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Stresscopin (SCP) & Stresscopin-related Peptide (SRP)
Urocortin II & III (UCN II & III)

Selective Agonist for the Type 2 Corticotropin-Releasing Factor Receptor

Urocortin III Is Expressed in Pancreatic -Cells and Stimulates Insulin and Glucagon Secretion
Urocortin (Ucn) III, or stresscopin, is a high affinity ligand for the type 2 corticotropin-releasing factor (CRFR2) receptor recently identified in rodents and human. Ucn III was initially identified as a neuropeptide expressed in discrete areas in the brain. In the present study, we demonstrate that Ucn III is expressed in pancreatic -cells and in a mouse -cell line, MIN6. Ucn III secretion from the cells was measured using a highly specific RIA, and we found that high potassium, forskolin, or high glucose can stimulate Ucn III secretion from these cells. In vivo studies showed that rats receiving an iv Ucn III injection had a significant elevation of plasma glucagon followed by plasma glucose levels compared with rats receiving vehicle. Ucn III injections also result in an increase in plasma insulin levels. The observed effects of Ucn III were blocked by pretreatment with a CRFR2 antagonist, astressin2-B. Furthermore, Ucn III stimulated glucagon and insulin release from isolated rat islets, and astressin2-B abolished the effects of Ucn III, in keeping with a CRFR2-mediated mechanism.Taken together, the present studies suggest pancreatic Ucn III acting through CRFR2 is involved in the local regulation ofglucagon and insulin secretion. Chien Li, et al. Endocrinology Vol. 2003,144(7), 3216-3224
Human Urocortin II, a Selective Agonist for the Type 2 Corticotropin-Releasing Factor Receptor, Decreases Feeding and Drinking in the Rat

Corticotropin-releasing factor (CRF) has been hypothesized to modulate consummatory behavior through the Type 2 CRF (CRF(2)) receptor. However, behavioral functions subserved by the CRF(2) receptor remain poorly understood. Recently, human urocortin II (hUcn II), a selective CRF(2) receptor agonist, was identified. To study the effects of this neuropeptide on ingestive behavior, we examined the effects of centrally infused hUcn II (i.c.v. 0, 0.01, 0.1, 1.0, 10.0 &mgr;g) on the microstructure of nose-poke responding for food and water in nondeprived, male rats. Malaise-inducing properties of the peptide were monitored using conditioned taste aversion (CTA) testing. To identify potential sites of action, central induction of Fos protein expression was examined. hUcn II dose dependently reduced the quantity and duration of responding for food and water at doses lower (0.01-1.0 &mgr;g) than that forming a CTA (10 &mgr;g). Effects were most evident during hours 4 to 6 of the dark cycle. Meal pattern analysis showed that hUcn II potently (0.1 &mgr;g) increased the satiating value of food. Rats ate and drank smaller and shorter meals without changing meal frequency. Rats also ate more slowly. hUcn II induced Fos in regions involved in visceral sensory processing and autonomic/neuroendocrine regulation and resembling those activated by appetite suppressants. hUcn II is a promising neuropeptide for investigating the role of the CRF(2) receptor in ingestive behavior.

Inoue K,et al. J Pharmacol Exp Ther 2003 Apr 1;305(1):385-393

Human Urocortin II (Urocortin Related Peptide, URP) is identical with Stresscopin Related Peptide (SRP) (6-43)-NH2 (Human).

References:
Lewis, K. et al. Proc. Natl. Acad. Sci. USA  98, 7570-7575, 2001 (June 19)
Hsu, S.Y. & Hsueh, A.J.W. Nature Medicine, 7 605-611, 2001
Review by Jun Yang on June 21, 2001

Human urocortin II, a new CRF-related peptide, displays selective CRF(2)-mediated action on gastric transit in rats

Human urocortin (hUcn) II is a new member of the corticotropin-releasing factor (CRF) family that selectively binds to the CRF(2) receptor. We investigated the CRF receptors involved in mediating the effects of hUcn II and human/rat CRF (h/rCRF) on gut transit. Gastric emptying, 4 h after a solid meal, and distal colonic transit (bead expulsion time) were monitored simultaneously in conscious rats. CRF antagonists were given subcutaneously 30 min before intravenous injection of peptides or partial restraint (for 90 min). hUcn II (3 or 10 microg/kg i.v.) inhibited gastric emptying (by 45% and 55%, respectively) and did not influence distal colonic transit. The CRF(2) peptide antagonist astressin(2)-B blocked hUcn II action. h/rCRF, rat Ucn, and restraint delayed gastric emptying while accelerating distal colonic transit. The gastric response to intravenous h/rCRF and restraint was blocked by the CRF(2) antagonist but not by the CRF(1) antagonist CP-154,526, whereas the colonic response was blocked only by CP-154,526. None of the CRF antagonists influenced postprandial gut transit. These data show that intravenous h/rCRF and restraint stress-induced delayed gastric emptying involve CRF(2) whereas stimulation of distal colonic transit involves CRF(1). The distinct profile of hUcn II, only on gastric transit, is linked to its CRF(2) selectivity.

Million M, et al. Am J Physiol Gastrointest Liver Physiol 2002 Jan;282(1):G34-40

Differential actions of peripheral corticotropin-releasing factor (CRF), urocortin II, and urocortin III on gastric emptying and colonic transit in mice: role of CRF receptor subtypes 1 and 2
Peripheral CRF inhibits gastric emptying and stimulates colonic motor function in rats. We investigated the role of CRF(1) and CRF(2) receptors in i.p. CRF-induced alterations of gut transit in conscious mice using selective CRF(1) and CRF(2) ligands injected i.p. Gastric emptying 2 h after ingestion of a solid chow meal and colonic transit (time to expel a bead inserted into the distal colon) were determined simultaneously. Rat/human (r/h)CRF, which has CRF(1) > CRF(2) binding affinity, decreased distal colonic transit time at lower doses (6-12 microg/kg) than those inhibiting gastric emptying (20-60 microg/kg). Ovine CRF, a preferential CRF(1) receptor agonist (6-60 microg/kg), reduced significantly the colonic transit time without altering gastric emptying, whereas the selective CRF(2) receptor agonists mouse urocortin II (20-60 microg/kg) and urocortin III (120 microg/kg) inhibited significantly gastric emptying without modifying colonic transit. The CRF(1)/CRF(2) receptor antagonist, astressin (30-120 microg/kg), dose dependently prevented r/hCRF (20 microg/kg)-induced inhibition of gastric emptying and reduction of colonic transit time. The selective CRF(1) receptor antagonists, NBI-27914 (C(18)H(20)Cl(4)N(4)C(7)H(8)O(3)S) and CP-154,526 (butyl-[2,5-dimethyl-7-(2,4,6-trimethylphenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl]e thylamine) (5-30 mg/kg), dose dependently blocked r/hCRF action on the colon without influencing the gastric response, whereas the CRF(2) receptor antagonist, antisauvagine-30 (30-100 microg/kg), dose dependently abolished r/hCRF-induced delayed gastric emptying and had no effect on colonic response. These data show that i.p. r/hCRF-induced opposite actions on upper and lower gut transit in conscious mice are mediated by different CRF receptor subtypes: the activation of CRF(1) receptors stimulates colonic propulsive activity, whereas activation of CRF(2) receptors inhibits gastric emptying.

Martinez V,et al. J Pharmacol Exp Ther 2002 May;301(2):611-7

 

Urocortin-Related Peptides Increase Interleukin-6 Output via Cyclic Adenosine 5'-Monophosphate-Dependent Pathways in A7r5 Aortic Smooth Muscle Cells

Corticotropin-releasing factor receptor type 2, expressed in the rodent cardiovascular system, is a member of the G protein-coupled receptor family. This receptor is coupled positively to adenylate cyclase and is bound preferentially by the urocortin (Ucn)-related peptides (Uncs): Ucn, Ucn II, and Ucn III. In the present study, we investigated the effects of Ucns on IL-6 levels in A7r5 aortic smooth muscle cells. In this cell line, both Ucn and Ucn II induced accumulation of intracellular cAMP via corticotropin-releasing factor receptor type 2 and also caused a significant increase in IL-6 output levels. The adenylate cyclase inhibitor, MDL-12330A, inhibited this Ucn- or Ucn II-induced increase in IL-6 levels. Although H89 (10 M), a protein kinase A inhibitor, had no effect on the increase in IL-6 concentration, bisindolylmaleimide I (10 nM), a protein kinase C inhibitor, was found to significantly inhibit IL-6 output levels. Blockade of Ucn- or Ucn II-induced increases in IL-6 levels by SB203580 (100 nM), a p38 MAPK inhibitor, suggested that the p38 MAPK pathway was involved in this regulation. The cAMP-mediated increase in IL-6 levels was suppressed synergistically by both bisindolylmaleimide I and SB203580. These findings demonstrate that both protein kinase C and p38 MAPK signaling cascades are involved downstream of the Ucns-cAMP pathway in A7r5 aortic smooth muscle cells.  Kazunori Kageyama and Toshihiro Suda. Endocrinology 2003, 144(6) 2234-2241

Figure 1. Expression and localization of Ucn III in mouse pancreatic islets and MIN6 cells. A, Representative electrophoretic analysis of the RT-PCR products of Ucn, Ucn II, and Ucn III in mouse pancreas and MIN6 cells. S16 RNA was used as positive control. -RT, RNA samples were used for PCR without RT; -cDNA, no RT product added to the PCR. B, Representative autoradiogram showing the detection of Ucn II and Ucn III mRNAs by RNase protection assay. Total RNA isolated from mouse brain stem (for Ucn II), hypothalamus (for Ucn III), or whole pancreas was hybridized with antisense probes specific to mouse Ucn II or Ucn III and mouse glyceraldehyde-3-phosphate dehydrogenase. Ucn II probe protects a 592-nt band in the brain stem and the pancreas and Ucn III probe protects a 414-nt band in the hypothalamus and the pancreas. C, Representative dark-field photomicrograph showing a mouse pancreas section probed with mouse Ucn III riboprobe. Ucn III mRNA-positive signals (silver grain clusters) were found in the pancreatic islets. D, Bright-field photomicrograph of panel C with nissl staining showing the islets. *, Blood vessel. E and F, Representative photomicrographs showing Ucn III immunostaining in the mouse pancreatic sections without (E) or with (F) preabsorption of the anti-Ucn III serum with 30 M of mouse Ucn III. Ucn III-positive cells (dark purple staining) were observed only in the islets. Scale bar, 50 m. Chien Li, et al. Endocrinology Vol. 2003,144(7), 3216-3224

Figure 2. Colocalization of Ucn III and insulin in mouse pancreatic islets. Stacked serial confocal images of insulin (A) and Ucn III (B) cells in the islet. C, Visualization of both substances simultaneously showing the majority of insulin and Ucn III colocalize in the same cells. High magnification of confocal images showing insulin (D) and Ucn III (E) in a single -cell. F, Combined image of panels D and E to visualize insulin and Ucn III simultaneously. Note that little colocalization is observed. G–I, Stacked serial confocal images of glucagon (G), Ucn III (H), and combined image (I) of panels G and H showing no colocalization of the two substances. Scale bar, 25 m for A–C and G–I and 1 m for D–F. Chien Li, et al. Endocrinology Vol. 2003,144(7), 3216-3224

Table 2. Effects of Ucn III and rat/human CRF on glucagon and insulin secretion from isolated rat islets

Ucn III


Rat/human CRF


0.1 1 10 100 (nM) 1 10 100 (nM)

Glucagon 103.6 38a 157.3 18a 169.7 26a1 284.1 771 119.5 29a 95.3 15a1 281.9 791
Insulin 180.8 66d 296.4 51e 231.8 39e 258.3 43e 132.6 24d 113.3 14d 362.7 87e

Data are expressed as percent of control (2.8 mM glucose). Values in same row with different superscripts (a, c, d, e) are significantly different with P < 0.05.  1 Significantly different from each other with P < 0.05. Chien Li, et al. Endocrinology Vol. 2003,144(7), 3216-3224

Figure 3. Displacement of 125I-labeled mUcn III binding to rabbit anti-mUcn III by mUcn III ({square}) and partially purified rat pancreatic ({oplus}) or isolated rat islet () extract. Rat pancreas or isolated rat islets were acid-extracted and partially purified using octadecyl silica cartridges. Human Unc III (h Ucn III, {boxplus}) shows approximately 10% cross-reactivity. Closely related family peptides including rat Ucn (r Ucn, {circ}), rat CRF (r CRF, {diamond}), or mouse Ucn II (m Ucn II, {triangleup}) showed little or no cross-reactivity. B/B0, Bound to maximum-bound ratio. Chien Li, et al. Endocrinology Vol. 2003,144(7), 3216-3224

Figure 4. Ucn III-like ir in secreted media from MIN6 cells. MIN6 cells (106 cells per well) were incubated in media containing various concentrations of glucose, 30 M forskolin, or 30 mM KCl and supplemented with BSA (0.2% wt/vol) for 4 h. Cultured supernatants were collected and assayed for Ucn III. Cells were treated in triplicate, and the average of duplicate experiments is shown. *, P < 0.05 vs. 5 mM glucose; **, P < 0.01 vs. 5 mM glucose. Chien Li, et al. Endocrinology Vol. 2003,144(7), 3216-3224

Figure 6. Plasma glucose (A) and glucagon (B) levels in male rats treated with vehicle or Ucn III (9 or 90 nmol/kg). Basal glucose and glucagon concentrations are 5.1 0.2 mmol/liter and 52.3 3.2 pg/ml, respectively. C, Glucagon levels at 5 min after vehicle or Ucn III (90 nmol/kg) injection with or without Ast2-B (90 nmol/kg) pretreatment. B, *, P < 0.05 vs. vehicle. C, *, P < 0.05 vs. the rest of groups. Chien Li, et al. Endocrinology Vol. 2003,144(7), 3216-3224 Figure 5. The effects of either vehicle or Ucn III (0.09–9 nmol/kg, iv) on plasma glucose (A) and insulin (B) levels in male rats. C, Insulin levels at 30 min after iv vehicle or Ucn III (90 nmol/kg) with or without Ast2-B (90 nmol/kg) pretreatment. Basal glucose level, 4.25 0.18 mmol/liter. B, For 9 nmol/kg group: +, P < 0.05 vs. vehicle; for 90 nmol/kg group: *, P < 0.05; **, P < 0.01 vs. vehicle. C, *, P < 0.05 vs. the rest of groups. Chien Li, et al. Endocrinology Vol. 2003,144(7), 3216-3224

Figure 8. Effects of a glucagon antagonist on Ucn III-induced insulin secretion from isolated rat islets. Ucn III (1 nM) and 10 nM glucagon stimulate insulin secretion. Pretreatment with 1 M des-glucagon abolishes the effect of glucagon but not Ucn III on insulin secretion. Values from islets incubated in medium with 2.8 mM glucose serve as controls. *, P < 0.05 compared with control. +, P < 0.05 vs. control and glucagon plus des-glucagon. Chien Li, et al. Endocrinology Vol. 2003,144(7), 3216-3224

An illustrated scheme of the pathways involved in Ucn/Ucn II, cAMP induction, and IL-6 output. Ucns increase intracellular cAMP levels via the stimulation of adenylate cyclase. To increase IL-6 output, both PKC and p38 MAPK signaling cascades are involved downstream of the Ucn/Ucn II-cAMP pathway in A7r5 cells. Kazunori Kageyama and Toshihiro Suda. Endocrinology 2003, 144(6) 2234-2241

Effects of CRF family peptides on interleukin-6 levels in A7r5 aortic smooth muscle cells. Cells were incubated for 48 h with medium alone (control) or with medium containing CRF (open triangle), Ucn (open circle), Ucn II (open square), antisauvagine-30 (closed triangle), 100 nM of Ucn with increasing concentrations of antisauvagine-30 (closed circle), and 100 nM of Ucn II with increasing concentrations of antisauvagine-30 (closed square). IL-6 levels in the medium were measured by ELISA. Results shown are representative of three independent experiments. Statistical analyses were performed using one-way ANOVA, followed by Scheff’s F post hoc test.

%019-30%;%019-29%;%019-27%;%019-28%

 Related Urocortin Products

Protocol for Urocortin II Immunohistochemistry

Tissue Sample Mouse brain
Fixative 10% formalin
Embedding paraffin
Negative Control No primary antibody
Pretreatment Target retrieval 25 min (Steam)
Blocking 2% Normal Goat Serum
Primary Antibody Anti-Urocortin II (Mouse),  Antibody (Catalog No.:H-019-24)
Optimal Dilution 1:50
Secondary Antibody Goat Anti-Rabbit IgG, Biotinylated (1:400)
Amplification ABC (Vector)
Detection System HRP
Substrate DAB (Sigma)
Counterstained Hematoxylin

 


Binding Properties and Functional Activities of Selective CRG Receptor Ligand

 
 
  CRF-R2 CRF-R1
  EC50, nM(cAMP) ki, nM (binding) EC50, nM(cAMP) ki, nM (binding)
Ucn II (Mouse) 0.14 0.66 < 100 < 100
URPI (Human) 0.42 0.50 < 100 < 100
Ucn (Rat) 0.17 0.62 0.29 0.32
 

 


Amino Acid Sequence of Prepro-Urocortin II (Mouse)

 
1   M T R W A L V V F V V L M L D R I L F V   20
21   P G T P I P T F Q L L P Q N S L E T T P   40
41   S S V T S E S S S G T T T G P S A S w S   60
61   N S K A S P Y L D T R V I L S L D V P I   80
81   G L L R I L L E Q A R Y K A A R N Q A A   100
101   T N A Q I L A H V G R R                   112
 

 

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