Effect of potassium depletion on proximal tubule AT1 receptor localization in normal and remnant rat kidney.

BACKGROUND: Since both potassium depletion and renal ablation result in proximal tubule hypertrophy and the angiotensin II type 1 (AT1) receptor has been localized in rat proximal tubules, we explored the possibility that the AT1 receptor intracellular distribution is modulated by potassium depletion in proximal tubular cells of 5/6 nephrectomized (Nx) rats. METHODS: Four groups of rats were studied: sham operated, potassium-depleted sham-operated rats, 5/6 Nx rats two weeks postsurgery, and potassium-depleted 5/6 Nx rats two weeks postsurgery. After the morphometry of proximal tubular cells was defined, by using immmunogold electron microscopy techniques the subcellular distribution of AT1 receptors were visualized and quantitated. RESULTS: Hypertrophy of proximal tubule cells due to both 5/6 Nx and potassium depletion was documented. Furthermore, to our knowledge for the first time, the results showed that in potassium depletion, with and without superimposed 5/6 Nx, the AT1 receptor density in proximal tubular cells was dramatically enhanced in the apical membrane, the basal membrane, and in nuclei. CONCLUSION: In normal rats and those subjected to renal ablation, these immunocytochemical data provide intracellular proximal tubule AT1 receptor localization and demonstrate loci of increased receptor density after potassium depletion.

Surviving rat distal tubule bicarbonate reabsorption: effects of chronic AT(1) blockade.

To determine the in vivo effects of chronic ANG II type 1 (AT(1))-receptor blockade by losartan (Los) on enhanced unidirectional bicarbonate reabsorption (J(HCO(3))) of surviving distal tubules, nephrectomized rats drank either water or a solution of Los, 7 days before microperfusion. J(HCO(3)) was suppressed by 50% after Los without further reduction by 5 nM concanamycin A (Conc), suggesting that Los suppresses all Conc-sensitive H(+)-ATPase pumping. Indeed, ultrastructural analysis of A-type intercalated cells revealed a 50% reduction of H(+)-ATPase immunogold labeling of the apical plasma membrane, whereas Western blotting showed that H(+)-ATPase protein levels were also reduced by one-half by Los treatment. To identify other transporters sustaining J(HCO(3)), we perfused three inhibitors simultaneously [5-(N, N-dimethyl) amiloride hydrochloride, Conc, Schering 28080] with or without prior Los treatment: J(HCO(3)) was unchanged despite marked reduction of water reabsorption. We conclude enhanced distal tubule J(HCO(3)) of surviving nephrons is largely mediated by AT(1) receptor-dependent synthesis and insertion of apical H(+)-ATPase pumps in A-type intercalated cells.

Downregulation of neuronal nitric oxide synthase in the rat remnant kidney.

Chronic renal failure is associated with disturbances in nitric oxide (NO) production. This study was conducted to determine the effect of 5/6 nephrectomy (5/6 Nx) on expression of intrarenal neuronal nitric oxide synthase (nNOS) in the rat. In normal rat kidney, nNOS protein was detected in the macula densa and in the cytoplasm and nuclei of cells of the inner medullary collecting duct by both immunofluorescence and electron microscopy. Western blot analysis revealed that 2 wk after 5/6 Nx, there were significant decreases in nNOS protein expression in renal cortex (sham: 95.42+/-15.60 versus 5/6 Nx: 47.55+/-12.78 arbitrary units, P<0.05, n = 4) and inner medulla (sham: 147.70+/-26.96 versus 5/6 Nx: 36.95+/-17.24 arbitrary units, P<0.005, n = 8). Losartan treatment was used to determine the role of angiotensin II (AngII) AT1 receptors in the inhibition of nNOS expression in 5/6 Nx. Losartan had no effect on the decreased expression of nNOS in the inner medulla, but partially increased nNOS protein expression in the cortex of 5/6 Nx rats. In contrast, in sham rats losartan significantly inhibited nNOS protein expression in the cortex (0.66+/-0.04-fold of sham values, P<0.05, n = 6) and inner medulla (0.74+/-0.12-fold of sham values, P<0.05, n = 6). nNOS mRNA was significantly decreased in cortex and inner medulla from 5/6 Nx rats, and the effects of losartan on nNOS mRNA paralleled those observed on nNOS protein expression. These data indicate that 5/6 Nx downregulates intrarenal nNOS mRNA and protein expression. In normal rats, AngII AT1 receptors exert a tonic stimulatory effect on expression of intrarenal nNOS. These findings suggest that the reduction in intrarenal nNOS expression in 5/6 Nx may play a role in contributing to hypertension and altered tubular transport responses in chronic renal failure.

K depletion stimulates in vivo HCO3 reabsorption in surviving rat distal tubules.

To evaluate whether K depletion enhances in vivo bicarbonate reabsorption (JtCO2) in surviving distal tubules (DT), we compared DT JtCO2 in five-sixths nephrectomized rats (Nx) with and without dietary K depletion (Nx-K). Furthermore, to identify possible mechanisms of increased JtCO2, we perfused inhibitors of proton secretion in both Nx and Nx-K rats. JtCO2 (102 +/- 8 pmol.min-1.mm-1) was significantly increased in Nx-K vs. Nx rats (65 +/- 7 pmol.min-1.mm-1, P < 0.05) but unaffected by 10(-6) M losartan perfusion (94 +/- 6 pmol.min-1.mm-1, P = not significant). Although 10(-5) M Sch-28080 also had no significant effect, 5 x 10(-9) M concanamycin A perfusion significantly decreased JtCO2 in Nx-K rats to 65 +/- 8 pmol.min-1. mm-1 (P < 0.05). Morphometric evaluation and H(+)-ATPase immunogold labeling of Nx-K A-type intercalated cells revealed cellular hypertrophy, elaborated apical microplicae, and enhanced H(+)-ATPase apical polarization. Accordingly, these combined studies confirm that K depletion enhances JtCO2 in surviving DT by stimulating H(+)-ATPase activity, independent of the AT1 receptor.

Gill morphology and acid-base regulation in freshwater fishes.

This review examines the recent advances in our understanding of the mechanisms of ion transport and acid-base regulation in the freshwater fish gill. The application of a combination of morphological, immunocytochemical and biochemical techniques has yielded considerable insight into the field. An important mechanism for regulation of Cl- uptake/base excretion is by morphological modification of the gill epithelium. During acidosis, the chloride cell associated Cl-/HCO3- exchanger is effectively removed from the apical epithelium because of a covering by adjacent pavement cells; this mechanism reduces base excretion and contributes to the compensation of the acidosis. In addition, acidosis induces changes in both the surface structure and ultrastructure of pavement cells. Evidence is accumulating to support the hypothesis that Na+ uptake/H+ excretion is accomplished by the pavement cell. Further, specific localization of a V-type H+-ATPase on the pavement cell epithelium and an increased expression during acidosis provides support for the model originally proposed, that this exchange is accomplished by an electrochemically coupled H+-ATPase/Na+ channel mechanism.

Expression of urokinase-type plasminogen activator and its receptor during ovarian follicular development.

Although tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1) are believed to be involved in the biochemical cascade leading to extracellular matrix degradation during ovulation, the presence and possible role of urokinase-type PA (uPA) and its receptor (uPAR) in follicular wall remodeling during follicular development are poorly understood. In the current studies, we have examined their presence in the rat ovary and compared the changes in both uPA and uPAR expression with those of tPA and PAI-1 during follicular growth in vivo. The presence of these proteins in various follicular cells at different stages of maturation was evaluated by immunolocalization and ELISA. Abundance of respective messenger RNA in granulosa cells from preantrallearly antral, midantral and preovulatory follicles and the residual ovaries was determined by Northern blot analysis. Whereas uPA transcript and protein levels were highest at the earliest stage of follicular growth examined and decreased markedly before the expected time of ovulation, the opposite was true for uPAR. In addition, tPA and PAI-1 messenger RNA abundance and protein contents were low in both granulosa and residual ovarian tissue during early follicular development but increased thereafter, reaching highest levels at the preovulatory period. These findings demonstrate for the first time the presence of uPAR in ovarian follicles and its developmental expression. The coincidental rise in uPAR and PAI-1 proteins during the preovulatory period may be important for the regulation of extracellular matrix remodelling before ovulation. The reciprocal expression of uPA and tPA during follicular development are consistent with the notion that these proteases have different biological functions in the ovary, i.e. tPA is involved in follicular wall remodelling before ovulation whereas uPA is important in extracellular matrix degradation during cell proliferation and migration that accompany follicle growth.

ANG II-dependent HCO3- reabsorption in surviving rat distal tubules: expression/activation of H(+)-ATPase.

Distal tubules (DT) from sham or five-sixths nephrectomized (Nx) rats were perfused in vivo to evaluate the hypothesis that, after Nx, endogenous angiotensin II (ANG II) modulates DT in vivo bicarbonate reabsorption (JtCO2) via H(+)-adenosinetriphosphatase (H(+)-ATPase) and Na+/H+ exchange. In Nx rats JtCO2 was increased (65 +/- 7 vs. -24 +/- 21 pmol.min-1.mm-1, P < 0.01). Both luminal and intravenous AT1-receptor blockade by losartan reduced Nx DT JtCO2 (41 +/- 6 and 34 +/- 4 pmol.min-1.min-1, respectively, P < 0.05), whereas neither 10(-9) M nor 10(-11) M ANG II luminal perfusion increased JtCO2, suggesting preexisting high endogenous ANG II levels. The Na+/H+ antiporter inhibitors 5-(N-ethyl-N-isopropyl)-amiloride and 5-(N,N-dimethyl)-amiloride were without effect. Luminal perfusion of 5 nM concanamycin A, a V-type H(+)-ATPase inhibitor, reduced Nx DT JtCO2 (45 +/- 8 pmol.min-1.mm-1, P < 0.05). In Nx A-type intercalated cells, we demonstrated cellular hypertrophy, elaboration of apical microplicae, and enhanced expression/apical polarization of H(+)-ATPase. Thus ANG II is an important determinant in sustaining brisk DT JtCO2 following Nx and is associated with enhanced expression and A-type intercalated cell apical polarization of H(+)-ATPase.

Hormonal regulation of the ligand for c-kit in the rat ovary and its effects on spontaneous oocyte meiotic maturation.

Kit ligand (KL, c-kit ligand) mRNA was detected in the ovaries of 26-day-old prepubertal rats using in situ hybridization. In antral follicles there was a gradient in the intensity of the hybridization signal across the layers of granulosa cells, with greatest intensity observed in the cumulus granulosa cells enclosing the oocyte, and less signal occurring in the granulosa cells furthest from the oocyte. In age-matched rats 40 hr after injection of pregnant mare serum gonadotropin (PMSG), the pattern of distribution of KL resembled that in the untreated ovaries, although the intensity of the hybridization signal was greater in the PMSG-primed ovaries. This morphological observation was confirmed using Northern blot analysis, which indicated that granulosa cells of PMSG-treated rats had 3.5-fold greater abundance of KL mRNA compared to untreated rats. The abundance of KL mRNA further increased to 7-fold over control levels at 6 hr after PMSG-primed rats were treated with human chorionic gonadotropin (hCG). By contrast, treatment of rats with diethylstilbestrol to stimulate follicular growth did not cause any change in the abundance of KL transcripts. To investigate a potential role for KL in oocyte meiotic maturation, fully grown oocytes were cultured for 24 hr with or without KL (50 or 500 ng/ml). The presence of KL resulted in a significant, albeit transient, delay in the progression of spontaneous meiotic maturation, using the indices of germinal vesicle breakdown and polar body formation. The inhibitory effects of KL were specifically blocked by ACK2, an antibody to the extracellular domain of the c-kit receptor. These results indicate that KL is produced in rat granulosa cells at particularly high levels in the cells closest to the oocyte and that this production may be regulated directly by gonadotropic hormones. Furthermore, KL inhibits the progression of meiosis in cultured oocytes, which suggests a possible role in the maintenance of meiotic arrest that occurs throughout oocyte growth.

The detection of the mRNAs of procollagen types I, II and III in human fetal fingers by in situ hybridization using digoxigenin-labelled oligonucleotide probes.

Messenger RNAs (mRNAs) encoding procollagen alpha 1 type I,alpha 1 type II and alpha 1 type III have been localized in paraffin sections of human fetal fingers using digoxigenin-labelled synthetic oligonucleotide probes. The probe-mRNA hybrids were visualized using an anti-digoxin antibody amplified with sandwich techniques. These protocols provided an excellent hybridization signal with minimal background noise. The sensitivity of the protocols was nearly equivalent to that seen when using isotopic cDNA probes. In human fetal fingers, intense hybridization signals for procollagen alpha 1 type I mRNA were detected in the osteoblasts and the fibroblasts of periosteum and perichondrium, the tenocytes of tendons, fibroblasts of ligaments, the synovial membrane and deeper layers of the dermis. In contrast, positive hybridization signals for procollagen alpha 1 type II mRNA were visualized in chondrocytes and the cambial layer of perichondrium. The signals for procollagen alpha 1 type III mRNA were detected in the fibroblasts of the dermis and perichondrium. The probes which have lower melting temperatures (Tm) could not detect the corresponding mRNAs.

Immunocytochemical localization of granulin-1 to mononuclear phagocytic cells of the teleost fish Cyprinus carpio and Carassius auratus.

A new class of low-molecular-weight cysteine-rich regulatory growth factors, designated granulins, has been isolated from hematopoietic tissues of a teleost fish (Cyprinus carpio) and structurally characterized. Granulin-1, the predominant form found in carp spleen, was used to raise polyclonal antibodies in rabbits and to establish a radioimmunoassay. This permitted preliminary tissue distribution studies of granulin-1 to be undertaken in carp (Cyprinus carpio) and goldfish (Carassius auratus). Granulin-1 immunoreactivity was found in the melanomacrophage centers of the spleen and head kidney. Carp tissues anatomically involved in the first line of defense against infection, including skin, gills, gut, and also heart, showed intense granulin-1 immunoreactive staining within presumptive macrophage cells. Granulin-1 immunoreactive macrophages prepared from goldfish spleen and head kidney adhered to glass slides, actively phagocytosed carbon particles, and contained granulin-1 immunoreactivity as well as abundant endogenous peroxidase activity. This study demonstrates that granulin-1 is synthesized and stored in macrophages/monocytes of spleen, head kidney, and peripheral tissues of teleost fish.

Localization of mRNA of procollagen alpha 1 type I in torn supraspinatus tendons. In situ hybridization using digoxigenin labeled oligonucleotide probe.

A tendon is predominantly composed of collagen Type I. To determine the synthesis of collagen Type I after a rotator cuff tear, an in situ hybridization technique was applied to localize cells containing procollagen alpha 1 Type I in the proximal stump of five torn supraspinatus tendons obtained during surgery. Specimens were fixed in 10% buffered formalin, embedded in paraffin, and sectioned at 6 microns. A 22 mer oligonucleotide corresponding to a sequence coding a part of human procollagen alpha 1 Type I messenger RNA (mRNA) was used as a hybridization probe. The probe was 3'-end labeled with digoxigenin-11-dUTP, and the probe-mRNA hybrids were enzymatically visualized using conventional chromogens for alkaline phosphatase. The procollagen alpha 1 type I mRNA was clearly observed in the cells near the margin of the tear. However, they were not consistently found in the vicinity of the intratendinous extension of the tear and in cells of the subacromial bursa. It is concluded that this method should be used to study the characteristics of collagen synthesis in a torn rotator cuff tendon.

Distributions and colocalization of neuropeptide Y and somatostatin in the goldfish brain.

The distributions of single- and double-labelled neuropeptide Y- (NPY-) and somatostatin-immunoreactive (SOM-IR) perikarya and processes were determined in the goldfish brain using immunoperoxidase and immunofluorescence techniques, respectively. In double-labelled material, it was evident that although these two peptides showed markedly similar distributions, they were colocalized in very few instances. A high degree of colocalization of NPY and SOM was noted in the neurons of the ventrolateral telencephalon (VI), the entopenduncular nucleus (NE) and, to a lesser extent, in the dorsocentral nucleus of the telencephalon (Dc). In Vl and NE, neurons showing NPY-IR displayed SOM-IR and vice versa. The only other instance of colocalization was that noted in the brainstem, where SOM and NPY were colocalized in the large cell bodies of the medial column of the vagal motor complex. Single-labelled SOM- and NPY-IR neurons shared a very similar distribution in various nuclei in the diencephalon and in the optic tectum. Colocalization was also noted within fibers throughout many nuclei of the telencephalon and within fibers innervating the swim bladder, one of the peripheral organs to which neurons of the medial column of the vagal motor complex project. Processes in the torus semicircularis and vagal lobe showed single-labelled immunoreactivity for both SOM and NPY in distinct laminar patterns. Large single-labelled SOM-IR terminals appeared to form pericellular baskets in the eminentia granularis of the cerebellum. Single-labelled NPY- or SOM-IR fibers were also found in the secondary gustatory nucleus and tract, the facial lobe, descending trigeminal tract, reticular formation and spinal cord. As in mammalian species, select groups of neurons in teleosts colocalize the neuropeptides SOM and NPY.

Histological changes in insulin-immunoreactive pancreatic ß-cells, and suppression of insulin secretion and somatotrope activity in brook trout (Salvelinus fontinalis) maintained on reduced food intake or exposed to acidic environment.

Immature brook trout (Salvelinus fontinalis) were randomly divided into a pH control, a pH and food control and an acid-stressed group. Fish in the first two groups were held at neutral pH and those in the last group were maintained at pH 4.2 for up to two months. The food supply to the pH and food control group was restricted to simulate the reduction in food intake demonstrated for acid-stressed trout. Plasma insulin levels were significantly decreased from 5-20 ng/ml to 1-2 ng/ml and plasma cortisol levels were significantly increased from 5-10 ng/ml to as high as 70 ng/ml in the acid-stressed brook trout. Concomitantly, a significant decrease of 21-39% in the proportion (volume density) of insulin immunoreactive ß-cells was observed within the principal pancreatic islets. Somatic growth was stunted and ultrastructural morphometry revealed the suppression of somatotrope secretory activity in the acid-stressed fish. Restriction of food supply induced a smaller but still significant decrease in circulating levels of insulin which was however not accompanied by a reduction in insulin immunoreactive ß-cells. The rise in plasma cortisol levels was not significant, and the plasma levels of glucose and protein were unaffected. Nevertheless, somatotrope secretory activity was suppressed and somatic growth was stunted. This study demonstrates for the first time the complexity of the endocrine response to acid stress and that some of the response to acid stress can be attributed to the lowering of food intake.

In situ hybridization of putative somatostatin mRNA in the brain of electric gymnotiform fish.

The distribution of somatostatin mRNA in the brain of Apteronotus leptorhynchus, a weakly electric gymnotiform fish, has been mapped by employing in situ hybridization with an oligodeoxynucleotide probe corresponding to nucleotides 1-26 of catfish somatostatin mRNA; this probe was end-labelled with digoxigenin. Most labelled cell groups were found in the telencephalon, followed by the diencephalon. This distribution is in good agreement with previous immunohistochemical investigations. In the complex of the diencephalic central-posterior/prepacemaker nucleus, a cluster of neurons controlling electrocommunicatory behavior, somatostatin mRNA could be detected only in lateral aspects, although many medial cells also stain positively with immunohistochemical techniques. This difference may be related to the growth of these neurons during adulthood.

Effects of excess iodine in chick embryo thyroid follicles: initial inhibition and subsequent hypertrophy.

The effects of excess iodine on the development of the thyroid gland of chick embryos was assessed following injections of potassium iodide prior to incubation. Iodide injection resulted in a significantly greater thyroid gland weight (goitre) on Day 18 of incubation and a delay in hatching. Histological studies of the thyroid gland on Day 12 of incubation revealed that iodide injection had inhibited thyroid follicle development. On Day 14, however, the thyroid glands of the iodide-treated embryos were indistinguishable from controls and on Day 18 the thyroid follicles of the iodide-injected embryos were clearly hypertrophied. In agreement with these light microscopical observations, electron microscopical examination showed conspicuous development of rough endoplasmic reticulum in the follicle cells of both iodide-treated 14 and 18 days old embryos and in those of the corresponding controls. Immunocytochemical studies of the pituitary of 18 days old embryos revealed a depletion of immunoreactive TSH suggesting that the iodide-induced hypertrophy of the thyroid was mediated by an activation of the thyrotropes. Iodide treatment was without effect on plasma levels of T3 and T4 for Day 18 embryos suggesting that the compensatory hypertrophy of the thyroid gland was sufficient to maintain circulating levels of thyroid hormones. The present results demonstrate that, in the embryonic chick thyroid, excess iodine produces effects which occur in two phases. The first phase consists of a transitory inhibition of the formation of follicles; it is followed by a second phase of compensatory hypertrophy resulting in goitre. The first phase probably results from a direct inhibitory effect of iodine on the developing thyroid whereas the second phase probably reflects a stimulation of the thyroid by TSH.

CRF, urotensin I, and sauvagine stimulate the release of POMC-derived peptides from goldfish neurointermediate lobe cells.

In teleost fishes, the melanotropes of the neurointermediate lobe of the pituitary gland release numerous peptides--adrenocorticotropin (ACTH), melanotropin (MSH), lipotropin (LPH), corticotropin-like intermediate lobe peptide (CLIP), and endorphin--which are derived from the precursor molecule proopiomelanocortin. Superfused, isolated, dispersed goldfish neurointermediate lobe cell columns were used to investigate the release of immunoreactive (ir) alpha-MSH and ir ACTH from goldfish melanotropes. Stimulation of neurointermediate lobe cell columns with pulses of the structurally homologous peptides, Catostomus urotensin I (UI), ovine corticotropin-releasing factor (oCRF), or sauvagine, produced a significant increase in the concomitant release of ir alpha-MSH and ir ACTH. UI was two to three times as potent as ovine CRF or sauvagine. These studies suggest that CRF- and UI-like peptides stimulate the secretory activity of teleost melanotropes.

Isolation, Amino-Acid Sequence, Synthesis and Biological Properties of Urotensin I from Hippoglossoides elassodon.

Abstract A 41-residue urotensin I neuropeptide (H-UI) was isolated from urophyses of the marine teleost Hippoglossoides elassodon (the flathead sole). The peptide was recognized by its partial cross-reactivity in a radioimmunoassay developed for Catostomus (sucker) Ul (S-UI), and was purified by reversed-phase high-performance liquid chromatography. The amino-acid sequence was shown to be H-Ser-Glu-Glu-Pro-Pro-Met-Ser-lle-Asp-Leu-Thr-Phe-His-Met-Leu-Arg-Asn-Met-lle-His-Arg-Ala-Lys-Met-Glu-Gly-Glu-Arg-Glu-Gln-Ala-Leu-lle-Asn-Arg-Asn-Leu-Leu-Asp-Glu-Val-NH(2). H-UI is 66% homologous with S-UI and 63% homologous with Cyprinus (carp) Ul (C-UI). Like S- and C-UI, H-UI is about 50% homologous with the frog skin peptide sauvagine and with Catostomus and mammalian corticotropin-releasing factors. H-UI had similar vasodilatory effects in mammals, and similar adrenocorticotropin-releasing effects (in rat and goldfish) to S-UI, C-UI, sauvagine and the corticotropin-releasing factors, but had relatively low potency (e.g. 10% to 30% of the vasodilatory potency of S- and C-UI) in all the bioassay systems studied.

TRH stimulates the release of POMC-derived peptides from goldfish melanotropes.

The release of immunoreactive (ir) alpha-MSH and ir ACTH from goldfish (Carassius auratus) melanotropes was investigated using superfused isolated dispersed neurointermediate lobe cell columns. Stimulation of neurointermediate lobe cell columns with pulses of TRH evoked dose-dependent increases in the concomitant release of ir alpha-MSH and ir ACTH. Reversed-phase high performance liquid chromatography (RP-HPLC) was used to characterize the alpha-MSH and ACTH immunoreactivities released from a neurointermediate cell column under spontaneous release conditions. Six peaks of ir alpha-MSH were revealed. Three of these peaks were identified as des-acetyl alpha-MSH, mono-acetyl alpha-MSH and di-acetyl alpha-MSH. Seven peaks of ir ACTH were revealed. Four of these peaks were tentatively identified as ACTH variants. These studies suggest that TRH stimulates the release of peptide hormones from teleost melanotropes and that the goldfish neurointermediate lobe in vitro releases numerous peptides derived from POMC.

Neuropeptides regulating the activity of goldfish corticotropes and melanotropes.

The goldfish (Carassius auratus) has proven an advantageous model for investigations of the neuroendocrine regulation of pituitary hormone secretion in teleost fishes. Investigations examining the secretion of adrenocorticotropin (ACTH) and melanocyte-stimulating hormone (MSH) from pituitary cellsin vitro have been used to identify neuropeptides influencing goldfish corticotrope and melanotrope activity. Ovine CRF, urotensin I (UI), arginine vasotocin (AVT), isotocin and angiotensins I and II stimulate the release of ACTH from corticotropesin vitro. Thyrotropin-releasing hormone (TRH), oCRF, UI and neuropeptide Y stimulate the release of MSH from melanotropesin vitro. Immunocytochemical studies have revealed the presence of separate CRF- and UI-immunoreactive perikarya in the hypothalamus suggesting the existence of two structurally similar, yet distinct, hypothalamic CRF-UI-like peptides. Interactions of AVT and CRF in the regulation of ACTH secretion is suggested from studies demonstrating the co-localization of AVT- and CRF-immunoreactivities in perikarya of the preoptic-hypophyseal system. These investigations demonstrate that the secretory activity of goldfish corticotropes and melanotropes is influenced by a diversity of neuropeptides of hypothalamic origin.

Angiotensin II stimulation of teleost adrenocorticotropic hormone release: interactions with urotensin I and corticotropin-releasing factor.

The release of adrenocorticotropic hormone (ACTH) from dispersed goldfish anterior pituitary cells was examined in order to determine whether human angiotensin II (AII) would potentiate the ACTH-releasing activity of urotensin I (UI) and ovine corticotropin-releasing factor (oCRF), peptides with a sequence homology of greater than 50%. In mammals, AII has a slightly enhancing or potentiating effect on CRF-stimulated ACTH release. In the present investigations, concentrations of AII (0.5 and 1 nM), and of UI (1 nM) or oCRF (3 nM), which elicit moderate increases in ACTH release, were tested alone and in combination. The ACTH-releasing activities of AII and UI combined, or of AII and oCRF combined, showed no potentiation and in fact were less than additive. It was concluded that AII does not potentiate the ACTH-releasing activity of either UI or oCRF observed with goldfish anterior pituitary cells in vitro.