TFF3 induced Fos protein expression in the magnocellular oxytocin neurons of the hypothalamus

TFF3 is synthesized in magnocellular oxytocin neurons of the supraoptic (SON) and paraventricular nuclei (PVN) of the rat and human hypothalamus. Here we investigated whether intracerebroventricular (i.c.v.) injection of TFF3 stimulates oxytocin release into the blood and activates Fos protein immunoreactivity in oxytocin neurons of the SON and PVN in rats. The results show that plasma oxytocin concentrations were not altered after i.c.v. injection of TFF3 or vehicle. Fos protein expression was significantly increased in both the SON and PVN after TFF3 injections and double labeling studies showed that the Fos signal was predominantly in oxytocin neurons.     

Calcitonin gene-related peptide in the human hypothalamus

Calcitonin gene-related peptide (CGRP)-like immunoreactivity (LI) in the human hypothalamus was investigated by radioimmunoassay and by immunocytochemistry. CGRP-LI was detected from two hypothalami obtained at autopsy (2.1 and 7.0 ng/g wet tissue) by radioimmunoassay. Reverse phase high performance liquid chromatography revealed that most of the CGRP-LI in the human hypothalamus was eluted in an identical position with synthetic human CGRP. For immunocytochemistry, human hypothalami obtained at autopsy were fixed and cryostat-sectioned at 40 microns. Free floating sections were immunostained with antibody to CGRP. CGRP-immunoreactive cell bodies were found in the supraoptic nucleus, paraventricular nucleus and infundibular nucleus. These findings indicate that CGRP exists in the cell bodies of the supraoptic nucleus, paraventricular nucleus and infundibular nucleus in the human hypothalamus and CGRP may play some roles in the endocrine and other functions of the human hypothalamus.     

Immunocytological evidence for oxytocin neurons in the human fetal hypothalamus

Summary The use of antibodies against oxytocin or neurophysin enabled the detection by immunocytochemistry of oxytocin-neurophysin neurons in the hypothalamus in the human fetus. The perikarya of these neurons are located in the paraventricular and supraoptic nuclei. Immunoreactive neurons occur in the median eminence. The neurophysin immunoreactive neurons were more numerous than the oxytocin immunoreactive neurons. The specificity of the immunocytological reaction was controlled. The first oxytocin-neurophysin neurons are seen as early as the 14th week of gestation.     

Ultrastructural co-localization of TFF3-peptide and oxytocin in the neural lobe of the porcine pituitary

TFF-peptides (formerly P-domain peptides, trefoil factors) are typical secretory products of many mucous epithelial cells. TFF3 is also synthesized in oxytocinergic neurons in the paraventricular and supraoptic nuclei of the human hypothalamus. Here, TFF3 and oxytocin are shown to be co-localized within the same secretory vesicles in the neural (posterior) lobe of the procine pituitary by means of immunoelectron microscopy. Relatively large amounts of TFF3, but not TFF1 and TFF2, are present in the neural lobe of the porcine pituitary, where it is probably released into the bloodstream.     

Expression of corticosteroid-binding protein in the human hypothalamus, co-localization with oxytocin and vasopressin.

Corticosteroid-binding globulin, a specific steroid carrier in serum with high binding affinity for glucocorticoids, is expressed in various tissues. In the present study, we describe the immunocytochemical distribution of this protein in neurons and nerve fibers in the human hypothalamus. CBG immunoreactive perikarya and fibers were observed in the paraventricular, supraoptic, and sexual dimorphic nuclei in the perifornical region, as well as in the lateral hypothalamic and medial preoptic areas, the region of the diagonal band, suprachiasmatic and ventromedial nuclei, bed nucleus of the stria terminalis and some epithelial cells from the choroid plexus and ependymal cells. Stained fibers occurred in the median eminence and infundibulum. Double immunostaining revealed a partial co-localization of corticosteroid-binding globulin with oxytocin and, to a lesser extent, with vasopressin in the paraventricular and the supraoptic nuclei. Double immunofluorescence staining showed coexistence of these substances in axonal varicosities in the median eminence. We conclude that neurons of the human hypothalamus are capable of expressing corticosteroid-binding globulin, in part co-localized with the classical neurohypophyseal hormones. The distribution of CBG immunoreactive neurons, which is widespread but limited to specific nuclei, indicates that CBG has many physiological functions that may include neuroendocrine regulation and stress response.     

Localization of TFF3 peptide to porcine conjunctival goblet cells

TFF-peptides (formerly P-domain peptides, trefoil factors) form a new family of mucin-associated peptides mainly in the gastrointestinal tract. TFF3 is a typical secretory product of intestinal goblet cells and occurs also in the respiratory tract. Here, polyclonal antisera specific for TFF3 were used in Western blot analysis and immunofluorescence to determine the presence and distribution of TFF3 in the porcine conjunctiva, which is the primary source for ocular mucins. Significant accumulation of TFF3 was detected in conjunctival goblet cells but not in the lacrimal glands. This peptide, together with ocular mucins, may play a role in the rheological function of the tear film.     

Coexistence of intestinal trefoil factor (hITF) and oxytocin in magnocellular neurons in the human hypothalamus.

Human intestinal trefoil factor hITF, a polypeptide of the P-domain family, was found to occur in hypothalamic neurons. With combined immunofluorescence and immunoperoxidase technique we investigated the coexistence of hITF with the neurohypophysial peptide oxytocin and the associated neurophysin I in sections of the human hypothalamus. In the supraoptic nucleus, 39.2% of magnocellular oxytocinergic perikarya show hITF immunoreactivity. A similar distribution was observed in perivascular hypothalamic oxytocinergic neurons, whereas in the paraventricular nucleus, 99% of the oxytocinergic neurons show hITF coexpression. In the periventricular nucleus (PEV), single, scattered neurons with both immunoreactivities occur. Our findings indicate that hITF and oxytocin are coexpressed in a portion of the magnocellular neurons in the human hypothalamus, and that hITF is among the neurohypophysial peptides.     

High-level expression of human TFF3 in Escherichia coli

A strategy for expression and purification of recombinant N-terminal human trefoil factor family-domain peptide 3 (hTFF3) in Escherichia coli was established. The gene of hTFF3 was synthesized to substitute the low-usage condons with corresponding high-usage synonymous condons. At the same time, the signal peptide of DsbC was added to the N-terminus of the hTFF3 gene. The mature recombinant hTFF3 was located in the periplasm of E. coli, which can be released by sonication. The protein was further purified by a two-step cation exchange chromatography mentod. The yield is about 14-15 mg/l of culture. The biological activity of purified hTFF3 was analyzed by cell-based apoptosis assay, which shows that the recombinant hTFF3 is biologically active.     

Design of peptide oxytocin antagonists with strikingly higher affinities and selectivities for the human oxytocin receptor than atosiban.

The peptide oxytocin (OT) antagonist atosiban, approved for tocolytic use in Europe (under the tradename Tractocile), represents an important new therapeutic advance for the treatment of premature labor. This paper presents some new peptide OT antagonists which offer promise as superior tocolytics. The solid phase synthesis is reported of four pairs of L and D-2-naphthylalanine (L/D-2Nal) position-2 modified analogs of the following four oxytocin (OT) antagonists: des-9-glycinamide [1-(beta-mercapto-beta,beta-pentamethylene propionic acid), 2-O-methyltyrosine, 4-threonine]ornithine-vasotocin (desGly-NH(2),d(CH(2))(5)[Tyr(Me)(2),Thr(4)]OVT) (A); the Tyr-NH(2) (9) analog of (A), d(CH(2))(5)[Tyr(Me)(2),Thr(4),Tyr-NH(2) (9)]OVT (B); the Eda(9) analog of (A), d(CH(2))(5)[Tyr(Me)(2),Thr(4),Eda(9)]OVT (C); and the retro COCH(2)Ph(4-0H)(10) modified analog of (C), d(CH(2))(5)[Tyr(Me)(2),Thr(4),Eda(9)<-- COCH(2)Ph(4-0H)(10)]OVT (D). The eight new analogs of A-D are (1) desGly-NH(2),d(CH(2))(5)[D-2Nal(2),Thr(4)]OVT, (2) desGly-NH(2),d(CH(2))(5)[2-Nal(2),Thr(4)]OVT, (3) d(CH(2))(5)[D-2Nal(2),Thr(4),Tyr-NH(2) (9)]OVT, (4) d(CH(2))(5)[2Nal(2),Thr(4),Tyr-NH(2) (9)]OVT, (5) d(CH(2))(5)[D-2Nal(2),Thr(4),Eda(9)]OVT, (6) d(CH(2))(5)[2Nal(2),Thr(4),Eda(9)]OVT, (7) d(CH(2))(5)[D-2Nal(2),Thr(4),Eda(9)<-- COCH(2)Ph(4-0H)(10)]OVT, (8) d(CH(2))(5)[2Nal(2),Thr(4),Eda(9)<-- COCH(2)Ph(4-OH)(10)]OVT. Peptides 1-8 were evaluated for agonistic and antagonistic activities in in vitro and in vivo rat bioassays, in rat OT receptor (rOTR) binding assays and in human OT receptor (hOTR) and human vasopressin (VP) vasopressor (V(1a)) receptor (hV(1a)R) binding assays. Also reported are the hOTR and hV(1a)R affinity data for atosiban and for B. None of the eight peptides exhibit oxytocic or vasopressor agonism. Peptides 1-8 exhibit weak antidiuretic agonism (activities in the range 0.014-0.21 U/mg). Peptides 1-6 exhibit potent in vitro (no Mg(2+)) OT antagonism (anti-OT pA(2) values range from 7.63 to 8.08). Peptides 7 and 8 are weaker OT antagonists. Peptides 1-6 are all OT antagonists in vivo (estimated in vivo anti-OT pA(2) values in the range 6.94-7.23). Peptides 1-8 exhibit vasopressor antagonism, anti-V(1a) pA(2) values in the range 5.1-7.65. Peptides 1-8 exhibit high affinities for the rOTR (K(i) values = 0.3-7.8 nM). Peptides 1-4 and B exhibit surprisingly very high affinities for the hOTR; their K(i) values are 0.17, 0.29, 0.07, 0.14 and 0.59 nM, respectively. Peptides 1-4 and B exhibit respectively 449, 263, 1091, 546 and 129 times greater affinity for the hOTR than atosiban (K(i) = 76.4 nM). Peptides 1-4 exhibit high affinities for the hV(1a)R (K(i)s = 1.1 nM, 1.3 nM, 0.19 nM and 0.54 nM, all higher than the hV1(a)R affinities exhibited by atosiban (K(i) = 5.1 nM) and by B (K(i) = 5.26 nM). Because of their strikingly higher affinities for the hOTR than atosiban, peptides 1-4 and B exhibit gains in anti hOT/anti hV(1a) receptor selectivity compared with atosiban of 93, 64, 39, 56 and 127, respectively. These OT antagonists are thus promising candidates for development as potential new tocolytic agents.     

Co-localization of hypocretin-1 and hypocretin-2 in the cat hypothalamus and brainstem

Hypocretin-1 (hcrt-1) and hypocretin-2 (hcrt-2) are two recently discovered hypothalamic neuropeptides. In the present study, using double immunofluorescent techniques, the co-localization of hcrt-1 and hcrt-2 was examined in neuronal soma and fibers/terminals located, respectively, in the cat hypothalamus and brainstem. In the hypothalamus, all hcrt-1 positive neuronal soma also displayed hcrt-2 immunoreactivity. In the brainstem, both hcrt-1 and hcrt-2 antibodies labeled the same fibers/terminals, indicating that hcrt-1 and hcrt-2 co-localize not only in the neuronal soma (hypothalamus) but also in their fibers/terminals (brainstem). If both peptides are released following neuronal activity, then the distinct effects of these peptides in the brain are likely to depend on the types of postsynaptic receptors that are activated.     

Co-localization of putative vasopressin receptors and vasopressinergic neurons in rat hypothalamus

Summary Vasopressin and oxytocin are synthesized by neurons in the paraventricular and supraoptic nuclei of hypothalamus. Dense concentrations of vasopressin binding sites have also been localized in these nuclei. Using a vasopressin anti-idiotypic antiserum, a dual immunocytochemical labeling procedure has been employed to elucidate the distribution of putative vasopressin receptors in anatomical relation to vasopressin and oxytocin immunoreactive cells in rat brain. Putative vasopressin receptors are observed in relation to magnocellular neurons in hypothalamus that are vasopressin immunoreactive. They do not appear to be associated with parvocellular vasopressinergic cells or oxytocin immunoreactive neurons. The presence of these presumed autoreceptors would support evidence that vasopressin may autoregulate the activity of magnocellular vasopressinergic neurons in hypothalamus.     

Co-localization of putative vasopressin receptors and vasopressinergic neurons in rat hypothalamus

Vasopressin and oxytocin are synthesized by neurons in the paraventricular and supraoptic nuclei of hypothalamus. Dense concentrations of vasopressin binding sites have also been localized in these nuclei. Using a vasopressin anti-idiotypic antiserum, a dual immunocytochemical labeling procedure has been employed to elucidate the distribution of putative vasopressin receptors in anatomical relation to vasopressin and oxytocin immunoreactive cells in rat brain. Putative vasopressin receptors are observed in relation to magnocellular neurons in hypothalamus that are vasopressin immunoreactive. They do not appear to be associated with parvocellular vasopressinergic cells or oxytocin immunoreactive neurons. The presence of these presumed autoreceptors would support evidence that vasopressin may autoregulate the activity of magnocellular vasopressinergic neurons in hypothalamus.     

Study of oxytocin receptor in human myometrium using highly specific 3H-labeled oxytocin.

A highly specific tritium labeled oxytocin (3H-OT) was synthesized utilizing the method of catalytic substitution of halogen for hydrogen. The specific activity of 3H-OT was 19 Ci/mM and the biologic activity was 350 U/mg, which was sufficient for the OT radioreceptor assay. The maximum % uptake of 3H-OT in the human myometrium was observed in 20,000 X g pellets under the optimal conditions of pH 7.4, at 20 degrees C and the incubation time of 90 min and it was augmented in the presence of Mn++. It was observed from the Scatchard plot, that the binding site of OT in the human myometrial specimens was a single type within the range of OT concentration of 0.4 nM to 1.6 nM. The dissociation constants (Kd) and the number of binding sites (NBS) showed a relative increase as gestation advance. The apparent Kd of term pregnancies was 1.25 X 10(-9) M regardless of the presence or absence of labor pains, while the NBS of term pregnancies with and without labor pain was 1.2 X 10(-12) and 4.7 X 10(-12) moles/mg, protein, respectively.     

Injected TFF1 and TFF3 bind to TFF2-immunoreactive cells in the gastrointestinal tract in rats

Peptides of the trefoil factor family (TFF1, TFF2 and TFF3) are co-secreted with mucus in most organ systems and are believed to interact with mucins to produce high-viscosity, stable gel complexes. We have previously demonstrated that cells in the GI tract possess binding sites to TFF2 and that injected TFF2 ends up in the mucus layer. In the present study, tissue binding and metabolism of parenterally administered human TFF1 and TFF3 in rats were described and compared to the immunohistochemical localization of the TFF peptides. 125I-TFF1 monomer and 125I-TFF3 mono- and dimer were given intravenously to female Wistar rats. The tissue distribution was assessed by gamma counting of organ samples and by autoradiography of histological sections. The degradation of 125I-TFF3 was studied by means of trichloracetic acid (TCA) precipitation and the saturability of the binding by administration of excess unlabelled peptide. The TFF peptides were localized in histologic sections from the GI tract by immunohistochemistry. Injected TFF3 dimer (12%) was taken up by the GI tract. At autoradiography, grains were localized to the same cells that were immunoreactive to TFF2. The binding could be displaced by excess TFF3. Similar binding was observed for the TFF1 and TFF3 monomers apart from binding in the stomach, where the uptake was only 15% in comparison to the dimer. There was no specific binding outside the GI tract and no binding to TFF1 or TFF3 immunoreactive cells. In conclusion, the TFF2-binding cells in the gastrointestinal tract seem to have basolateral, receptor-like activity to all three TFF peptides. The mucous neck cells of the stomach predominantly take up TFFs with two trefoil domains, indicating a different receptor-like activity in the stomach compared to the rest of the GI tract.     

Studies on the vasopressin and oxytocin storage in the hypothalamus and neurohypophysis

The effects of modified adrenergic transmission on the bioassayed storage of vasopressin and oxytocin in the hypothalamus and neurohypophysis under conditions of stress (cold or immobilization), disturbed water balance and pinealectomy are reviewed. Alpha-adrenergic mechanisms seem to be included in the response of vasopressinergic and oxytocinergic neurones to stress; on the other hand, impulses of osmoreceptor origin are of importance in regulatory processes affecting the functional response of these neurones to altered alpha-adrenergic transmission and also to melatonin. The beta-adrenergic (and, to some extent, also the alpha-adrenergic) transmission is probably involved in the neural mechanisms of the pineal-neurohypophysial relationship. Furthermore, a possible regulatory role of cholecystokinin in water metabolism and release of neurohypophysial hormones is suggested.     

Suppressor of cytokine signaling 3 in the human hypothalamus

In rodents, the mediobasal hypothalamus and the hypothalamic paraventricular nucleus (PVN) are implicated in leptin signaling. Surprisingly little data is available on the human hypothalamus. We set out to study the expression of suppressor-of-cytokine-signaling 3 (SOCS3), α-melanocyte stimulating hormone (αMSH) and agouti-related protein (AgRP) in the infundibular nucleus (IFN) and to investigate the relationship between these neuropeptide expressions and serum leptin concentrations in a blood sample taken within 24h before death. We studied post-mortem human brain material by means of quantitative immunocytochemistry. We found that SOCS3 immunoreactivity was widely distributed throughout the hypothalamus, and most prominent in the PVN, whereas expression levels in the IFN were low. Surprisingly, SOCS3 expression in the PVN was inversely related to serum leptin. A significant positive correlation was observed between AgRP and NPY expression in the IFN. The inverse correlation between SOCS3 expression in the PVN and serum leptin was unexpected and may be related to the hypothalamic adaptation to fatal illness rather than to nutritional status, or may represent an interspecies difference.     

Co-localization of serine/threonine kinase 33 (Stk33) and vimentin in the hypothalamus

We investigate the immunoreactivity of serine/threonine kinase 33 (Stk33) and of vimentin in the brain of mouse, rat and hamster. Using a Stk33-specific polyclonal antibody, we show by immunofluorescence staining that Stk33 is present in a variety of brain regions. We found a strong staining in the ependymal lining of all cerebral ventricles and the central canal of the spinal cord as well as in hypothalamic tanycytes. Stk33 immunoreactivity was also found in circumventricular organs such as the area postrema, subfornical organ and pituitary and pineal glands. Double-immunostaining experiments with antibodies against Stk33 and vimentin showed a striking colocalization of Stk33 and vimentin. As shown previously, Stk33 phosphorylates recombinant vimentin in vitro. Co-immunoprecipitation experiments and co-sedimentation assays indicate that Stk33 and vimentin are associated in vivo and that this association does not depend on further interacting partners (Brauksiepe et al. in BMC Biochem 9:25, 2008). This indicates that Stk33 is involved in the dynamics of vimentin polymerization/depolymerization. Since in tanycytes the vimentin expression is regulated by the photoperiod (Kameda et al. in Cell Tissue Res 314:251–262, 2003), we determine whether this also holds true for Stk33. We study hypothalamic sections from adult Djungarian hamsters (Phodopus sungorus) held under either long photoperiods (L:D 16:8 h) or short photoperiods (L:D 8:16 h) for 2 months. In addition, we examine whether age-dependent changes in Stk33 protein content exist. Our results show that Stk33 in tanycytes is regulated by the photoperiod as is the case for vimentin. Stk33 may participate in photoperiodic regulation of the endocrine system.