John L. Krstenansky

Activation of neuropeptide Y1 and neuropeptide Y2 receptors by substituted and truncated neuropeptide Y analogs: identification of signal epitopes.

Neuropeptide Y (NPY-(1-36)) acts on Y1 and Y2 receptors at the sympathetic neuroeffector junction. Various truncated NPY analogs were tested in the isolated guinea-pig caval vein where NPY is a vasoconstrictor (Y1 receptors) and in isolated rat vas deferens, by monitoring the suppression of electrically evoked contractions (Y2 receptors). The aim of this study was to define which parts of the NPY-(1-36) molecule were required to activate these receptors. NPY-(1-36), [Pro34]NPY and [Glu16,Ser18,Ala22,Leu28,31]NPY (ESALL-NPY), the latter being an analog with increased alpha-helicity in the 14-31 region, evoked vasoconstriction with similar potency and efficacy. Cyclic as well as linear NPY analogs having the 4 to 7 N-terminal amino acid residues linked to the 9 to 19 C-terminal residues by an 8-aminooctanoic acid (Aoc) residue were 25-50 times less potent than NPY-(1-36) itself. In the cyclic analogs, a disulfide bond was introduced to bring the N- and C-termini close together. Linear Aoc-2-27-NPY was virtually inactive. The Y1 receptor needs an intact N-terminal end of NPY in order to become fully activated. The requirements for the C-terminus are less stringent, since substitutions in this part of the molecule resulted in fully active analogs. The central portion of the molecule may impose steric constraints on the N- and C-terminal ends, thereby facilitating Y1 receptor activation, but it does not seem to be essential for receptor recognition. NPY-(2-36) and NPY-(5-36) were only slightly less potent than the parent molecule in suppressing electrically evoked twitches in the vas deferens.(ABSTRACT TRUNCATED AT 250 WORDS)

The dogfish peptides scyliorhinin I and scyliorhinin II bind with differential selectivity to mammalian tachykinin receptors

The dogfish intestinal, linear tachykinin scyliorhinin I (SCYI) and cyclic tachykinin scyliorhinin II (SCYII) bound with differential selectivity to mammalian tachykinin, membrane receptor sites. SCYI bound with highest affinity to NK-1 sites in rat submandibular gland (KI = 0.9 nM) and to NK-2 sites in hamster urinary bladder (KI = 2 nM) whereas SCYII bound with highest affinity to NK-3 sites in rat cerebral cortex (KI = 2.5 nM). These results suggest that SCYI is a dual NK-1/NK-2 tachykinin receptor agonist while SCYII is an NK-3 selective tachykinin receptor agonist.

Neuropeptide Y and truncated neuropeptide Y analogs evoke histamine release from rat peritoneal mast cells. A direct effect on G proteins

Several regulatory peptides, including neuropeptide Y, can release histamine from mast cells. In the present study we investigated which parts of the neuropeptide Y molecule are required to evoke the release of histamine from isolated rat peritoneal mast cells. In addition, we examined whether the histamine release evoked by neuropeptide Y (and by compound 48/80) is sensitive to the G protein inhibitors pertussis toxin and benzalkonium chloride. Neuropeptide Y released histamine in a concentration-dependent manner. Also a neuropeptide Y analog with the center part substituted by 8-aminooctanoic acid, [Aoc2-27]neuropeptide Y, and the cyclic form of the C-terminal hexapeptide, cyclic neuropeptide Y-(31-36), released histamine. The three peptides were equally effective and equally potent. Neuropeptide Y-(1-24)NH2 also released histamine, but its efficacy was low. The rank order of potency of the analogs tested did not agree with that of any of the previously known or postulated neuropeptide Y receptors. Pretreatment of mast cells with pertussis toxin or benzalkonium chloride markedly inhibited the histamine release evoked by neuropeptide Y, [Aoc2-27]neuropeptide Y and compound 48/80. In conclusion, most of the histamine-releasing activity of neuropeptide Y resides in the six C-terminal amino acid residues. The release appears to be G protein-dependent and is probably not receptor mediated.

Cyclic hexapeptide antagonists of the bradykinin B2 receptor: Receptor binding and solution backbone conformation

Cyclic hexapeptide analogs of bradykinin, based on a folded receptor-bound model of bradykinin, were found to be able to antagonize the action of bradykinin at its B2 bradykinin receptor. The best of these, cyclo(d-Lys(Arg)-Phe-Ser-d-Tic-Oic- Arg) [compound 17], has affinities at the human and rat B2 bradykinin receptors of 230 and 8.5 nM, respectively. This potency is significant, since the analogs lack the C-terminal carboxylate group, residues 2–4 and the important interaction of Phe5. These constrained analogs may serve as tools for the determination of the receptor-bound conformation of antagonists at the bradykinin receptor and for the design of even smaller and more potent antagonist analogs.

Heparin Binding Properties of the Carboxyl Terminal Domain of [A103,106,108] Antistasin 93–119

Antistasin is a 119 amino acid protein with anticoagulant, antimetastatic and heparin-binding properties derived from the salivary glands of the leech Haementaria officinalis (1). This protein contains a specific consensus sequence for heparin binding at its carboxyl terminal end and a region between residues 32 and 48 putatively involved in glycosaminoglycan interactions. The cyclic peptide antistasin 37-48 (C-P-H-G-F-Q-R-S-R-Y-G-C) and the carboxyl terminal fragment [A103,106,108] antistasin 93-119 (P-N-G-L-K-R-D-K-L-G-A-E-Y-A-E-A-R-P-K-R-K-L-I-P-R-L-S) were synthesized by solid-phase peptide chemistry and their interactions with 125I-labeled heparin were investigated. Heparin binding to [A103,106,108] antistasin 93-119 was specific and saturable as binding was blocked by addition of the unlabeled glycosaminoglycan. The rank order of potency of various glycosaminoglycans in blocking 125I-labeled heparin binding to [A103,106,108] antistasin 93-119 was dextran sulfate greater than heparin much greater than dermatan sulfate greater than or equal to chondroitin sulfate A and C indicating a specificity of the peptide for the glycosaminoglycan structure. Moreover, heparin binding increased linearly with increasing salt and was optimal at 0.15 M NaCl and physiological pH. In contrast, binding of heparin to the basic peptide antistasin 37-48 decreased linearly as the ionic strength of the medium was increased to physiological concentration (0.15 M) thus showing a greater specificity of heparin for [A103,106,108] antistasin 93-119. These studies indicate that residues 93-119 of antistasin mediate this inhibitors interaction with heparin.

Demonstration that [A103,106,108] antistasin 93–119 inhibits the specific binding of antistasin to sulfatide [Gal(3-SO4)β1-1Cer]

Antistasin is a 119 amino acid heparin-binding protein from the leech Haementaria officinalis which has anticoagulant and antimetastatic properties. A series of peptides representing the basic amino acid-rich domains of the amino- and carboxyl-terminal regions of the inhibitor were synthesized by solid-phase peptide chemistry and their ability to bind sulfated glycolipids was investigated. The findings show that [A103,106,108] antistasin 93-119 has high affinity for sulfatide and inhibits the specific interaction of whole antistasin with [Gal(3-SO4)beta 1-1Cer]. We conclude that the 93-119 region is a critical domain that mediates the interaction of antistasin with sulfated glycolipids.