Preparation of tri(ethylene glycol) grafted core-shell type polymer support for solid-phase peptide synthesis.

A core-shell type polymer support for solid-phase peptide synthesis has been developed for high coupling efficiency of peptides and versatile applications such as on-bead bioassays. Although various kinds of polymer supports have been developed, they have their own drawbacks including poor accessibility of reagents and incompatibility in aqueous solution. In this paper, we prepared hydrophilic tri(ethylene glycol) (TEG) grafted core-shell type polymer supports (TEG SURE) for efficient solid-phase peptide synthesis and on-bead bioassays. TEG SURE was prepared by grafting TEG derivative on the surface of AM PS resin via biphasic diffusion control method and subsequent acetylation of amine groups which are located at the core region of AM PS resin. The performance of TEG SURE was evaluated by synthesizing several peptides. Three points can be highlighted: (1) easy control of loading level of TEG, (2) improved efficiency of peptide synthesis compared with the conventional resins, and (3) applicability of on-bead bioassays.

Replacement of Thr32 and Gln34 in the C-terminal neuropeptide Y fragment 25-36 by cis-cyclobutane and cis-cyclopentane ß-amino acids shifts selectivity toward the Y(4) receptor.

Neuropeptide Y (NPY) and pancreatic polypeptide (PP) control central and peripheral processes by activating the G protein coupled receptors YxR (x = 1, 2, 4, 5). We present analogs of the C-terminal fragments 25-36 and 32-36 of NPY and PP containing (1R,2S)-cyclobutane (ßCbu) or (1R,2S)-cyclopentane (ßCpe) ß-amino acids, which display exclusively Y4R affinity. In particular, [ßCpe(34)]-NPY-(25-36) is a Y4R selective partial agonist (EC50 41 ± 6 nM, Emax 71%) that binds Y4R with a Ki of 10 ± 2 nM and a selectivity >100-fold relative to Y1R and Y2R and >50-fold relative to Y5R. Comparably, [Y(32), ßCpe(34)]-NPY(PP)-(32-36) selectively binds and activates Y4R (EC50 94 ± 21 nM, Emax 73%). The NMR structure of [ßCpe(34)]-NPY-(25-36) in dodecylphosphatidylcholine micelles shows a short helix at residues 27-32, while the C-terminal segment R(33)ßCpe(34)R(35)Y(36) is extended. The biological properties of the ßCbu- or ßCpe-containing NPY and PP C-terminal fragments encourage the future application of these ß-amino acids in the synthesis of selective Y4R ligands.

Manipulating Y receptor subtype activation of short neuropeptide Y analogs by introducing carbaboranes.

Short selective neuropeptide Y (NPY) analogs are highly attractive because of their facile synthesis. Based on the reduced-size NPY analog [Pro(30), Nle(31), Bpa(32), Leu(34)]NPY 28-36 position 32 was identified as a key position to alter the preferential activation pattern of the human neuropeptide Y receptors (hYRs). By replacing benzoylphenylalanine (Bpa) by a biphenylalanine (Bip) the photostability was first improved while the biological activity was maintained. SAR-studies showed that both aromatic rings have a high influence on the preferential hYR subtype activation. Interestingly, replacement of Bpa(32) by a strongly hydrophobic moiety changed the hYR subtype preference of the analog. Whereas the parent compound is able to activate the human neuropeptide Y1 receptor (hY1R) subtype, the introduction of an N(ε)-ortho-carbaboranyl propionic acid modified lysine resulted in a loss of activity at the hY1R but in an increased activity at both the hY2R and the hY4R. However, subsequent receptor internalization studies with this novel analog revealed that receptor internalization can neither be triggered at the hY2R nor at the hY4R suggesting a biased ligand. Surprisingly, investigations by (1)H NMR spectroscopy revealed structural changes in the side chains of residues Pro(30) and Leu(34) which nicely correlates with the shift from hY1R/hY4R to hY2R/hY4R activation preference. Thus, position 32 has been identified to switch the bioactive conformation and subsequently influences receptor subtype activation behavior.

Effects of galanin message-associated peptide and neuropeptide Y against various non-albicans Candida strains.

Antimicrobial peptides (AMPs) could represent promising therapeutic agents against fungal pathogens, especially in cases of pathogen resistance to common antifungal substances. The neuropeptides galanin message-associated peptide (GMAP) and neuropeptide Y (NPY) are both potent AMPs against certain microbes. The objective of this study was to test clinically relevant non-albicans Candida strains (C. glabrata, C. krusei, C. lusitaniae, C. parapsilosis, C. pelliculosa, C. tropicalis and C. utilis) with regard to their susceptibilities to NPY and GMAP. GMAP showed a higher potency than NPY, which only inhibited growth of some isolates of C. krusei, C. tropicalis and C. utilis. Interestingly, the fluconazole-resistant C. krusei was susceptible to both AMPs. In summary, we show that these neuropeptides have Candida strain-dependent antifungal activity, which in some cases does not match the susceptibility of the strains to the positive controls fluconazole and magainin I. Thus, the findings demonstrate the therapeutic potential of these AMPs in cases of resistance to traditional antifungal substances. This study also confirms the research on neuropeptides as potential fungicides, which are still in the early stages. The results also suggest that testing of strain-specific susceptibility is mandatory.

Introduction of lipidization-cationization motifs affords systemically bioavailable neuropeptide Y and neurotensin analogs with anticonvulsant activities.

The neuropeptides galanin (GAL), neuropeptide Y (NPY) or neurotensin (NT) exhibit anticonvulsant activities mediated by their respective receptors in the brain. To transform these peptides into potential neurotherapeutics, their systemic bioavailability and metabolic stability must be improved. Our recent studies with GAL analogs suggested that an introduction of lipoamino acids in the context of oligo-Lys residues (lipidization-cationization motif) significantly increases their penetration into the brain, yielding potent antiepileptic compounds. Here, we describe an extension of this strategy to NPY and NT. Rationally designed analogs of NPY and NT containing the lipidization-cationization motif were chemically synthesized and their physicochemical and pharmacological properties were characterized. The analogs NPY-BBB2 and NT-BBB1 exhibited increased serum stability, possessed log D > 1.1, retained high affinities toward their native receptors and produced potent antiseizure activities in animal models of epilepsy following intraperitoneal administration. Our results suggest that the combination of lipidization and cationization may be an effective strategy for improving systemic bioavailability and metabolic stability of various neuroactive peptides.

Novel chemically modified analogues of neuropeptide Y for tumor targeting.

The successful use of peptides as potential radiopharmaceuticals essentially requires the modification of the bioactive peptide hormones to introduce chelators for radiolabeling. In this study, four Y 1/Y 2 receptor-selective NPY analogues with different receptor subtype specificities have been investigated. For in vitro studies, the cold metal surrogate was used. Gallium and indium complexes were introduced by using 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid as bifunctional chelator. The peptides were synthesized by solid-phase peptide synthesis (SPPS), the chelator was coupled either at the N-terminus or at the N(epsilon) side chain of Lys(4) of the resin-bound peptide, and the labeling was performed in solution after cleavage. Competitive binding assays showed high binding affinity of the receptor-selective analogues at NPY receptor expressing cells. To test internalization of the novel peptide analogues and the metabolic stability in human blood plasma, the corresponding 5(6)-carboxyfluorescein (CF) analogues were prepared and investigated. One of the most promising analogues, the Y 1-receptor selective [Lys(DOTA)(4), Phe(7), Pro(34)]NPY was labeled with (111)In and injected into nude mice that bear MCF-7 breast cancer xenografts, and biodistribution studies were performed. In vitro and in vivo studies suggest that receptor-selective analogues of NPY have promising characteristics for future applications in nuclear medicine for breast tumor diagnosis and therapy.

Facile and selective nanoscale labeling of peptides in solution by using photolabile protecting groups.

For the selective labeling of peptides, a novel strategy was developed that combines the advantages of solid-phase peptide synthesis with the flexibility of labeling reactions in solution. To direct a label at a distinct position within the peptide sequence, other reactive positions are blocked with photolabile protecting groups that could be easily removed after the labeling reaction. Therefore selective labeling may become feasible for the first time even in nanomol amounts.

Semisynthesis and characterization of the first analogues of pro-neuropeptide y.

Enzymatic cleavage of prohormone neuropeptide Y (proNPY) leads to mature neuropeptide Y (NPY), a widely distributed neuropeptide with multiple functions both peripherally and centrally. A single dibasic pair of amino acids, Lys38-Arg39, represents the recognition motif for a class of hormone-processing enzymes known as prohormone convertases (PCs). Two members of this PC family, PC1/3 and PC2, are involved in proNPY cleavage. The aim of this work was to establish an effective method for the generation of full-length 69-amino acid proNPY analogues for further studies of prohormone convertase interaction. We have chosen two ligation sites in order to perform the semisynthesis of proNPY analogues by expressed protein ligation (EPL). By using the intein-mediated purification system (IMPACT) with improved conditions for intein splicing, we were able to isolate proNPY 1-40 and proNPY 1-54 fragments as Cterminal thioesters. Peptides bearing Nterminal cysteine instead of the naturally occurring Ser41 and Thr55 residues, respectively, were generated by solid-phase peptide synthesis. Moreover, labels (carboxyfluorescein and biotin) were inserted into the peptide sequences. The synthesis of the [C41]proNPY 41-69 fragment, which proved to be a difficult peptide sequence, could be achieved by the incorporation of two pseudo-proline derivatives. Western blot analysis revealed that all five proNPY analogues are recognized by monoclonal antibodies directed against NPY as well as against the Cflanking peptide of NPY (CPON).

Synthesis and binding affinity of neuropeptide Y at opiate receptors.

Neuropeptide Y and several metabolic fragments were synthesized and evaluated for binding affinity at non-selective opiate receptors. Neuropeptide Y and several C-terminal fragments were shown to bind to non-selective opiate receptors with an affinity similar to that of Leu-enkephalin.

Ala31-Aib32: identification of the key motif for high affinity and selectivity of neuropeptide Y at the Y5-receptor.

The turn-inducing sequence Ala-Aib introduced into positions 31 and 32 of neuropeptide Y (NPY) and its analogues has been identified as the key structure for Y(5)-receptor selectivity. Analogues of NPY and PP/NPY chimera containing the motif Ala-Aib were prepared; these peptides turned out to be selective for the Y(5)-receptor. The affinity of the NPY-based peptides was in the range of 6-150 nM, while the affinity of three (Ala-Aib)-containing PP/NPY chimera was in the range of 0.2-0.9 nM. The circular dichroism spectra of the Aib analogues in aqueous solution were all characteristic of an alpha helix; however, they had different intensities of the two negative bands at 220 and 208 nm. Affinity and selectivity for the Y(5)-receptor were correlated with the ratio of the ellipticity at 220 nm versus the one at 208 nm (R), which indicates the presence of a pronounced helix (R > 1) versus a less stabile one (R < 1). When R was in the range 0.74-0.96, the affinity at the Y(5)-receptor was in the range >5 nM, while there was complete loss of affinity at the Y(4)-receptor. R > 1.15 was associated with very high affinity at the Y(5)-receptor and weak affinity at the Y(4)-receptor. These results suggest that the selectivity of the Ala(31)-Aib(32) motif for the Y(5)-receptor derives from a specific conformation that must be correlated with the bioactive conformation of NPY at this subtype.

Novel peptide conjugates for tumor-specific chemotherapy.

One of the major problems in cancer chemotherapy are the severe side effects that limit the dose of the anticancer drugs because of their unselectivity for tumor versus normal cells. In the present work, we show that coupling of anthracyclines to peptides is a promising approach to obtain selectivity. The peptide-drug conjugate was designed to bind to specific receptors expressed on the tumor cells with subsequent internalization of the ligand-receptor complex. Neuropeptide Y (NPY), a 36-amino acid peptide of the pancreatic polypeptide family, was chosen as model peptide because NPY receptors are overexpressed in a number of neuroblastoma tumors and the thereof derived cell lines. Daunorubicin and doxorubicin, two widely used antineoplastic agents in tumor therapy, were covalently linked to NPY via two spacers that differ in stability: an acid-sensitive hydrazone bond at the 13-keto position of daunorubicin and a stable amide bond at the 3'-amino position of daunorubicin and doxorubicin. Receptor binding of these three conjugates ([C(15)]-NPY-Dauno-HYD, [C(15)]-NPY-Dauno-MBS, and [C(15)]-NPY-Doxo-MBS) was determined at the human neuroblastoma cell line SK-N-MC, which selectively expresses the NPY Y(1) receptor subtype, and cytotoxic activity was evaluated using a XTT-based colorimetric cellular cytotoxicity assay. The different conjugates were able to bind to the receptor with affinities ranging from 25 to 51 nM, but only the compound containing the acid-sensitive bond ([C(15)]-NPY-Dauno-HYD) showed cytotoxic activity comparable to the free daunorubicin. This cytotoxicity is Y(1) receptor-mediated as shown in blocking studies with BIBP 3226, because tumor cells that do not express NPY receptors were sensitive to free daunorubicin, but not to the peptide-drug conjugate. The intracellular distribution was investigated by confocal laser scanning microscopy. We found evidence that the active conjugate [C(15)]-NPY-Dauno-HYD releases daunorubicin, which is localized close to the nucleus, whereas the inactive conjugate [C(15)]-NPY-Dauno-MBS is distributed distantly from the nucleus and does not seem to release the drug within the cell.

Molecular characterization of the human neuropeptide Y Y2-receptor.

Five neuropeptide Y receptors, the Y1-, Y2-, Y4-, Y5- and y6-subtypes, have been cloned, which belong to the rhodopsin-like G-protein-coupled, 7-transmembrane helix-spanning receptors and bind the 36-mer neuromodulator NPY (neuropeptide Y) with nanomolar affinity. In this study, the Y2-receptor subtype expressed in a human neuroblastoma cell line (SMS-KAN) and in transfected Chinese hamster ovary cells (CHO-hY2) was characterized on the protein level by using photoaffinity labeling and antireceptor antibodies. Two photoactivatable analogues of NPY were synthesized, in which a Tyr residue was substituted by the photoreactive amino acid 4-(3-trifluoromethyl)-3H-diazirin-3-ylphenylalanine ((Tmd)Phe), [Nalpha-biotinyl-Ahx2,(Tmd)Phe36]NPY (Tmd36), and the Y2-receptor subtype selective [Nalpha-biotinyl-Ahx2,Ahx5-24,(Tmd)Phe27]N PY (Tmd27). Both analogues were labeled with [3H]succinimidyl-propionate at Lys4 and bind to the Y2-receptor with affinity similar to that of the native ligand. A synthetic fragment of the second (E2) extracellular loop was used to generate subtype selective antireceptor antibodies against the Y2-receptor. Photoaffinity labeling of the receptor followed by SDS-PAGE and detection of bound radioactivity and SDS-PAGE of solubilized receptors and subsequent Western blotting revealed the same molecular masses. Two proteins correspondingly have been detected for each cell line with molecular masses of 58 +/- 4 and 50 +/- 4 kDa, respectively.

Activity of centrally truncated analogues of neuropeptide Y at Y1 and Y2 receptor subtypes in vivo.

Neuropeptide Y (NPY), a sympathetic cotransmitter, has both prejunctional and postjunctional actions in the cardiovascular system. In anaesthetized rats, the bioassay system used here, NPY attenuates cardiac vagal action (a prejunctional or Y2 action) and increases blood pressure (a postjunctional or Y1 action). Several NPY analogues were tested against NPY. In these, centrally located amino acid sequences of various lengths were removed, and replaced with simpler 'spacers'. As the parent NPY molecule is considered to exist in a U-shape, these central truncations were intended to shorten the depth of the U, while maintaining the integrity of its two ends. The centrally truncated NPY analogues examined here retain activity at both receptor subtypes in vivo. These findings indicate that the U-shape of the parent molecule probably exists to assist stability, but that receptor binding occurs through sequences closer to the termini.

Characterization of Y3 receptor-mediated synaptic inhibition by chimeric neuropeptide Y-peptide YY peptides in the rat brainstem.

1. Neuropeptide Y (NPY) and peptide YY (PYY) act at receptors referred to as Y1 and Y2, while the Y3 receptor is specific to NPY and does not recognize PYY. The effects of NPY, its related peptides and a series of newly constructed chimeric NPY-PYY peptides were examined on excitatory and inhibitory postsynaptic currents (e.p.s.cs and i.p.s.cs, respectively) in rat dorsomedial nucleus tractus solitarius (NTS) neurones recorded in coronal brainstem slices. Monosynaptic activity was evoked by electrical stimulation in the region of the tractus solitarius. 2. NPY (5-500 nM) inhibited e.p.s.cs and i.p.s.cs in a concentration-dependent manner. In contrast, PYY (500 nM) failed to affect either e.p.s.cs or i.p.s.cs. The N- and C-terminal parts of a series of chimeric NPY-PYY peptides were joined at positions where NPY and PYY sequences differ. In binding experiments the chimeric peptides were all about equipotent with NPY and PYY in displacing [125I]-PYY from Y1 and Y2 binding sites on SK-N-MC cells and rat hippocampus respectively. 3. In the whole cell voltage clamp recordings of NTS neurones, NPY(1-23)-PYY(24-36) and NPY(1-14)-PYY(15-36) evoked a concentration-dependent inhibition of e.p.s.cs and i.p.s.cs, while NPY(1-7)-PYY(8-36) and NPY(1-3)-PYY(4-36) were inactive. The only differences in amino acid residues between NPY(1-14)-PYY(15-36) and NPY(1-7)-PYY(8-36) reside in positions 13 and 14. 4. Furthermore, [Pro34]NPY (500 nM) was equivalent in potency to NPY itself at inhibiting monosynaptic transmission in NTS, while [Leu31,Pro34]NPY and pancreatic polypeptide (both at 500 nM) failed to affect synaptic transmission. 5. The present study has shown that NPY acts at Y3 receptors to suppress both excitatory and inhibitory currents in the NTS. The different efficacy of the chimeric NPY-PYY peptides suggests that positions 13 and 14 are of great importance for Y3 receptor recognition. Finally, this receptor type readily recognizes [Pro34]NPY, but not [Leu31,Pro34]NPY.

Crudes of synthesis of neuropeptide Y and beta-amyloid peptide examined by liquid chromatography/electrospray tandem mass spectrometry.

The two peptides, neuropeptide Y (M(r) = 4254) and beta (1-39) amyloid (M(r) = 4230) are attracting pharmaceutical and biomedical interest for different yet equally important reasons. The first is recognized for its various physiological functions in the peripheral and central nervous system, while the second has been identified as one of the components of the cerebral amyloid deposits characteristic of Alzheimer's disease. High level purity of both peptides is considered of prime importance for correct interpretation of data obtained by biological and pharmaceutical assays and binding tests. Solid-phase synthesis of both peptides resulted in crudes of reaction containing both the target peptides as well as a number of undesired side products. The use of liquid chromatography/electrospray-tandem mass spectrometry (LC/ESI-MS/MS) furnished reliable information on the target peptides and provided sequencing information on a number of side products. Furthermore, LC/MS/MS data of doubly and triply protonated sequences yielded a number of C-terminal fragment ions exhibiting the loss of NH3 considered to be an important process for the understanding of fragmentation mechanism(s) of multiply protonated peptides.

Solid-phase synthesis of caged peptides using tyrosine modified with a photocleavable protecting group: application to the synthesis of caged neuropeptide Y.

A simple method for the synthesis of caged peptides using a novel derivative of tyrosine, N-Fmoc-O-(2-nitrobenzyl)-tyrosine, is described. The derivative of tyrosine can be incorporated at any position in an amino acid sequence by solid-phase peptide synthesis under the condition for Fmoc chemistry, and caged peptides that contain nitrobenzyl group on the side chain of tyrosine residue can be obtained. The nitrobenzyl group can be photocleaved by UV irradiation and the half life of the intermediate during photolysis is approximately 7 microseconds. The method was successfully applied to the synthesis of caged neuropeptide Y (NPY). The binding affinity of the caged NPY for the Y1 receptor was one or two orders of magnitude lower than that of intact NPY, but it increased to the value for intact NPY upon irradiation by UV light.

Antagonistic properties of centrally truncated analogs of [D-Trp(32)]NPY.

We have previously shown that [D-Trp(32)]NPY can competitively antagonize NPY-induced feeding in rats (Balasubramaniam et al. J. Med. Chem. 1994, 37, 811-815). This peptide, however, did not bind to SK-N-MC cells with Y-1 receptors. Since centrally truncated NPY analogs have been shown to bind Y-1 receptors, we synthesized similar analogs of [D-Trp(32)]NPY and investigated their Y-1 (SK-N-MC) and Y-2 (SK-N-BE2) receptor affinities and their properties in human erythroleukemia (HEL) cells. None of the analogs with D-Trp(32) mobilized intracellular calcium, [Ca2]i, in HEL cells. Although Des-AA(6-24)[Aoc(6)]NPY and the corresponding D-Trp(32) analog exhibited no affinity to Y-1 receptors, Des-AA(7-24)[Aoc(6),D-Trp(32)] NPY(6) exhibited weak binding. Replacing Pro(5) in 6 with D-Ala to stabilize the central chain reversal, and hence the antiparallel alignment of the N- and C-terminal regions known to be important for Y-1 binding, resulted in an analog, Des-AA(7-24)[D-Ala(5),Aoc(6),D-Trp(32)]NPY (7), which exhibited moderate antagonist potency in attenuating NPY effects on cAMP and [Ca2+]i, in SK-N-MC and HEL cells, respectively. This analog also shifted the dose-response curve of NPY on blood pressure in anesthetized rats. Deletion of only the 7-17 and/or the incorporation of N-Me-Ala(5), superior beta-turn stabilizer, in 7 did not improve the Y-1 receptor affinity. Des-AA(7-24)[D-Ala(5), Gly(6),D-Trp(32)]NPY exhibited an affinity similar to that of 7, suggesting that a long spacer arm is not necessary for efficient Y-1 receptor interaction. Locking the antiparallel alignment via a 2/26 or 2/27 lactam bridge did not improve the binding. Finally, replacement of D-Ala(5) in 7 with D-Trp dramatically increased both the binding and the antagonistic potencies. Modeling based on the avian pancreatic polypeptide X-ray structure suggested that analogs which have the N- and C-terminal regions in close proximity might exhibit good binding, and that the D-Trp(32) substitution may induce a beta-turn that could be important for exhibiting antagonism. A systematic investigation has resulted in the development of relatively potent Y-1 receptor antagonists. Further structure-activity studies with these compounds and those previously reported by us and other investigators should result in the development of long-acting and receptor selective antagonists.

Synthesis, structure, and antagonistic properties of des-Asn29[D-Trp28,32]NPY(27-36).

We have previously reported that [D-Trp32]NPY and its centrally truncated analogues such as des-AA7-24[D-Trp5,32,Aoc6]NPY can competitively antagonize NPY effects on rat hypothalamus and Y1 (SK-N-MC AND HEL) cells, respectively. In continuation of this work, we performed structure-activity studies with C-terminal decapeptide sequence keeping D-Trp at position 32 to develop lower molecular weight Y1-selective antagonists. This study led to the development of des-Asn29[D-Trp28,32]NPY(27-36), which bound to both Y1 (SK-N-MC, Ki > or = 10 microM) and Y2 (SK-N-BE2, Ki = 1.01 +/- 0.03 microM) receptors. This peptide did not exhibit any agonist activity at Y1 receptors, and exhibited comparable potencies in antagonizing the effects of NPY on the synthesis of cAMP and mobilization of [Ca2+]i in HEL cells. However, in SK-N-MC cells, it was more potent in antagonizing the mobilization of [Ca2+]i than inhibition of cAMP synthesis. Substitution of Nva for Gln34 to increase the hydrophobicity without altering the carbon skeleton substantially increased Y1 affinity (Ki = 0.33 +/- 0.15 microM) and imparted Y1 selectivity (Ki for Y2 affinity = 3.16 +/- 0.50). Moreover, this peptide exhibited good antagonistic potency in HEL cells. 2D NMR studies of des-Asn29[D-Trp28,32]NPY(27-36) revealed the existence of a fairly stable loop-like structure between residues 27 and 32 and a less stable one between residues 32 and 36. The increased Y1 affinity of des-Asn29[D-Trp28,32,Nva34]NPY(27-36) may be due to the stabilization of the 32-36 loop by Nva34. It appears therefore that stabilization of the loop structures in these peptides should result in the development of more potent Y1 receptor antagonists. Our investigations also suggest that HEL cells express a homogeneous population of NPY Y1 receptors whereas SK-N-MC cells express high- and low-affinity Y1 receptors coupled to Ca2+ and cAMP, respectively.

Intravenous NPY (27-36)-D decreases cardiac output in conscious Sprague-Dawley rats.

Intravenous (IV) administration of NPY (27-36)-D, a substituted carboxyterminal fragment of neuropeptide Y (NPY), decreases mean arterial pressure (MAP) in normo-and hypertensive rats by a mechanism partially involving histamine receptors. The purpose of this study is to further characterize the cardiovascular effects of NPY (27-36)-D. NPY (27-36)-D dose-dependently decreased MAP, cardiac output, and stroke volume without significantly altering peripheral resistance. Myocardial contractility diminished by 151.2 +/- 31.8, 529.6 +/- 182.5, and 495.4 +/- 66.7 mmHg/s2 in rats treated with 300, 500, and 750 nmol/kg NPY (27-36)-D, respectively. Therefore, NPY (27-36)-D modifies MAP, in part, by a reversible negative inotropic effect on the heart.