K. Lederis

Distribution and efferent projections of corticotropin-releasing factor-like immunoreactivity in the rat amygdaloid complex.

Using cobalt-enhanced immunohistochemistry, the tracing of retrograde transport of horseradish peroxidase (HRP) and experimental manipulations, a widespread localization of corticotropin-releasing factor-like immunoreactive (CRFI) structures in the rat amygdaloid complex, and CRFI-containing pathways from the amygdala to the lower brainstem, bed nucleus of the stria terminalis (bst) and ventromedial nucleus of the hypothalamus (VMH) have been demonstrated. By means of cobalt-enhanced immunohistochemistry, CRFI cells were detected in almost all the regions of the amygdala, including the central amygdaloid nucleus (Ce), basolateral amygdaloid nucleus (B1), intra-amygdaloid bed nucleus of the stria terminalis (Abst), medial amygdaloid nucleus (Me), amygdalohippocampal area (Ahi), posterior cortical amygdaloid nucleus (Aco), lateral amygdaloid nucleus (La), anterior amygdaloid area (AAA) and basomedial amygdaloid nucleus (Bm). Neural processes with CRFI were found in all of the above areas. The greatest density of CRFI fibres was observed in the Ce, the Me and Ahi. Unilateral lesions located in the Ce and adjacent areas caused an ipsilateral decrease in CRFI fibre number in the lateral hypothalamic area (LH), mesencephalic reticular formation (RF), dorsal (Dpb) and ventral (Vpb) parabrachial nuclei, mesencephalic nucleus of the trigeminal nerve (MeV) and in the lateral division of the bst (bstl). In addition, ipsilateral CRFI fibres decreased in number in the core and shell of the VMH after unilateral lesions of the corticomedial amygdala (CoM) and ventral subiculum (S). These findings suggest that the CRFI cells in the Ce and adjacent areas innervate the Dpb, Vpb and MeV through the LH and RF; the CRFI fibres in the bstl are supplied by the Ce and adjacent areas; and the CoM and S give rise to the CRFI fibres to the VMH. The distribution of retrogradely transported HRP has confirmed these projections. Furthermore, combined HRP and immunohistochemical staining has demonstrated double labeled cells in the Ce following HRP injection into the Dpb, Vpb, MeV and bstl. This provides direct evidence for the amygdalofugal CRF-containing projections to the lower brainstem and bstl. Double-labeled cells were not seen in the CoM and S after HRP injection into the VMH.

Isolation and amino acid sequence of urotensin I, a vasoactive and ACTH-releasing neuropeptide, from the carp (Cyprinus carpio) urophysis

Urotensin I (UI), a 41-residue mammalian hypotensive and fish or mammalian corticotropin-releasing peptide, isolated from 0.1 N HCI extracts of urophyses of the carp (Cyprinus carpio) was purified and the amino acid sequence was determined to be: H-Asn-Asp-Asp-Pro-Pro-Ile-Ser-Ile-Asp-Leu-Thr-Phe-His-Leu-Leu- Arg-Asn-Met-Ile-Glu-Met-Ala-Arg-Asn-Glu-Asn-Gln-Arg-Glu-Gln-Ala-Gly-Leu-Asn-Arg-Lys-Tyr-Leu-Asp-Glu-Val-NH2. When the extraction procedure included heating at 100 degrees C for 15 min, UI was cleaved at a highly acid labile Asp-Pro bond to give the fully active UI (4-41). Urotensin I shows close structural and biological homology with the recently isolated ovine hypothalamic corticotropin-releasing factor (CRF) and the frog skin peptide sauvagine and thus may be considered an evolutionary prototype of unique mammalian-hypotensive and vertebrate corticotropin-releasing factors.

Functional receptors for fish neuropeptide urotensin II in major rat arteries

Receptor binding of fish neuropeptide urotensin II (UII) was characterized in membranes isolated from major rat arteries. Monoiodinated UII radioligand (125I-UII) was prepared and purified using high-pressure liquid chromatography (HPLC). The contractile potency of iodinated UII (I-UII) on rat thoracic aorta strips was somewhat lower than that of native UII. The binding of 125I-UII to the membrane preparations of rat thoracic aorta was saturable, specific and time-dependent. Scatchard analysis indicated a single population of binding sites with an apparent dissociation constant of 5.9 x 10(-9) M. The calculated maximal number of binding sites was about 155 fmol/mg protein. The specific binding to the membrane preparations from the abdominal aorta and mesenteric artery was about 27 and 8%, respectively, of those in the thoracic aorta, which corresponds to the order of contractile potency of UII on rat blood vessels: thoracic aorta greater than abdominal aorta greater than mesenteric artery. The displacement of 125I-UII binding by the UII peptide or its fragments (UII-(5-12), UII-(6-12) and UII-(6-11] were also comparable to their contractile effects on rat thoracic aorta strips (UII greater than UII-(5-12) greater than UII-(6-12) much greater than UII-(6-11]. These results suggest that the fish neuropeptide, UII, can induce contraction of rat vascular tissue by interacting with its functional receptors.

In vitro and in vivo ACTH-releasing activity of ovine CRF, sauvagine and urotensin I

The ability of three homologous peptides, ovine corticotropin-releasing factor (CRF), sauvagine and urotensin I, to release ACTH was examined in vitro and in vivo. All three peptides exhibit statistically equivalent potencies to stimulate ACTH secretion both by cultured pituitary cells and in pharmacology blocked rats. These results suggest that all three peptides evolved from a common mammalian precursor that possessed high hypophysiotropic potency.

Characterization of the relaxant effects of vasoactive intestinal peptide (VIP) and PHI on isolated brain arteries

We have studied the vasorelaxant properties of vasoactive intestinal peptide (VIP) using helical strips of bovine, porcine and human brain arteries in vitro. The resting tension of the arterial strips was increased during experiments by prostaglandin F2 alpha or KCl so as to increase the magnitude of the relaxant response to VIP. Arteries supplying different regions of the bovine brain responded potently to VIP with ED50 values of 1.8 nM, 2.3 nM, 6.8 nM and 9.0 nM for the middle, anterior and posterior cerebral arteries and the basilar artery, respectively. The porcine basilar artery and branches of the human middle cerebral artery responded to VIP with ED50 values of 4.2 nM and 1.6 nM, respectively. The homologous neuropeptide, PHI, relaxed the bovine middle cerebral and porcine basilar arteries less potently than did VIP, with ED50 values for PHI being 11 nM and 43 nM, respectively. However, PHI elicited in the two arteries a maximal vasodilatory response of similar magnitude as did VIP. The other homologous peptides, human pancreatic growth hormone releasing factor 1-40 [hpGRF 1-40], secretin, and glucagon, and the VIP fragments, VIP 1-12 and VIP 10-28, were completely inactive. In contrast, VIP, which had been oxidized to VIP-(Met17 sulfoxide) or VIP-(Met17 sulfone), retained full activity. These structure-activity relationships for relaxation of brain arteries are consistent with previous studies of other biological responses to VIP.

Co-localization of the immunoreactivities of corticotropin-releasing factor and arginine vasotocin in the brain and pituitary system of the teleost Catostomus commersoni

SummaryThe peroxidase-antiperoxidase immunocytochemical procedure was used to study the distribution of ovine corticotropin-releasing factor (CRF) and arginine vasotocin (AVT) immunoreactivities sequentially in the same sections or in adjacent sections of the brain and pituitary of Catostomus commersoni. It was found that all CRF-immunoreactive (IR) neurons in the nucleus preopticus (NPO) also contained AVT immunoreactivity. Co-localization of both immunoreactivities was also observed in fibres forming the preoptic-pituitary tract and in the neurohypophyseal digitations, the IR-CRF and IR-AVT fibres projecting mainly to the neurointermediate lobe (NIL) of the pituitary. An additional population of exclusively IR-AVT neurons and fibres in the NPO, preoptic-pituitary tract and NIL was also observed. Exclusive CRF-immunostaining was found in neurons of the nucleus lateralis tuberis (NLT), in fibres distributed in some diencephalic nuclei and in the neurohypophyseal digitations in the region of the rostral pars distalis (RPD). These results suggest (i) that CRF- and AVT-like substances, present in NIL fibres (probably originating in the NPO), may have an integrated role in the release of the cell products from the pars intermedia, and (ii) that the control of corticotrops in the rostral pars distalis, innervated exclusively by IR-CRF fibres (probably originating in the NLT), does not require a simultaneous presence of CRF- and AVT-like substances.

TELEOST ISOTOCIN RECEPTOR : STRUCTURE, FUNCTIONAL EXPRESSION, MRNA DISTRIBUTION AND PHYLOGENY

A cDNA encoding a receptor for the oxytocin‐related peptide isotocin has been identified by screening a lambda gt11 library constructed from poly(A)+ RNA of the hypothalamic region of the teleost Catostomus commersoni. The probe used was obtained by PCR amplification of white sucker genomic DNA using degenerate primers based on conserved sequences in the mammalian receptor counterparts. The full‐length cDNA specifies a polypeptide of 390 amino acid residues that displays the typical hydrophobicity profile of a seven transmembrane domain receptor and which exhibits greatest similarity to mammalian oxytocin receptors. Oocytes that express the cloned receptor respond to the application of isotocin by an induction of membrane chloride currents indicating that it is coupled to the inositol phosphate/calcium pathway. The isotocin receptor (ITR) can also be activated by vasotocin, mesotocin, oxytocin and Arg‐vasopressin, although these have lower potencies than isotocin. ITR‐encoding mRNA has been detected in brain, intestine, bladder, skeletal muscle, lateral line, gills and kidney indicating that this receptor may mediate a variety of physiological functions.

Localization of urotensin I- and corticotropin-releasing factor-like immunoreactivity in the central nervous system of Catostomus commersoni

The distribution of urotensin I (UI) and corticotropin-releasing factor (CRF) immunoreactive (IR) structures was studied in the central nervous system (CNS) of the white sucker using the peroxidase-antiperoxidase immunocytochemical procedure. The close sequence homology between both peptides resulted in a high degree of crossreactivity. This was resolved by saturating the antisera solutions with heterologous antigens and specificity tests were done by adding excess of homologous peptides. UI immunoreactivity was seen in all of the identifiable caudal spinal cord neurosecretory cells, in their processes projecting to the urophysis, in thin beaded fibres coursing along the spinal cord, in brain stem, hypothalamus, proximal pars distalis and, especially, in the telencephalon. Some IR-UI specific and IR-CRF specific parvocellular neurons were also identified in the caudo-ventral tuberal region and ventral telencephalon. The IR-CRF was mainly present in parvocellular and magnocellular perikarya of the nucleus preopticus and in the preoptic-neurohypophysial pathway. Dense networks of IR-CRF reacting beaded fibres were also located in the lateral and posterior recessus nuclei. In the pituitary, IR-CRF fibre bundles were seen mainly in the neurointermediate lobe and in the rostral pars distalis. The cells of origin of the extraurophyseal system of IR-UI fibres in the sucker CNS have not been identified. The distribution of CRF immunostaining correlates well with the documented knowledge of CNS structures involved in the control of ACTH secretion in the goldfish. The probability of the occurrence of two UI-CRF related molecules, or of two different forms resulting from a common precursor molecule, forming two separate neuronal systems in the sucker CNS seems likely.

Amunine (Ovine CRF), Urotensin I and Sauvagine, Three Structurally-Related Peptides, Produce Selective Dilation of the Mesenteric Circulation

Three synthetic peptides, urotensin I, sauvagine and ovine CRF, have less than 50% sequence homology. In the dog, all three produce selective mesenteric dilation, which appears to be solely responsible for a decrease in peripheral resistance and systemic arterial blood pressure. Urotensin I and sauvagine were found to be 5-10 times more potent than CRF.

Subcellular localization and characterization of vasopressin binding sites in the ventral septal area, lateral septum, and hippocampus of the rat brain.

[Arg8]‐Vasopressin (AVP) has been shown to exert characteristic central physiological actions in the ventral septal area of the rat brain. This study reports the characterization of receptors for AVP in synaptic plasma membranes prepared from the ventral septal area, the lateral septum, and the hippocampus. Binding of [3H]AVP was temperature and time dependent, linearly related to protein concentration, saturable, and specific. Scatchard plot analysis suggested the presence of a population of binding sites in the three brain areas with dissociation constants and maximal binding capacities, respectively, of 1.06 ± 0.39 nM and 24.0 ± 7.01 fmol/mg of protein (mean ± SEM; n = 3) for the ventral septal area, 0.92 ± 0.13 nM and 47.0 ± 4.96 fmol/mg of protein (n = 3) for the lateral septum, and 0.91 ± 0.14 nM and 25 ± 5.02 fmol/mg of protein (n = 3) for the hippocampus. In all three brain regions, the rank order of potencies of several vasopressin analogs, unrelated peptides, and other compounds for competitive displacement of ligand indicated a receptor with properties resembling those of the V1‐like receptor for AVP. These data document the presence of a high‐affinity, V1‐like vasopressin receptor in the rat ventral septal area for which the pharmacological properties are similar to those previously reported in physiological studies.