Peptides (v.23, #6)

A purification scheme involving ion exchange chromatography on DEAE–cellulose, affinity chromatography on Affi-gel blue gel, and ion exchange chromatography on CM–Sepharose and Mono S was employed to isolate a peptide with a molecular weight of 7.8 kDa from sponge gourd seeds. The peptide, which was designated luffacylin, exhibited an N-terminal sequence with pronounced resemblance to that of the 6.5 kDa arginine–glutamate rich polypeptide previously isolated from sponge gourd seeds. Luffacylin inhibited translation in a rabbit reticulocyte lysate system with an ic 50 of 140 pM and reacted positively in the N-glycosidase assay for ribosome inactivating proteins. Luffacylin exerted anti-fungal activity against Mycosphaerella arachidicola and Fusarium oxysporum.
Keywords: Sponge gourd; Seeds; Anti-fungal; Ribosome inactivation;

An isolation procedure comprising ion exchange chromatography on DEAE–cellulose, affinity chromatography on Affi-gel blue gel, ion exchange chromatography on SP–Sepharose and gel filtration on Superdex 75 was used to isolate an anti-fungal peptide from the bulbs of the shallot Allium ascalonicum. The peptide demonstrated a molecular weight of 9.5 kDa, and possessed an N-terminal sequence YQCGQGG somewhat similar to chitinases from other Allium species which are however much larger in molecular weight. The peptide designated ascalin manifested a unique specific anti-fungal activity. It inhibited mycelial growth in the fungus Botrytis cinerea but not in the fungi Mycosphaerella arachidicola and Fusarium oxysporum. Ascalin inhibited HIV-1 reverse transcriptase with an ic 50 of 10 μM, much more potently than Allium tuberosum anti-fungal protein and other anti-fungal proteins.
Keywords: Ascalin; HIV type 1; Shallot bulbs;

This paper demonstrates for the first time the occurrence of tetrapeptides related to APGWamide in the mollusk cephalopod Sepia officinalis. LC-ESI-MS/MS analysis allowed the identification of the APGWamide-related peptides predicted by the two genes cloned previously in Lymnaea stagnalis and in Mytilus edulis, as well as the dipeptide GWamide released from the processing of the tetrapeptides by a dipeptidyl aminopeptidase (DPAP). TPGWamide and GWamide appeared to be exclusively located in the CNS, and the APGWamide in both the CNS and the nerve endings. The RPGWamide and the KPGWamide were not detected by LC-ESI-MS/MS suggesting they could be totally processed into GWamide. The in vitro processing of the tetrapeptides into GWamide by optic lobe extract revealed a differential processing for each, with APGWamide (44.7%)>RPGWamide  (24.3%)>KPGWamide  (19.3%)>TPGWamide (11.7%). The tissue mapping results, together with the processing efficiency data suggest that the GWamide is mainly produced from the M. edulis gene products RPGWamide and KPGWamide.
Keywords: LC-ESI-MS/MS; APGWamide; Neuropeptide; Cephalopods; Cuttlefish; Sepia officinalis; Oviduct; Egg laying; DPAP;

Allatostatins of the tiger prawn, Penaeus monodon (Crustacea: Penaeidea) by Hanne Duve; Anders H Johnsen; Alan G Scott; Alan Thorpe (1039-1051).
More than 40 peptides belonging to the -Y/FXFGL-NH2 allatostatin superfamily have been isolated and identified from the central nervous system (CNS) of the tiger prawn, Penaeus monodon (Crustacea: Penaeidea). The peptides can be arranged in seven sub-groups according to the variable post-tyrosyl residue represented by Ala, Gly, Ser, Thr, Asn, Asp, and Glu. Two of the residues (Thr and Glu) have not been observed in this position previously in either insects or crustaceans. Also reported for the first time for allatostatins, two of the peptides are N-terminally blocked by a pyroglutamic acid residue. The yields of certain peptides with similar amino acid sequences to each other were, in some instances, very different. As an example, the yield of ANQYTFGL-NH2 was 2 pmol, compared with ASQYTFGL-NH2, with a yield of 156 pmol. There are several possibilities to account for this. If, as in all species so far investigated, there is a single allatostatin gene in P. monodon, then it would appear that different sub-populations have contributed mutant forms of particular peptides to the extract. Another, less likely possibility is that this species has more than one allatostatin gene, producing a variable array of peptides albeit in different molar ratios. Several peptides were present apparently as a result of the loss of one or more residues at the N-terminus of a larger form, either due to N-terminal degradation or specific post-translational processing. The number of peptides identified exceeds that for any other insect or crustacean species previously investigated. None is identical to any of the 60–70 insect allatostatins so far identified, and only three are common to other crustaceans. Immunohistochemical study of the CNS of P. monodon, with the same antisera as used to monitor the purification, confirms the widespread nature and complexity of allatostatinergic neural pathways in arthropods. Thus, all neuromeres of the brain, and all except one of the ventral cord ganglia, possess allatostatin neurons and extensive areas of allatostatin-innervated neuropile. In addition to the cytological evidence that the allatostatins act as neurotransmitters, associated with tissues as varied as eyes and legs, their presence in neurohemal areas such as the sinus gland and the perineural sheath of the thoracic ganglia suggests a neuroendocrine function. As well as posing a challenge to physiologists assigning specific functions to the allatostatins, their extensive intra-species multiplicity, linked to their inter-species variability, also presents a complex problem to geneticists and evolutionists.
Keywords: Neuropeptide; Allatostatin; Penaeus monodon;

The quantitative distribution of corticotropin-releasing hormone (CRH) in the brain and pituitary of the fish Oreochromis mossambicus (tilapia) was studied following the validation of a radioimmunoassay. Compared to the pituitary content, the brain contained 20 times more CRH. Eighty percent of the total brain content was located outside the hypothalamus, particularly in the telencephalon. Substantial amounts of CRH were also present in other regions devoid of hypophysiotropic neurons, such as the vagal lobe and optic tectum. Telencephalic and pituitary CRH co-eluted with the tilapia CRH1–41standard on reverse phase HPLC. In vitro CRH release by the telencephalon amounted to 5% of its content per hour, whereas release from the pituitary was negligible. We conclude that CRH in the brain of tilapia regulates pituitary and non-pituitary related functions, probably as a neurotransmitter or neuromodulator.
Keywords: CRF; Pituitary; Stress; RIA; HPLC; Cichlids; Teleost; Superfusion; Telencephalon;

Diet-induced changes in hypothalamic pro-opio-melanocortin mRNA in the rat hypothalamus by Carla Torri; Patrizia Pedrazzi; Giuseppina Leo; Eugenio E Müller; Daniela Cocchi; Luigi F Agnati; Michele Zoli (1063-1068).
Hypothalamic mRNA and peptide levels of pro-opio-melanocortin (POMC) and other neuropeptides were studied in rats that either develop obesity (diet-induced obese, DIO), when fed a palatable and hypercaloric diet (cafeteria diet, caf) or do not develop obesity (diet resistant, DR), when fed the same diet. cafDIO rats showed a significant increase in POMC, but not in melanin concentrating hormone, mRNA levels as determined by semiquantitative in situ hybridization. cafDR and cafDIO rats showed no change in POMC-derived peptide levels, whereas neuropeptide Y immunoreactivity was significantly increased in cafDR rats. POMC mRNA levels were also studied in high-fat diet-fed rats but no significant change was observed. Altered hypothalamic transmission by POMC-derived peptides may contribute to the susceptibility of cafDIO rats to the weight promoting action of caf diet.
Keywords: Obesity; Cafeteria diet; High-fat diet; Pro-opio-melanocortin; In situ hybridization; Rat;

Sustained orexigenic effect of Agouti related protein may be not mediated by the melanocortin 4 receptor by Min-Seon Kim; Michela Rossi; Caroline R Abbott; Samaher H AlAhmed; David M Smith; Stephen R Bloom (1069-1076).
Intracerebroventricular (ICV) injection of Agouti related protein (AgRP), an endogenous melanocortin 3 and 4 receptor (MC3/4-R) antagonist, produces a prolonged increase in food intake. To clarify the roles of the MC3-R and MC4-R in AgRP-induced hyperphagia, the feeding effect of AgRP (83–132) was compared with that of the selective MC4-R antagonist, JKC-363 (cyclic [Mpr11, D-Nal14, Cys18, Asp22-NH2]-beta-MSH11-22). Single ICV administration of AgRP (83–132) increased food intake for 48 h whilst ICV JKC-363 increased food intake for 8 h. An increase in body weight at 24 and 48 h was observed following AgRP (83–132) but not JKC-363 treatment. These data suggest that the sustained orexigenic action of AgRP (83–132) may not be through MC4-R antagonism.
Keywords: Agouti related protein; Melanocortin 4 receptor; Appetite; Hypothalamus; Intracerebroventricular;

The structure–activity relationship and signal transduction properties of the pro-opiomelanocortin (POMC)-derived γ-MSH peptides in the GH3 cell line was compared with that described for the known melanocortin receptors (MCRs). Single alanine replacements showed that, unlike the classical MCRs, the His5–Phe6–Arg7–Trp8 sequence in γ2-MSH is not a core sequence for activating the γ-MSH receptor in GH3 cells, whereas Met3 is essential. γ2-MSH increased binding of [ 35 S ]GTPγS to membrane preparations of GH3 cells. Blockade of protein kinase A abolished the [Ca2+] i responses to γ3-MSH, and low nanomolar doses of γ3-MSH increased intracellular cAMP levels, which could be blocked by pertussis toxin (PTX). We conclude that the putative novel γ-MSH receptor in GH3 cells is a GPCR, but with structure–activity and signal transduction features different from those of the classical MCRs.
Keywords: Melanocortin; Receptor; γ-MSH; GH3 cells; Intracellular calcium; cAMP; G protein;

Attenuation of hypercholesterolemia and hyperglycemia in ob/ob mice by NPY Y2 receptor ablation by Philippe Naveilhan; Lennart Svensson; Susanne Nyström; A.Jonas Ekstrand; Patrik Ernfors (1087-1091).
Neuropeptide Y (NPY) is a 36 amino acid peptide well known for its role in regulating food intake and energy homeostasis. It has previously been shown that the NPY Y2 receptor is required for a full biological response to leptin in the central nervous system. We have examined the impact of this receptor on plasma levels of lipid and cholesterol in wild type and obese (ob/ob) mice. The results show that an absence of Y2 in female mice has no effect on cholesterol level in normal lean mice but profoundly decreases serum cholesterol and glucose levels in ob/ob mice. We conclude that NPY, interacting with the Y2 receptor, participates in cholesterol and glucose homeostasis of obese mice.
Keywords: Y2 receptor knock out; Mice; Obesity;

Immunotoxic catecholamine lesions attenuate 2DG-induced increase of AGRP mRNA by G.S. Fraley; T.T. Dinh; S. Ritter (1093-1099).
Injections of the immunotoxin, saporin conjugated to anti-dopamine-β-hydroxylase (DSAP), into the hypothalamic paraventricular nucleus (PVH) selectively destroy norepinephrine (NE) and epinephrine (E) terminals in the medial hypothalamus and abolish glucoprivic feeding. We utilized PVH DSAP injections to examine the role of NE/E neurons in the previously reported 2-deoxy-d-glucose (2DG)-induced increases in mRNA levels for the orexigenic peptides, AGRP and NPY. Northern blot analysis revealed that DSAP lesions elevated basal but blocked 2DG-induced increases in AGRP mRNA levels. Changes in NPY mRNA were not detectable. AGRP neurons may contribute to circuitry activated by NE/E neurons for elicitation of glucoregulatory responses.
Keywords: 2-Deoxy-d-glucose; Norepinephrine; Epinephrine; Hindbrain afferents; Paraventricular nucleus of the hypothalamus; Arcuate nucleus; Anti-dopamine-β-hydroxylase saporin; Glucoprivic feeding;

Pharmacological characterization of β-endorphin- and dynorphin A1–17-induced feeding using G-protein α-subunit antisense probes in rats by Robert M Silva; Henya C Grossman; Grace C Rossi; Gavril W Pasternak; Richard J Bodnar (1101-1106).
Antisense (AS) oligodeoxynucleotides targeting G-protein α-subunits distinguish feeding responses of morphine and its metabolite, as well as nocturnal and deprivation-induced feeding. The present study examined whether feeding elicited by β-endorphin (βEND) or dynorphin A1–17 was altered by ventricularly-applied Giα1, Giα2, Giα3, Gsα, Goα, Gqα or Gx/zα AS probes, or a nonsense (NS) control. The βEND-induced feeding was reduced by the Giα1 and Gx/zα AS probes, and increased by Giα2 or Giα3 AS treatment. Dynorphin-induced feeding was attenuated by Giα1 and Goα AS treatment. Yet, Gsα or Gqα AS and NS treatments failed to alter opioid agonist-induced feeding. These data provide initial characterization of potential effector signaling pathways mediating βEND and dynorphin-induced feeding.
Keywords: Feeding; β-Endorphin; Dynorphin A1–17; G-protein α-subunit antisense;

Peptide nucleic acids targeted to the mouse proNPFFA reveal an endogenous opioid tonus by Elisabeth Bonnard; Honoré Mazarguil; Jean-Marie Zajac (1107-1113).
Pharmacological studies have implicated the anti-opioid neuropeptide FF (NPFF) in the modulation of pain transmission. Since its physiological role has not yet been fully elucidated, the present study examined whether antisense peptide nucleic acid (PNA) complementary to the NPFF precursor (proNPFFA) modified pain sensitivity. Mice received three intraperitoneal (i.p.) injections (10 mg/kg) of antisense PNA (As-proNPFFA) over a period of 24 h. As-proNPFFA treatment significantly increased the basal tail withdrawal latency in the tail-flick test. This analgesia persisted during 2 days and was completely reversed by naloxone. Thus, antisense PNAs, by decreasing anti-opioid effects, revealed a basal endogenous opioid activity. Our results evidence a physiological interplay between NPFF and opioid systems and further support the use of PNA as effective antisense agents, for studying gene function in vivo.
Keywords: Peptide nucleic acids; Antisense; Neuropeptide FF; Anti-opioid; Endogenous opioid activity; Pain sensitivity;

Synthetic peptide SLTCLVKGFY competes with β-endorphin for naloxone-insensitive binding sites on rat brain membranes by Elena V Navolotskaya; Tatyana A Zargarova; Natalia V Malkova; Svetlana B Krasnova; Vladimir P Zav’yalov; Valery M Lipkin (1115-1119).
The synthetic decapeptide Ser–Leu–Thr–Cys–Leu–Val–Lys–Gly–Phe–Tyr (termed immunorphin) corresponding to the sequence 364–373 of the CH3 domain of human immunoglobulin G heavy chain and its synthetic fragment VKGFY were found to compete with 125 I -labeled β-endorphin for high-affinity naloxone-insensitive binding sites on membranes isolated from the rat brain cortex (K i=1.18±0.09 and 1.58±0.11 nM, respectively). The binding specificity study revealed that these binding sites were insensitive not only to naloxone but to [Met5]enkephalin and [Leu5]enkephalin as well. The K d values characterizing the specific binding of 125 I -labeled immunorphin and its fragment Val–Lys–Gly–Phe–Tyr to these binding sites were determined to be 2.93±0.27 nM and 3.17±0.29 nM, respectively.
Keywords: β-Endorphin; Receptor; Peptide; T lymphocytes;

Oxytocin stimulates proliferation of human osteoblast-like cells by Maria Petersson; Alena Lagumdzija; André Stark; Elisabet Bucht (1121-1126).
Oxytocin receptors have recently been demonstrated in human osteoblast-like (hOB) cells. In this study, oxytocin 100–1000 pmol/l increased cell proliferation of primary cultures of hOB cells, measured by [ 3 H ]thymidine incorporation, (P<0.01). In human osteosarcoma cell-line (SaOS-2), oxytocin 100 pmol/l increased cell proliferation (measured by [ 3 H ]thymidine incorporation and a commercially available kit) and protein synthesis ([ 3 H ]proline incorporation) (P<0.05). The increase in cell proliferation was abolished when SaOS-2 cells were incubated with an oxytocin antagonist and oxytocin. Oxytocin 100 pmol/l decreased interleukin-6 (IL-6) production of the hOB cells (23.4±1.96 versus 33.4±2.65 pg/well; P<0.001). These findings indicate that oxytocin may affect bone metabolism in humans.
Keywords: Oxytocin; Osteoblast; hOB cell; SaOS-2; Oxytocin antagonist; Interleukin-6;

Expression of growth hormone-releasing hormone (GHRH) and splice variants of GHRH receptors in human experimental prostate cancers by Artur Plonowski; Andrew V Schally; Rebeca Busto; Magdalena Krupa; Jozsef L Varga; Gabor Halmos (1127-1133).
The expression of mRNA for GHRH and splice variants (SVs) of GHRH receptors in LNCaP, MDA-PCa-2b and PC-3 human prostate cancers grown in nude mice was investigated by RT-PCR. The expression of mRNA for GHRH was detected in LNCaP and PC-3, but not in MDA-PCa-2b prostatic carcinoma. RT-PCR analyses of mRNA isolated from LNCaP, MDA-PCa-2b and PC-3 cancers, revealed the presence of 720 and 566 bp products, corresponding to SV1 and SV2 isoforms of GHRH receptors. In PC-3 tumor membranes a radiolabeled GHRH antagonist [ 125 I ]-JV-1-42 was bound to one class of high-affinity binding sites (K d=1.81±0.47 nM) and maximum binding capacity of 332.7±27.8 fmol/mg membrane protein. The in vivo uptake of [ 125 I ]-JV-1-42 was observed in all xenografts of human prostate cancer, the tracer accumulation being the highest in PC-3 tumors. These results indicate that GHRH and SVs of its receptors, different from those found in the pituitary, are present in experimental human prostate cancers and may form a local mitogenic loop. The antiproliferative effects of GHRH antagonists on growth of prostate cancer could be exerted in part by an interference with this local GHRH system.
Keywords: Growth hormone-releasing hormone; Receptors for growth hormone-releasing hormone; Prostate cancer;

Expression of prolactin-releasing peptide and its receptor in the human adrenal glands and tumor tissues of adrenocortical tumors, pheochromocytomas and neuroblastomas by Kazuhiro Takahashi; Kazuhito Totsune; Osamu Murakami; Masahiko Sone; Takao Noshiro; Yutaka Hayashi; Hironobu Sasano; Shigeki Shibahara (1135-1140).
Adrenal tumors, such as pheochromocytomas, are known to express various peptides and their receptors. Prolactin-releasing peptide (PrRP) is a novel neuropeptide isolated from bovine hypothalamic tissues. In the present study, expression of PrRP receptor was studied in the human brain, pituitaries, adrenal glands and tumor tissues of adrenocortical tumors, pheochromocytomas, a ganglioneuroblastoma and neuroblastomas by reverse transcriptase polymerase chain reaction (RT-PCR) and Northern blot analysis. The presence of immunoreactive-PrRP in the adrenal glands and in these tumor tissues was studied by radioimmunoassay. Human brain tissues and pituitaries were obtained at autopsy. Normal portions of adrenal glands and tumor tissues were obtained at surgery. RT-PCR analysis showed expression of PrRP receptor in the human brain, pituitaries, normal portions of adrenal glands and various tumor tissues. Northern blot analysis showed high expression of PrRP receptor only in tumor tissues of pheochromocytomas, indicating that PrRP receptor expression is high in pheochromocytomas. Immunoreactive-PrRP was detected in normal portions of adrenal glands (0.162±0.024 pmol/g wet weight, n=4, mean±S.E.M.), four out of six cases of pheochromocytomas (0.050–7.9 pmol/g wet weight), one neuroblastoma and some adrenocortical tumors. The present study has shown that PrRP receptor mRNA was widely expressed in the brain tissues, pituitaries, adrenal glands and various tumors. The high expression of PrRP receptor in pheochromocytomas suggests potential pathophysiological roles of PrRP in these tumors.
Keywords: Prolactin-releasing peptide; Brain; Pheochromocytoma; Receptor; Adrenal;

Effects of different peptide fragments derived from proadrenomedullin on gene expression of adrenomedullin gene by Yong Fen Qi; Ding Fang Bu; Da Di Niu; Yan Rong Shi; Shu Heng Wang; Yong Zheng Pang; Chao Shu Tang; Jun Bao Du (1141-1147).
Primary culture of vascular smooth muscle cells (VSMC) from rat aorta was used for the study of the effect of different peptides derived from proadrenomedullin on the expression of adrenomedullin (ADM) gene. ADM and preproADM22–41 (PAMP) secreted by VSMC were measured by radioimmunoassay, and ADM mRNA in VSMC was determined by quantitative RT-PCR. After the incubation of VSMC in 10−7  M ADM for 24 h, PAMP in the medium and ADM mRNA in the VSMC were decreased by 34 and 41.3%, respectively, and cAMP concentration in the VSMC was increased by 385%. After the incubation of VSMC in 10−7  M PAMP for 24 h, ADM in the medium and ADM mRNA in the VSMC were decreased by 12.2 and 39.1%, respectively, and cAMP concentration in the VSMC was increased by 67%. The decreased ADM mRNA in VSMC induced by the ADM and PAMP treatment was completely reversed by the pre-treatment of the cells in 10−7  M protein kinase inhibitor for 30 min. After the incubation of VSMC in 10−7  M preproADM153–185 (ADT) for 24 h, however, ADM in the medium and ADM mRNA in the VSMC were increased by 21 and 35.2%. The increased ADM mRNA in VSMC induced by the ADT treatment was partially blocked by the co-incubation in ADM and ADT, and was totally blocked by the co-incubation in PAMP+ADM and ADT, but was not blocked by the co-incubation in PAMP and ADT. Our results suggest that the four peptides derived from proadrenomedullin may have different effects, possibly through a cAMP-dependent pathway, on the expression of ADM gene.
Keywords: Proadrenomedullin N-terminal 20 peptide (PAMP); Adrenomedullin (ADM); Adrenotensin (ADT); Adrenomedullin gene;

Gastroprotective effect of adrenomedullin administered subcutaneously in the rat by Giuseppe Clementi; Antonina Caruso; Vincenza Maria Catena Cutuli; Nunzio Guido Mangano; Salvatore Salomone; Laurence Lempereur; Agata Prato; Mario Matera; Matilde Amico-Roxas (1149-1153).
Subcutaneous injections of adrenomedullin prevented reserpine-induced gastric mucosal damage in a dose-dependent manner (1–1000 ng/kg), but did not interfere with the lesions produced by ethanol administration. In pylorus-ligated rats adrenomedullin significantly reduced gastric volume, total and free acid output as well as ulcer formation. The gastroprotective activity of adrenomedullin was not present in rats pretreated with cysteamine. These results suggest that adrenomedullin exerts its antiulcer effect, when it is administered subcutaneously (s.c.), probably by a mechanism which involves somatostatin related transmission.
Keywords: Adrenomedullin; Gastroprotective activity; Subcutaneous administration; Somatostatin transmission;

The tachykinin NK-2 receptor antagonist SR48968 does not block noncholinergic contractions in unstable human bladder by Kate H Moore; David S.H Lam; William Lynch; Elizabeth Burcher (1155-1160).
Concentration–response curves to acetylcholine, and responses to electrical field stimulation (EFS) were compared in detrusor muscle strips, from control patients and those with idiopathic detrusor instability (IDI). Responses were similar in both groups. However, atropine abolished responses to EFS in 80% of control but only 33% of IDI patients (P>0.05), with the residual atropine-resistant response in most IDI patients abolished by tetrodotoxin. The post-atropine residual response was unaffected by the tachykinin NK-2 receptor antagonist SR48968. Despite the known existence of NK-2 receptors in the human detrusor, there was no evidence for tachykinin contribution to EFS-induced contractions.
Keywords: Detrusor; Electrical field stimulation; Atropine; Tetrodotoxin; Tachykinin receptors; Neurokinin A; SR48968;

The mechanism of the angiotensin-converting enzyme inhibitor quinapril is not related to bradykinin level in heart tissue by Christiane Barthelemy; Joelle Eurin; Philippe Lechat; Francoise Masson; Micheline Cortines; Nathalie Mougenot; Hayet Soualmia; Alain Carayon (1161-1169).
In order to examine the effect of the angiotensin-converting enzyme inhibitor (ACEi) quinapril, we performed a sensitive and specific radioimmunoassay (RIA) to quantify bradykinin, BK-(1–9), in heart and kidney tissues. The BK-(1–9) level was unaffected in the heart of sham and water-deprived rats treated for 2 h with quinapril (10 mg/kg), but was significantly higher in the kidneys in the two groups. In these conditions, circulating and tissue angiotensin II (Ang II) levels were significantly decreased by quinapril. Moreover, our results indicated that acute treatment with this dose of quinapril induced kinin-mediated effects which were not related to its action on bradykinin degradation in rat hearts.
Keywords: Bradykinin; Angiotensins; Angiotensin-converting enzyme inhibition; Quinapril; Heart; Kidney; Tissue distribution;

Metabolism of angiotensin I in the coronary circulation of normal and diabetic rats by Asim Mahmood; Herbert L Jackman; Linda Teplitz; Rajko Igić (1171-1175).
Formation of metabolites from angiotensin I that passed the coronary vessels in the isolated working rat hearts of normal and streptozotocin-induced diabetes was evaluated. HPLC analysis showed that the levels of angiotensin II and angiotensin 1–7 were unaltered in the diabetic hearts, but the perfusates of the diabetic hearts contained smaller amounts of angiotensin 1–9. It is not clear why the perfusates of diabetic hearts contain less amount of angiotensin 1–9. It is possible that the peptide is metabolized faster or greater internalization takes place in the diabetic heart. The amount of angiotensin II in the perfusates of normal hearts was 5.8 times greater at the perfusion rate of 2 than at 10 ml/min/g wet heart weight. At such conditions, the amount of angiotensin 1–9 and angiotensin 1–7 in the perfusates were increased 2.4 and 1.5 times, respectively. A higher amount of angiotensin II during myocardial hypoperfusion may lead to constriction of the coronary vessels. As a result, myocardial damage may be facilitated.
Keywords: Angiotensin I; Angiotensin II; Angiotensin 1–9; Angiotensin 1–7; Diabetes mellitus; Streptozotocin; Isolated perfused heart; Rat;

Sexually dimorphic expression of prepro-orexin mRNA in the rat hypothalamus by Olaf Jöhren; Steffi J Neidert; Marco Kummer; Peter Dominiak (1177-1180).
The neuropeptides orexin A and B are expressed in the lateral hypothalamic area and are involved in the regulation of energy homeostasis and arousal. Recent results showed gender differences in the expression of orexin receptor subtypes in rats. In the present study, we analyzed the mRNA expression of prepro-orexin (PPO) in the hypothalamus of male and female rats using quantitative real-time PCR. We found significantly higher levels of PPO mRNA in the hypothalamus of female rats compared to male rats. Our study indicates a sex-dependent regulation of hypothalamic PPO expression and suggests gender-specific functions of orexins.
Keywords: Prepro-orexin; mRNA expression; Real-time PCR; Hypothalamus;

The PAR-1-activating peptide attenuates carrageenan-induced hyperalgesia in rats by Atsufumi Kawabata; Naoyuki Kawao; Ryotaro Kuroda; Atsuko Tanaka; Chiho Shimada (1181-1183).
We examined if thrombin or a receptor-activating peptide for protease-activated receptor-1 (PAR-1), a thrombin receptor, could modulate nociception at peripheral levels. Intraplantar administration of PAR-1 activators, thrombin or TFLLR-NH2, but not its inactive control FTLLR-NH2 or a PAR-2 activator SLIGRL-NH2, significantly attenuated the hyperalgesia in rats treated with carrageenan, although they had no effect on nociception in naı̈ve rats. The thrombin-PAR-1 system might thus act to attenuate nociception during inflammatory hyperalgesia.
Keywords: Protease-activated receptor-1 (PAR-1); Thrombin; Pain; Hyperalgesia;

Erratum to (1185).

Characterization of a growth hormone-releasing hormone binding site in the rat renal medulla by Luce Boulanger; Nathalie Girard; Julie Strecko; Pierrette Gaudreau (1187-1194).
Receptor binding analysis was performed in the renal medulla from 2-month-old rats, an extrapituitary tissue containing the highest level of GHRH receptor mRNA. At 4 °C, in the presence of a cocktail of protease inhibitors, binding of [ 125 I -Tyr10]hGHRH (1–44)NH2 to medullary homogenates was specific, time-dependent, reversible and saturable (K d: 28 nM; B max: 30 fmol/mg prot.). In these experimental conditions, no change of binding parameters could be detected in the course of aging. The structure–affinity profile was different in the two tissues and chemical cross-linking revealed the presence of 65-, 55- and 38-kDa 125 I -GHRH-labeled complexes in the renal medulla compared to 65-, 47- and 28-kDa radioactive complexes in the anterior pituitary. It is suggested that GHRH binding sites, and possibly the receptor, may be different in the two tissues.
Keywords: Growth hormone-releasing hormone; 125 I -GHRH; Binding site; Structure–affinity; Kidney; Aging;

Erratum to “Predominant role by CaM kinase in NPY Y1 receptor signaling: Involvement of CREB” [Peptides (2002) 87–96] by Sulaiman Sheriff; Asbah F Qureshy; William T Chance; John W Kasckow; Ambikaipakan Balasubramaniam (1195).