Transcriptional and posttranscriptional regulation of inhibin alpha-subunit gene expression in rat Sertoli cells by 8-bromo-3',5'-cyclic-adenosine monophosphate.

FSH is a major regulator of inhibin production in the testis. FSH effects on Sertoli cell inhibin production are believed to be mediated, at least in part, via the cAMP second messenger system. Previously, it has been shown that 8-bromo-cAMP (8-Br-cAMP) stimulates inhibin-alpha mRNA levels. This study examines whether the cAMP-induced increase in inhibin-alpha mRNA levels results from increased alpha mRNA synthesis, decreased degradation of mRNA, or both. The effects of cAMP on inhibin-alpha gene transcription were examined using nuclear run-on assays. Furthermore, the ability of 8-Br-cAMP to drive the transcription of chimeric constructs containing a 2.2-kilobase (kb) segment of the 5'-regulatory region of the alpha gene placed upstream of the coding region of the luciferase reporter gene was also examined. Data from nuclear run-on assays demonstrated rapid induction of alpha gene transcription by cAMP within 2 h and maximal 4- to 5-fold increase within 4-8 h in primary Sertoli cells. Transfection of TM.4 and JEG.3 cells with an alpha (2.2 kb):luciferase chimeric construct (containing 2.2 kb of the alpha gene 5'-flanking DNA) revealed rapid time-dependent induction of luciferase activity by 8-Br-cAMP in these cell types. To examine the effects of 8-Br-cAMP on alpha mRNA stability, cells were pretreated with medium or 50 micrograms/ml 8-Br-cAMP for 24 h before addition of 5 microM actinomycin D to arrest new RNA synthesis, and the decay of alpha mRNA transcripts was assessed over 24 h by Northern analysis and nonlinear regression.(ABSTRACT TRUNCATED AT 250 WORDS)

The brain renin-angiotensin system: location and physiological roles.

Angiotensinogen, the precursor molecule for angiotensins I, II and III, and the enzymes renin, angiotensin-converting enzyme (ACE), and aminopeptidases A and N may all be synthesised within the brain. Angiotensin (Ang) AT(1), AT(2) and AT(4) receptors are also plentiful in the brain. AT(1) receptors are found in several brain regions, such as the hypothalamic paraventricular and supraoptic nuclei, the lamina terminalis, lateral parabrachial nucleus, ventrolateral medulla and nucleus of the solitary tract (NTS), which are known to have roles in the regulation of the cardiovascular system and/or body fluid and electrolyte balance. Immunohistochemical and neuropharmacological studies suggest that angiotensinergic neural pathways utilise Ang II and/or Ang III as a neurotransmitter or neuromodulator in the aforementioned brain regions. Angiotensinogen is synthesised predominantly in astrocytes, but the processes by which Ang II is generated or incorporated in neurons for utilisation as a neurotransmitter is unknown. Centrally administered AT(1) receptor antagonists or angiotensinogen antisense oligonucleotides inhibit sympathetic activity and reduce arterial blood pressure in certain physiological or pathophysiological conditions, as well as disrupting water drinking and sodium appetite, vasopressin secretion, sodium excretion, renin release and thermoregulation. The AT(4) receptor is identical to insulin-regulated aminopeptidase (IRAP) and plays a role in memory mechanisms. In conclusion, angiotensinergic neural pathways and angiotensin peptides are important in neural function and may have important homeostatic roles, particularly related to cardiovascular function, osmoregulation and thermoregulation.

Tissue distribution and regulation of insulin-like growth factor (IGF)-binding protein-3 messenger ribonucleic acid (mRNA) in the rat: comparison with IGF-I mRNA expression.

Insulin-like growth factor-binding protein-3 (IGFBP-3) is the most abundant IGFBP in rat and human sera. The present study demonstrates the expression of the rat IGFBP-3 gene in a large number of tissues and coexpression, but not necessarily equal expression, with IGF-I mRNA. Tissues with a major abundance of IGFBP-3 were kidney, antrum of stomach, placenta, uterus, and liver. Changes in hepatic and renal levels of IGFBP-3 mRNA were analyzed after hypophysectomy (with and without GH treatment) and in the developing postnatal rat. These results were compared to changes in IGF-I mRNA levels under the same physiological conditions. Using S1 nuclease analysis, IGFBP-3 mRNA was present in the kidney and liver of 1-day-old rats and rose significantly in both organs by week 1. Thereafter, levels remained relatively constant, particularly in the liver. This is in marked contrast to the hepatic IGF-I pattern, which showed a continual rise up to 8 weeks. Hepatic IGFBP-3 gene expression was partially GH dependent, with IGFBP-3 mRNA levels falling (approximately 50%) after hypophysectomy and rising slightly after GH treatment. These changes were much less dramatic than those in IGF-I mRNA. In contrast, the renal levels of IGFBP-3 mRNA increased after hypophysectomy, (approximately 100%), but did not decrease with GH treatment. These data suggest that IGFBP-3 mRNA abundance is regulated differently in different tissues, and in at least some tissues is less sensitive to regulation than is IGF-I mRNA.

Cloning and characterization of the promoter for the rat insulin-like growth factor-binding protein-3 gene.

Insulin-like growth factor-binding protein-3 (IGFBP-3), the major IGFBP in the adult circulation, is produced by a wide range of cell and tissue types. IGFBP-3 appears to be regulated by transcriptional and/or posttranslational mechanisms in a species-, cell-, and development-specific manner. In vitro and in vivo studies suggest that a number of factors (e.g. cAMP, GH, insulin-like growth factor-I, epidermal growth factor, TSH, and FSH) can act as transcriptional regulators of IGFBP-3 in particular cell types. To address the mechanistic basis for these observations, we isolated the rat IGFBP-3 gene and began characterization and analysis of the hormonal regulation of its promoter. The rat IGFBP-3 gene is located within 2 adjacent EcoRI fragments spanning about 10 kilobases. Southern analysis indicated a single copy gene. A 1.18-kilobase fragment 5' to the translation initiation codon has been sequenced and showed 65% homology with the corresponding human IGFBP-3 sequence. The region between -100 and -1 bp relative to the transcription start site showed 85% homology. The transcription start site was 118 basepairs (bp) up-stream of the initiation codon, and a TATA box consensus was located 27 bp 5' to this CAP site. No CAAT box was present, but a CpG island was identified. Consensus sequences for a number of putative response elements (e.g. activating protein-2, insulin, TSH/insulin-like growth factor, and GH) were present within -700 bp of the CAP site. A series of 5'-truncated chloramphenicol acetyltransferase reporter constructs has been transfected into both COS-1 cells and the rat thyroid cell line FRTL-5. Both basal and hormonally responsive (TSH and phorbol ester) promoter activities have been localized within the first 472 bases of the promoter region. These data indicate that suitable transfected cell systems can be established in which additional investigations can be undertaken into the mechanisms of cell- and species-specific hormonal regulation of IGFBP-3 gene expression.

Rat ovarian insulin-like growth factor binding protein-3: a growth hormone-dependent theca-interstitial cell-derived antigonadotropin.

To further the identification and characterization of insulin-like growth factor binding proteins at the level of the immature rat ovary, we have set out to study the ovarian expression, cellular localization, and hormonal regulation of the insulin-like growth factor binding protein (IGFBP)-3. To this end, use was made of a solution hybridization/RNAse protection assay wherein ovarian total RNA from immature (21-23 days old) female rats was hybridized with a 343 bases-long [32P]-labeled rat IGFBP-3 riboprobe. As in liver, a single protected fragment (315 bases-long) corresponding to IGFBP-3 transcripts was identified in whole ovarian material. Cellular localization studies revealed the IGFBP-3 gene to be exclusively expressed in the theca-interstitial rather than the granulosa cell compartment. To confirm presence and cellular distribution of the IGFBP-3 protein, media conditioned by cultured granulosa cells, theca-interstitial cells, and whole ovarian dispersates were subjected to Western Ligand Blotting. Importantly, media conditioned by cultured theca-interstitial (but not granulosa) cells displayed an IGFBP the size of rat IGFBP-3 (46kDa) as determined by comigration with a rat serum standard. A similarly-sized band was apparent in media conditioned by cultured whole ovarian dispersates reflecting in all likelihood the contribution of the theca-interstitial cell component. Significantly, deglycosylation of media conditioned by cultured theca-interstitial cells revealed the glycosylated nature of the 46kDa IGFBP species as judged by the apparent reduction in its molecular size to 35kDa. Similar alterations were noted in corresponding rat serum samples. Hypophysectomy of immature rats resulted in a modest but statistically insignificant decrease in the relative (densitometrically-quantified) abundance of ovarian IGFBP-3 transcripts, an effect further augmented by the systemic provision of either FSH or diethylstilbestrol (DES). In contrast, systemic treatment of hypophysectomized rats with GH produced a marked (3.2-fold) increase (P less than 0.05) in the steady state levels of ovarian (as well as hepatic) IGFBP-3 transcripts. However, the concurrent provision of either FSH or DES resulted in substantial (P less than 0.05) attenuation (78 and 57% inhibition, respectively) of the upregulatory GH effect. These findings document the highly compartmentalized expression of the IGFBP-3 gene at the level of the immature rat ovary, implicate the theca-interstitial cell as the sole source of its generation, reveal its pituitary dependence, and disclose its diametrically-opposed (indeed antagonistic) regulation by FSH (or estrogens) and GH.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of I.C.V. injection of AT4 receptor ligands, NLE1-angiotensin IV and LVV-hemorphin 7, on spatial learning in rats.

Central administration of angiotensin IV (Ang IV) or its analogues enhance performance of rats in passive avoidance and spatial memory paradigms. The purpose of this study was to examine the effect of a single bolus injection of two distinct AT4 ligands, Nle1-Ang IV or LVV-haemorphin-7, on spatial learning in the Barnes circular maze. Mean number of days for rats treated with either Nle1-Ang IV or LVV-haemorphin-7 to achieve learner criterion is significantly reduced compared with controls (P < 0.001 and P < 0.05 respectively). This is due to enhanced ability of the peptide-treated rats to adopt a spatial strategy for finding the escape hatch. In all three measures of learning performance, (1) the number of errors made, (2) the distance travelled and (3) the latency in finding the escape hatch, rats treated with either 100 pmol or 1 nmol of Nle1-Ang IV or 100 pmol LVV-haemorphin-7 performed significantly better than the control groups. As early as the first day of testing, the rats treated with the lower dose of Nle1-Ang IV or LVV-haemorphin-7 made fewer errors (P < 0.01 and P < 0.05 respectively) and travelled shorter distances (P < 0.05 for both groups) than the control animals. The enhanced spatial learning induced by Nle1-Ang IV (100 pmol) was attenuated by the co-administration of the AT4 receptor antagonist, divalinal-Ang IV (10 nmol). Thus, administration of AT4 ligands results in an immediate potentiation of learning, which may be associated with facilitation of synaptic transmission and/or enhancement of acetylcholine release.

Sex- and tissue- specific regulation of 11 beta-hydroxysteroid dehydrogenase mRNA.

In the present study we have investigated the effect of hypophysectomy on 11 beta HSD1 gene expression in a variety of tissues from male and female rats. Of the tissues analysed significant changes were observed only in the male and female livers, and in the female kidney. Hypophysectomy (hypox) increased hepatic message levels in the female 20-fold, and male levels rose 2-fold. Male renal 11 beta HSD1 message levels remained constant following hypophysectomy, whereas female renal 11 beta HSD1 mRNA levels increased approximately 2-fold. In intact animals hepatic 11 beta HSD1 mRNA levels are 18-fold lower in the female than the male, while renal levels are 1.4-fold higher in the male than the female. Hepatic 11 beta HSD1 levels in the female rat appear critically responsive to the pattern of growth hormone (GH) administration. Twice daily injections of GH had no significant effect on hepatic message levels in the hypox female rat, whereas constant infusions of GH, via an osmotic mini-pump produced a marked decrease. Renal 11 beta HSD1 mRNA levels were demonstrated to be under the regulation of estrogen, and 11 beta HSD1 enzyme activity in both liver and kidney correlated with the observed changes in mRNA levels. 11 beta HSD1 gene expression is thus regulated in a tissue-specific and sexually dimorphic manner.

Cloning and characterization of the rat alpha-inhibin gene.

The gene for the rat glycoprotein hormone alpha-inhibin has been cloned and characterized. The entire gene was found to be contained within a 5.5 kilobase EcoRI fragment. It is composed of two exons separated by a 1.5 kb intron. Primer extension and S1 nuclease analysis showed that the major transcription initiation site in the ovary was 77 bp from the start of translation. The promoter region of the gene did not contain a conventional TATA box but instead a number of GA rich repeated sequences were found to be present. Other potential regulatory elements found included a repeating purine-pyrimidine tract (TG)28, cAMP and phorbol ester response elements and a putative glucocorticoid response element. Southern blot analysis of rat genomic DNA indicated that there is a single gene for alpha-inhibin in the rat.

Divergent ileal IGF-I and IGFBP-3 gene expression after small bowel resection: a novel mechanism to amplify IGF action?

Changes in the levels of ileal insulin-like growth factor-I (IGF-I) and insulin-like growth factor binding protein-3 (IGFBP-3) mRNA in the rat following massive small bowel resection (MSBR) have been investigated with a sensitive S1 nuclease assay. IGF-I mRNA levels vary little over 7 days; in contrast IGFBP-3 mRNA levels decreased to one-third 7 h post-MSBR, and remained suppressed for the length of this study. We postulate that decreased ileal synthesis of IGFBP-3 enhances the ability of IGF-I to stimulate the adaptive response.

Cloning and tissue distribution of the human 11 beta-hydroxysteroid dehydrogenase type 2 enzyme.

The enzyme 11 beta-hydroxysteroid dehydrogenase (11 beta HSD) is thought to protect the non-selective mineralocorticoid receptor from occupation by glucocorticoids, and to modulate access of glucocorticoids to glucocorticoid receptors resulting in protection of the fetus and gonads. A ubiquitous low affinity NADP+ dependent enzyme (11 beta HSD1) and a tissue specific, high affinity NAD+ dependent form (11 beta HSD2) of 11 beta HSD exist. We now report the isolation of a cDNA coding for human 11 beta HSD2. The new isoform is NAD+ dependent, exclusively dehydrogenase in directionality, inhibited by glycyrrhetinic acid and metabolizes the synthetic glucocorticoid dexamethasone; it displays Km values for corticosterone and cortisol of 5.1 nM and 47 nM, respectively. Sequence alignment shows that 11 beta HSD2 shares 35% identity with 17 beta HSD2, but is only 14% identical with 11 beta HSD1. The 11 beta HSD2 gene is highly expressed in kidney, colon, pancreas and placenta and the message is also present in the ovary, prostate and testis. These data suggest that 11 beta HSD2 plays an important role in modulating mineralocorticoid and glucocorticoid receptor occupancy by glucocorticoids.

Distribution of angiotensin IV binding sites (AT4 receptor) in the human forebrain, midbrain and pons as visualised by in vitro receptor autoradiography.

Angiotensin IV and other AT4 receptor agonists, improve memory retention and retrieval in the passive avoidance and swim maze learning paradigms. Angiotensin IV binding sites (also known as the AT4 receptors) are widely distributed in guinea pig and monkey (Macaca fascicularis) brains where high densities of the binding sites have been detected in the hippocampus, neocortex and motor nuclei. However, the distribution of the binding sites in the human brain is not known. We have recently localised the angiotensin IV binding sites (AT4 receptors) in post-mortem human brain using iodinated Nle-angiotensin IV, a higher affinity and more stable analogue of angiotensin IV. This radioligand bound with relatively high affinity and specificity to angiotensin IV binding sites. In competition studies on consecutive sections through the prefrontal cortex and claustrum, angiotensin IV, Nle-angiotensin IV and LVV-hemorphin 7 competed for the binding of 125I[Nle]-angiotensin IV with nanomolar affinities. Angiotensin II and the AT1 and AT2 receptor antagonists were ineffective in competing for the binding at concentrations of up to 10 microM. We found high densities of 125I[Nle]-angiotensin IV binding sites throughout the cerebral cortex including the insular, entorhinal, prefrontal and cingulate cortices. Very high densities of the binding sites were observed in the claustrum, choroid plexus, hippocampus and pontine nucleus. Some thalamic nuclei displayed high densities of binding including the anteroprincipal, ventroanterior, anteromedial, medial dorsal and ventrolateral nuclei. The caudate nucleus, putamen, many amygdaloid nuclei and the red nucleus all displayed moderate densities of binding with a higher level detected in the substantia nigra pars compacta. In the hypothalamus, high densities binding sites were found in the ventromedial nucleus with lower levels in the dorsomedial and paraventricular nuclei. The distribution of 125I[Nle]-angiotensin IV binding sites in the human brain is similar to that found in other species and supports multiple roles for the binding sites in the central nervous system, including facilitation of memory retention and retrieval.

Changes in the levels of 11 beta-hydroxysteroid dehydrogenase mRNA over the oestrous cycle in the rat.

The NADP dependent enzyme 11 beta-hydroxysteroid dehydrogenase (11 beta HSD) metabolizes glucocorticoids to their inactive 11-keto-metabolites in a wide range of tissues. To date very little is known about the regulation of this enzyme at the level of gene transcription. In this study we show significant changes in the uterine, renal, ovarian and hepatic levels of 11 beta HSD1 mRNA over the oestrous cycle. Uterine and renal message levels followed the same pattern, with the highest levels observed at dioestrus and the lowest levels at oestrus, a pattern that correlates with plasma oestrogen levels during the cycle. In both the uterus and kidney 11 beta HSD1 message levels more than halved from dioestrus to oestrus, while renal levels than doubled at metoestrus. In contrast, hepatic 11 beta HSD1 message levels at prooestrus were twice those observed at metoestrus. Ovarian levels remain constant until metoestrus when a marked decrease in message levels was seen. 11 beta HSD1 mRNA levels are thus differentially regulated in a tissue specific manner throughout the oestrous cycle.

The human 11 beta-hydroxysteroid dehydrogenase type II enzyme: comparisons with other species and localization to the distal nephron.

Effective glucocorticoid inactivation is currently thought to be an indispensable feature of mineralocorticoid target cells. The enzyme 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) inactivates glucocorticoids and prevents them from binding to the non-selective mineralocorticoid receptor. In the kidney it is the NAD dependent high affinity isoform (11 beta-HSD2) which is thought to endow specificity on the receptor. The recent cloning of the human, sheep and rabbit 11 beta-HSD2 enzymes permits a comparison of the enzyme from the three species. Human and rabbit enzymes are 87% identical and of similar length, while the human and sheep enzymes have only 75% identity. The last 12 residues in all three species were found to be highly divergent, but most of the ovine dishomology can be accounted for by the deletion of a single nucleotide toward the C-terminus of the protein resulting in a shift in reading frame generating a protein 27 residues longer than the human isoform. Numerous other deletions were also observed in this region of the sheep cDNA sequence. Furthermore, the rabbit cDNA also displayed a large degree of dishomology with the human sequence a short distance downstream from the termination codon. Conserved overlapping cytoplasmic translocation signals were observed in all three species, suggesting a topology whereby the enzyme is anchored into the endoplasmic reticulum by multiple hydrophobic regions in the N-terminus and the bulk of the 11 beta-HSD2 peptide is sited in the cytoplasm. A polyclonal antibody generated against the C-terminus of human 11 beta-HSD2 was used to localize the enzyme within the kidney. A high level of immunoreactive was observed in distal tubules and collecting ducts, localizing the enzyme to the same part of the nephron as the mineralocorticoid receptor. Moderate levels of staining were also seen in vascular smooth muscle cells. These results support the notion that 11 beta-HSD2 is an autocrine protector of the mineralocorticoid receptor and that it plays an important role in cardiovascular homeostatic mechanisms.

Up regulation of AT4 receptor levels in carotid arteries following balloon injury.

Angiotensin IV, (V-Y-I-H-P-F), binds to AT4 receptors in blood vessels to induce vasodilatation and proliferation of cultured bovine endothelial cells. This latter effect may be important not only in developing tissues but also in injured vessels undergoing remodelling. In the present study, using normal rabbit carotid arteries, we detected AT4 receptors in vascular smooth muscle cells and in the vasa vasorum of the adventitia. Very low receptor levels were observed in the endothelial cells. In keeping with the described binding specificity of AT4 receptors, unlabelled angiotensin IV competed for [125I]angiotensin IV binding in the arteries, with an IC50 of 1.4 nM, whereas angiotensin II and angiotensin III were weaker competitors. Within the first week following endothelial denudation of the carotid artery by balloon catheter, AT4 receptor binding in the media increased to approximately 150% of control tissue. AT4 receptor binding further increased in the media, large neointima and re-endothelialized cell layer to 223% at 20 weeks after injury. In view of the known trophic effects of angiotensin IV, the elevated expression of AT4 receptors, in both the neointima and media of arteries, following balloon injury to the endothelium, suggests a role for the peptide in the adaptive response and remodelling of the vascular wall following damage.

Isozymes of 11 beta-hydroxysteroid dehydrogenase: which enzyme endows mineralocorticoid specificity?

Intracellular enzymes which interconvert circulating hormones between active and inactive forms aid in regulating the biological activity of the ligand in a cell-specific manner. This is particularly important in mineralocorticoid target tissues where glucocorticoids and mineralocorticoids have equivalent affinity for the mineralocorticoid receptor. Inactivation of glucocorticoids at the 11-hydroxyl position by the action of 11 beta-hydroxysteroid dehydrogenase (11 beta-OHSD) permits the occupation of the mineralocorticoid receptor by aldosterone in the presence of much higher levels of circulating cortisol. The suppression of dehydrogenase activity allows glucocorticoids to activate the mineralocorticoid receptor, leading to classical mineralocorticoid type effects such as sodium retention and potassium excretion. A number of 11 beta-OHSDs are currently candidate protectors of the mineralocorticoid receptor. This review examines the attributes of these 11 beta-hydroxysteroid dehydrogenase isozymes and suggests reasons why a high affinity, NAD-dependent enzyme appears to be the most likely candidate to endow specificity on the mineralocorticoid receptor.

Cloning and characterization of the growth hormone-dependent insulin-like growth factor binding protein (IGFBP-3) in the rat.

We report for the first time the complete amino acid sequence for the growth hormone dependent insulin-like growth factor binding protein (IGFBP-3) in the rat. A human IGFBP-3 clone was generated using the polymerase chain reaction (PCR) and used to screen a rat liver cDNA library. cDNA clones of the rat IGFBP-3 were isolated and the full amino acid sequence deduced. The sequence begins with a putative, 26 amino acid signal peptide followed by a 265 amino acid binding protein. The amino acid sequence is over 80% homologous with the equivalent human IGFBP-3 form and shows complete conservation of 18 cysteine residues that are clustered at the amino and carboxy ends of the protein. IGFBP-3 is the binding subunit of the major circulating IGFBP in the rat, and hence the availability of precise structural data and cDNA probes provides an important opportunity for a detailed study of the control of IGFBP-3 synthesis at the level of gene expression.

Increased ACTH levels do not alter renal 11beta-hydroxysteroid dehydrogenase type 2 gene expression in the sheep.

The regulation of renal 11beta-hydroxysteroid dehydrogenase type 2 (11betaHSD2) gene expression is poorly understood. Inhibition of expression can result in hypertension. An example of this is in ectopic adrenocorticotropin (ACTH) syndrome (EAS). Inhibition of 11betaHSD2 activity is suggested by the observed increased ratio of cortisol to cortisone in both plasma and urine. To investigate whether ACTH or ACTH-dependent steroids can modulate renal 11betaHSD2 gene expression we analysed renal 11betaHSD2 mRNA levels after treatment with ACTH of 1 H and 24 H and demonstrated no change in the levels of gene expression. We have demonstrated in this study that the expression of 11betaHSD2 in the kidney is unaltered by ACTH. The reduced inactivation of cortisol by 11betaHSD2 observed in EAS is likely to be in part due to end product inhibition or substrate overload of the enzyme by endogenous substrates (cortisol, corticosterone, etc) rather than inhibition of 11betaHSD2 at the transcriptional level by either ACTH or ACTH regulated steroids.

Cloning of the 11 beta HSD type II enzyme from human kidney.

The enzyme 11 beta-hydroxysteroid dehydrogenase (11 beta HSD) converts glucocorticoids to receptor inactive metabolites. Two isoforms of the enzyme exist. 11 beta HSD1 is a low affinity NADP dependent enzyme, while 11 beta HSD2 is a high affinity NAD dependent species thought to be responsible for endowing specificity on the mineralocorticoid receptor and for protecting the fetus from high circulating levels of maternal glucocorticoids. We have recently cloned the human renal 11 beta HSD2 enzyme. In this report we show that 11 beta HSD2 potently inactivates the synthetic glucocorticoid dexamethasone, producing a single product thought to be the 11-dehydrodexamethasone metabolite. Sequence analysis shows that the new isoform is a member of the short-chain alcohol dehydrogenase superfamily (SCAD), most closely related to 17 beta HSD2 and distantly related to 11 beta HSD1.

A globin fragment, LVV-hemorphin-7, induces [3H]thymidine incorporation in a neuronal cell line via the AT4 receptor.

The AT4 receptor was characterized initially as a specific binding site for angiotensin IV, a C-terminal fragment of the vasoactive peptide angiotensin II. Recently, we found that LVV-hemorphin-7, a fragment of beta globin, is an abundant peptide in the brain and binds to the AT4 receptor with high affinity and specificity. In the neuroblastoma/glioma hybrid cell line, NG108-15, LVV-hemorphin-7 and angiotensin IV competed for 125I-angiotensin IV binding in a biphasic fashion with IC50 values of 1.2 x 10(-10) and 1.1 x 10(-9) M for the high-affinity site, respectively, and 6.7 x 10(-8) and 1.5 x 10(-8) M for the low-affinity site, respectively. Both peptides were internalized rapidly by the cells. However, LVV-hemorphin-7, but not angiotensin IV, elicited a 1.8-fold increase in DNA synthesis in a dose-dependent manner. Furthermore, co-incubation of the cells with an excess of angiotensin IV (10(-6) M) inhibited LVV-hemorphin-7-stimulated DNA synthesis. Therefore, whereas LVV-hemorphin-7 and angiotensin IV were capable of binding to the AT4 receptor, only LVV-hemorphin-7 elicited [3H]thymidine incorporation in NG108-15 cells. In contrast, angiotensin IV behaved as an antagonist. The current finding suggests that LVV-hemorphin-7 is a functional peptide in the central nervous system and in view of its abundance in neural tissue, compared with angiotensin IV, may be of significant physiological importance.