Actions of intracerebroventricular administration of kyotorphin and an analog on thermoregulation in the mouse

Intracerebroventricular (ICV) administration of kyotorphin (L-Tyr-L-Arg) and cyclo (N-methyl-L-Tyr-L-Arg), its analog, produced significant dose-dependent hypothermic responses in mice at an ambient temperature of 24°C. The hypothermic action of kyotorphin was much greater than that of Met-enkephalin (Met-ENK) but less than that of cyclo NMTA. This action was slightly but not significantly reversed by intraperitoneally administered naloxone (8 mg/kg), an opioid receptor antagonist. Met-ENK utilized as a control peptide in this study also produced a dose-dependent hypothermia which was slightly antagonized by naloxone (8 mg/kg, IP). Thyrotropin releasing hormone (TRH) injected ICV produced hyperthermia dose-dependently. The hypothermia induced by kyotorphin, its cyclic analog and Met-ENK was prevented by a small dose of TRH (0.18 μg=0.5 nmol/animal) which by itself had little effect on body temperature. A TRH neuronal system in the brain may explain the mechanism of kyotorphin-induced hypothermia. However, there was little evidence of involvement of opioid receptors. The present study demonstrates a potent action of kyotorphin and its analog on thermoregulation.     

Topochemical analysis of morphiceptin and dermorphin bioactivities.

To elucidate the topochemical requirements for bioactivities of morphiceptin (Tyr-Pro-Phe-Pro-NH2) and dermorphin (Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2), we have designed and synthesized two diastereomers Tyr-(L and D)-(NMe)Ala-Phe-D-Pro-NH2. Both the analogs display high activities in the guinea pig ileum assay. The only difference in the composition of these two diastereomers arises from the chirality at residue 2. The high mu-receptor activities are attributed to structures where the Tyr1-L-(NMe)Ala2 amide bond assumes a cis configuration while the Tyr1-D-(NMe)Ala2 amide bond assumes a trans configuration. Accessible space studied for the second residues of these molecules confirms the fact that the L-(NMe)Ala2 analog belongs to the morphiceptin family of opioids while the D-(NMe)Ala2 analog belongs to the dermorphin class of opioids. The similarity in the spacial array of the analogs explains their high mu-receptor activities and indicates that they are likely recognized at the same opioid receptor.     

Biological activity of parathyroid hormone antagonists substituted at position 13.

Lysine occupies position 13 in the parathyroid hormone (PTH) antagonist, [Nle8,18,Tyr34]bPTH(7-34)NH2. Acylation of the epsilon-amino group in lysine 13 by a hydrophobic moiety is well tolerated in terms of bioactivity: the analog [Nle8,18, D-Trp12,Lys 13 (epsilon-3-phenylpropanoyl),Tyr34]bPTH(7-34)NH2 is equivalent to the parent peptide in its affinity for PTH receptors and its ability to inhibit PTH-stimulated adenylate cyclase in both kidney- and bone-based assays. Truncation of this peptide by deletion of phenylalanyl7 with concomitant removal of the amino-terminal alpha-amino group yielded the analog desamino[Nle8,18,D-Trp12,Lys13 (epsilon-3-phenylpropanoyl),Tyr34]bPTH(8-34)NH2, an antagonist of high potency in vitro (Kb = 4 and 9 nM, Ki = 73 and 3.5 nM in kidney- and bone-based assays, respectively). Also this analog is potentially stable to aminopeptidases present in many biological systems.     

The kyotorphin (tyrosine-arginine) receptor and a selective reconstitution with purified Gi, measured with GTPase and phospholipase C assays

We attempted to identify the kyotorphin receptor and the post receptor mechanisms mediated by GTP-binding proteins (G-proteins), using reconstitution techniques. The specific binding of [3H]kyotorphin in rat brain membranes was composed of high affinity (Kd = 0.34 nM) and low affinity (Kd = 9.07 nM) binding. As the high affinity binding disappeared in the presence of guanosine 5'-O-(3-thiotriphosphate) and MgCl2, we investigated the kyotorphin receptor-mediated changes in membrane G-protein activity by measuring low Km GTPase activity. Kyotorphin produced a stimulation of low Km GTPase, and this stimulation was antagonized by Leu-Arg, a synthetic dipeptide which showed a potent displacement of [3H]kyotorphin binding, yet in itself had no effect on the low Km GTPase. The kyotorphin stimulation of low Km GTPase was abolished by pretreating membranes with islet-activating protein, pertussis toxin, and was recovered by reconstitution with purified G-protein, Gi, but not with Go. Similar evidence of selective coupling of kyotorphin receptor to Gi was obtained with the phospholipase C assay. Kyotorphin-induced stimulation of phospholipase C was also abolished by islet-activating protein-treatment and recovered by reconstitution with Gi but not with Go. These findings indicate that specific high and low affinity kyotorphin receptors exist in the rat brain and that the kyotorphin receptor is functionally coupled to stimulation of phospholipase C, through Gi. This study provides the first evidence of a selective involvement of Gi in the receptor-mediated activation of phospholipase C.     

Uptake and release of kyotorphin in rat brain synaptosomes

Uptake and release of kyotorphin (TyrArg) in rat brain synaptosomes were studied. Synthetic kyotorphin was taken up into crude synaptosomes (P₂), in a temperature-dependent manner. The Km and Vmax of the uptake were 1.31 ± 0.12 × 10⁻⁴M and 5.9 ± 0.5 pmol/mg protein/min, respectively. Metabolic inhibitors such as dinitrophenol and iodoacetamide and ouabain which is known as an inhibitor of Na⁺ dependent uptake mechanism significantly inhibited the uptake. When the synaptosomes previously preloaded with synthetic kyotorphin at 10⁻⁴M were exposed to high K⁺ medium, kyotorphin was released in a Ca²⁺-dependent manner. These findings support the view that kyotorphin plays a role as neurotransmitter/neuroregulator.     

High-performance liquid chromatographic separation of kyotorphin, a basic TyrArg dipetide, in rat brain tissue and quantification using fluorimetric detection

Two HPLC procedures based on sample derivatization at the N-terminal Tyr moiety with agents yielding fluorescent derivatives were applied to the selective and sensitive detection as well as quantification of the basic kyotorphin, TyrArg dipeptide, in rat brain tissue. The first one is a post-column fluorescence derivatization method, whereby the peptides extracted from the brain tissue are separated on an octadecylsilyl-silica gel column, followed by on-line fluorescence derivatization for detection. The other one is a pre-column derivatization method, where the extracted peptides are first reacted with fluorogenic agents at the N-terminal Tyr moiety to their corresponding fluorescent derivatives, subsequently separated on an octadecyl-poly(vinyl alcohol) copolymer gel column, and signal responses are measured fluorimetrically. Both methods permitted the quantification of the synthetic kyotorphin added to the rat brain tissues. The concentration range of kyotorphin-like biogenic peptide was 60–100 pmol/g in the cortex, striatum and hypothalamus tissues.     

Kyotorphin (tyrosine-arginine) synthetase in rat brain synaptosomes

Kyotorphin (Tyr-Arg) is a unique neuropeptide which produces analgesia by releasing Met-enkephalin from slices of the brain and spinal cord. Recent studies revealed that kyotorphin possesses the properties of neurotransmitter/neuroregulator. In the present study, we identified a kyotorphin synthetase in the soluble fraction of rat brain synaptosomes (synaptosol) and characterized it. The enzyme partially purified with Sephacryl S-300 showed an absolute requirement for ATP, MgCl2, tyrosine, and arginine. The optimal pH was 7.5-9.0 and the pI was determined to be 6.1-6.2 by isoelectric focusing. The Km was 25.6 microM for tyrosine, 926 microM for arginine, 294 microM for ATP, and 442 microM for MgCl2. The Vmax was 34.0 pmol/mg of protein/h. The apparent molecular size of this "kyotorphin synthetase" further purified by the DE52 column was 240,000-245,000 daltons, estimated using TSKgel G4000SW column chromatography. The enzyme reaction is represented by the following equation: Tyr + Arg + ATP + MgCl2 + kyotorphin synthetase----Tyr-Arg (kyotorphin) + AMP + PPi + MgCl2 + kyotorphin synthetase. The regional distribution and subcellular localization of the synthetase showed a close correlation to that of kyotorphin levels in the rat brain. The amounts of kyotorphin formed from amino acids by the synthetase in the dialyzed synaptosol was 3.0-4.0 times higher than that from precursor proteins by processing enzymes within the 30 min incubation.     

Synthesis and characterization of an immobilized phenylethanolamine N-methyltransferase liquid chromatographic stationary phase

Norepinephrine is N-methylated to epinephrine by the catalytic effect of the terminal enzyme in catecholamine biosynthesis, phenylethanolamine N-methyltransferase (PNMT). PNMT has been covalently immobilized onto a silica-based liquid chromatographic support, glutaraldehyde-P (Glut-P). The resulting PNMT-Glut-P stationary phase (PNMT-SP) was enzymatically active, stable, and reusable. Standard Michaelis-Menten kinetic studies were performed with both free and immobilized PNMT and known substrates and inhibitors were examined. The results demonstrate that the PNMT-SP can be utilized for the rapid screening of potential PNMT substrates as well as the screening of compounds for PNMT inhibitory activity.     

High-performance liquid chromatographic separation of kyotorphin, a basic Tyr---Arg dipetide, in rat brain tissue and quantification using fluorimetric detection

Two HPLC procedures based on sample derivatization at the N-terminal Tyr moiety with agents yielding fluorescent derivatives were applied to the selective and sensitive detection as well as quantification of the basic kyotorphin, Tyr---Arg dipeptide, in rat brain tissue. The first one is a post-column fluorescence derivatization method, whereby the peptides extracted from the brain tissue are separated on an octadecylsilyl-silica gel column, followed by on-line fluorescence derivatization for detection. The other one is a pre-column derivatization method, where the extracted peptides are first reacted with fluorogenic agents at the N-terminal Tyr moiety to their corresponding fluorescent derivatives, subsequently separated on an octadecyl-poly(vinyl alcohol) copolymer gel column, and signal responses are measured fluorimetrically. Both methods permitted the quantification of the synthetic kyotorphin added to the rat brain tissues. The concentration range of kyotorphin-like biogenic peptide was 60–100 pmol/g in the cortex, striatum and hypothalamus tissues.     

Effects of hydrophobic substitutions at position 18 on the potency of parathyroid hormone antagonists

Position 18 in a parathyroid hormone (PTH) antagonist, [Nle8,18,Tyr34]bPTH(7-34)NH2 (ii), was shown to tolerate substitutions by a range of amino acids with retention of inhibitory activity. The effects of hydrophobic substitutions at this position as a means of enhancing binding interactions with the receptor were evaluated. Substitution of Nle at position 18 with either D-Ala, D-Trp, or L-Trp in analog ii or with Trp (D or L) in the recently reported, highly potent antagonist, [Nle8,18,D-Trp12,Tyr34]bPTH(7-34)NH2 (in vitro activities; Kb = 15 nM and Ki = 125 nM), was performed. In terms of activity on renal receptors, one antagonist, [Nle8,D-Trp12,18,Tyr34]bPTH(7-34)NH2, is the most active in vitro PTH antagonist yet reported (Kb = 4 nM; Ki = 30 nM). The rationale for design of this antagonist and the conclusions regarding PTH-receptor interactions are discussed.     

Kyotorphin has a novel action on rat cardiac muscle

Several endogenous peptides for G-protein-coupled receptors have been found to play physiological roles in muscle contraction in addition to their well-demonstrated actions in other tissues. To further identify such peptides, we screened over 400 peptides using an isometric tension assay of rat papillary muscle. Here, we report that kyotorphin, which is known as an analgesic dipeptide, has a cardiac effect. Although kyotorphin had no effect on the twitch tension itself, it inhibited beta-adrenergic agonist isoprenaline-induced increases in twitch tension in a dose-dependent manner. Leu-Arg, a selective antagonist of kyotorphin, reversed this inhibitory effect. The inhibitory effect was also reversed by naloxone, an opioid receptor antagonist. These results suggest that kyotorphin may release opioid peptides from rat cardiac muscle and have an indirect regulatory role in beta-adrenergic action through cross-talk with opioid receptors.     

Role of tyrosine phosphorylation in the regulation of cleavage secretion of angiotensin-converting enzyme

Both germinal (gACE) and somatic (sACE) isozymes of angiotensin-converting enzyme (ACE) are type I ectoproteins whose enzymatically active ectodomains are cleaved and shed by a membrane-bound protease. Here, we report a role of protein tyrosine phosphorylation in regulating this process. Strong enhancements of ACE cleavage secretion was observed upon enhancing protein Tyr phosphorylation by treating gACE- or sACE-expressing cells with pervanadate, an inhibitor of protein Tyr phosphatases. Secreted gACE, cell-bound mature gACE and its precursors were all Tyr-phosphorylated, as was the endoplasmic reticulum protein, immunoglobulin heavy chain-binding protein, that co-immunoprecipitated with ACE. The enhancement of cleavage secretion by pervanadate did not require the presence of the cytoplasmic domain of ACE, and it was not accomplished by enhancing the rate of intracellular processing of the protein. The observed enhancement of cleavage secretion of ACE in pervanadate-treated cells was specifically blocked by an inhibitor of the p38 mitogen-activated protein (MAP) kinase but not by inhibitors of many other Ser/Thr and Tyr protein kinases, including a specific inhibitor of protein kinase C that, however, could block the enhancement of cleavage secretion elicited by phorbol ester. These results indicate that ACE Tyr phosphorylation, probably in the endoplasmic reticulum, enhances the rate of its cleavage secretion at the plasma membrane using a regulatory pathway that may involve p38 MAP kinase.     

Gonadotropin-releasing hormone analog structural determinants of selectivity for inhibition of cell growth: support for the concept of ligand-induced selective signaling

GnRH and its receptor are expressed in human reproductive tract cancers, and direct antiproliferative effects of GnRH analogs have been demonstrated in cancer cell lines. The intracellular signaling responsible for this effect differs from that mediating pituitary gonadotropin secretion. The GnRH structure-activity relationship is different for the two effects. Here we report a structure-activity relationship study of GnRH agonist antiproliferative action in model cell systems of rat and human GnRH receptors stably expressed in HEK293 cells. GnRH II was more potent than GnRH I in inhibiting cell growth in the cell lines. In contrast, GnRH I was more potent than GnRH II in stimulating inositol phosphate production, the signaling pathway in gonadotropes. The different residues in GnRH II (His(5), Trp(7), Tyr(8)) were introduced singly or in pairs into GnRH I. Tyr(5) replacement by His(5) produced the highest increase in the antiproliferative potency of GnRH I. Tyr(8) substitution of Arg(8) produced the most selective analog, with very poor inositol phosphate generation but high antiproliferative potency. In nude mice bearing tumors of the HEK293 cell line, GnRH II and an antagonist administration was ineffective in inhibiting tumor growth, but D-amino acid stabilized analogs (D-Lys(6) and D-Arg(6)) ablated tumor growth. Docking of GnRH I and GnRH II to the human GnRH receptor molecular model revealed that Arg(8) of GnRH I makes contact with Asp(302), whereas Tyr(8) of GnRH II appears to make different contacts, suggesting these residues stabilize different receptor conformations mediating differential intracellular signaling and effects on gonadotropin and cell growth. These findings provide the basis for the development of selective GnRH analog cancer therapeutics that directly target tumor cells or inhibit pituitary gonadotropins or do both.     

PTH-receptors regulate norepinephrine release in human heart and kidney

Recent data suggests that chronic renal failure and hyperparathyroidism are associated with sympathetic overactivity. Since peptide hormones are known to modulate norepinephrine (NE) release by activating prejunctional receptors, this study investigates whether parathyroid hormone fragment (1-34) (hPTH(1-34)) increases neuronal NE release in human heart and kidney. Using specific PTH-receptor agonists and antagonists, this study furthermore highlights functional differences between PTH1 and PTH2 receptors. Human atrial and renal tissues were incubated with [(3)H]-NE and superfused. Three electrical stimulations (5Hz, 1min) induced a stable [(3)H]-NE release which was taken as an index of endogenous NE release. RT-PCR with specific primers for PTH1- and PTH2-receptor was performed in heart and kidney. hPTH(1-34) (0.01-0.1μmol/L) and a stable analog of its second messenger cAMP (8-bromo-cAMP) increased [(3)H]-NE release in human atria. This facilitatory effect of PTH was also observed in human renal cortex. The PTH1-receptor antagonist (D-Trp(12), Tyr(34))-pTH-(7-34) (0.5μmol/L) abolished the effect of hPTH(1-34). This data was verified using isolated perfused mouse kidneys. Tuberoinfundibular peptide of 39 residues (TIP-39) (0.1nmol/L-0.1μmol/L) decreased [(3)H]-NE release in atria. PTH1- and PTH2-receptor expressions were demonstrated in human heart and kidney. Moreover, a splice variant of the PTH2-receptor was detected in human kidney. In conclusion, PTH is able to facilitate NE release in human atria and renal cortex by activation of PTH1-receptors. The highly increased PTH levels that can be observed in chronic renal failure might be one contributor for the elevated sympathetic nerve activity and the associated cardiovascular mortality in patients with end stage renal disease.     

The spinal antinociceptive effect of FR140423 is mediated through kyotorphin receptors

We investigated the antinociceptive effect of a novel anti-inflammatory and analgesic drug, 3-(difluoromethyl)-1-(4-methoxyphenyl)-5-[4-(methylsulfinyl)phenyl]pyraz ole (FR140423), in the tail-pinch test in mice, and evaluated the mechanism of action of FR140423 using L-leucyl-L-arginine (Leu-Arg), a kyotorphin (endogenous Met-enkephalin releaser) receptor antagonist, L-NG-nitroarginine methylester (L-NAME), an inhibitor of nitric oxide (NO) synthase, and methylene blue (MB), an inhibitor of activation of guanylate cyclase. Oral administration of FR140423, at doses of 5-80 mg/kg, produced a dose-dependent antinociceptive effect with an ED50 value of 18 mg/kg. This antinociception was reversed by intrathecal (i.t.) (10 microg/mouse), but not by intracerebroventricular (i.c.v.) (100 microg/mouse), injection of Leu-Arg. Moreover, the antinociceptive effect of i.t. injection of FR140423 with an ED50 value of 3.7 microg/mouse was completely antagonized by co-administered Leu-Arg 10 microg/mouse. However, L-NAME (2000 mg/kg s.c.) and MB (200 mg/kg s.c.) did not antagonize the antinociception of FR140423. These findings suggest that FR140423 plays a role in nociceptive modulation in the spinal cord, being antinociceptive via the kyotorphin-Met-enkephalin pathway but not via the peripheral NO-cyclic GMP pathway.     

Hindlimb paralytic effects of arginine vasopressin and related peptides following spinal subarachnoid injection in the rat

Intrathecal (IT) injection of arginine vasopressin (AVP) in rats caused a transient (<30 min), dose-related paralysis of the hindlimbs, loss of hindlimb and tail nociceptive responsiveness, and increased mean arterial pressure. Motor dysfunction was produced with comparable potency by lysine vasopressin (LVP) and arginine vasotocin (AVT); oxytocin (OXY) was approximately 1000 times less potent. Paralysis induced by these peptides was selectively blocked following IT pretreatment with 0.5 nmoles of the vasopressin V₁ receptor antagonist [1-(β-mercapto-β,β-cyclopentamethylene propioinic acid), 2-(O-methyl)tyrosine] Arg⁸-vasopressin (d(CH₂)₅[Tyr(Me²)]AVP). Pressor and antinociceptive responses to AVP were also blocked by this compound. However, at higher doses (2–5 nmoles, IT), d(CH₂)₅[Tyr(Me²)]AVP produced hindlimb paralysis, antinociception, and pressor responses by itself. In contrast to the fiber degeneration, cell loss, and necrosis found in lumbosacral cords of rats persistently paralyzed by other peptides (dynorphin A, somatostatin, and ICI 174864), neuropathological changes were not evident in spinal cords of rats transiently paralyzed by IT AVP. These results indicate that AVP-related peptides affected diverse spinal cord functions through interactions with a V₁-like receptor. The similar pattern of cardiovascular and antinociceptive responses to other peptides (dynorphin A, somatostatin, and ICI 174864), which also caused hindlimb paralysis, suggests that the former responses may actually reflect the nonselective consequences of a peptide-induced disruption of spinal cord function, rather than specific shared pharmacological effects.     

The pH-dependent conformational states of kyotorphin: a constant-pH molecular dynamics study

An extensive conformational study of the analgesic dipeptide kyotorphin (L-Tyr-L-Arg) at different pH values was performed using a constant-pH molecular dynamics method. This dipeptide showed a remarkable pH-dependent conformational variety. The protonation of the N-terminal amine was identified as a key element in the transition between the more extended and the more packed conformational states, as monitored by the dihedral angle defined by the atoms 1Cbeta-1Calpha-2Calpha-2Cbeta. The principal-component analysis of kyotorphin identified two major conformational populations (the extended trans and the packed cis) together with conformations that occur exclusively at extreme pH values. Other, less stable conformations were also identified, which help us to understand the transitions between the predominant populations. The fitting of kyotorphin's conformational space to the structure of morphine resulted in a set of conformers that were able to fulfill most of the constraints for the mu-receptor. These results suggest that there may be strong similarities between the kyotorphin receptor and the structural family of opioid receptors.     

GRK2 protein-mediated transphosphorylation contributes to loss of function of μ-opioid receptors induced by neuropeptide FF (NPFF2) receptors

Neuropeptide FF (NPFF) interacts with specific receptors to modulate opioid functions in the central nervous system. On dissociated neurons and neuroblastoma cells (SH-SY5Y) transfected with NPFF receptors, NPFF acts as a functional antagonist of μ-opioid (MOP) receptors by attenuating the opioid-induced inhibition of calcium conductance. In the SH-SY5Y model, MOP and NPFF(2) receptors have been shown to heteromerize. To understand the molecular mechanism involved in the anti-opioid activity of NPFF, we have investigated the phosphorylation status of the MOP receptor using phospho-specific antibody and mass spectrometry. Similarly to direct opioid receptor stimulation, activation of the NPFF(2) receptor by [D-Tyr-1-(NMe)Phe-3]NPFF (1DMe), an analog of NPFF, induced the phosphorylation of Ser-377 of the human MOP receptor. This heterologous phosphorylation was unaffected by inhibition of second messenger-dependent kinases and, contrarily to homologous phosphorylation, was prevented by inactivation of G(i/o) proteins by pertussis toxin. Using siRNA knockdown we could demonstrate that 1DMe-induced Ser-377 cross-phosphorylation and MOP receptor loss of function were mediated by the G protein receptor kinase GRK2. In addition, mass spectrometric analysis revealed that the phosphorylation pattern of MOP receptors was qualitatively similar after treatment with the MOP agonist Tyr-D-Ala-Gly (NMe)-Phe-Gly-ol (DAMGO) or after treatment with the NPFF agonist 1DMe, but the level of multiple phosphorylation was more intense after DAMGO. Finally, NPFF(2) receptor activation was sufficient to recruit β-arrestin2 to the MOP receptor but not to induce its internalization. These data show that NPFF-induced heterologous desensitization of MOP receptor signaling is mediated by GRK2 and could involve transphosphorylation within the heteromeric receptor complex.     

Spatial separation of beta-sheet domains of beta-amyloid: disruption of each beta-sheet by N-methyl amino acids

In a recent model of beta-amyloid (Abeta) fibrils, based mainly on solid-state NMR data, a molecular layer consists of two beta-sheets (residues 12-23 and 31-40 of Abeta1-40), folded onto one another by a connecting "bend" structure (residues 25-29) in the side-chain dimension. In this paper, we use two N-methyl amino acids to disrupt each of the two beta-sheets individually (2NMe(NTerm), residues 17 and 19; and 2NMe(CTerm), residues 37 and 39), or both of them at the same time (4NMe, with the above four N-methylated residues). Our data indicate that incorporation of two N-methyl amino acids into one beta-sheet is sufficient to disrupt that sheet while leaving the other, unmodified beta-sheet intact and able to form fibrils. We show, however, that disruption of each of the two beta-sheets has strikingly different effects on fibrillogenesis kinetics and fibril morphology. Both 2NMe(NTerm) and 2NMe(CTerm) form fibrils at similar rates, but more slowly than that of unmodified Abeta1-40. Electron microscopy shows that 2NMe(NTerm) forms straight fibrils with fuzzy amorphous material coating the edges, while 2NMe(CTerm) forms very regular, highly twisted fibrils-in both cases, distinct from the morphology of Abeta1-40 fibrils. Both 2NMe peptides show a "CMC" approximately four times greater than that of Abeta1-40. CD spectra of these peptides also evolve differently in time: whereas the CD spectra of 2NMe(NTerm) evolve little over 10 days, those of 2NMe(CTerm) show a transition to high beta-sheet content at about day 4-5. We also show that disruption of both beta-sheet domains, as in 4NMe, prevents fibril formation altogether, and renders Abeta1-40 highly water soluble and monomeric, and with solvent-exposed side chains. In summary, our data show (1) that the two beta-sheet domains fold in a semiautonomous manner, since disrupting each one still allows the other to fold; (2) that disruption of the N-terminal beta-sheet has a more profound effect on fibrillogenesis than disruption of the C-terminal beta-sheet, suggesting that the former is the more critical for the overall structure of the fibril; and (3) that disruption of both beta-sheet domains renders the peptide monomeric and unable to form fibrils.     

Dual effects of [Tyr6]‐γ2‐MSH(6–12) on pain perception and in vivo hyperalgesic activity of its analogues

[Tyr⁶]‐γ2‐MSH(6–12) with a short effecting time of about 20 min is one of the most potent rMrgC receptor agonists. To possibly increase its potency and metabolic stability, a series of analogues were prepared by replacing the Tyr⁶ residue with the non‐canonical amino acids 3‐(1‐naphtyl)‐L ‐alanine, 4‐fluoro‐L ‐phenylalanine, 4‐methoxy‐L ‐phenylalanine and 3‐nitro‐L ‐tyrosine. Dose‐dependent nociceptive assays performed in conscious rats by intrathecal injection of the MSH peptides showed [Tyr⁶]‐γ2‐MSH(6–12) hyperalgesic effects at low doses (5–20 nmol) and analgesia at high doses (100–200 nmol). This analgesic activity is fully reversed by the kyotorphin receptor‐specific antagonist Leu–Arg. For the two analogues containing in position 6, 4‐fluoro‐L ‐phenylalanine and 3‐nitro‐L ‐tyrosine, a hyperalgesic activity was not observed, while the 3‐(1‐naphtyl)‐L ‐alanine analogue at 10 nmol dose was found to induce hyperalgesia at a potency very similar to γ2‐MSH(6–12), but with longer duration of the effect. Finally, the 4‐methoxy‐L ‐phenylalanine analogue (0.5 nmol) showed greatly improved hyperalgesic activity and prolonged effects compared to the parent [Tyr⁶]‐γ2‐MSH(6–12) compound. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.