Peptides (v.87, #C)

Hypothalamic oxytocin (OXT) and arginine vasopressin (AVP) are known to act oppositely on hypothalamic-pituitary-adrenal (HPA) axis, stress response and gastrointestinal (GI) motility. In rodents, exposure to restraint stress (RS) delays gastric emptying (GE), however, repeated exposure to the same stressor (chronic homotypic stress (CHS)), the delayed GE is restored to basal level, while hypothalamic OXT is upregulated. In contrast, when rats are exposed to chronic heterotypic stress (CHeS), these adaptive changes are not observed. Although the involvement of central OXT in gastric motor adaptation is partly investigated, the role of hypothalamic AVP in CHeS-induced maladaptive paradigm is poorly understood. Using in-vivo brain microdialysis in rats, the changes OXT and AVP release from hypothalamus were monitored under basal non-stressed (NS) conditions and in rats exposed to acute stress (AS), CHS and CHeS. To investigate the involvement of central endogenous OXT or AVP in CHS-induced habituation and CHeS-induced maladaptation, chronic central administration of selective OXT receptor antagonist L-371257 and selective AVP V1b receptor antagonist SSR-149415 was performed daily. OXT was measured higher in AS and CHS group, but not in CHeS-loaded rats, whereas AVP significantly increased in rats exposed to AS and CHeS. Additionally, the response of the hypothalamic OXT- and AVP-producing cells was amplified following CHS and CHeS, respectively. In rats exposed to AS for 90 min solid GE significantly delayed. The delayed-GE was completely restored to the basal level following CHS, however, it remained delayed in CHeS-loaded rats. The CHS-induced restoration was prevented by L-371257, whereas SSR-149415 abolished the CHeS-induced impaired GE. A significant correlation was observed between GE and (i) OXT in CHS-loaded rats (rho = 0.61, p < 0.05, positively), (ii) AVP in CHeS-loaded rats (rho = 0.69, p < 0.05, negatively). Under long term stressed conditions, the release of AVP and OXT from hypothalamus may vary depending on the content of the stressors. Central AVP appears to act oppositely to OXT by mediating CHeS-induced gastric motor maladaptation. Long term central AVP antagonism might be a pharmacological approach for the treatment of stress-related gastric motility disorders.
Keywords: Oxytocin; Arginine vasopressin; Chronic stress; Gastric emptying; Maladaptation;

Intracerebroventricular injection of ghrelin decreases wheel running activity in rats by Yumiko Miyatake; Tetsuya Shiuchi; Kazuaki Mawatari; Satomi Toda; Yasuko Taniguchi; Akari Futami; Fukiko Sato; Masashi Kuroda; Mayu Sebe; Rie Tsutsumi; Nagakatsu Harada; Yasuhiko Minokoshi; Tadahiro Kitamura; Koro Gotoh; Masaki Ueno; Yutaka Nakaya; Hiroshi Sakaue (12-19).
There is an increasing interest in elucidating the molecular mechanisms by which voluntary exercise is regulated. In this study, we examined how the central nervous system regulates exercise. We used SPORTS rats, which were established in our laboratory as a highly voluntary murine exercise model. SPORTS rats showed lower levels of serum ghrelin compared with those of the parental line of Wistar rats. Intracerebroventricular and intraperitoneal injection of ghrelin decreased wheel-running activity in SPORTS rats. In addition, daily injection of the ghrelin inhibitor JMV3002 into the lateral ventricles of Wistar rats increased wheel-running activity. Co-administration of obestatin inhibited ghrelin-induced increases in food intake but did not inhibit ghrelin-induced suppression of voluntary exercise in rats. Growth hormone secretagogue receptor (GHSR) in the hypothalamus and hippocampus of SPORTS rats was not difference that in control rats. We created an arcuate nucleus destruction model by administering monosodium glutamate (MSG) to neonatal SPORTS rats. Injection of ghrelin into MSG-treated rats decreased voluntary exercise but did not increase food intake, suggesting that wheel-running activity is not controlled by the arcuate nucleus neurons that regulate feeding. These results provide new insights into the mechanism by which ghrelin regulates voluntary activity independent of arcuate nucleus neurons.
Keywords: Ghrelin; Voluntary exercise; Central nervous system;

Region-specific bioconversion of dynorphin neuropeptide detected by in situ histochemistry and MALDI imaging mass spectrometry by Erik Bivehed; Robert Strömvall; Jonas Bergquist; Georgy Bakalkin; Malin Andersson (20-27).
Display OmittedBrain region-specific expression of proteolytic enzymes can control the biological activity of endogenous neuropeptides and has recently been targeted for the development of novel drugs, for neuropathic pain, cancer, and Parkinson’s disease. Rapid and sensitive analytical methods to profile modulators of enzymatic activity are important for finding effective inhibitors with high therapeutic value.Combination of in situ enzyme histochemistry with MALDI imaging mass spectrometry allowed developing a highly sensitive method for analysis of brain-area specific neuropeptide conversion of synthetic and endogenous neuropeptides, and for selection of peptidase inhibitors that differentially target conversion enzymes at specific anatomical sites. Conversion and degradation products of Dynorphin B as model neuropeptide and effects of peptidase inhibitors applied to native brain tissue sections were analyzed at different brain locations. Synthetic dynorphin B (2 pmol) was found to be converted to the N-terminal fragments on brain sections whereas fewer C-terminal fragments were detected. N-ethylmaleimide (NEM), a non-selective inhibitor of cysteine peptidases, almost completely blocked the conversion of dynorphin B to dynorphin B(1–6; Leu-Enk-Arg), (1–9), (2–13), and (7–13). Proteinase inhibitor cocktail, and also incubation with acetic acid displayed similar results.Bioconversion of synthetic dynorphin B was region-specific producing dynorphin B(1–7) in the cortex and dynorphin B (2–13) in the striatum. Enzyme inhibitors showed region- and enzyme-specific inhibition of dynorphin bioconversion. Both phosphoramidon (inhibitor of the known dynorphin converting enzyme neprilysin) and opiorphin (inhibitor of neprilysin and aminopeptidase N) blocked cortical bioconversion to dynorphin B(1–7), wheras only opiorphin blocked striatal bioconversion to dynorphin B(2–13).This method may impact the development of novel therapies with aim to strengthen the effects of endogenous neuropeptides under pathological conditions such as chronic pain. Combining histochemistry and MALDI imaging MS is a powerful and sensitive tool for the study of inhibition of enzyme activity directly in native tissue sections.
Keywords: Neuropeptide; Dynorphin; Bioconversion; Enzyme; Enzyme inhibitor; Histochemistry; MALDI imaging mass spectrometry; Mass spectrometry; Parkinson’s disease; Neuropathic pain;

Neuropeptide glutamic acid-isoleucine (NEI)-induced paradoxical sleep in rats by Moe Fujimoto; Satoru Fukuda; Hidetoshi Sakamoto; Junko Takata; Shigehito Sawamura (28-33).
Neuropeptideglutamic acid-isoleucine (NEI) as well as melanin concentrating hormone (MCH) is cleaved from the 165 amino acid protein, prepro-melanin concentrating hormone (prepro-MCH). Among many physiological roles of MCH, we demonstrated that intracerebroventricular (icv) injection of MCH induced increases in REM sleep episodes as well as in non REM sleep episodes. However, there are no studies on the effect of NEI on the sleep-wake cycle. As for the sites of action of MCH for induction of REM sleep, the ventrolateral periaqueductal gray (vlPAG) has been reported to be one of its site of action. Although MCH neurons contain NEI, GABA, MCH, and other neuropeptides, we do not know which transmitter(s) might induce REM sleep by acting on the vlPAG. Thus, we first examined the effect of icv injection of NEI on the sleep-wake cycle, and investigated how microinjection of either NEI, MCH, or GABA into the vlPAG affected REM sleep in rats. Icv injection of NEI (0.61 μg/5 μl: n = 7) significantly increased the time spent in REM episodes compared to control (saline: 5 μl; n = 6). Microinjection of either NEI (61 ng/0.2 μl: n = 7), MCH (100 ng/0.2 μl: n = 6) or GABA (250 mM/0.2 μl: n = 7) into the vlPAG significantly increased the time spent in REM episodes and the AUC. Precise hourly analysis of REM sleep also revealed that after those microinjections, NEI and MCH increased REM episodes at the latter phase, compared to GABA which increased REM episodes at the earlier phase. This result suggests that NEI and MCH may induce sustained REM sleep, while GABA may initiate REM sleep. In conclusion, our findings demonstrate that NEI, a cleaved peptide from the same precursor, prepro-MCH, as MCH, induce REM sleep at least in part through acting on the vlPAG.
Keywords: Neuropeptide glutamic acid-isoleucine; Melanin concentrating hormone; Gamma aminobutyric acid; Lateral periaqueductal gray;

Functional and immuno-reactive characterization of a previously undescribed peptide from the venom of the scorpion Centruroides limpidus by Timoteo Olamendi-Portugal; Rita Restano-Cassulini; Lidia Riaño-Umbarila; Baltazar Becerril; Lourival D. Possani (34-40).
A previously undescribed toxic peptide named Cl13 was purified from the venom of the Mexican scorpion Centruroides limpidus. It contains 66 amino acid residues, including four disulfide bonds. The physiological effects assayed in 7 different subtypes of voltage gated Na+-channels, showed that it belongs to the β-scorpion toxin type. The most notorious effects were observed in subtypes Nav1.4, Nav1.5 and Nav1.6. Although having important sequence similarities with two other lethal toxins from this scorpion species (Cll1m and Cll2), the recently developed single chain antibody fragments (scFv) of human origin were not capable of protecting against Cl13. At the amino acid sequence level, in 3 stretches of peptide Cl13 (positions 7–9, 30–38 and 62–66) some differences with respect to other similar toxins are observed. Some of these differences coincide with contact points with the human antibody fragments.
Keywords: Antibody; Centruroides limpidus; Scorpion venom; toxinToxin;

Exercise modulates the aortic renin-angiotensin system independently of estrogen therapy in ovariectomized hypertensive rats by Patrick W. Endlich; Erick R.G. Claudio; Leandro C.F. Lima; Rogério F. Ribeiro Júnior; Antonio A.B. Peluso; Ivanita Stefanon; Nazaré S. Bissoli; Virginia S. Lemos; Robson A.S. dos Santos; Glaucia R. de Abreu (41-49).
The renin-angiotensin-system is an important component of cardiovascular control and is up-regulated under various conditions, including hypertension and menopause. The aim of this study was to evaluate the effects of swimming training and estrogen therapy (ET) on angiotensin-II (ANG II)-induced vasoconstriction and angiotensin-(1-7) [ANG-(1-7)]-induced vasorelaxation in aortic rings from ovariectomized spontaneously hypertensive rats. Animals were divided into Sham (SH), Ovariectomized (OVX), Ovariectomized treated with E2 (OE2), Ovariectomized plus swimming (OSW) and Ovariectomized treated with E2 plus swimming (OE2 + SW) groups. ET entailed the administration of 5 μg of 17β-Estradiol three times per week. Swimming was undertaken for sixty minutes each day, five times per week. Both, training and ET were initiated seven days following ovariectomy. Forty-eight hours after the last treatment or training session, the animals’ systolic blood pressures were measured, and blood samples were collected to measure plasma ANG II and ANG-(1-7) levels via radioimmunoassay. In aortic rings, the vascular reactivity to ANG II and ANG-(1-7) was assessed. Expression of ANG-(1-7) in aortic wall was analyzed by immunohistochemistry. The results showed that both exercise and ET increased plasma ANG II levels despite attenuating systolic blood pressure. Ovariectomy increased constrictor responses to ANG II and decreased dilatory responses to ANG-(1-7), which were reversed by swimming independently of ET. Moreover, it was observed an apparent increase in ANG-(1-7) content in the aorta of the groups subjected to training and ET. Exercise training may play a cardioprotective role independently of ET and may be an alternative to ET in hypertensive postmenopausal women.
Keywords: Angiotensin II; Angiotensin-(1-7); Swimming; Estrogen therapy; SHR; Vascular reactivity;

In vitro and in vivo delivery of therapeutic proteins using cell penetrating peptides by Azam Bolhassani; Behnaz Sadat Jafarzade; Golnaz Mardani (50-63).
Display OmittedThe failure of proteins to penetrate mammalian cells or target tumor cells restricts their value as therapeutic tools in a variety of diseases such as cancers. Recently, protein transduction domains (PTDs) or cell penetrating peptides (CPPs) have been shown to promote the delivery of therapeutic proteins or peptides into live cells. The successful delivery of proteins mainly depends on their physicochemical properties. Although, linear cell penetrating peptides are one of the most effective delivery vehicles; but currently, cyclic CPPs has been developed to potently transport bioactive full-length proteins into cells. Up to now, several small protein transduction domains from viral proteins including Tat or VP22 could be fused to other peptides or proteins to entry them in various cell types at a dose-dependent approach. A major disadvantage of PTD-fusion proteins is primary uptake into endosomal vesicles leading to inefficient release of the fusion proteins into the cytosol. Recently, non-covalent complex formation (Chariot) between proteins and CPPs has attracted a special interest to overcome some delivery limitations (e.g., toxicity). Many preclinical and clinical trials of CPP-based delivery are currently under evaluation. Generally, development of more efficient protein transduction domains would significantly increase the potency of protein therapeutics. Moreover, the synergistic or combined effects of CPPs with other delivery systems for protein/peptide drug delivery would promote their therapeutic effects in cancer and other diseases. In this review, we will describe the functions and implications of CPPs for delivering the therapeutic proteins or peptides in preclinical and clinical studies.
Keywords: Cell penetrating peptide; Therapeutic protein/peptide; Cancer; Disease; Preclinical and clinical trials;

Antidepressant-like effects exerted by the intranasal administration of a glucagon-like peptide-2 derivative containing cell-penetrating peptides and a penetration-accelerating sequence in mice by Sachie Sasaki-Hamada; Ryuji Nakamura; Yusuke Nakao; Toshiki Akimoto; Emi Sanai; Mio Nagai; Michiko Horiguchi; Chikamasa Yamashita; Jun-Ichiro Oka (64-70).
The intracerebroventicular (i.c.v.) administration of glucagon-like peptide-2 (GLP-2) to rodents was shown to have antidepressant-like effects in imipramine-resistant depression-model mice. In order to utilize GLP-2 as a clinical treatment tool for depression, we herein focused on the intranasal delivery that is non-invasive approach, because the i.c.v. administration is invasive and impractical. In the present study, we prepared a GLP-2 derivative containing cell penetrating peptides (CPPs) and a penetration accelerating sequence (PAS) (PAS-CPPs-GLP-2) for the intranasal (i.n.) administration. PAS-CPPs-GLP-2 (i.n.) exhibited antidepressant-like effects in the forced-swim test (FST) and tail suspension test (TST) in naïve mice as well as adrenocorticotropic hormone (ACTH) treated-mice. However, PAS-CPPs-GLP-2 (i.v.) and the GLP-2 derivative containing CPPs without a PAS (CPPs-GLP-2) (i.n.) did not affect the immobility time in the mouse FST. Moreover, fluorescein isothiocyanate (FITC)-labeled PAS-CPPs-GLP-2 (i.n.), but not FITC-labeled CPPs-GLP-2 (i.n.) was distributed through the mouse brain after the FST session. These results suggest that PAS-CPPs-GLP-2 is effective for i.n. delivery to the brain, and may be useful in the clinical treatment of major depression.
Keywords: Glucagon-like peptide-2; Antidepressant; Intranasal administration; Brain drug delivery and targeting;

Recent studies have shown that UVB irradiation induces primary and secondary hyperalgesia in rats and humans peaking about 24 h after UVB exposure. In the present study we investigated the changes in galanin, substance P and c-fos immunoreactivity in rat DRG and spinal cord at the L5 level 2–96 h after UVB irradiation. UVB irradiation of the heel area in rats almost increased the skin blood flow two-fold 24 h after irradiation as measured by laser Doppler technique. UVB irradiation induced a significant reduction of the proportion of galanin positive DRG neurons for all time points, except at 12 h. In the spinal cord, UVB irradiation induced increased immunoreactivity for galanin in the dorsal horn, the area around the central canal and interestingly also in the lateral spinal nucleus 12–96 h after exposure. For substance P the proportion of substance P positive neurons was unchanged but UVB irradiation induced increased substance P immunoreactivity in the dorsal part of the spinal cord 48 h after irradiation. UVB irradiation also induced c-fos immunoreactivity in the dorsal horn and the area around the central canal 24 and 48 h after exposure. This translational model of UVB irradiation will induce rapid changes of neuropeptides implicated in nociceptive signaling in areas known to be of importance for nociception in a time frame, about 24 h after exposure, where also neurophysiological alteration have been described in humans and rats.
Keywords: Lateral spinal nucleus; Dorsal horn; Inflammation; Neuropeptides; Pain translational model; Immunoreactivity;