin and reduced by weak bases and metabolic inhibitors. Therefore, the NTR-mediated process entailed transferrin adsorption to membrane-bound proteins, internalization, and release of iron from transferrin by a pH-dependent step followed by the intracellular transport of iron into ferritin and heme by a saturable carrier-mediated mechanism.

Uptake of iron from N-terminal half-transferrin by isolated rat hepatocytes. Evidence of transferrin-receptor-independent iron uptake.

The aim of the present study was to determine if human N-terminal half-transferrin (N- fragment), prepared by thermolysin cleavage of diferric transferrin, would bind to the rat hepatocyte transferrin receptor and donate iron to the cell. Competition experiments between 125I-labelled N-fragment and diferric transferrin revealed no receptor binding of the half-transferrin. Still, the N-fragment delivered iron to the cells in amounts approximately 30-fold above what could be accounted for by uptake of the fragment itself. The rate of cellular iron uptake from the fragment was comparable to what is seen with the intact transferrin. The uptake of 125I-labelled N-fragment was not inhibited by excess non-radioactive diferric transferrin. By comparison, the uptake of 59Fe from the N-fragment was inhibited 70% by excess nonradioactive diferric transferrin. This suggests that iron derived from diferric transferrin competes with the iron derived from the N-fragment for a common transport pathway. Although some cellular degradation of the N-fragment occurred, the extent of degradation was too low to explain the amount of iron accumulated by the cells. The results show that the hepatocyte has an effective transferrin-receptor-independent mechanism for accumulation of iron from transferrin.

Primary receptor-recognition site of human transferrin is in the C-terminal lobe.

The role of the transferrin receptor in capturing and conveying transferrin through the cell during the iron-donating cycle of receptor-mediated endocytosis has been studied extensively. Nevertheless, almost nothing is known of how human transferrin binds to its receptor. In an initial approach toward delineating the receptor-recognition site(s) of human transferrin, we have studied the interactions of proteolytically-cleaved, single-sited fragments of transferrin, representing the N- and C-lobes of the molecule respectively, with cells expressing the transferrin receptor on their plasma membranes. Only the C-fragment was found capable of donating iron to hepatoma-derived HuH-7 cells or of binding to surface receptors of HuH-7 and leukemic K562 cells. Although no association of N- and C-fragments could be demonstrated by gel chromatography, the presence of excess N-fragment strengthened the binding of C-fragment by an order of magnitude. An explanation of these observations is that the primary receptor recognition site of human transferrin is on the C-lobe of the protein, but that prior binding of this lobe to receptor enables the N-lobe to respond to receptor as well, either directly or by interaction with the bound C-lobe.

Androgen manipulation and vasopressin binding in the rat brain and peripheral organs.

It is now widely recognized that there is a sexual dimorphism in the development of arginine vasopressin (AVP) immunoreactivity in certain parts of the brain, and that changes in brain AVP immunoreactivity change with manipulation of androgen status. The aim of this experiment was to determine specifically any AVP receptor changes in response to manipulation of androgen levels using a selective V1 antagonist radioligand. Following castration, plasma testosterone levels fell and AVP immunoreactivity was reduced in the lateral septum and bed nucleus of the stria terminalis. With testosterone supplementation in castrated animals, the immunoreactivity in these regions was restored to a higher degree than in sham-operated animals. Central and peripheral V1 AVP receptor binding (as determined using the selective AVP V1 antagonist radioligand [125I](d(CH2)5,sarcosine7)AVP was not changed in any of the brain regions studied or in liver or kidney membranes from the three groups. This study demonstrates that there is no change in brain AVP receptor binding despite changes in regional AVP immunoreactivity in the brain, and excludes any confounding interaction with changes in oxytocin receptors.

Vasopressin V1 and V2 receptors in diabetes mellitus.

Diabetes mellitus causes hypertonicity, increased plasma arginine vasopressin (AVP), polydipsia, and polyuria. Downregulation of AVP V2 receptors may contribute to the polyuria through diminished V2 receptor-mediated free water retention. After 2 wk of streptozotocin-induced diabetes mellitus, the diabetic rats had raised plasma glucose, AVP, and osmolality levels (P < 0.001) compared with nondiabetic controls (Sham). Insulin treatment (4 U long-acting insulin sc, daily) partially lowered these values (P < 0.01). There was a reduction in the number of renal and hepatic V1 receptors in the diabetic and diabetic+insulin animals compared with the sham animals (P < 0.05). The receptor affinity remained unchanged. In parallel, there was a reduction in maximum AVP-activated total inositol phosphate production in the liver and kidney of the diabetic and diabetic+insulin animals compared with the sham animals (P < 0.05). The density and affinity of renal V2 receptors and AVP-stimulated adenosine 3',5'-cyclic monophosphate production in the diabetic and diabetic+insulin animals were unchanged compared with the sham. These results demonstrate differential regulation of AVP receptors and suggest that downregulation of renal V2 receptors does not contribute to the polyuria of diabetes. In contrast, downregulation of V1 receptors might contribute to diminished V1 receptor-mediated biological responses to AVP seen in diabetes mellitus.

Monoclonal antibodies to arginine vasopressin receptor bind to liver, kidney and pituitary membranes.

1. A vasopressin binding protein purified from rat liver membranes was used to immunize Balb/c mice and, subsequently, for the screening of hybrids raised in two different cell fusions. 2. Three hybrids were obtained which secreted monoclonal antibodies (MoAb) that bound to the purified solubilized receptor as detected by an enzyme-linked immunosorbent assay technique. All three MoAb immunoprecipitated the purified receptor. 3. In addition, the MoAb bound in a concentration-dependent manner to crude liver, kidney and anterior pituitary membranes, tissues known to contain arginine vasopressin (AVP) receptors but not to cardiac ventricle membranes which lack AVP receptors. 4. However, the binding of [125I]-[d(CH2)5,Sar7]AVP (a specific radiolabelled V1 antagonist) to the membrane-bound receptor was not inhibited by these antibodies. 5. These results suggest that MoAb recognize epitopes which are common to rat liver, kidney and anterior pituitary membranes but are not at the ligand binding site.

Regulation of vasopressin receptors in deoxycorticosterone acetate-salt hypertension.

Since arginine vasopressin may play a role in mineralocorticoid hypertension, we examined the effects of deoxycorticosterone acetate (DOCA)-salt on vasopressin V1 and V2 receptor binding and their second messengers, inositol phosphate and adenylate cyclase, respectively, in liver and kidney to determine whether altered vasopressin receptor binding is pathogenetic in mineralocorticoid hypertension. The mean arterial blood pressure of mineralocorticoid (DOCA-salt)-treated rats (163 +/- 1 mm Hg) was increased compared with control salt-treated rats (salt) (122 +/- 1 mm Hg) and water-treated rats (120 +/- 1 mm Hg; p less than 0.001). Mineralocorticoid treatment also increased plasma sodium, osmolality, and vasopressin concentration (p less than 0.001). In the hypertensive animals, there was a reduction in hepatic V1 (DOCA-salt, 91 +/- 12; salt, 132 +/- 13; and water, 145 +/- 13 fmol/mg protein; p less than 0.05) and renal V2 receptor binding density (DOCA-salt, 53 +/- 5; salt, 93 +/- 9; and water, 95 +/- 9 fmol/mg protein; p less than 0.01), although receptor affinities remained unaltered. In contrast, the density of renal V1 receptors was increased by mineralocorticoid treatment (DOCA-salt, 24 +/- 2; salt, 16 +/- 2; water, 18 +/- 1 fmol/mg protein; p less than 0.05), although the affinity was unchanged. Downregulation of V2 receptors was associated with a decrease in maximum cyclic adenosine monophosphate levels (DOCA-salt, 19 +/- 4; salt, 49 +/- 6; water, 53 +/- 9 pmol.mg protein-1.10 min-1; p less than 0.05), whereas changes in V1 receptor levels were not associated with changes in maximum inositol phosphate levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Presence of functional vasopressin V1 receptors in rat vagal afferent neurones.

The nucleus of the solitary tract (NTS) is one of the brain regions by which arginine vasopressin (AVP) influences blood pressure. This series of experiments in adult male rats was designed to determine whether the AVP binding sites which have been demonstrated in the NTS by in vitro autoradiography might be presynaptic on vagal afferents from the nodose ganglion; whether the AVP binding sites on vagal afferent neurones are functional receptors; and whether vagal transport of AVP receptors to other organs also occurs. High affinity binding sites (using the selective V1 antagonist radioligand [125I][d(CH2)5,Sar7]AVP and in vitro autoradiography) with characteristics of V1 receptors in the medial subnucleus of the NTS were reduced by 40% ipsilateral to nodose ganglionectomy. The nodose ganglion itself also contained high affinity V1 AVP binding sites that localised over cell bodies of vagal sensory neurones. That these binding sites were functional receptors was apparent when low concentrations of AVP but not oxytocin were found to depolarize the isolated nodose ganglion utilizing the 'silicone grease gap' technique. Furthermore, the actions of AVP were antagonised by low concentrations of a selective V1 receptor antagonist. However, there was no accumulation of AVP binding sites adjacent to either the proximal or distal vagal ligations suggesting that peripheral vagal transport of AVP receptors may not occur. Therefore these results are consistent with functional AVP V1 receptors occurring in the nodose ganglion. These receptors may occur on central terminals of vagal sensory neurones in the medial subnucleus of the NTS, but there was no evidence for peripheral transport of AVP V1 receptors.