Additive effects of epinephrine and corticotropin-releasing factor (CRF) on adrenocorticotropin release in rat anterior pituitary cells

Rabbit antibody was prepared against NADPH-cytochrome c reductase of Tetrahymena microsomes. When examined by the Ouchterlony double diffusion test, anti-NADPH-cytochrome c reductase immunoglobulin formed a single precipitation line with Tetrahymena reductase but not rat liver one. The antibody inhibited the NADPH-cytochrome c reductase activity of Tetrahymena microsomes, but it did not affect either NADH-ferricyanide or NADH-cytochrome c reductase activity of Tetrahymena microsomes. The NADPH-dependent desaturation of stearoyl-CoA in Tetrahymena microsomes was inhibited by anti-reductase immunoglobuline, while the NADH-dependent desaturation was affected by neither anti-reductase nor control immunoglobuline. It was suggested that the temperature associated-alteration of NADPH-cytochrome c reductase activities would be important for regulation of microsomal NADPH-dependent desaturase activities in Tetrahymena which contains no cytochrome P-450.     

Comparison of the biologic actions of corticotropin-releasing factor and sauvagine

Corticotropin-releasing factor (CRF), a peptide isolated from ovine hypothalamus, and sauvagine, a peptide isolated from frog skin, share significant structural homology and elicit a number of similar biological responses. CRF is more potent than sauvagine in stimulating pituitary ACTH secretion. Sauvagine, however, is 5–10 times more potent than CRF to act within the brain to increase plasma levels of catecholamines and glucose and to elevate mean arterial pressure. Sauvagine is likewise more potent than CRF to act outside the brain to increase superior mesenteric artery flow and plasma glucose concentrations and to decrease mean arterial pressure. CRF and sauvagine produce important effects representative of biologically active peptides.     

Role of cyclic AMP in corticotropin releasing factor mediated ACTH release

To elucidate the role of cAMP in the secretion of ACTH, the effect of (1) three phosphodiesterase inhibitors, (2) forskolin, and (3) 8Bromo-cAMP, on CRF mediated ACTH release was studied in rat pituitary cell culture. The action of glucocorticoids on CRF induced cAMP accumulation and ACTH release was investigated. Isobutyl-methylxanthine (IBMX), caffeine, and forskolin augmented the release of ACTH induced from CRF 1.0 nM by 17%, 39%, and 20%, respectively. Also IBMX and caffeine potentiated CRF 10 nM stimulated ACTH release by 32% and 20%. Doses of forskolin and 8Bromo-cAMP, which alone stimulate large amounts of ACTH release, did not increase the amount of ACTH released from CRF 100 nM stimulated cells. Cortisol (500 nM) and corticosterone (500 nM) inhibited CRF induced intracellular cAMP by 39% and 26% while inhibiting pituitary ACTH release by 40% and 52%. In conclusion, cAMP plays an important role in the mechanism of ACTH secretion and it appears the final intracellular mechanism of CRF stimulated ACTH is via cAMP. Also, glucocorticoids exert their inhibitory influence prior to cAMP generation.     

Isolation and characterization of caprine corticotropin-releasing factor

The hypophysiotropic peptide, corticotropin-releasing factor (CRF) was isolated from caprine hypothalamic median eminence tissue by means of acid extraction, immunoaffinity chromatography, gel filtration and two steps of reverse-phase high-performance liquid chromatography (RPLC). Amino acid sequence determination using a gas-phase sequencer established the primary structure of the first 39 residues from the NH2-terminus. The nature of the COOH-terminal dipeptide was elucidated by Staphylococcus aureus V8 protease digestion of the native peptide, dansylation of the digest and comparative RPLC studies with the dansylated dipeptides Ile-Ala-NH2, Ile-Ala-OH, Ala-Ile-NH2 and Ala-Ile-OH. The complete structure of the peptide was established as: H-Ser-Gln-Glu-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu- Val-Leu-Glu-Met-Thr-Lys-Ala-Asp-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn- Arg-Lys-Leu-Leu-Asp-Ile-Ala-NH2, which is identical to that of ovine CRF.     

Effect of angiotensin II on myocardial collagen gene expression

Molecular and Cellular Biochemistry - Recent studies suggest that angiotensin II (angiotensin) may be involved in the regulation of metabolism of the cardiac extracellular matrix (ECM). Two major...     

Intrarenal localization of angiotensin II specific binding in rat fetuses

Specific binding sites for angiotensin II were localized in the developing rat kidney (18th day of pregnancy and immediately before birth) by autoradiography using [125I]-ileu-5-angiotensin II either perfused in vivo through the fetal aorta or added in vitro to frozen sections in an incubation mixture. Specific binding was localized in the walls of the afferent and efferent arterioles, in the intraglomerular cells and in the peritubular arterioles of the subcapsular cortical zone. The immunohistochemical analysis, carried out on receptors saturated with unlabelled angiotensin II perfused through the mother's aorta, confirmed the autoradiographical localization. Antisera against ileu-5-angiotensin II were used in the indirect immunofluorescence technique and in the PAP method. Immunolocalization of angiotensin II was also found in the proximal tubule and in the thick ascending limb of Henle's loop.     

Effects of angiotensin II microinjected into medial amygdala on male sexual behavior in rats

Research was undertaken to study the role of central angiotensin in the modulation of male sexual behavior, testing the effect of angiotensin II (Ang II) injections into the medial amygdaloid nucleus (MeA). The sexual behavior of adult male Wistar rats was evaluated, 15 min after bilateral intra-amygdaloid microinjection (0.3 microl) of saline and 5 doses of Ang II: 10; 25; 50; 100, and 150 fmol. The effects of the Ang II receptor blockade were also studied. We tested the effect of coinjection of Ang II (50 fmol) with the AT1 antagonist, losartan (20 pmol) and the AT2 antagonist, CGP 42112 (1 pmol). Ang II inhibited sexual behavior and this inhibition was prevented by the coinjection of AT1 antagonist, losartan, or the AT2 antagonist, CGP 42112. Results show that Ang II has a powerful effect on male sexual behavior, which may be mediated by both AT1 and AT2 receptors.     

Chronic corticotropin-releasing factor type 1 receptor antagonism with antalarmin regulates the dopaminergic system of Fawn-Hooded rats

Corticotropin-releasing factor is a neuropeptide associated with the integration of physiological and behavioural responses to stress and also in the modulation of affective state and drug reward. The selective, centrally acting corticotropin-releasing factor type 1 receptor antagonist, antalarmin, is a potent anxiolytic and reduces volitional ethanol consumption in Fawn-Hooded rats. The efficacy of antalarmin to reduce ethanol consumption increased with time, suggestive of adaptation to reinforcement processes and goal-directed behaviour. The aim of the present study was to examine the effects of chronic antalarmin treatment on reward-related regions of Fawn-Hooded rat brain. Bi-daily antalarmin treatment (20 mg/kg, i.p.) for 10 days increased tyrosine hydroxylase messenger RNA expression throughout the ventral mesencephalon. Following chronic antalarmin the density of dopaminergic terminals within the basal ganglia and amygdaloid complex were reduced, as was dopamine transporter binding within the striatum. Receptor autoradiography indicated an up-regulation of dopamine D2, but no change in D1, binding in striatum, and Golgi-Cox analysis of striatal medium spiny neurones indicated that chronic antalarmin treatment increased spine density. Thus, chronic antalarmin treatment modulates dopaminergic pathways and implies that chronic treatment with drugs of this class may ultimately alter postsynaptic signaling mechanisms within the basal ganglia.     

Oxidative stress in the infarcted heart: role of de novo angiotensin II production

This study aimed to elucidate whether angiotensin (Ang) II generated de novo at the infarct site regulates the redox state of inflammatory cells participating in cardiac repair. On days 3-28 following ligation of the rat left coronary artery, we addressed at the infarct site: (a) the appearance and cellular origin of oxidative stress by monitoring the expression (mRNA and protein) of gp91phox (a subunit of superoxide producing NADPH oxidase) and the antioxidant superoxide dismutase (SOD), together with the presence of 3-nitrotyrosine (a marker of oxidative stress); (b) the presence of components requisite to Ang peptide generation; and (c) response to treatment with losartan (Los, 10mg/kg/day). We found at the infarct site, macrophage-derived oxidative stress appears during week 1 coincident with the appearance of components requisite to AngII generation and activity in these cells. Based on observed response to AT1 receptor antagonism with Los, we would suggest de novo AngII, in an autocrine manner, participates in the induction of oxidative stress while suppressing the expression of antioxidant defenses.     

Stable expression of a functional rat angiotensin II (AT1A) receptor in CHO-K1 cells: Rapid desensitization by angiotensin II

The octapeptide angiotensin II mediates the physiological actions of the renin-angiotensin system through activation of several angiotensin II receptor subtypes; in particular the AT₁. In many tissues, the presence of multiple angiotensin II receptor subtypes, together with a low number of receptors, makes it difficult to study biological responses to physiological concentrations (10^–11–10^–9 M) of angiotensin II. Also, cultured cells show diminished angiotensin II receptor binding with respect to time in culture and passage number. To address these problems, we expressed the recombinant AT_1A receptor in CHO-K1 cells. The stably transfected receptor was characterized using radioligand binding studies and functional coupling to cytosolic free calcium. Radioligand binding of [¹²⁵I] angiotensin II to the angiotensin II receptor was specific, saturable, reversible and modulated by guanine nucleotides. Like the endogenous AT_1A receptor, reported in a variety of tissues, the specific, noncompetitive, nonpeptide AII receptor antagonist, EXP3174, blocked binding of [¹²⁵I] angiotensin II to the transfected receptor. Scatchard analysis demonstrated that the transfected receptor had a dissociation constant of 1.9 nM with a density of 3.4 pmol/mg protein.An important feature of many of the responses to angiotensin II is the rapid desensitization that occurs following agonist occupancy and the development of tachyphylaxis. In AT_1A receptor transfected CHO-K1 cells, angiotensin II (10^–9 M) stimulated a rapid increase in cytosolic free calcium that was completely desensitized within 50 sec following receptor occupancy. Agonist induced desensitization was unaffected when receptor internalization was blocked by pretreatment with concanavalin A or incubation at 4°C, and no changes in AT_1A receptor affinity or number were observed. Receptor desensitization was also unaffected by inhibition or activation of protein kinase C. Thus, we have established a permanent, high-level transfectant of the AT_1A receptor in CHO-K1 cells and have shown that these receptors rapidly desensitize following exposure to physiological concentrations of agonist. The mechanism of rapid desensitization is not related to receptor sequestration, internalization or controlled by PKC phosphorylation. This provides an excellent model for studying AII actions mediated through a specific receptor subtype, at subnanomolar concentrations.     

Effect of angiotensin II on transepithelial potential in the isolated perfused flounder (Platichthys flesus) gill

Angiotensin II (Asp¹, Val⁵) perfused through isolated flounder gills inhibited the transepithelial potential by up to 25 per cent at a concentration of 10⁻⁹M. There was no effect on gill haemodynamics and the subsequent response to 10⁻⁵ M adrenaline was normal.     

Acute abdominal vagotomy reduces drinking to peripheral but not central angiotensin II

Rats were tested two or three days after bilateral abdominal vagotomy or a laparotomy control procedure for their drinking responses to subcutaneous (1 mg-kg-1) or intracerebroventricular (100 ng) injections of angiotensin II. Vagotomy delayed the initiation of drinking and decreased 60-min water intake after subcutaneous, but not after intracerebroventricular, angiotensin II. This is the shortest postoperative interval in which the decrease in drinking after systemic injection of angiotensin II by abdominal vagotomy has been observed. The failure of vagotomy to decrease the response to intracerebroventricular angiotensin II demonstrates that the deficit after subcutaneous injection was not a nonspecific effect of recent vagotomy. These results, therefore, suggest that the abdominal vagus is necessary for normal drinking in response to circulating angiotensin II. Furthermore, the selective and acute onset of the deficit is consistent with the loss of a specific, rather than tonic facilitatory, vagal mechanism for drinking after elevation of circulating angiotensin II levels. Finally, the results imply that the physiological mechanisms which mediate the drinking responses to central and peripheral angiotensin are not identical.     

Reduction of cardiac hypertrophy in TGR(mREN2)27 by angiotensin II receptor blockade

TGR(mREN2)27 is a transgenic rat harboring the murine Ren-2 gene and exhibit fulminant hypertension and marked heart hypertrophy. In order to study the role of angiotensin II in the increase of cardiac mass, these animals were treated with anti-hypertensive and non-antihypertensive doses of the angiotensin II receptor AT₁ antagonist Telmisartan for 9 weeks. All doses led to significant reductions of heart hypertrophy detected by the evaluation of the diameter of cardiac muscle bundles. We conclude from this study that cardiac hypertrophy in TGR(mREN2)27 is characterized by an increased volume of cardiomyocytes and an unchanged amount of fibrous tissue and that angiotensin II plays an important role in the mechanisms leading to this phenotype.     

Regulation of angiotensinogen by angiotensin II in mouse primary astrocyte cultures

Astrocytes are the major source of angiotensinogen in the brain and play an important role in the brain renin-angiotensin system. Regulating brain angiotensinogen production alters blood pressure and fluid and electrolyte homeostasis. In turn, several physiological and pathological manipulations alter expression of angiotensinogen in brain. Surprisingly, little is known about the factors that regulate astrocytic expression of angiotensinogen. There is evidence that angiotensinogen production in both hepatocytes and cardiac myocytes can be positively regulated via the angiotensin type 1 receptor, but this effect has not yet been studied in astrocytes. Therefore, the aim of this project was to establish whether angiotensin II modulates angiotensinogen production in brain astrocytes. Primary astrocyte cultures, prepared from neonatal C57Bl6 mice, expressed angiotensinogen measured by immunocytochemistry and real-time PCR. Using a variety of approaches we were unable to identify angiotensin receptors on cultured astrocytes. Exposure of cultured astrocytes to angiotensin II also did not affect angiotensinogen expression. When astrocyte cultures were transduced with the angiotensin type 1A receptor, using adenoviral vectors, angiotensin II induced a robust down-regulation (91.4% ± 1.8%, p < 0.01, n = 4) of angiotensinogen gene expression. We conclude that receptors for angiotensin II are present in extremely low levels in astrocytes, and that this concurs with available data in vivo. The signaling pathways activated by the angiotensin type 1A receptor are negatively coupled to angiotensinogen expression and represent a powerful pathway for decreasing expression of this protein, potentially via signaling pathways coupled to Gα(q/11) .     

Similarities and differences between effects of angiotensin III and angiotensin II on human prostate cancer cell migration and proliferation

Proliferation plays a critical role in tumor growth when cell migration is essential to invasion. The effect of Ang III and Ang II was evaluated on these important processes. Changes in the migration potential of prostate cancer cells were investigated using Wound Healing Test and a Transwell Migration Chamber with a 3μm pore size. Cell proliferation was measured with a BrdU Assay and Countess Automated Cell Counter, thus determining the influence of angiotensins on hormone-dependent (LNCaP) and hormone-independent (DU-145) human prostate cancer lines. The influence of Ang III and Ang II on classic receptors may be inhibited by Losartan or PD123319. Test peptide modulation of the AT1 and AT2 receptors was examined by Western Blot and fluorescent immunocytochemistry. The results indicate that Ang III promotes the migration of both LNCaP and DU-145 lines, whereas Ang II stimulates this process only in androgen-independent cells. Both angiotensin peptides can induce prostate cancer cell proliferation in a time- and dose-dependent manner. The obtained results show that Ang III and Ang II can modify the expression of classic receptors, particularly AT2. These results suggest that the investigated peptide can modulate cell migration and proliferation in prostate cancer cells. Angiotensins probably have a greater influence on proliferation in the early-stage prostate cancer model than hormone-independent cell lines. Assume also that Ang II can enhance the migration tendency aggressive prostate cancer cells, while Ang III does so more effective in non-metastatic cells.