Nephroblastoma overexpressed gene (NOV) codes for a growth factor that induces protein tyrosine phosphorylation.

NOV (nephroblastoma overexpressed gene) is a member of the CCN (connective tissue growth factor [CTGF], Cyr61/Cef10, NOV) family of proteins. These proteins are cysteine-rich and are noted for having growth-regulatory functions. We have isolated the rat NOV gene, and the DNA sequence shares 90% identity with the mouse and 80% identity with the human sequences. The rat NOV gene was expressed in all rat tissues examined, including brain, lung, heart, kidney, liver, spleen, thymus and skeletal muscle. Higher levels of rat NOV mRNA were seen in the brain, lung and skeletal muscle compared to the other tissues. Examination of NOV expression in various human cell lines revealed that NOV was expressed in U87, 293, T98G, SK-N-MC and Hs683 but not in HepG2, HL60, THP1 and Jurkat. The human NOV gene was transfected into 293 cells and the expressed protein purified. When 3T3 fibroblasts were treated with this recombinant NOV protein, a dose-dependent increase in proliferation was observed. Analysis of tyrosine-phosphorylated proteins revealed that when 3T3 cells were treated with NOV, a 221 kDa protein was phosphorylated. These data suggest that NOV can act as a growth factor for some cells and binds to a specific receptor that leads to the phosphorylation of a 221 kDa protein.

Expression and characterization of a putative high affinity human soluble leptin receptor.

Leptin, a circulating 16-kDa protein secreted by adipocytes, decreases body weight by reducing food intake and enhancing energy utilization. Leptin receptors that share homology to the glycoprotein gp130 have been recently cloned. In addition, differentially spliced leptin receptor messenger RNAs have been identified. Functional mutations in either the leptin or leptin receptor gene cause obesity. In the present study, expression of the full length human leptin receptor complementary DNA encoding the long cytoplasmic domain of leptin receptor in COS7 cells resulted in high affinity membrane binding of 125I-leptin (Ki approximately 200 pM); no detectable binding was present in the medium. In addition, we expressed the extracellular domain of human leptin receptor in COS7 cells and identified a soluble leptin receptor in the conditioned medium that binds human and mouse leptin with high affinity comparable with the full length membrane receptor. Transfected COS7 cells expressing the soluble leptin receptor also demonstrated modest specific 125I-leptin binding in whole cells, presumably due to association of the soluble leptin receptor to cell membrane proteins. Data from cross-linking studies identified two specific bands in the 125I-leptin/soluble leptin receptor complex with molecular masses of approximately 130-150 kDa and 300 kDa. The 130-150 kDa molecular mass was confirmed in Western blot analysis and Coomassie staining of the purified soluble receptor and probably represents the glycosylated form of the receptor. The 300-kDa band most likely represents a homodimer of the soluble leptin receptor complex because HPLC gel filtration analysis of the 125I-leptin/soluble leptin receptor complex identified a single peak corresponding to a molecular mass of approximately 340 kDa. The soluble leptin receptor antagonized 125I-leptin binding to the membrane receptor, suggesting its potential utility as a functional tool for determining the role of endogenous leptin.

[Studies on chemical constituents of Polygala arillata buch-ham].

Two new compounds were isolated from the roots of Polygala arillata Buch-Ham. On the basis of chemical reactions and spectral (UV, IR, MS, 1HNMR, DIFNOE, 13CNMR) analysis, they were identified as 1,3-dihydroxy-2-methoxyxanthone(I) and 7-hydroxy-1-methoxy-2,3-methylenedioxyxanthone(II). Pharmacological study indicated that I and II have inhibitory effect on aldose reductase activity.

Immunohistochemical localization of novel CART peptides in rat hypothalamus, pituitary and adrenal gland.

CART peptide specific polyclonal antisera were raised in rabbits. The antisera were raised to CART peptide fragments that span most of the predicted CART protein. The specificity of each antisera was demonstrated by blockade of immunostaining by the immunizing peptide but not by the other CART peptide fragments. In the hypothalamus and pituitary of colchicine and noncolchicine treated rats, immunostaining was observed in cell bodies, fibers and varicosities. Clusters of cells were also stained in the adrenal medulla. It is noteworthy that cellular immunostaining was only found in areas previously shown to express CART mRNA. These findings indicate the presence of CART peptide(s) in the hypothalamus, pituitary, and adrenal gland. Furthermore, we also present evidence for the possible processing of the CART pro-peptide into smaller peptide fragments. These neuroanatomical findings suggest a role of CART peptides in hypothalamic, pituitary and adrenal function.

Hypothalamic regulation of growth hormone secretion during food deprivation in the rat.

Suppressed pulsatile GH secretion in food-deprived rats has been hypothesized to be due to an increase in hypothalamic somatostatin secretion. We investigated this hypothesis and the role of GHRH in regulating GH secretion during food deprivation using two different models. In experiment one, rats were food deprived for 72h during which time they received a saline infusion (n = 5). At the same time rats were normal fed for 72h during which time they received a somatostatin infusion (5 micrograms/h, n = 7). After the 72h infusion period, all rats received two iv injections of GHRH (1 microgram/rat) at 2h intervals. GH concentrations in food-deprived rats rose from approximately 10 ng/ml to 400-800 ng/ml in response to both GHRH injections. This increase was significantly greater (p < 0.01) than the GH response (100-400 ng/ml) observed in somatostatin-infused animals. The significantly higher GH response observed in food-deprived rats as compared to somatostatin-infused, normal-fed rats suggests that somatostatin concentrations may decrease during food deprivation. In experiment two, rats were infused for 5h with either saline (n = 6) or GHRH (10 micrograms/h, n = 9) at the end of a 72h fast. GH concentrations did not change in saline-infused animals. In contrast, GH concentrations significantly increased (p < 0.01) upon initiation of the continuous GHRH infusion. Yet, this release of GH was pulsatile in nature. Pulsatile GH secretion in the presence of a constant GHRH infusion suggests that pulsatile somatostatin release from the hypothalamus is maintained during food deprivation. These studies suggest that during food deprivation in the rat 1) absolute concentrations of somatostatin decrease, but its pattern of secretion remains pulsatile, and 2) decreased GHRH release may be responsible for the absence of spontaneous GH pulses.

Characterization of an insulin-like growth factor binding protein (IGFBP-4) produced by the B104 rat neuronal cell line: chemical and biological properties and differential synthesis by sublines.

A previous report from our laboratory described an approximately 30 kilodalton (kDa) insulin-like growth factor binding protein (IGFBP) that inhibited the binding of insulin-like growth factor I (IGF-I) by its receptor and was secreted by a subline of the B104 rat neuronal cell line. To better understand the biology of this IGFBP, it was purified from media conditioned by these B104 cells, and the chemical and biological properties of the protein were examined. The IGFBP existed as a 24 kDa form and a 28 kDa form when the conditioned media were analyzed by ligand blot. Deglycosylation studies indicated the 28 kDa species was the N-linked glycosylated form of the 24 kDa IGFBP. Multiple forms at both mol wts were found using two-dimensional electrophoresis, suggesting that there were posttranslational modifications in addition to glycosylation. The amino acid sequence of the 12 amino-terminal residues was identical to that of rat IGFBP-4. Increased synthesis of IGFBP-4 by the subline contrasted with neglible production by other B104 cells. Blot hybridization with rat IGFBP-4 complementary DNA showed differential expression of a 2.6 kilobase transcript among B104 cell lines that correlated with quantities of IGFBP-4 secreted in media. The difference persisted even when the cells were xenografted into athymic nude mice. Purified IGFBP-4 inhibited the binding of [125I]IGF-I by its receptor and blunted stimulation of [3H]thymidine incorporation by IGF-I. These findings suggest a role for IGFBP-4 in neural cell function and indicate the B104 cell lines may be a useful model for further examination of IGFBP-4 biology.

In vitro release of growth hormone-releasing factor (GRF) from the hypothalamus: somatostatin inhibits GRF release.

The manner of release of growth hormone-releasing factor (GRF) from the rat hypothalamus was studied in a perifusion system using a highly sensitive radioimmunoassay for rat GRF. The recovery of GRF in this system was 50-60%. The release of GRF from the rat hypothalamic blocks was almost stable for 20-240 min after the start of the perifusion and was stimulated by depolarization induced by high K+ concentration. The release of GRF was inhibited by somatostatin at concentrations of 10(-11) to 10(-8) M with maximum inhibition to 52.5% of the basal release at a concentration of 10(-9) M. These results suggest that this system is useful in studying the regulatory mechanism of GRF release and that, in addition to its action on the pituitary, somatostatin appears to act at the level of the hypothalamus in inhibiting GRF release in the regulation of GH secretion.

Immunocytochemical demonstration of GAP-like immunoreactive neuronal elements in the human hypothalamus and pituitary.

GnRH-associated peptide (GAP)-like immunonreactive elements located in the human hypothalamus were investigated by PAP immunocytochemistry using specific antiserum against [pro-GnRH (14-69) OH]. Immunoreactive neuronal perikarya were distributed in the MPOA, PVN and infundibular nucleus, with the largest numbers of GAP-like immunoreactive perikarya found in the infundibular nucleus. We also detected the coexistence of GAP-like and GnRH-like immunoreactivities in the same neuronal perikarya in the MPOA by using a double immunolabelling procedure. In addition to the above regions immunoreactive neuronal perikarya were present in the region dorsal to the medial mammillary nucleus. GAP-like immunoreactive fibers were distributed in same areas that immunoreactive perikarya were observed. Many immunoreactive terminals were found adjacent to capillaries in the infundibulum. Immunoreactive dots, presumably terminals, were observed in the posterior pituitary and these were particularly evident along the margin adjacent to the anterior pituitary. The distribution pattern and density of GAP-like immunoreactive neuronal elements are compared with those of other mammalian species. We also compared GAP-like immunoreactive elements with that of GnRH as has been previously observed in the human hypothalamus.

Myelin basic protein present in the acid extracts of rat hypothalami releases insulin and glucagon from isolated rat pancreatic islets.

The insulinotropic and glucagon-releasing activity of acid extracts of rat hypothalami were tested in two bioassay systems using short-time incubation of isolated rat pancreatic islets and a rat insulinoma (RIN) cell line. Release of insulin and glucagon into the incubation medium was measured by radioimmunoassay. The major insulin-releasing and glucagon-releasing activity eluted in a broad zone in Sephacryl S-200 gel filtration in 30% acetic acid corresponding to the molecular weights between approximately 10 and 40 kD. The highest activity was eluted in a zone corresponding to 14 kD and was purified to homogeneity by means of two steps of reversed-phase HPLC. Amino acid analysis and SDS polyacrylamide gel electrophoresis indicated that the biologically active material was the rat small (myelin) basic protein characterized previously by Dunkley & Carnegie (1974). The purified rat hypothalamic material showed insulinotropic and glucagon-releasing activity indistinguishable from that of purified porcine myelin basic protein, and lost its insulinotropic activity when incubated with anti-myelin basic protein immunosorbent. We conclude that the major insulin-releasing and glucagon-releasing activity in acid extracts of the high-molecular-weight fractions of rat hypothalami is myelin basic protein.

Ontogeny of pituitary responsiveness to corticotropin-releasing hormone in rat.

The ontogeny of the pituitary's responsiveness to synthetic rat corticotropin-releasing hormone (CRH) in the late prenatal and early postnatal periods of rats was studied by a superfusion system using whole pituitaries. A significant increase of immunoreactive beta-endorphin (IR-beta-Ep) secretion in response to 10(-10) M CRH but not to 10(-11) M CRH was observed in pituitaries from the 15th day of gestation, the earliest day that we tested, whereas 10(-11) M CRH stimulated IR-beta-Ep release from the pituitaries of 17.5-day-old fetuses. Dose-related IR-beta-Ep secretions induced by 10(-12) M to 10(-10) M CRH were observed in pituitaries of 19.5- and 21.5-day-old fetuses, and 1-, 3- and 9-day-old newborn pups. CRH stimulated not only IR-beta-Ep and IR-adrenocorticotropic hormone (ACTH) but also IR-alpha-melanocyte-stimulating hormone (IR-alpha-MSH) secretions from fetal pituitaries. The content of IR-CRH in the hypothalamic extract from 15-day-old fetus was 6.6 +/- 3.6 pg/hypothalamus (mean +/- S.E.M.) and it gradually increased to reach 212.7 +/- 20.3 pg/hypothalamus on the 21.5th day of gestation. However, the content of IR-CRH in the hypothalamus dramatically decreased just after birth and then rapidly increased again from the 5th day after birth. These data indicate that the responsiveness of corticotrophs to CRH is already present on the 15th day of gestation, when the content of IR-CRH in the hypothalamus is extremely low and that the amount of hypothalamic IR-CRH dramatically dropped for several days just after birth in rats.

Effect of sodium ion on atrial natriuretic factor release from rat hypothalamic fragments.

The effects of Na ion and choline chloride on the release of atrial natriuretic factor (ANF) and growth hormone-releasing factor (GHRF) from rat hypothalamic fragments including the organum vasculosum of the lamina terminalis (OVLT) were examined in vitro. Although the release of ANF was stimulated by Na ion, choline chloride, and glucose in concentration-dependent manners, the release was more sensitive to a change in concentration of Na ion than to those of choline chloride and glucose. On the other hand, the change in Na ion concentration did not affect the release of GHRF. It can be therefore proposed that Na ion is the first candidate controlling ANF release from the brain tissue and that ANF in the hypothalamus and/or OVLT may play some role in the regulation of the Na ion and water balance in the central nervous system.

In vitro release of growth hormone-releasing factor from rat hypothalamus: effect of insulin-like growth factor-1.

The release of growth hormone-releasing factor (GHRF) from rat hypothalamus was investigated in vitro. After 60 min preincubation the released GHRF from sliced rat hypothalamic fragments during 60 min incubation was detected by a highly specific and sensitive radioimmunoassay for rat GHRF. The release of GHRF was Ca2+-dependent and enhanced by high concentration of K+. Insulin-like growth factor-1 (IGF-1) significantly decreased GHRF release to 65% and 84% of the control at concentrations of 10(-8) M and 10(-7) M, respectively. These results suggest that this in vitro system is useful for the investigation of the mechanism of GHRF release from the hypothalamus and that IGF-1 is probably involved in the feedback inhibition of growth hormone secretion by attenuating GHRF release from the hypothalamus besides countering the effect of GHRF on the pituitary.

Effects of neonatal administration of monosodium glutamate on plasma growth hormone (GH) response to GH-releasing factor in adult male and female rats.

Female and male rats were treated with monosodium glutamate (MSG; 4 mg/g b. wt.) as neonates and the capability of the pituitary gland to secrete growth hormone (GH) in response to an intravenous injection of human GH-releasing factor (GRF) was evaluated under pentobarbital anesthesia on 109 days of life. Immunoreactive GRF content in the pituitary stalk-median eminence tissue in MSG-treated rats was less than 20% of that of control. A significant dose-dependent plasma GH response was observed after the administration of two doses of human GRF (0.25 and 1 microgram/kg b. wt., i.v.) in both control and MSG-treated rats. The responses between MSG-treated and control rats were comparable in female rats, but they were significantly reduced in male MSG-treated rats. These results show that the pituitary's responsiveness to exogenous GRF is well preserved in MSG-treated rats despite prolonged and severe depletion of endogenous GRF and there exists a sex difference in the effect of MSG on GH secretion elicited by GRF.

Immunohistochemical evidence that growth hormone-releasing factor (GRF) neurons contain an amidated peptide derived from cleavage of the carboxyl-terminal end of the GRF precursor.

Antisera were raised against synthetic replicates of the carboxyl-terminal (C-terminal) fragment of the precursor to human GH-releasing factor (GRF) (pre-proGRF) whose structure was predicted from the complementary DNA cloned from a pancreatic tumor. These antisera were used along with antisera to human GRF itself to search for the presence of related molecules in the human hypothalamus, with an immunohistochemical approach. The antisera to pre-proGRF that recognize specifically the C-terminal amidated form of pre-proGRF stain GRF neurons in their cell bodies, fibers, and nerve endings that are in contact with portal capillaries of the median eminence. Antisera against the nonamidated form of the molecule did not give any staining in the hypothalamus. These results strongly suggest that human hypothalamic GRF derives from a precursor immunologically related (and probably identical) to the tumorous one and that this precursor is cleaved inside GRF cell bodies to give, in addition to the GRF-44-NH2 a second amidated peptide, the C-terminal pre-proGRF that is transported distally to nerve endings and most probably coreleased with GRF into portal capillaries.

Growth hormone-releasing factor: a new chapter in neuroendocrinology.

The isolation and characterization of growth hormone-releasing factor (GRF) has initiated a new and exciting era in our understanding of the neuroendocrine regulation of pituitary growth hormone (GH) secretion. This report briefly describes the isolation and characterization of GRF, factors which modulate the GH response to GRF and the effects of chronic administration and deprivation of GRF on somatic growth. The intent of this report is to serve as a general introduction on biochemical and physiological aspects of GRF. The following reports from this symposium will then cover many of these topics in much greater detail.

Immunohistochemical localization and radioimmunoassay of corticotropin-releasing factor in the forebrain and hypophysis of the frog Rana ridibunda.

The distribution of immunoreactive corticotropin-releasing hormone (CRF) in the forebrain and pituitary of the frog Rana ridibunda was studied by means of specific radioimmunoassay and immunohistochemistry using the indirect immunofluorescence and the peroxidase-antiperoxidase techniques. Relatively high concentrations of CRF-like material were found in both chiasmatic and infundibular regions of the hypothalamus (352 +/- 11 and 422 +/- 36 pg, respectively). Large amounts of CRF were also found in neurointermediate lobe extracts. Standard curves of synthetic CRF and the dilution curves for hypothalamic or neurointermediate lobe extracts were parallel. After Sephadex G-75 gel filtration, CRF-like immunoreactivity eluted in a single peak, in the same position as synthetic ovine CRF. Reversed-phase high-performance liquid chromatography of the material purified on Sephadex G-75 revealed 5 components with CRF-like immunoreactivity. The major peak had a retention time of 22 min as compared to 25.4 min for ovine CRF and 36 min for rat CRF. The detection of CRF-like immunoreactivity in neurons was facilitated by colchicine pretreatment of the frogs. The great majority of the CRF-positive perikarya were seen in the ventral region of the preoptic nucleus. A few scattered perikarya were also observed in the dorsal preoptic nucleus and in the retrochiasmatic region. Immunoreactive fibers were found in the infundibular nucleus and in various extrahypothalamic zones. CRF-containing neurons were apparently distinct from mesotocinergic and vasotocinergic neurons. A large number of immunoreactive nerve fibers were observed in the median eminence in close contact with the capillaries of the pituitary portal plexus and in the neural lobe. A few CRF-positive fibers were detected in the intermediate lobe, whereas the distal lobe was totally negative. These results show that the diencephalon and pars intermedia-nervosa of the frog contain a peptide immunologically related to mammalian CRF.

Somatostatin-28 [1-12]-like peptides.

The search for a peptide corresponding to the NH2-terminus of somatostatin-28 (SS-28) in tissues has led to the isolation and characterization of somatostatin-28[1-12] from pancreas and hypothalamus. Somatostatin-28[1-12]-like immunoreactivity [SS-28 [1-12]-LI] is widely distributed throughout the central nervous system and the digestive system of rodents and primates, reaching levels comparable to those of somatostatin-14 (SS-14). Antibodies directed against the C-terminal end of the dodecapeptide are more specific and constitute excellent markers for the "prosomatostatin" system in mammalian tissues. In rat brain, SS-28[1-12]-LI material is highly concentrated in nerve fibres and terminals, especially in the median eminence, layer I of neocortex, the outer molecular layer of the dentate gyrus and the striatum. Additionally, immunoreactivity is observed in large multipolar or occasionally pyramidal-like neurons of the neocortex. SS-28[1-12] is secreted from hypothalamus and amygdala by a calcium dependent mechanism. No biological role is presently known for the dodecapeptide. Two other peptides of Mr = 8000 (8 K) and Mr = 5000 (5 K) which contain SS-28[1-12] at their carboxy-termini are present in acid extracts from rat pancreas, brain and spinal cord. These two peptides were isolated from an acid extract of rat brains using ion-exchange chromatography, gel permeation chromatography and reverse-phase HPLC. Results from amino acid analysis and partial sequencing were compared to the sequence of the cDNA encoding rat pre-prosomatostatin (prepro-SS) and revealed that the 8 K peptide is a 76 amino acid molecule corresponding to prepro-SS[25-100] and that the 5K peptide, which contains 44 amino acids, corresponds to prepro-SS [57-100]. The 5 K peptide was generated after cleavage of a Leu-Leu bond at position 56-57 of prepro-SS. The four most predominant peptides of the "prosomatostatin system" presently characterized are: SS-14, SS-28[1-12], SS-28 and prepro-SS[25-100]. Studies on pooled perfusates from rat hypothalamic tissue show that prepro-SS[25-100] is released with SS-28[1-12] in vitro and accounts for 22% of the total SS-28[1-12]-like immunoreactive material released during depolarization. The 5 K peptide is apparently not secreted. The presence of prepro-SS[25-100] in brain implies that, first, prosomatostatin can serve as an immediate precursor for SS-14 without going through SS-28 as an intermediate step and second, other peptides could conceivably be derived from the cryptic portion of the precursor.