CONFORMATIONAL ENERGY CALCULATIONS ON ENKEPHALINS AND ENKEPHALIN ANALOGS:

Conformational energy calculations were carried out on the peptide enkephalins (ENK) and selected analogs to find those conformers of low energy. The analogs studied include [D-Ala²]Enk-NH₂, [D-Ala²]Enk, [D-Met², Pro⁵]Enk-NH₂, [D-Ala², D-Phe⁵]Enk, [D-Ala², D-Leu⁵]Enk, [D-Ala², (N-Me)Phe⁴, Met⁵] Enk-NH₂ and [D-Ala², (N-Me)Met⁵]Enk-NH₂. When the low-energy conformers for all the analogs are compared, different allowed backbone conformations are found which orient the functional side-chains such that three classifications of structures appear. Each classification shows a unique configuration of side- chain positions in space even though different backbone conformations are found within each classification.     

β-ENDORPHIN: SYNTHESIS AND MORPHINE-LIKE ACTIVITY OF ANALOGS WITH D-AMINO ACID RESIDUES IN POSITIONS 1, 2, 4 AND 5

The solid-phase syntheses of [D-Tyr¹]-, [D-Ala²]-, [D-Phe⁴]-, and [D-Met⁵]-β_c-endorphins are described. A comparison of certain methods of purification and criteria of homogeneity is made with the use of these compounds. Bioassay of these synthetic analogs both in vitro and in vivo show that [D-Ala²]-β_c-endorphin possesses significant opiate activity whereas the other analogs have minimal activity.     

Cystamine-enkephalin dimer

A cystamine-enkephalin dimer, containing two molecules of [D-Ala², Leu⁵] enkephalin cross-linked at the COOH-terminal leucine residue with cystamine, (NH₂-CH₂-CH₂-S-)₂, has been synthesized in order to examine directly the dimerization effect of an enkephalin molecule on the opiate receptor interactions. In a comparison of potencies against [³H]-[D-Ala²,D-Leu⁵]enkephalin (³H-DADLE) and [³H]-[D-Ala²,MePhe⁴,Gly-ol⁵]enkephalin (³H-DAGO) as delta and mu tracers, respectively, enkephalin dimer showed a very high affinity, especially for the delta opiate receptors. Dimer was almost threefold more potent than DADLE, which is one of the most utilized delta ligand to date. When the binding affinity of cystamine-dimer was compared with that of its reduced thiol-monomer, namely [D-Ala²,Leu⁵, cysteamine⁶]enkephalin, the increment in affinity was four to fivefold for both delta and mu receptors. The results strongly indicate that the dimeric enkephalin is more potent presumably due to the simultaneous interaction with the two binding sites of the opiate receptors.     

Synthesis and in vitro and in vivo activity of analogs of growth hormone-releasing hormone (GH-RH) with C-terminal agmatine

In the search for more active analogs of human growth hormone-releasing hormone (GH-RH), 37 new compounds were synthesized by solid phase methodology, purified, and tested biologically. Most of the analogs contained a sequence of 27 amino acids and N-terminal desaminotyrosine (Dat) and C-terminal agmatine (Agm), which are not amino acids. In addition to Dat in position 1 and Agm in position 29, the majority of the analogs had Ala¹⁵ and Nle²⁷ substitutions and one or more additional L- or D-amino acid modifications. [Dat¹, Ala¹⁵, Nle²⁷]GH-RH(1-28)Agm (MZ-2-51) was the most active analog. Its in vitro GH-releasing potency was 10.5 times higher than that of GH-RH(1-29)NH₂ and in the i.v. in vivo assay, MZ-2-51 was 4-5 times more active than the standard. After s.c. administration to rats, MZ-2-51 showed an activity 34 times higher at 15min and 179 times greater at 30min than GH-RH(1-29)NH₂ and also displayed a prolonged activity. D-Tyr¹⁰, D-Lys¹², and D-Lys¹² homologs of MZ-2-51 also showed enhanced activities. Thus, [Dat¹, D-Tyr¹⁰, Ala¹⁵, Nle²⁷]GH-RH(1-28)Agm (MZ-2-159), [Dat¹, D-Lys¹², Ala¹⁵, Nle²⁷]GH-RH(1-28)AGM (MZ-2-57), and [Dat¹, Ala¹⁵, D-Lys²¹, Nle²⁷]GH-RH(1-28)Agm (MZ-2-75) were 4-6 times more active in vitro than GH-RH(1-29)NH₂. In vivo, after i.v. administration, analog MZ-2-75 was equipotent and analogs MZ-2-159 and MZ-2-57 about twice as potent as the standard. After S.C. administration, the potencies of MZ-2-57 and MZ-2-75 were 10-14 times higher than the standard at 15 min and 45-89 times greater when determined at 30 min. Most of the analogs containing two or more D-amino acid substitutions were less active than GH-RH(1-29)NH₂ or inactive. Our studies indicate that very potent GH-RH analogs can result from the combination of agmatine in position 29 with other substitutions.     

Radioiodinated [D-Ala2, Met 5] enkephalin

¹²⁵I[D-Ala², Met⁵] enkephalin with high specific activity (122–185 Ci/mmol) was prepared and purified by Sep-Pak C₁₈ reverse phase cartridge followed by high performance liquid chromatography (HPLC). HPLC at pH 3.0 resolved ¹²⁵I[D-Ala², Met⁵] enkephalin into two fractions, which ran as a single spot in thin-layer chromatography with the same R_f values. Alkaline hydrolysates of the HPLC-purified fractions showed a single spot corresponding to monoiodotyrosine standard when analysed by thin-layer chromatography. Binding kinetics of the tracer was found to approach equilibrium after 30 min at 24d?. Scatchard analysis of the saturation equilibrium binding studies gave an equilibrium dissociation constant of 3.58 nM and the number of binding site of 30 fmol/mg protein. Enkephalin analogs were capable of displacing ¹²⁵I[D-Ala², Met⁵] enkephalin binding from the rat brain plasma membrane. The effective concentration of [D-Ala², Met⁵] enkephalin and [D-Ala², Leu⁵] enkephalin for 50% inhibition of ¹²⁵I[D-Ala², Met⁵] enkephalin binding was estimated to be 79 nM and 23 nM, respectively. Both substance P and gastrin tetrapeptide failed to displace the ¹²⁵I[D-Ala², Met⁵] enkephalin binding to any significant extent. The ¹²⁵I[d-Ala², Met⁵] enkephalin prepared by the present procedure is therefore a useful tracer. This method of preparing radioiodinated peptide may be applicable to other enkephalin analogs or neuropeptides in general.     

Preparation and opioid activities of N-methylated analogs of [D-Ala2, Leu5]enkephalin

Analogs of opioid pentapeptide [D-Ala², Leu⁵]enkephalin were prepared using two kinds of N-methylation reactions, namely quaternization and amide-methylation. Quaternization reaction with CH³I-KHCO³ in methanol was applied to the deprotected N-terminal group of the pentapeptide derivatives affording trimethylammonium group-containing analogs. [Me₃⁺ Tyr¹,D-Ala², Leu⁵]enkephalin and its amide were found to show opioid activity on guinea pig ileium assay only slightly lower than the parent unmethylated peptides. Application of amide-methylation reaction using CH₃I-Ag₂O in DMF to the protected pentapeptide yielded a pentamethyl derivative in which all of the five N atoms were methylated. Deprotection of the derivative gave pentamethyl analogs of [D-Ala², Leu⁵]enkephalin, which showed no significant activity on the guinea pig ileum assay and opiate-receptor binding assay.     

Synthesis and properties of dermorphin and an analog of β-endorphin containing the dermorphin sequence

Dermorphin (I) and [D-Ala², Phe³, Gly⁴, Tyr⁵, Pro⁶]-β_c-EP (II) have been synthesized by the solid-phase method (β-EP, camel β-endorphin). Positions I through 7 of II correspond to the sequence of I. Relative potencies of synthetic peptides in the mouse tail-flick test for analgesia by the intracerebroventricular route were: human β-endorphin, 100; camel β-endorphin, 164; I, 450; II, 440. The dermorphin was about 670 times more potent than morphine in the assay. Peptide II represents a rare instance where the enkephalin moiety of β-endorphin has been altered to produce a more potent analgesic.     

Dehydro-enkephalins. III. Synthesis and biological activity of [ΔAla2, Leu5]-enkephalin

[ΔAla², Leu⁵]-enkephalin has been prepared and shown to be more active than the parent saturated enkephalin in a binding assay using rat brain membranes and [³H] dihydromorphine as a tracer. In a comparison of potencies against [³H] dihydromorphine and [³H]-[d -Ala², d -Leu⁵]-enkephalin as tracers, [ΔAla², Leu⁵]-enkephalin showed preference for μ opiate receptors, possibly due to the hydrophobicity of the ΔAla² residue. A synthetic tetrapeptide enkephalin [ΔAla²]-desLeu⁵-enkephalin had weak activity and high selectivity for the μ receptors. O-Acylation of a serine residue in the peptide was achieved by coupling between the peptide and a carboxylic acid using DCC and a catalytic amount of 4-dimethylaminopyridine.     

Effect of secondary structure on the rate of deamidation of several growth hormone releasing factor analogs

The objective of this study was to determine whether the rates of deamidation of Asn⁸ in selected growth hormone releasing factor (GRF) analogs were related to the peptide's secondary structures in solution. Bovine or human [Leu²⁷]GRF(1–32)NH₂ (both having Gly at position 15), [Ala¹⁵ Leu²⁷]bGRF(1–32)NH₂ and [Pro¹⁵ Leu²⁷]bGRF(1–32)NH₂ were used as model peptides. The peptide helical content (assessed by CD) increased with the increasing methanol concentration and was as follows: 7, 12 and 18% in 0% MeOH; 24, 48 and 52% in 40% MeOH; and 41, 77 and 81% in 80% MeOH for Pro¹⁵ Leu²⁷ bGRF(1–32)NH₂, [Leu²⁷]hGRF(1–32)NH₂, and Ala¹⁵ Leu²⁷ bGRF(1–32)NH₂, respectively. 2D NMR studies done in the presence of 40% CD₃OH indicated more helical structure for the Ala¹⁵ analog as compared to [Len²⁷]hGRF(1–32)NH₂. In both these peptides Asn⁸ was included in the helical region. In contrast, the lack of conformational information for the Pro¹⁵ analog indicated little helical structure around Asn⁸. The peptides’ deamidation rates decreased and their half-lives increased with increasing MeOH concentrations. At 40% MeOH, the least helical Pro¹⁵ bGRF analog (t_1/2= 10.78 h) deamidated 1.5 and 2 times faster than its Gly¹⁵ (t_1/2= 15.74 h) and Ala¹⁵ (t_1/2= 21.53 h) counterparts, respectively. This study indicates that helical environment around Asn⁸ in GRF makes this residue less prone to deamidation.     

Degradation of growth hormone releasing factor analogs in neutral aqueous solution is related to deamidation of asparagine residues

The incubation of a solution of the human growth hormone releasing factor analog, [Leu²⁷] hGRF(1-32)NH₂ at pH 7.4 and 37°, resulted in extensive degradation of the sample. The major degradation products were identified as the peptides [β-Asp⁸, Leu²⁷] hGRF(l-32)NH₂ and [α-Asp⁸, Leu²⁷] hGRF(1-32)NH₂, produced by deamidation of the Asn⁸ residue. When tested as growth hormone (GH) secretagogues in cultured bovine anterior pituitary cells, [β-Asp⁸, Leu²⁷] hGRF(l-32)NH₂ was estimated to be 400-500 times less potent than the parent Asn⁸ peptide, while [2-Asp⁸, Leu²⁷] hGRF(l-32)NH₂ was calculated to be 25 times less potent than the parent Asn⁸ peptide. Three additional analogs of [Leu²⁷] hGRF(1-32)NH₂ containing either Ser or Asn at positions 8 and 28 were prepared and evaluated for their GH releasing activity and stability in aqueous phosphate buffer (pH 7.4, 37°). Based on disappearance kinetics, [Leu²⁷] hGRF(1-32)NH₂ had a half-life of 202 h while the other analogs had the following half-lives: [Leu²⁷, Asn²⁸] hGRF(1-32)NH_: (150h); [Ser⁸, Leu²⁷, Asn²⁸] hGRF(l-32)NH₂ (746h); and [Ser⁸, Leu²⁷] hGRF(1-32)NH₂ (1550 h). After 14 days, incubated samples of the Asn⁸ analogs lost GH releasing potency, while the Ser⁸ analogs retained full potency. The potential for loss of biological activity brought about by deamidation of other engineered peptides and proteins should be considered in their design.     

Importance of the leucine side-chain to the spasmogenic activity and binding of Substance P analogues

Previous studies with Substance P (SP) antagonists (GR 71251, [d Pro⁹, Pro¹⁰, Trp¹¹]SP and d Pro⁹, MeLeu¹⁰, Trp¹¹]SP) have suggested the existence in the guinea-pig ileum (GPI) of two distinct tachykinin receptors associated with the contractile responses of [Pro⁹]SP and septide. In addition [Apa^9-10]SP, a glycine-substituted analogue of SP with a carba bond between residues 9 and 10, [Gly⁹-ψ(CH₂-CH₂)-Gly¹⁰SP = [Apa^9-10]SP, was shown to belong to the ‘septide family’ (low affinity for NK-1 specific binding sites and high potency in the GPI). In order to establish the importance of the isopropyl side-chain in position 10, the binding potencies and activities of [Gly⁹-ψ(CH₂-CH₂)-Gly¹⁰]SP, [Ala¹⁰]SP, [Gly⁹-ψ(CH₂-CH₂)-Leu¹⁰]SP and [Gly⁹-ψ(CH₂-CH₂)-d Leu¹⁰]SP were compared. Conformational behaviour of active peptides with a carba bond was analyzed by NMR and modelisation studies. This study with agonists demonstrated that undecapeptides substituted in position 10 in the SP sequence also enabled discrimination of NK-1 receptors from receptors responsible for the spasmogenic activities of peptides belonging to the ‘septide family’. [Gly⁹-ψ(CH₂-CH₂)- Leu¹⁰]SP is ahighly potent NK-1 agonist, [Gly⁹-ψ(CH₂-CH₂)-Gly¹⁰]SP acts on the septide-sensitive receptor, and [Ala¹⁰]SP is a mixed agonist.     

Synthesis and assay of tyrosinated analogs of a crustacean pigment-dispersing neuropeptide hormone

In an effort to obtain biologically active tyrosine analogs of the octadecapeptide pigment-dispersing hormone (β-PDH) of the fiddler crab Uca pugilator, we have synthesized [Tyr¹⁵]- and [Nle¹⁸]-β-PDH, as well as eight tyrosine analogs of [Nle¹⁵]-β-PDH by the solid-phase method. Each analog was purified to homogeneity by gel filtration, ion-exchange chromatography, and partition chromatography. When tested for melanophore pigment-dispersing activity in destalked Uca, [Nle¹⁵, Tyr¹⁶]-β-PDH and [Nle¹⁵]-β-PDH were found to be 31-fold and 16-fold more potent, respectively, than β-PDH. Somewhat reduced potency was displayed by [N-Tyr,Nle¹⁵]-β-PDH (81%) and [Nle¹⁵, Tyr¹⁷]-β-PDH (26%). All other analogs, including [Nle¹⁵, Tyr¹⁹]-β-PDH and the four derivatives of [Nle¹⁵]-β-PDH obtained through substitution for nonpolar amino acid residues at positions 4, 5, 8, and 11 in the primary structure, showed marked decrease in biological activity (0.01 to 2% potency).     

Pegylated peptides V. Carboxy-terminal PEGylated analogs of growth hormone-releasing factor (GRF) display enhanced duration of biological activity in vivo

In the present study, human growth hormone-releasing factor (hGRF) and analogs were successfully pegylated at the carboxy-terminus using a novel solid- and solution-phase strategy. Following synthesis, these pegylated hGRF analogs were evaluated for in vitro and in vivo biological activity. Specifically, hGRF(1-29)-NH2, [Ala¹⁵-hGRF(1-29)-NH₂, [desNH₂ 2Tyr1, o-Ala2, Ala15-hGRF(1-29)-NH2 and [His1, Val2, Gin8, Ala15, Leu27-hGRF(1-32)-OH were each C-terminally extended using a Gly-Gly-Cys-NH2 spacer (previously demonstrated not to alter intrinsic biological activity), and then monopegylated via coupling to an activated dithiopyridyl-PEG reagent. PEG moieties of 750, 2000, 5000 or 10000 molecular weight (MW) were examined to determine the effect of polymer weight on activity. Initial biological evaluations in vitro revealed that all C-terminally pegylated hGRF analogs retained high growth hormone (GH)-releasing potencies, regardless of the MW of PEG polymer employed. Two of these pegylated hGRF analogs, [desN₂Tyr, D-Ala², Ala¹⁵-hGRF(1-29)-Gly-Gly-Cys(NH₂)-S-Nle- PEG₅₀₀₀ and [His, Val², Gin⁸, Ala¹⁵, Leu²⁷-hGRF(1-32)-Gly-Cys(NH2)-S-Nle-PEG5000, were subsequently evaluated in both pig and mouse models and found to be highly potent (in vivo potency range = 12-55-fold that of native hGRF). Relative to their non-pegylated counterparts, these two pegylated hGRF analogs exhibited enhanced duration of activity. © Munksgaard 1997.     

Effect of three angiotensin II antagonists, [Sar1, Thr8]-, [Sar1, Ile8]-and [Sar1, Ala8]angiotensin II on blood pressure and endocrine factors in normal subjects

Summary The biological effects of 1-Sarcosine, 8-Threonine angiotensin II ([Sar¹, Thr⁸]ANG II) on blood pressure, plasma aldosterone concentration (PAC) and plasma renin activity (PRA) were investigated in six normal subjects on an unrestricted diet, and compared with those of 1-Sarcosine, 8-Isoleucine ANG II ([Sar¹, Ile⁸]ANG II) and 1-Sarcosine, 8-Alanine ANG II ([Sar¹, Ala⁸]ANG II). All three ANG II analogues (A II A) showed agonistic pressor activity, that of [Sar¹, Ile⁸]ANG II being greater than that of [Sar¹, Thr⁸]ANG II or [Sar¹, Ala⁸]ANG II. The antagonistic effect of [Sar¹, Thr⁸]ANG II on blood pressure was less than [Sar¹, Ile⁸]ANG II or [Sar¹, Ala⁸]ANG II. Both [Sar¹, Ile⁸]ANG II and [Sar¹, Ala⁸]ANG II increased PAC and blocked the steroidogenic action of ANG II, while [Sar¹, Thr⁸]ANG II showed little effect on PAC. All three A II A caused similar suppression of PRA and showed no inhibitory effect on the decrease in PRA produced by ANG II. These results indicate that [Sar¹, Thr⁸] ANG II is an A II A with weak agonistic pressor action and that it has vascular selective properties. It is also suggested that ANG II receptors in a variety of target organs are heterogeneous.     

Spectrum of the μ-, δ- and κ-binding sites in homogenates of rat brain

1 In homogenates of rat brain, the binding characteristics of tritiated opiates and opioid peptides were examined and the relative capacities of μ-, δ- and κ-binding sites of the opiate receptor determined by saturation analysis.

2 In competition experiments, binding of the selective μ-ligand [³H]-[D-Ala²,MePhe⁴,Gly-ol⁵]enkephalin at the μ-site was displaced by [D-Ala²,D-Leu⁵]enkephalin with rather low affinity (K_I = 12.6 nM) and more readily by the ketazocine-like compounds (-)-ethylketazocine (K_I = 3.1 nM) and (-)-bremazocine (K_I = 0.32 nM), which also displaced the binding of [³H]-[D-Ala²,D-Leu⁵]enkephalin from the δ-site. In contrast, the binding to the κ-site was easily displaced by ethylketazocine (1.0 nM) and bremazocine (0.37 nM) but not by the μ-ligand [D-Ala²,MePhe⁴,Gly-ol⁵]enkephalin (K_I = 2000-3000 nM) or the δ-ligand [D-Ala²,D-Leu⁵]enkephalin (K_I > 20,000 nM).

3 The dissociation equilibrium constant (K_D) and the binding capacity (pmol/g) of the μ-binding site were determined with the selective μ-ligand [³H]-[D-Ala²,MePhe⁴,Gly-ol⁵]enkephalin. For the δ-site, [³H]-[D-Ala²,D-Leu⁵]enkephalin was used in the presence of unlabelled [D-Ala²,MePhe⁴,Gly-ol⁵]enkephalin in order to suppress cross-reactivity to the μ-binding site. For the estimation of κ-binding, [³H]-(±)-ethylketazocine or [³H]-(-)-bremazocine were used in the presence of unlabelled μ- and δ-ligands for the suppression of cross-reactivities to the μ- and δ-binding sites.

4 In rat brain the capacity of the μ-binding site was 7.3 pmol/g brain, that of the δ-binding site 6.7 pmol/g brain and that of the κ-binding site 2.0 pmol/g brain. Thus, the κ-binding site had the lowest value whereas in the guinea-pig brain the capacity of the μ-binding site was lower than that of the δ- or κ-binding site.

     

Dehydro-enkephalins

The Gly³ residue in Gly²- and D-Ala²-enkephalins has been replaced by ΔAla³ and Ser³ in order to examine the effect on binding to the Δ and μ opiate receptors. [D-Ala², ΔAla³, Leu⁵]-enkephalin maintains most of its receptor binding affinities for both sites. It is suggested that the proper conjunctions of positions 2 and 3 of the enkephalin sequence are important to receptor preference and that position 3 may have a very specific interaction with the Δ receptor. The in vivo analgesic and CNS activities of the peptides are also discussed.     

Studies of peptide antibiotics. XLVI. Syntheses of gramicidin S analogs containing D-α, β-diaminopropionic acid or α,β-dehydroalanine*

A gramicidin S (GS) analog ([d -Dpr^4,4] GS) containing d -α,β-diaminopropionic acid (D-Dpr) in place of D-Phe at 4,4′ positions was derived from [l -Orn(δ-formyl)^2,2, d -Dpr(β-Z)^4,4′]GS, which was synthesized by conventional method in solution. An analog [ΔAla^4,4′]GS was synthesized from [l -Orn(δ-Boc)^2,2′, d -Dpr^4,4′]GS through Hofmann degradation of the d -Dpr residues. Antimicrobial activities of these analogs were tested; [d -Dpr(β-Z)^4,4′]GS and [ΔAla^4,4′]GS showed high antimicrobial activities against Gram-positive bacteria. [d -Dpr^4,4′]-GS showed an appreciable activity against Gram-negative bacteria such as Escherichia coli. Four semigramicidin S (semiGS) analogs such as [ΔAla⁴]semiGS were synthesized; these had no antimicrobial activity. Analogs containing ΔAla residues were hydrogenated, and the formation of l -Ala or d -Ala residues was determined. The ΔAla residues in [ΔAla^4,4′] GS were reduced to dl -Ala, and ΔAla in [ΔAla⁴]semiGS mostly to l -Ala. The relationships of the antimicrobial activity, CD curves and asymmetric hydrogenation to the structure were discussed.     

Importance of the C‐terminal phenylalanine of gastrin for binding to the human CCK2 receptor

Abstract: The importance of the C‐terminal Phe of gastrin and structural requirements at position 17 for binding to the human CCK₂ receptor were assessed using analogs of [Leu¹⁵]G(11?17). The following peptides were synthesized, Ac[Leu¹⁵]G(11?17), Ac[Leu¹⁵]G(11?16)NH₂, [Leu¹⁵]G(11?17), [Leu¹⁵,Ala¹⁷]G(11?17), [Leu¹⁵,Abu¹⁷]G(11?17), [Leu¹⁵,Val¹⁷]G(11?17), [Leu¹⁵,Leu¹⁷]G(11?17), [Leu¹⁵,Cha¹⁷]G(11?17), [Leu¹⁵,Trp¹⁷]G(11?17), [Leu¹⁵,Tic¹⁷]G(11?17), [Leu¹⁵, d ‐Phe¹⁷]G(11?17) and [Leu¹⁵,p‐X‐Phe¹⁷]G(11?17), where X = F, Cl, Br, I, OH, CH₃, NH₂ and NO_2. Competition binding experiments with [³H]CCK‐8 were performed using human CCK₂ receptors stably expressed in CHO cells. Phe¹⁷ was shown to be important for binding. A hydrophobic side‐chain larger than Leu is required at position 17 but aromaticity does not appear to be essential. Constraint of the aromatic side‐chain either in the g(+) or g(–) conformation, as in the case of Tic, results in a significant decrease in affinity. In addition, the peptide conformation induced by incorporation of d ‐Phe decreases binding. The size and electron withdrawing/donating properties of the para substituent are not important for interaction with the receptor. The current study shows that the use of des‐Phe analogs of gastrin is not a viable strategy for development of antagonists for the human CCK₂ receptor.     

Effect of cyclopiazonic acid on contractions produced by tachykinin NK1 and NK2 receptor agonists in the circular muscle of guinea-pig colon

1 This study aimed to assess the effect of cyclopiazonic acid (CPA), an inhibitor of sarcoplasmic reticulum calcium (Ca) pump, against contractile responses produced by selective tachykinin NK₁ and NK₂ receptor agonists, [Sar⁹]substance P (SP) sulfone and[βAla⁸]neurokinin A (NKA) (4–10), respectively, on the circular muscle of guinea-pig colon. All experiments were performed in the presence of atropine (1 μm ) and indomethacin (10 μm ). 2 In organ bath experiments, a submaximally equieffective concentration of the two agonists (10 nm ) was selected: [Sar⁹]SP sulfone (10 nm ) produced a biphasic contraction, the two amplitudes averaging 75 ± 2 and 43 ± 3% of the maximal response to KCl (80 mm ) at 1 and 15 min from application of the agonist, respectively. CPA (3 μm for 60 min) slightly reduced the phasic response to [Sar⁹]SP sulfone (16 ± 4% inhibition) and markedly suppressed the tonic component (89 ± 3% inhibition). 3 The contraction produced by [βAla⁸]NKA (4–10) (10 nm ) was more sustained than that induced by the NK₁ receptor agonist: it averaged 69 ± 5 and 73 ± 4% of the response to KCl at 1 and 15 min from application of the agonist, respectively. CPA slightly and evenly depressed the response to [βAla⁸]NKA (4–10) (18 ± 7 and 21 ± 5% inhibition at 1 and 15 min). 4 In the presence of tachykinin NK₁ and NK₂ receptor antagonists (SR 140333 and MEN 10627, respectively, 1 μm each) and of l -nitroarginine (100 μm ), KCl (40 mm ) produced a distinct phasic and tonic contraction which was suppressed by 1 mm nifedipine. CPA (3 μm ) did not affect the phasic contraction to KCl but abolished the tonic component of the response. 5 In the presence of 1 μm nifedipine, the response to [βAla⁸]NKA (4–10) was slightly depressed (32 ± 6% inhibition) in its early component only, while the response to [Sar⁹]SP sulfone was abolished. CPA produced a slight inhibition (15 ± 9 and 33 ± 10% at 1 and 15 min, respectively) of the nifedipine-resistant response to [βAla⁸]NKA (4–10), an effect similar to that observed in the absence of nifedipine. Therefore, a large part of the response to [βAla⁸]NKA (4–10) persisted in the presence of both CPA and nifedipine. 6 In the sucrose gap, a prolonged superfusion with [Sar⁹]SP sulfone (0.1 μm for 5 min) produced sustained depolarization with superimposed spikes and contraction. CPA (3 μm ) produced transient depolarization and contraction. In the presence of CPA, the initial responses (depolarization, spikes and contraction) to [Sar⁹]SP sulfone were unaffected but the sustained component of contraction was absent; the latter effect was accompanied by a suppression of spikes while the sustained depolarization was present. 7 We conclude that, during sustained depolarization produced by the NK₁ receptor agonist, blockade of the sarcoplasmic reticulum Ca pump by CPA produces a faster Ca-dependent inactivation of Ca channels, thereby eliminating spikes and abolishing the tonic component of contraction. Ca mobilization/reuptake from a CPA-sensitive store seems to be of minor importance for regulating the NK₂ receptor-mediated contractile responses.     

Photoreactive enkephalin analogue: [D-Ala2, p-N3-Phe 4-Met 5] enkephalin

A photoreactive [D-Ala², p-N₃-Phe⁴-Met⁵]enkephalin was synthesized by classical solution peptide synthetic methods. The hydroxysuccinimide ester was used in all the coupling steps in the presence of a weak base, triethylamine. The deprotected enkephalin analogue was purified on high performance liquid chromatography using a Waters, C₁₈μBondapak reverse phase column and its purity was assessed by thin-layer chromatography. The composition of the analogue was determined and confirmed by elemental analysis and amino acid analysis. Its photoreactivity was demonstrated by the time dependent ultraviolet spectral changes on exposure to light. Competition receptor binding showed that [D-Ala², p-N₃-Phe⁴-Met⁵]enkephalin was 4-fold more potent than [D-Ala², Met⁵]-enkephalin in competing for binding to the enkephalin binding site. The data presented suggest that this photoreactive enkephalin analogue may be suitable for use in the in situ photoaffinity labeling of the enkephalin receptor.