The biological activity of diuretic factors in Rhodnius prolixus.

The rapid post-feeding diuresis of Rhodnius prolixus is under neurohormonal control and involves the integrated activity of the crop, Malpighian tubules and hindgut. One of the factors which is involved in this rapid diuresis is serotonin, however a peptide(s) is also considered to be involved. In other insects, corticotropin releasing factor (CRF)-like and kinin-like, calcitonin-like peptides and CAP(2b) have been demonstrated to be diuretic factors/hormones. In the present study, serotonin and CRF-like peptides increased secretion rate and cAMP content of Rhodnius Malpighian tubules, while the kinin-like peptides tested did not increase secretion rate or cAMP content of the tubules. Extracts of the CNS were processed and several HPLC fractions revealed kinin-like immunoreactivity but these fractions did not increase secretion rate when tested on Malpighian tubules. However, these same fractions did possess activity when tested on the hindgut contraction assay. In addition, material eluting at higher acetonitrile concentrations from the HPLC increased secretion and cAMP content of Rhodnius Malpighian tubules. This material eluted at concentrations of acetonitrile consistent with the elution time of CRF-like peptide standards. Synergism was demonstrated using the pharmacological agent forskolin and serotonin, tested on the rate of secretion of Rhodnius Malpighian tubules, in agreement with data of Maddrell et al. As well, synergism could be demonstrated using mesothoracic ganglionic mass (MTGM) homogenates and serotonin at some concentrations of serotonin. However, combinations of CRF-like material and serotonin increased secretion additively, not synergistically. Kinin-like peptides, tested along with CRF-like material and serotonin, at low concentrations, did not increase secretion above that of those factors tested alone.

Differential colocalization of estrogen receptor beta (ERbeta) with oxytocin and vasopressin in the paraventricular and supraoptic nuclei of the female rat brain: an immunocytochemical study.

Evidence exists for the localization of the newly identified estrogen receptor beta (ERbeta) within the rat paraventricular nucleus (PVN) and supraoptic nucleus (SON), regions which lack ERalpha. Presently, we investigate whether ERbeta-like-immunoreactivity (-ir) is found within cells of several major neuropeptide systems of these regions. Young adult Sprague-Dawley rats were ovariectomized (OVX), and 1 week later half of the animals received estradiol-17beta (E). Dual-label immunocytochemistry was performed on adjacent sections by using an ERbeta antibody, followed by an antibody to either oxytocin (OT), arginine-vasopressin (AVP), or corticotropin releasing hormone. Nuclear ERbeta-ir was identified within SON and retrochiasmatic SON, and in specific PVN subnuclei: medial parvicellular part, ventral and dorsal zones, dorsal and lateral parvicellular parts, and in the posterior magnocellular part, medial and lateral zones. However, the ERbeta-ir within magnocellular areas was noticeably less intense. OT-/ERbeta-ir colocalization was confirmed in neurons of the parvicellular subnuclei, in both OVX and OVX+E brains ( approximately 50% of OT and 25% of ERbeta-labeled cells between bregma -1.78 and -2.00). In contrast, few PVN parvicellular neurons contained both AVP- and ERbeta-ir. As well, very little overlap was observed in the distribution of cells containing corticotropin releasing hormone- or ERbeta-ir. In the SON, most nuclear ERbeta-ir colocalized with AVP-ir, whereas few OT-/ERbeta-ir dual-labeled cells were observed. These findings suggest that estrogen can directly modulate specific OT and AVP systems through an ERbeta-mediated mechanism, in a tissue-specific manner.

Isolation and identification of a diuretic hormone from the mealworm Tenebrio molitor.

A diuretic hormone of unusual structure was isolated from extracts of whole heads of the mealworm Tenebrio molitor. The hormone is a 37-aa peptide of 4371 Da, with the sequence SPTISITAPIDVLRKTWEQERARKQMVKNREFLNSLN. This peptide increases cAMP production in Malpighian tubules of T. molitor. The amino acid sequence reveals that this peptide is a member of the family of sauvagine/corticotropin-releasing factor/urotensin I-related insect diuretic hormones. The C-terminal sequence of this peptide is quite different from other members of this family, which have a hydrophobic C terminus (isoleucinamide or valinamide). When aligned comparably, T. molitor diuretic hormone has a more hydrophilic C terminus, leucylasparagine (free acid). In contrast to all other known diuretic hormones of this family, this peptide has exceptionally low stimulatory activity on cAMP production in Malpighian tubules of Manduca sexta. However, at nanomolar concentrations it stimulates cAMP production in Malpighian tubules of T. molitor. Diuretic hormones of this family have been isolated previously from Lepidoptera, Orthoptera, Dictyoptera, and Diptera. This appears to be the first diuretic hormone isolated from a coleopteran insect.

Plasma levels of corticotropin-releasing hormone in hypothalamic-pituitary-adrenal disorders and chronic renal failure.

Plasma levels of corticotropin-releasing hormone (CRH) were measured in hypothalamic-pituitary-adrenal disorders and chronic renal failure to investigate the clinical significance of plasma CRH. The mean plasma CRH level in normal subjects (N = 26) was 1.64 +/- 0.43 pmol/l (normal range 0.77-2.5 pmol/l). Four of six patients with hypothalamic disorders receiving hydrocortisone supplementation had a low plasma CRH level. Two of six patients with Sheehan's syndrome had a low plasma CRH level whereas one patient had a high plasma CRH level. Two patients with Cushing's syndrome had a low plasma CRH level whereas two patients with Cushing's disease had a normal plasma CRH level. Six of 19 patients receiving prednisolone therapy had a low plasma CRH level. The mean plasma CRH level in this group was 0.97 +/- 0.34 pmol/l, which is significantly lower than that in the normal group. In this group, significant correlation was seen between plasma CRH and adrenocorticotropin levels. Eleven of 21 patients with chronic renal failure undergoing hemodialysis had a high plasma CRH level. Just after hemodialysis the plasma CRH levels decreased in 15 of 20 patients, while plasma adrenocorticotropin and cortisol levels increased in 13 of 19 patients and in 15 of 20 patients, respectively. Immunoreactive CRH in plasma measured both before and after hemodialysis eluted similarly on reversed-phase high-performance liquid chromatography. These results suggest that the plasma CRH level is at least partially suppressed by a chronically elevated plasma glucocorticoid level and that CRH in plasma is partially removed by hemodialysis.

Structural characterization and localization of corticotropin-releasing factor in testis.

To sequence and thereby definitively characterize corticotropin-releasing factor (CRF)-like material from a representative peripheral tissue, CRF was obtained from 76 ovine testes. The novel extraction procedure involved use of an immunoaffinity column to which a high-affinity CRF monoclonal antibody was attached as well as fast protein liquid chromatography. The complete sequence was elucidated by gas-phase sequencing, carboxyamidopeptidase digestion and cyanogen bromide cleavage. Aside from microheterogeneity at position 39, all the other amino acids were identical to ovine hypothalamic CRF. Additionally, in immunohistochemical studies in the rat, CRF was localized to the Leydig cell. These findings along with related observations by ourselves and others are compatible with the hypothesis that CRF plays a significant local role, possibly by paracrine or autocrine mechanisms.

Isolation and characterization of a corticotropin-releasing hormone-like peptide from human placenta.

Immunoreactive CRH was detected in extracts of human term placentae [5.2 +/- 0.8 (+/- SE) pmol/g wet wt; n = 9]. Molecular sieve chromatography revealed three size classes of immunoreactive CRH. The major species eluted with the Kav of synthetic rat CRH; the minor species had apparent mol wt (MW) of 18,000 and 8,000. A placental CRH-(1-41)-sized peptide was isolated by fractional acetone precipitation, molecular sieve chromatography, and sequential reverse phase high performance liquid chromatography steps. This peptide had the same chromatographic behavior as did rat CRH in all high performance liquid chromatographic isolation steps as well as the same UV absorbance to immunoreactive CRH ratio after the final purification step. Purified placental CRH stimulated ACTH release from anterior pituitary tissue in a dose-dependent manner and was equipotent with synthetic rat CRH. Partial sequencing indicated that 32 amino acids of this peptide are identical to those of rat and human CRH (sequence deduced from genomic sequence), and comparative peptide mapping with rat CRH provided further evidence that the placental CRH-like peptide is very homologous if not identical to CRH. The high mol wt placental CRH fractions also were partially purified by acetone precipitation, immune affinity chromatography, and gel filtration. Neither of these materials [big form (MW, 18,000) or intermediate form (MWr, 8,000)] stimulated ACTH release from rat pituitary tissue in vitro. Limited trypsin digestion of the highest MW CRH, followed by gel filtration analysis, resulted in conversion to the smaller [8,000 MW-sized and CRH-(1-41)-sized] forms. The detection of a CRH-like peptide in placenta together with our previous demonstration of plasma immunoreactive CRH in pregnant women suggest that the placenta synthesizes and secretes CRH into the maternal circulation.

Synthesis, purification and biological evaluation of porcine corticotropin-releasing factor.

The 41-residue sequences of recently identified porcine corticotropin-releasing factors [Ile40]pCRF and [Asn40]pCRF were assembled on a benzhydrylamine resin support. Deprotection and cleavage from the resin were accomplished by HF treatment. The crude peptides were purified by HPLC. The homogeneity of the final materials, obtained in 0.2% and 0.4% overall yield for [Ile40]pCRF and [Asn40]pCRF respectively, was assessed after the isolation by HPLC and amino acid analysis. Both sequences of the synthetic 41-residue pCRF stimulated the release of corticotropin (ACTH) from superfused rat pituitary cells on a column, the responses being related to a log-dose of CRF in the range of 1-20 ng/ml. [Ile40]pCRF and [Asn40]pCRF also augmented the in vivo release of ACTH in rats pretreated with chlorpromazine, morphine and Nembutal. [Ile40]pCRF appeared to be equipotent to ovine CRF and about twice as active as [Asn40]pCRF. The data indicate that synthetic porcine [Ile40]pCRF and [Asn40]pCRF have high biological activity.

Purification and characterization of peptides with corticotropin-releasing factor activity from porcine hypothalami.

Ten polypeptides that stimulated the release of corticotropin from superfused rat pituitary cells and that are structurally related to porcine corticotropin-releasing factor were isolated from porcine hypothalami. The purification was carried out by gel filtration followed by reversed-phase HPLC using trifluoroacetic acid or heptafluorobutyric acid as the ion-pairing agent in water/acetonitrile solvent systems. The purified peptides were homogeneous by chromatography and by sequence analysis. One major polypeptide was characterized. Its structure is -H-Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Gl u-Val -Leu-Glu-Met-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys -Leu-Met-Glu-Asn-Phe-NH2 [Patthy, M., Horvath, J., Mason-Garcia, M., Szoke, B., Schlesinger, D. H. & Schally, A. V. (1985) Proc. Natl. Acad. Sci. USA 82, 8762-8766]. This 41-amino acid sequence is thought to represent porcine corticotropin-releasing factor. Based on automated gas-phase sequencing of the intact and CNBr-cleaved peptides, amino acid analysis, and carboxypeptidase Y digestion, the other nine polypeptides were found to be structurally similar to this 41-amino acid sequence. Modifications of this structure include deamidation of glutamine at position 26 or 29, oxidation of methionine at positions 21 and/or 38, a blocked N terminus, and deletion of phenylalanine amide at the C terminus. Eight of these nine modified peptides retained significant corticotropin-releasing factor activity as shown by the stimulation of corticotropin release from superfused rat and pig pituitary cells. Some of these peptides may be present in pig hypothalami, while the others could have been produced during the isolation.

Discordances in the temporal pattern of the ACTH/beta-endorphin releasing effects of synthetic CRFs and partially purified bovine CRF in a superfusion system.

Temporal characteristics of ACTH and beta-endorphin secretion induced by bovine hypothalamic CRF-A (void volume) and CRF-B (Kav = 0.583) separated by Sephadex G-100 were compared to those of synthetic ovine or rat CRF, sauvagine and vasopressin. Dispersed cells or minced fragments of rat adenohypophyses perifused in a column were exposed to various secretagogues, and ACTH and/or beta-endorphin concentrations of the effluent were measured by radioimmunoassays. CRF-A or CRF-B induced an immediate brisk rise of ACTH and beta-endorphin within 1 min after initiation of CRF perifusion. The maximum rate of ACTH or beta-endorphin secretion was reached 1-2 min later. Hormone secretion persisted throughout a 10-min exposure to these secretagogues. More than 80% of the total ACTH or beta-endorphin secretion induced by 10-min perifusion with bovine CRF occurred during exposure to CRF. With 10-min perifusion with 300 ng/ml ovine or rat CRF, the onset of the major CRF-stimulated ACTH or beta-endorphin secretion was markedly delayed compared to that following bovine CRF. During perifusion with ovine or rat CRF, a modest slow increase in ACTH or beta-endorphin secretion was observed. More than 60-70% of the total ACTH or beta-endorphin secretion induced by 10-min perifusion with rat or ovine CRF occurred after CRF withdrawal. The ACTH secretory patterns for sauvagine were very similar to those for synthetic rat or ovine CRF. These results suggest some qualitative differences between partially purified bovine CRF and synthetic ovine or rat CRF.

Isolation and amino acid sequence of corticotropin-releasing factor from pig hypothalami.

A polypeptide was isolated from acid extracts of porcine hypothalami on the basis of its high ability to stimulate the release of corticotropin from superfused rat pituitary cells. After an initial separation by gel filtration on Sephadex G-25, further purification was carried out by reversed-phase HPLC. The isolated material was homogeneous chromatographically and by N-terminal sequencing. Based on automated gas-phase sequencing of the intact and CNBr-cleaved peptide and on carboxypeptidase Y digestion, the primary structure of this 41-residue polypeptide was determined to be Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Val -Leu-Glu-Met-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys -Leu-Met-Glu-Asn-Phe-NH2. Porcine corticotropin-releasing factor (CRF) shares a common amino acid sequence (residues 1-39) with rat and human CRF and differs from these only in positions 40 and 41. However, isoleucine was also present at position 40 in porcine CRF, but in a smaller percentage than asparagine. The sequence of porcine CRF shows 83% homology with ovine CRF. Porcine CRF markedly stimulated the release of corticotropin from superfused rat and pig pituitary cells. The biological activity and close structural relationship to CRFs of other species indicate that the peptide isolated represents porcine CRF.

In vivo and in vitro comparisons of biological activities of bovine, ovine and rat CRF (corticotrophin-releasing factor).

The biological activity of partially purified bovine hypothalamic CRF (corticotrophin- releasing factor) was compared to those of synthetic CRFs (ovine, rat) sauvagine and vasopressin in vivo and in vitro. ACTH-primed hypophysectomized rats with heterotopically transplanted pituitaries and medial basal hypothalamic ablation (H-T + MBHA ), and intact rats pre-treated with chlorpromazine, morphine and Nembutal (C-M-N) were used for in vivo CRF assays. Perifused rat adenohypophyseal fragments were employed for in vitro studies. CRF-A (void volume fractions, 'big' CRF) and CRF-B (Kav = 0.583) purified from bovine hypophyseal stalk, synthetic ovine and rat CRF, and sauvagine all induced significant stimulation of ACTH and/or corticosterone secretion in these systems. Synthetic ovine and rat CRF and sauvagine showed comparable CRF potency. The CRF dose-response slopes for bovine CRF were somewhat steeper than those for ovine CRF or sauvagine in the in vitro system. Vasopressin had the least steep dose-response slope. Intravenous bolus administration of ovine CRF caused a more prolonged (greater than 20 min) elevation of plasma ACTH compared to a relatively short duration after bovine CRF-A. These data suggest that bovine hypothalamus contains substance(s) which exhibits different CRF characteristics from those of ovine CRF.

Immunoreactive corticotropin and corticotropin-releasing factor in human hypothalamus, adrenal, lung cancer, and pheochromocytoma.

Immunoreactive corticotropin-releasing factor (I-CRF) and ACTH (I-ACTH) were examined using RIA, immunoaffinity chromatography, and gel filtration chromatography in human hypothalamus, adrenal (cortex and medulla), lung cancer, and pheochromocytoma. I-CRF and I-ACTH were present in these tissues. Gel filtration of I-ACTH in the adrenal, pheochromocytoma, and lung cancer showed the presence of larger amounts of I-ACTH with large molecular weight forms in contrast to the hypothalamus. Gel filtration of I-CRF in these tissues showed the main peak eluted at the position of synthetic rat CRF. High performance liquid chromatography of this main peak showed two components which eluted in the positions of synthetic rat CRF and oxidized CRF. These elution positions were the same in all tissues and identical with those in the hypothalamus. These results suggest the presence of I-ACTH and I-CRF in these tissues and that CRF outside the brain is identical to hypothalamic CRF.

Characterization of rat hypothalamic corticotropin-releasing factor.

A polypeptide was purified from rat hypothalamic extracts on the basis of its high intrinsic activity to release corticotropin (ACTH) from cultured rat anterior pituitary cells and its immunoactivity in a radioimmunoassay directed against the NH2 terminus (residues 4-20) of ovine hypothalamic corticotropin-releasing factor (CRF). Based on Edman degradation, peptide mapping, and amino acid analysis, the primary structure of this rat CRF was established to be: H-Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Met-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-Met-Glu-Ile-Ile-NH2. The hypophysiotropic potency of synthetic rat CRF did not deviate significantly from the potencies of the isolated native peptide or of synthetic ovine CRF. The close structural relationship between rat and ovine hypothalamic CRF is indicated by an 83% sequence homology.