Effects of Microinjections of Cholecystokinin and Neurotensin into Lateral Hypothalamus and Ventral Mesencephalon on Intracranial Self-Stimulation

Changes in intracranial self-stimulation (ICSS) evoked from ventral tegmental area–substantia nigra (VTA-SN) and lateral hypothalamus–medial forebrain bundle (LH-MFB) before and after microinjections of sulfated cholecystokinin octapeptide (CCK-8S) and unsulfated cholecystokinin (CCK-8US), neurotensin tridecapeptide ([D-Tyr¹¹]NT_1–13 or [DTrp¹¹] NT_1–13) into either VTA-SN or LH-MFB were assessed. The current intensity was fixed at a level to obtain 60–70% of the maximal asymptotic rate. CCK-8S (0.10 μg/0.5 μl and 0.25 μg/0.5 μl) into VTA-SN resulted in dose-dependent decreases in VTA-SN ICSS of 38–42% and 78–92%, respectively, without affecting the ICSS of LH-MFB. Similar doses of CCK-8S injected into LH-MFB changed neither LH-MFB ICSS nor VTA-SN ICSS. CCK-8Us injected into VTA-SN or LH-MFB had no effect on ICSS in either site. Intra-VTA-SN injections of the neurotensin-1 (NT₁) receptor agonist [DTyr¹¹]NT_1–13 and the NT₁ receptor antagonist [D-Trp¹¹]NT_1–13 at doses of 5 μg/0.5 μl and 10 μg/0.5 μl decreased VTA-SN ICSS. NT₁ receptor agonist and antagonist injections did not alter LH-MFB ICSS in any significant manner. Similar injections of these peptides into LH-MFB did not change the responding rates for LH-MFB ICSS or VTA-SN ICSS. Increasing the current intensity reversed the inhibitory effect of CCK-8S and [D-Trp¹¹]NT_1–13 on VTA-SN ICSS and restored basal preinjection rates of responding. These results suggest that CCK_A and NT₁ receptor mechanisms in the ventral tegmentum in association with dopamine neurotransmission may be important in mediating the rewarding effects of VTA-SN ICSS but not LH-MFB ICSS.     

Selective blockade of neurotensin-induced coronary vessel constriction in perfused rat hearts by a neurotensin analogue

[D-Trp¹¹]-NT, an analogue of neurotensin (NT) in which Tyr¹¹ was replaced with D-Trp, was found to antagonize selectively NT-induced coronary vessel constriction in perfused rat hearts, in concentrations varying between 1.3 × 10^?7 and 1.1 × 10^?6 M. Higher concentrations of [D-Trp¹¹]-NT displayed NT-like activity. In rat stomach strips and guinea pig atria, [D-Trp¹¹]-NT exhibits full intrinsic activity, markedly reduced affinity for NT receptors, but no inhibitory effect against NT. These results suggest that the receptors mediating the constrictor action of NT in the coronary vessels of rat hearts are pharmacologically distinct from those subserving the stimulant effects of NT in rat stomach strips and guinea pig atria.     

Structure-activity studies of neurotensin on muscular rigidity and tremors induced by 6-hydroxydopamine lesions in the posterolateral hypothalamus of the rat

It has previously been reported that intracerebroventricular administration of neurotensin (30 micrograms) reduced muscular rigidity and tremors, induced by a neurochemical lesion with 6-hydroxydopamine in the posterolateral hypothalamus of rats. In the present study, the effects of two fragments (NT1-10 and NT8-13) and two analogues ([D-Tyr11]-NT and [Ala11]-NT) of neurotensin on the grasping time (index of muscle rigidity) and tremors in 6-hydroxydopamine-lesioned rats are reported. Intracerebroventricular administration with 120 micrograms of NT1-10 and [Ala11]-NT had no effect on the muscle rigidity and tremors induced by the neurochemical lesion. The administration of NT8-13 60 micrograms) significantly attenuated both behavioural responses. The analogue [D-Tyr11]-NT produced a much greater attenuation of the muscle rigidity and tremors. The dose of 1.8 micrograms of [D-Tyr11]-NT significantly reduced the grasping time, while the number of tremors was attenuated with the threshold dose of 0.9 micrograms. Together, these results suggest that the effects of neurotensin on muscle rigidity and tremors, induced by pretreatment with 6-hydroxydopamine injected into the posterolateral hypothalamus, were not caused by non-specific effects but largely depended on the carboxy terminal of the peptide. The tyrosine residue in position 11 of the molecule plays a critical role in the action of neurotensin, as shown with the high potency and duration of action of the analogue [D-Tyr11]-NT. As previously suggested, the greater effect with [D-Tyr11]-NT may be due to greater resistance of the analogue to enzymatic degradation because of the incorporation of the D-Tyr amino acid, in position 11 of neurotensin.     

Study of the cytotoxicity and antioxidant capacity of N/OFQ(1–13)NH2 and its structural analogues

The effect of side-chain shortening of N/OFQ(1–13)NH₂ at position 9 ([Orn⁹]N/OFQ(1–13)NH₂, [Dab⁹]N/OFQ(1–13)NH₂, [Dap⁹]N/OFQ(1–13)NH₂) was studied regarding potential toxicity and antioxidant capacity. Staurosporine- and H2O2-induced damage of SH-SY5Y neuroblastoma cells was not changed in the presence of N/OFQ(1–13)NH2 and [Orn⁹]N/OFQ(1–13)NH₂, but was strongly enhanced in the presence of [Dab⁹]N/OFQ(1–13)NH₂ and [Dap⁹]N/OFQ(1–13)NH₂. Moreover, treatment of cells with the latter two analogues alone led to cell injury. Neuropeptide-dependent differences in the viability of SH-SY5Y cells were also observed, i.e., a cytoprotective effect was observed only in the presence of N/OFQ(1–13)NH₂ and [Orn⁹]N/OFQ(1–13)NH₂. Compared to [Dab⁹]N/OFQ(1–13)NH₂ and [Dap⁹]N/OFQ(1–13)NH₂, the effects of N/OFQ(1–13)NH₂ and [Orn⁹]N/OFQ(1–13)NH₂ were more beneficial in systems generating free oxygen radicals (O2^? and OH), as well as on the antioxidant status of rat brain and liver. Taken together, our findings show that N/OFQ(1–13)NH₂ and its structural analogue [Orn⁹]N/OFQ(1–13)NH₂ possess more favorable profiles than the other two nociceptin (N/OFQ) analogues. The present results suggest that shortening of the side-chain of N/OFQ(1–13)NH2 might increase cell damage and reduce the viability of SH-SY5Y neuroblastoma cells. Moreover, such alterations may lead to changes in free-oxygen generating systems and in antioxidant status in animal tissues.     

Synthesis and biological activities of ψ(CH2NH) pseudopeptide analogues of the C-terminal hexapeptide of neurotensin

Each peptide CO-NH function in the biologically important C-terminal 8-13 sequence of neurotensin was replaced by the reduced CH₂-NH isostere using the rapid in situ solid phase procedure developed by Sasaki & Coy. In general this modification resulted in a drop in receptor affinity except for the [Argψ(CH₂NH)Arg]-NT_8–13 analogue (PIC₅₀ 9.23 vs. NT_8–13 PIC₅₀ 8.03). This analogue also showed enhanced enzymatic stability, but acted as a full agonist as shown by the observation of relaxations of guinea-pig colon ascendens.     

Chemical synthesis of human β-endorphin(1–27) analogs by peptide segment coupling

[Gly⁸]β_hEP(1–27)NH₂ and [l -Leu⁸]β_hEP(1–27)NH₂, two analogs of human β-endorphin, were synthesized by both all-stepwise solid phase synthesis and peptide segment coupling. For the peptide segment coupling method, two thiocarboxyl peptides, Msc-[Gly⁸]β_hEP(1–8)SH and Msc-[l -Leu⁸]β_hEP(1–8)SH, were synthesized by standard solid phase method on 4-[α-(Boc-Gly-S)benzyl]phenoxyacetamidomethy-resin and 4-[α-(Boc-l -Leu-S)benzyl]phenoxyacetamidomethy-resin. These two thiocarboxyl peptides were coupled to H-[Lys(Cit)^{9, 19, 24}]-β_hEP(9–27)NH₂ [Gly⁸]β_hEP(1–27)NH, and [l -Leu⁸]β_hEP(1–27)NH₂ were obtained after removal of Msc groups and citraconyl groups from products of the segment coupling reaction. The yields of both [Gly⁸]β_hEP(1–27)NH₂ and [l -Leu⁸]β_hEP(1–27)NH₂ in the segment coupling reaction were approximately 18%. Less than 1 % of racemization of Leu-8 occurred during coupling of Msc-[l -Leu⁸]β_hEP(1–8)SH to H-[Lys(Cit)^{9, 19, 24}]-β_h EP(9–27)NH₂. Results of amino acid composition analysis, analysis by reverse phase high pressure liquid chromatography and receptor binding activity assays of the analogs showed that peptide analogs prepared by segment coupling method and those prepared by all-stepwise solid phase synthesis were identical. Results of receptor binding activity assays suggested that the molecular charge properties of β-endorphin(1–27) and its analogs influenced the receptor binding activity.     

Central neurotensin receptor activation produces differential behavioral responses in Fischer and Lewis rats

Rationale Lewis (LEW) and Fischer (F344) rats exhibit marked differences in appetitive and consummatory responses to numerous drugs, including psychostimulants. Neurotensin (NT) produces psychostimulant-like actions, which sensitize with repeated exposure, and neuroleptic-like actions; effects that are dependent on the site of microinjection. The aim of the present experiments was to assess the behavioral sensitivity of these two strains of rats to NT receptor activation. Methods In expt 1, locomotor activity was assessed on alternate days following an ICV injection of NT, [d-Tyr¹¹]neurotensin (d-NT; 18 nmol/10 μl), or vehicle (days 1, 3, 5, and 7) in independent groups of LEW and F344 rats. On day 14, locomotor activity was assessed in all rats following an injection of d-amphetamine (1 mg/kg, IP). In expt 2, activity was assessed following injection into the ventral tegmental area of NT, or d-NT, (2.5 μg/hemisphere) or into the nucleus accumbens (2.5 and 5.0 μg/hemisphere). Results Repeated ICV injections of NT, or d-NT, produced differential behavioral effects in the two strains of rats on days 1–7; activity was initially suppressed in LEW, but less so in F344 rats, following NT. In F344, but not in LEW rats, d-NT produced a significant increase in activity. Neurotensin and d-NT sensitized LEW rats to amphetamine-induced ambulatory and non-ambulatory activity. Except for vertical activity, this effect was weaker or in the opposite direction in F344 rats. When injected into the ventral tegmental area, NT produced an increase in locomotor activity in both strains, an effect that was greater in F344 than LEW rats with d-NT. In the nucleus accumbens, NT marginally decreased activity in both strains, while d-NT produced a significant increase in F344 but not in LEW rats. Conclusions These results provide empirical evidence that endogenous NT neurotransmission within limbic circuitry differs in F344 and LEW rats.     

Syntheses,opioid binding affinities,and potencies of dynorphin A analogues substituted in positions 1, 6, 7, 8 and 10

Structural, stereochemical, stereoelectronic and conformational requirements for biological activity of dynorphin A_1–11-NH₂ analogues at opioid receptors were explored by substitution of Tyr¹, Arg⁶, Arg⁷, Ile⁸ and Pro¹⁰ with other amino acid residues. Interestingly, substitution of Tyr¹ with N^α-Ac-Tyr^l, D-Tyr¹, Phe¹ or p-BrPhe¹ led to analogues that were quite potent at κ opioid receptors, and additional substitution of Ile⁸with D-Ala⁸ and/or Pro¹⁰ with D-Pro¹⁰ retained high potency in brain binding assay: [N^α-Ac-Tyr¹]- (1), [D-Tyr¹]- (2) [Phe¹]- (3), [Phe¹. D-Ala⁸]- (5), [p-BrPhe¹, D-Ala^s]- (6), [Phe¹, D-Pro¹⁰]- (7) and [Phe¹, D-Ala⁸, D-Pro¹⁰]-Dyn A_1–11-NH₂ (8) had IC₅₀(nM) binding affinities of 13.2, 18.6, 1.64, 1.26, 1.84, 2.44 and 1.62 nM, respectively. The D-Phe¹ analogue 4, however, was only weakly active (610 nM). All of the analogues except 4 were modestly selective for κ vs. μ guinea pig brain opioid receptor (11- to 88–fold) and quite selective for κ vs. δ receptors (65–576). However, all of the analogues appeared to have very low or essentially no activity in the guinea pig ileum and mouse vas deference functional bioassays, and one analogue, 5, appeared to have weak antagonist activities. On the other hand, if constrained amino acids such as β-methylphenylalanine or 1,2,3,4-tetrahydroisoquinoline carboxylic acid, and hydroxyproline were placed in the 1 position, inactive analogues or analogues with greatly reduced potency and biological activity were obtained (compounds 12–14). It had previously been suggested that the Arg⁶ and Arg⁷ residues were critical for biological activity. However, when we replace either one of these residues, [Nle⁶]Dyn A_1–11 (9) and [Nle⁷]Dyn A_1–11-NH₂ (10) were both highly potent binders in κ receptor binding studies (IC₅₀= 0.95 and 0.43 nM, respectively), and interestingly also were potent in μ and δ binding studies. Furthermore, both of the analogues were modestly potent in the GPI and MVD assays (94, 65 nM; 31, 81 nM, respectively). These results demonstrate that basic residues at positions 6 and 7 in dynorphin are not very important for binding to κ opioid receptors. Finally, many of the compounds reported here showed high selectivity for central vs. peripheral κ opioid receptors, with compound 4 being the most selective (63 000-fold).     

Differentiation of the structural features of melanotropins important for biological potency and prolonged activity in vitro

Several α-melanotropin (α-MSH) analogues have been synthesized and tested for their melanotropin activities in order to determine the functional importance of certain amino acids near the primary active sequence of α-MSH, H-(Glu)-His-Phe-Arg-Trp-Gly-OH, on the biological activities of the hormone. In particular, we have examined the importance of the 4 and 11 positions in conjunction with the substitution of l -Phe in position 7 by d -Phe on potency and prolonged activity of the hormone. In the frog (Rana pipiens) skin system the relative potencies were found to be: [Nle⁴, d -Phe⁷]-α-MSH (60) > α-MSH (1.0) > Ac-[Nle⁴, d -Phe⁷]-α-MSH_4–11-NH₂ (0.16) > Ac-[Nle⁴, d -Phe⁷]-α-MSH_4–10-NH₂ (0.02) · Ac-[d -Phe⁷]-α-MSH_5–11-NH₂ (0.01) > Ac-[Nle⁴]-α-MSH_4–10-NH₂ (0.002) = Ac-[Nle⁴]-α-MSH_4–11-NH₂ > Ac-α-MSH_4–10-NH₂ (0.0003) · Ac-α-MSH_5–11-NH₂ (0.0002). On the other hand the relative potencies on the lizard (Anolis carolinensis) skin system were found to be: Ac-[Nle⁴, d -Phe⁷]-α-MSH_4–10-NH₂ (10) · Ac-[Nle⁴, d -Phe⁷]-α-MSH_4–11-NH₂ (8.0) · Ac-[Nle⁴, d -Phe⁷]-α-MSH (5.0) > α-MSH (1.0) = Ac-[Nle⁴]-α-MSH_4–11-NH₂ = Ac-[d -Phe⁷]-α-MSH_5–11-NH₂ > Ac-[Nle⁴]-α-MSH_4–10-NH₂ (0.06) > Ac-α-MSH_5–11-NH₂ (0.01) > Ac-α-MSH_4–10-NH₂ (0.004). Detailed analyses of these data suggest species-dependent differences in the stereostructural relationships of the residues in the 4, 7, and 11 positions for melanotropic potency in vitro. Particularly noteworthy is the observation that the 4–11 fragment analogue Ac-[Nle⁴]-α-MSH_4–11-NH₂ is equipotent to α-MSH in the lizard assay system, suggesting that the 1–3, 12, and 13 residues of α-MSH are not involved in the binding or transduction in this system. Examination of the ability of these α-melanotropin analogues to effect sustained biological activity (prolongation) following removal of exogenous peptide from the bioassay medium showed striking differences in the two systems. On the lizard skin assay, only [Nle⁴, d -Phe⁷]-α-MSH, Ac-[Nle⁴, d -Phe⁷]-α-MSH_4–11-NH₂ and Ac-[Nle⁴, d -Phe⁷]-α-MSH_4–10-NH₂ effect marked prolonged melanotropic activity as compared to α-MSH. In contrast, on the frog skin assay, only [Nle⁴, d -Phe⁷]-α-MSH, Ac-[Nle⁴, d -Phe⁷]-α-MSH_4–11-NH₂, Ac-α-MSH_5–11-NH₂, and Ac-[Nle⁴]-α-MSH_4–10-NH₂ exhibited significant prolonged activity. These results demonstrate that relative potency and prolongation of melanotropic activity are not directly related, but rather are the manifestation of different, species-dependent structural and topographical requirements for peptide-receptor interactions related to binding and signal transduction.     

Human β-endorphin

Three analogs of human β-endorphin (β_h-EP) were synthesized by the solidphase method: [Gln⁸, Trp²⁷]-β_h-EP (I), [Gln⁸,Arg⁹,Trp²⁷]-β_h-EP (II), and [Gln⁹,Arg¹¹,Trp²⁷]-β-EP (III). Radioreceptor binding assay with use of tritiated β_h-EP as primary ligand gave relative potencies as follows: β_h-EP, 100; I, 778; II, 467; III, 449. Relative potencies in an analgesic assay were: β-EP, 100; I, 114; II, 165; III, 83. The 8–11 segment of β_h-EP can tolerate a net increase in charge of + 2 without diminishing analgesic potency. The substitution of Glu⁸ may be one of the more dependable means of designing β-endorphin antagonists.     

Comparative biological activities of potent active-site analogues of α-melanotropin. Effect of tyrosine substitution at position-4

We have prepared several α-melanotropin (α-MSH) analogues with tyrosine substituted for methionine at the 4-position and determined their melanotropic activities on the frog (Rana pipiens), lizard (Anolis carolinensis) and S-91 (Cloudman) mouse melanoma adenylate cyclase bioassays. The potencies of Ac-[Tyr⁴]-α-MSH_4–10-NH₂ and Ac-[Tyr⁴]-α-MSH_4–11-NH₂ were compared with α-MSH and with their corresponding methionine and norleucine substituted analogues. The Tyr-4 analogues were found to be less active than the Nle-4 analogues on both the frog and lizard assays. Ac-[Tyr⁴]-α-MSH_4–10-NH₂ was found to be less active than Ac-[Tyr⁴]-α-MSH_4–11-NH₂ on the lizard bioassay, but more active than the longer fragment on the frog skin assay. Ac-[Tyr⁴]-α-MSH_4–10-NH₂ exhibited extremely prolonged biological activity on frog skin, but not on lizard skin, while the melanotropic activity of Ac-[Tyr⁴]-α-MSH_4–11-NH₂ was rapidly reversed on both assay systems. The increased potency of Ac-[Tyr⁴]-α-MSH_4–10-NH₂ over Ac-[Tyr⁴]-α-MSH_4–11-NH₂ on frog melanocytes may be related to the fact that the shorter 4–10 analogue exhibits prolonged biological activity. Interestingly, it was found that both Tyr-4 analogues were partial agonists on the mouse melanoma adenylate cyclase bioassay, and stimulated the enzyme to only about 50% of the maximal activity of α-MSH. We reported previously that replacement of L-Phe-7 by its D-enantiomer in [Nle⁴]-α-MSH and its Nle-4 containing analogues resulted in peptides with increased potency and in some instances prolonged activity. Similarly, incorporation of D-Phe-7 into Tyr-4 containing melanotropin fragments produced analogues Ac-[Tyr⁴, D-Phe⁷]-αMSH_4–10-NH₂ and Ac-[Tyr⁴, D-Phe⁷]-α-MSH_4–11-NH₂, which also exhibited greatly increased biological activity in all three assay systems. Both of these analogues were also found to have prolonged activity in the frog skin bioassay but little or no prolonged activity in the lizard skin bioassay. These two analogues turned out to be full agonists in the mouse melanoma adenylate cyclase bioassay and were equipotent to α-MSH. These results demonstrate that substitution of tyrosine for methionine at position-4 dramatically affects the potency and prolonged activity of these melanotropin analogues and the melanotropic activities observed as a result of such substitutions are themselves affected by concomitant substitutions at the 7(Phe) and 11 (Lys) positions of the analogues.     

Potent pseudopeptide bombesin-like agonists and antagonists

Bombesin-like pseudopeptides have been synthesized, and certain physicochemical properties and biological activities have been examined. Bombesin and the related peptide litorin were modified at positions 13–14 and 8–9, respectively, with ψ[CH₂S] and ψ[CH₂N(CH₃)]. [Phe¹³ψ[CH₂S]Leu¹⁴]bombesin and [Phe⁸ψ[CH₂S]-Leu⁹]litorin bound to the murine pancreatic bombesin gastrin releasing peptide receptor with similar dissociation constants (K_d= 3.9 and 3.4 nM. respectively). Increased potency was achieved by oxidation of the thiomethylene ether to two diastereomeric sulfoxides (isomer I, K_d= 1.6 nM and isomer II, K_d= 0.89nM. Further oxidation to the sulfone decreased potency ([Phe⁸ψ[CH₂SO₂]Leu⁹]litoin, K_d= 9.9nM). All five analogs were receptor antagonists as determined by phosphatidylinositol turnover in murine pancreas. In contrast to these peptide backbone substitutions, a ψCH₂N(CH₃)] at the 8–9 amide bond position resulted in an agonist. The analogs were compared with those of litorin (K_d= 0.1 nM) and [Leu⁹]litorin (K_d= 0.17 nM) by CD and fluorescence spectroscopy. The CD spectra demonstrated ordered conformation for all the peptides in TFE. Different conformations corresponding to agonist and antagonist peptides were suggested by CD. Based on the pH-dependence of the fluorescence spectra of the peptides in a zwitterionic detergent, two titratable groups were identified (pK_a= 6.3 and 8.5). The lower pK_a is found in the agonist analogs but not in the ψ[CH₂S]-containing antagonist.     

Distribution,localization and characteristics of neurotensin binding sites in the rat brain

Neurotensin binds reversibly to brain membranes (0.44 pmol/mg) with high affinity (K_D = 8 nM). Stereospecific membrane binding sites for ¹²⁵I-neurotensin are localized in the thalamus, hypothalamus and cerebrum of rat brain: the hypothalamus and cerebrum contained 38% and 32% of the number of binding sites, respectively, compared to thalamus. The values of ¹²⁵I-neurotensin binding in the medulla and pons, cerebellum and pituitary were only 13, 9 and 2% of that found for the thalamus. Fractionation of brain tissue homogenates on discontinuous sucrose gradients resulted in an enrichment of binding sites in the membrane fraction banding at the interphase of 0.4–0.6 m sucrose. Maximum binding occurred at pH 7.0 in 25 mm buffer in the absence of mono- and divalent cations which are inhibitory. The degree of binding of neurotensin and neurotensin analogs to membranes correlates well with their biological activity as measured by their production of hyperglycemia and hyperglucogonemia in rats.     

Effects of mescaline, [delta9]-tetrahydrocannabinol and pentobarbital on the auditory evoked responses in the cat

Effects of mescaline, [Δ⁹]-tetrahydrocannabinol ([Δ⁹]-THC) and pentobarbital were studied on the peak latency, area and amplitude of the waves (positive P₁ and negative N₁) of the averaged auditory evoked potentials in restrained conscious cats. Mescaline (5–15 mg/kg i.p.) caused a significant increase in peak latency (8–13%), area (77–195%) and amplitude (38–128%) of these waves. The onset of these changes was within 10–40 min and their duration ranged between 90–180 min. [Δ⁹]-THC (0.5–2 mg/kg in 4% Tween 80 i.p.) also caused a marked increase in peak latency, area and amplitude (10–25%, 113–370% and 66–310% respectively), while Tween 80 caused slight and variable effects. The effects of [Δ⁹]-THC had its onset within 40–105 min and lasted for more than 120–180 min. Pentobarbital (20 mg/kg i.p.) also caused an increase in the peak latency (8–23%), area (94–121%) and amplitude (119–132%) of these waves within 5–7 min. The peak effect occurred within 10–15 min and lasted for more than 100–120 min. These effects of the hallucinogens, mescaline and [Δ⁹]-THC thus appear to resemble those of pentobarbital, a central nervous system depressant; however the mechanism of their actions differs.     

Structure-activity studies of hydrophobic amino acid replacements at positions 9, 11 and 16 of glucagon

We have designed and synthesized eight compounds 2-9 which incorporate neutral, hydrophobic amino acid residues in positions 9, 11 and 16 of the glucagon molecule: (2) [desHis¹,Va1⁹,11e^11,16] glucagon amide, (3) [desHis¹,Val^9,11,16]glucagon amide, (4) [desHis¹,Va1⁹,Leu^11,16]glucagon amide, (5) [desHis¹,Nle⁹,11e^11,16]glucagon amide, (6) [desHis¹,Nle⁹,Val^11,16]glucagon amide, (7) [desHis¹,Nle⁹,Leu^11,16]glucagon amide, (8) [desHis¹,Val⁹,Leu^11,16,Lys^17,18,Glu²¹]glucagon amide and (9) [desHis¹,Nle⁹,Leu^11,16,Lys^17,18,Glu²¹]glucagon amide. The effect of neutral, hydrophobic residues at positions 9, 11 and 16 led to good binding to the glucagon receptor. Compared to glucagon (IC₅₀= 1.5 nM), analogues 2-9 were found to have IC₅₀ values of 6.0, 6.0, 11.0, 9.0, 2.5, 2.8, 6.5 and 7.0 nM, respectively. When these compounds were tested for their ability to block adenylate cyclase (AC) activity, they were found to be antagonists having no stimulation of adenyl cyclase, with PA₂, values of 6.15, 6.20, 6.30, 7.25, 6.10, 7.30, 6.25 and 7.25, respectively. © Munksgaard 1997.     

Cyclic melanotropins

The highly potent cyclic analogue of α-MSH, Ac-[Cys⁴, Cys¹⁰]-α-MSH_4–13-NH₂, was structurally modified in position 4. Four analogues were prepared and their biological activities in the in vitro frog and lizard skin bioassays were determined. It was shown that removing the terminal acetylamino group to give [Mpa⁴, Cys¹⁰]-α-MSH_4–13-NH₂ resulted in little change in the biological activity, but a change in the stereochemistry of cysteine in position 4 to give Ac-[D-Cys⁴, Cys¹⁰]-α-MSH_4–13-NH₂ led to a small decrease of activity in both bioassays. Decreasing the size of the intramolecular ring by removing one methylene group to give [Maa⁴, Cys¹⁰]-α-MSH_4–13-NH², resulted in an analogue with lower activities in both assays (about 3 times in the lizard and 500 times in the frog), and increasing the size of the ring by one methylene group to give Ac-[Hcy⁴, Cys¹⁰]-α-MSH_4–13-NH₂ led to much lower activities in the lizard system and similar effects were seen upon decreasing the ring size in the frog skin assay.     

Specificity of antisera obtained to Substance P and its C-terminal hexapeptide

Synthetic fragments and analogs were used to characterize specificity of antisera toSP and SP_6–11. [Tyr⁸]SP and [Lys⁶]SP_6–11 were both used as radioiodinated ligands. The latter was conjugated with Bolton-Hunter reagents before labelling. In both systems, the C-terminal pentapeptide SP_7–11 was the shortest fragment showing antigenic identity with Substance P molecule. Substitution of different amino acid residues in SP_6–11 by His or Gly showed that all but Glu⁶ take part in the structure of the antigenic determinant.     

The pressor response to substance P and hexapeptide [pGlu6]SP 6--11 injections into the cerebral ventricles in rats

Synthetic Substance P (SP_1–11) or hexapeptide, the pyroglutamic acid derivative of the C-terminal of Substance P (pGlu⁶)SP_6–11, were injected into the cerebral ventricles (i.c.v.) through cannulae implanted 8 days earlier or immediately before the injection into the lateral cerebral ventricle. Injected intraventricularly, in increasing doses from 500 fmol to 5 nmol, through cannulae implanted 8 days earlier, SP_1–11 increased, in all cases, the blood pressure recorded from the femoral artery of urethane-anaesthetized rats. The same doses of SP_1–11 injected through cannulae implanted immediately before the injection in most cases (n = 9) increased the blood pressure, but in one case decreased it. In doses of 15–500 pmol [pGlu⁶]SP_6–11 injected through cannulae implanted 8 days earlier or immediately before the injection increased the blood pressure in all cases. Repeated injections of (pGlu⁶)SP_6–11 (150 pmol i.c.v.) diminished the pressor response. This pressor response was not affected by cervical vagotomy but was significantly diminished by intraventricular injection of propranolol. This suggests that the arterial blood pressure responses are evoked by the action of (pGlu⁶)SP_6–11 on central receptors which are blocked by propranolol.     

Author index to volume 43

The effect of analogs of angiotensin (modified with an Ile-substituted for Phe) was studied in the isolated, retrogradely perfused adrenal of the cat. Continuous differential analysis of norepinephrine and epinephrine output was quantified with an automated trihydroxyindole procedure. [Ile⁸]-angiotensin I and [Ile⁷]-angiotensin III exhibited negligible secretory activity, in contrast to the stimulatory effects of [Ile⁸]-angiotensin II (10–20% activity relative to angiotensin II). [Ile⁸]-angiotensin I blocked angiotensin II-induced catecholamine secretion and a pA₂ value of 8.50 was obtained. [Ile⁷]-angiotensin III was an especially potent antagonist of angiotensin II and a pA₂ value of 10.4 was calculated for this heptapeptide analog. The pA₂ value for [Ile⁸]-angiotensin II, a partial agonist in the adrenal medulla was 9.33. These three analogs were equally effective against secretion induced by the corresponding unsubstituted homologs (Ang I and Ang III). These data suggest that all these angiotensin peptides interact with a common receptor. [Ile⁸]-angiotensin I and [Ile⁷]-angiotensin III had no effect on adrenal medullary responses induced by KCl, nicotine and bradykinin. These structural analogs of angiotensin are pure competitive antagonists of angiotensin in the cat adrenal chromaffin cell.     

Synthesis and properties of human β-endorphin-(1–9) and its analogs

Four analogs of human β-endorphin (β_h-EP) were synthesized by the solid-phase method: β_h-EP-(1–9) (I), [D-Ala²]-β_h-EP-(1–9) (II), [Gln⁸]-β_h-EP-(1–9) (III), and [D-Ala², Gln⁸]-β_h-EP-(1–9) (IV). Measurement in a radioreceptor binding assay with use of tritiated β_h-EP as primary ligand gave relative potencies as follows: Met-enkephalin, 100; I, 76; II, 100; III, 200; IV, 200. Two new amino acid derivatives were prepared and used for synthesis of the analogs: N^α-t-butyloxycarbonyl-O-(cyclopentyl) -tyrosine and N^α-t-butyloxycarbonyl-γ-(cyclopentyl)-glutamic acid.