Secretory actions of vasoactive intestinal polypeptide, peptide histidine isoleucine and helodermin in rat small intestine: the effects of putative VIP antagonists upon VIP-induced ion secretion

Vasoactive intestinal polypeptide (VIP), peptide histidine isoleucine (PHI), and helodermin stimulate electrogenic anion secretion in preparations of rat jejunum stripped of muscularis propria. Concentration-response curves to exogenously applied peptides yielded EC50 values of 12 nM, 12 nM and 100 nM for VIP, PHI and helodermin respectively. These secretory responses were most probably mediated via the same receptor population given that cross-desensitisation was observed between all 3 analogues. Four putative VIP antagonists, namely, two growth hormone releasing factors (GRF); [AcTyr1, D-Phe2]GRF-(1-29)-NH2 and [AcTyr1]hGRF-(1-40)-OH as well as [4Cl-D-Phe6,Leu17]VIP and VIP-(10-28) were tested for their ability to inhibit VIP induced electrogenic ion secretion. None of the above exhibited any intrinsic agonist activity nor were they competitive antagonists, although some inhibition was observed with [AcTyr1]hGRF-(1-40)-OH and VIP-(10-28). Their use as selective VIP antagonists is therefore limited in rat jejunal mucosa.     

Degradation of growth hormone releasing factor analogs in neutral aqueous solution is related to deamidation of asparagine residues. Replacement of asparagine residues by serine stabilizes

The incubation of a solution of the human growth hormone releasing factor analog, [Leu27] hGRF(1-32)NH2 at pH 7.4 and 37 degrees, resulted in extensive degradation of the sample. The major degradation products were identified as the peptides [beta-Asp8, Leu27] hGRF(1-32)NH2 and [alpha-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, [beta-Asp8, Leu27] hGRF(1-32)NH2 was estimated to be 400-500 times less potent than the parent Asn8 peptide, while [alpha-Asp8, Leu27] hGRF(1-32)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 degrees). 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)NH2 (150 h); [Ser8, Leu27, Asn28] hGRF(1-32)NH2 (746 h); 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.     

Semisynthesis of human growth hormone-releasing factor by trypsin catalyzed coupling of leucine amide to a C-terminal acid precursor.

Human growth hormone-releasing factor, GRF(1-44)-NH2, was synthesized by trypsin catalyzed coupling of Leu-NH2 to Arg43 of the precursor, GRF(1-43)-OH, prepared by solid phase peptide synthesis. The semisynthetic GRF(1-44)-NH2 was fully characterized and showed full potency in the rat pituitary in vitro bioassay. Conversion to GRF(1-44)-NH2 was limited to 60-70% in both 75% v:v N,N'-dimethylacetamide and 95% v:v 1,4-butanediol due to competing transpeptidations at Arg41 and Arg38 generating [Leu42]-GRF(1-42)-NH2 and [Leu39]-GRF(1-39)-NH2 side-products, respectively. The rates of formation and yields of GRF(1-44)-NH2 versus pH, Leu-NH2 concentration, and solvent composition were also studied.     

Kinetics of dipeptidyl peptidase IV proteolysis of growth hormone-releasing factor and analogs.

The kinetics and selectivity of proteolysis of synthetic human growth hormone-releasing factor and analogs by purified human placental dipeptidyl peptidase IV (DPP IV) were studied by HPLC. The initial rates of Ala2-Asp3 cleavage (pH 7.8, 37 degrees C, So = 0.15 mM) were all approx. 5 mumol min-1 mg-1 for the parent hormone, GRF(1-44)-NH2, and the fragments, GRF(1-29)-NH2 and GRF(1-20)-NH2. Lower activities observed for GRF(1-11)-OH, GRF(1-3)-OH, and cyclic lactam analogs indicate S1'-Sn' binding. Assays of [Trp6]-GRF(1-29)-NH2 versus [D-Trp6]-GFR(1-29)-NH2 indicate an S4' binding cavity. Peptides with D-configuration at P2, P1 or P1' and desNH2Tyr1 and N-MeTyr1 analogs of GRF were not cleaved. Catalytic parameters for the P1-substituted analogs [X2,Ala15]-GRF(1-29)-NH2 were found to vary with X as follows, Km: Abu less than Ala less than Pro less than Val less than Ser less than Gly much less than Leu; kcat: Pro greater than Ala greater than Abu greater than Ser greater than Gly much greater than Leu greater than Val; kcat/Km: Abu greater than Pro greater than Ala much greater than Ser greater than Gly = Val much greater than Leu. Km is at a minimum and kcat/Km at a maximum, for a hydrophobic P1 side-chain of about 0.25 nm in length, i.e., the ethyl side-chain of alpha-aminobutyric acid (Abu) is very close to optimal. These results further define the S1 selectivity of DPP IV and may be useful in the design of DPP IV resistant GRF analogs that can be produced by recombinant DNA methods and the design of DPP IV inhibitors.     

Vasoactive intestinal polypeptide VPAC1 and VPAC2 receptor chimeras identify domains responsible for the specificity of ligand binding and activation.

In order to identify the receptor domains responsible for the VPAC1 selectivity of the VIP1 agonist, [Lys15, Arg16, Leu27] VIP (1-7)/GRF (8-27) and VIP1 antagonist, Ac His1 [D-Phe2, Lys15, Arg16, Leu27] VIP (3-7)/GRF (8-27), we evaluated their binding and functional properties on chimeric VPAC1/VPAC2 receptors. Our results suggest that the N-terminal extracellular domain is responsible for the selectivity of the VIP1 antagonist. Selective recognition of the VIP1 agonist was supported by a larger receptor area: in addition to the N-terminal domain, the first extracellular loop, as well as additional determinants in the distal part of the VPAC1 receptor were involved. Furthermore, these additional domains were critical for an efficient receptor activation, as replacement of EC1 in VPAC1 by its counter part in the VPAC2 receptor markedly reduced the maximal response.     

Degradation of aspartic acid and asparagine residues in human growth hormone-releasing factor.

Products of the degradation of human growth hormone-releasing factor (GRF) in aqueous solutions (15-200 microM) have been isolated and fully characterized. The cleavage product, GRF(4-44)-NH2, and the isomerization product, [beta-Asp3]GRF(1-44)-NH2, from the degradation of GRF(1-44)-NH2 in acidic solution and the corresponding products, GRF(4-29)-NH2 and [beta-Asp3]GRF(1-29)-NH2, from the degradation of GRF(1-29)-NH2 have been isolated and characterized. The products, [beta-Asp8]GRF(1-44)-NH2 and [Asp8]GRF(1-44)-NH2, from the deamidation of GRF(1-44)-NH2 at pH 8.0 and the corresponding products, [beta-Asp8]GRF(1-29)-NH2 and [Asp8]GRF(1-29)-NH2, from the deamidation of GRF(1-29)-NH2 have been isolated and characterized. All the degradation products of GRF(1-44)-NH2 and GRF(1-29)-NH2 were evaluated for biological activity and found to have much lower in vitro potencies than the parent peptides. Degradation occurs at Asp3 and Asn8 and the kinetics of these various transformations versus pH and temperature have been studied. GRF is most stable at pH 4-5. At pH below the pKa of the Asp3 side-chain (pH less than 4), cleavage at Asp3-Ala4 is the major route of degradation. For pH greater than 4, isomerization of Asp3 to beta-Asp3 (iso-Asp3) predominates. The rates of cleavage and isomerization are simple first order and vary with pH, independent of buffer concentration, such that the protonated (COOH) form of Asp3 undergoes cleavage while the ionized (COO-) form isomerizes. The more rapid deamidation of Asn8 to generate beta-Asp8 and Asp8 in about a 4:1 ratio, presumably via a cyclic imide intermediate, occurs at pH greater than or equal to 5 and is general base-catalyzed. Evidence was also obtained for direct hydrolysis of protonated Asn8 in GRF(1-29)-NH2 at pH less than or equal to 2 to give exclusively [Asp8]GRF(1-29)-NH2. The deamidation of Asn8 in GRF(1-29)-NH2 at pH 8.0, 22-55 degrees C, is relatively insensitive to temperature for T less than 37 degrees C, possibly due to conformational constraints. Asp25 and Asn35 are sterically, conformationally, or otherwise hindered with respect to these changes as no degradation at these sites was observed under the conditions employed.     

Antagonism by GRP(18-27) and substance P analogues on insulin release stimulated by GRP(18-27).

The antagonistic effects of [D-Phe25]gastrin-releasing peptide (GRP)(18-27) and [D-Arg1,D-Pro2,D-Trp7,9,Leu11]substance P (SP) on the stimulation of insulin release by GRP(18-27) from isolated canine pancreas were compared with that of [Ala23]GRP(18-27). The stimulation of insulin release by 1 nM GRP(18-27) was reduced to 24.1% and 15.4% by the prior infusion of 1 microM of [D-Arg1,D-Pro2,D-Trp7,9,Leu11]SP and 10 microM of [D-Phe25]GRP(18-27), respectively. Glucagon release by GRP(18-27) was not affected by these peptides using the above concentrations. The results indicate that these peptides are antagonists of bombesin-like peptide receptors on pancreatic B-cells, although the inhibitory activities are lower than that of [Ala23]GRP(18-27).     

A covalently constrained congener of the Saccharomyces cerevisiae tridecapeptide mating pheromone is an agonist

An analog of alpha-factor, the Saccharomyces cerevisiae tridecapeptide mating pheromone (Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr), in which the side chains of Lys7 and Gln10 were covalently linked, was synthesized using solid phase methodologies. The yield of the purified cyclic analog cyclo7,10[Nle12]alpha-factor was 30%, and its structure was verified by amino acid analysis, peptide sequencing, fast atom bombardment-mass spectrometry, and proton nuclear magnetic resonance spectroscopy. Cyclo7,10[Nle12]alpha-factor caused growth arrest and morphological alterations in S. cerevisiae MATa cells qualitatively identical to those induced by linear pheromone and was one-fourth to one-twentieth as active as the linear alpha-factor depending upon the S. cerevisiae strain tested. Consistent with the relative activities of the linear and cyclic peptides, binding competition studies indicated that cyclo7,10[Nle12]alpha-factor had approximately 20-40-fold less affinity for the alpha-factor receptor. Hydrolysis of the cyclic peptide by the target cells did not lead to opening of the ring and was less rapid than that of linear alpha-factor. The alpha-factor antagonist des-Trp1-[Ala3,Nle12]alpha-factor reversed the activity of the cyclic analog, and cyclo7,10[Nle12]alpha-factor was not active at the restrictive temperature in a temperature-sensitive receptor mutant. These results support the conclusion that the cyclic alpha-factor occupies the same binding site within the receptor as is occupied by the natural pheromone. The cyclic alpha-factor represents a rare example of an agonist among covalently constrained congeners of small linear peptide messengers.     

Structure-activity relationships in the dodecapeptide alpha-factor of Saccharomyces cerevisiae: position 6 analogues are poor inducers of agglutinability

Five des-Trp1,Cha3,X6 analogues of alpha-factor, where X = Ala, Val, Ile, Nle, or D-Leu and X = Leu in the natural alpha-factor sequence, were prepared by solution-phase techniques utilizing isobutyl chloroformate or 1-hydroxybenzotriazole accelerated active esters as the coupling agents. Purification to 98% or greater homogeneity was accomplished by high-performance liquid chromatography on a reversed-phase muBondapak C18 column with methanol/water/trifluoroacetic acid as the mobile phase. Three of the synthesized analogues (X6 = Val, Ile, Nle) induced morphogenesis and increased agglutinability in a cells. These substitutions demonstrate that a gamma-branched side chain at position 6 is not essential for biological activity. All of the active analogues induced morphogenesis at lower concentrations than they induced enhanced agglutinability. These results and other structure-activity relationships [Baffi, R. A., Shenbagamurthi, P., Terrance, K., Becker, J. M., Naider, F., & Lipke, P. (1984) J. Bacteriol. 158, 1152-1156] indicate that the agglutination and morphological responses to alpha-factor can be varied independently. Replacement of Leu6 with Ala or D-Leu resulted in inactive analogues that were not antagonistic for alpha-factor activity. Cell-mediated hydrolysis experiments indicated that the biological activities of the alpha-factor analogues are independent of their rates of degradation. All position 6 analogues were hydrolyzed more slowly than the parent compound, suggesting that the enzyme which degrades alpha-factor is highly specific for the native structure.     

A new type of functional VIP receptor has an affinity for helodermin in human SUP-T1 lymphoblasts

A new type of VIP receptor was characterized in human SUP-T1 lymphoblasts. The order of potency of unlabeled peptides, in the presence of [125I]helodermin, was: helodermin(1-35)-NH2 = helodermin(1-27)-NH2 greater than helospectin greater than VIP = PHI greater than [D-Ser2]VIP greater than [D-Asp3]VIP greater than [D-His1]VIP greater than or equal to [D-Ala4]VIP greater than or equal to secretin = GRF. This specificity was distinct from that of all VIP receptors described so far in that: (i) the affinity for helodermin (Kd = 3 nM) was higher than that of VIP (Kd = 15 nM) and PHI (Kd = 20 nM); and (ii) position 4 played an important role in ligand binding. The labeled sites were likely to be functional receptors as adenylate cyclase in crude lymphoblastic membranes (200-10,000 x g pellets) was stimulated by peptides, in the presence of GTP, with the following order of potency: helodermin(1-35)-NH2 greater than helodermin(1-27)-NH2 greater than helospectin = VIP = PHI.     

Effects of surfactant-associated protein SP-B synthetic analogs on the structure and surface activity of model membrane bilayers.

The effect of several synthetic peptides based on the sequence of human pulmonary surfactant-associated protein B (SPB) on the molecular packing of model membrane lipids (7:1 dipalmitoyl phosphatidylcholine (DPPC)/dipalmitoyl phosphatidylglycerol (DPPG)) was studied using fluorescence anisotropy. This information was then correlated with complementary biophysical data obtained on both a modified Wilhelmy-Langmuir balance and a pulsating bubble surfactometer. The SP-B peptides examined in these studies are synthetic human SP-B Phe1-Ser78 (SP-B 1-78, full-length sequence), synthetic human SP-B Phe1-Thr60 (SP-B 1-60), synthetic human SP-B Phe1-Ala20 (SP-B 1-20), synthetic human SP-B Ala20-Thr60 (SP-B 20-60), synthetic human SP-B Leu27-Ser78 (SP-B 27-78), synthetic human SP-B Leu40-Thr60 (SP-B 40-60) and synthetic human SP-B Tyr53-Ser78 (SP-B 53-78). trans-parinaric acid was utilized to detect changes in ordering of lipids within the interior upon incorporation of synthetic SP-B peptide, whereas 1-hexadecanoyl-2-[N-(7-nitro-2-benzoxa-1,3-diazol-4-yl)-a min ohexanoyl] phosphatidylcholine (6-NBD-PC) and 1-acyl-2-[N-(7-nitro-2-benzoxa-1,3-diazol-4-yl)aminohexanoyl ] phosphatidylglycerol (6-NBD-PG) were utilized to determine alterations in lipid order at the surface of the model membrane bilayer. With the exception of SP-B 40-60, which corresponds to the most hydrophobic segment of the full-length SP-B, none of the other peptide significantly perturbed the interior bilayer as determined by fluorescence anisotropy of trans-parinaric acid. Incorporation of any of the peptides with the exception of SP-B 40-60, resulted in an increase in anisotropy of NBD-PC. The most significant enhancements resulted from the addition of SP-B 1-78, SP-B 1-20, SP-B 27-78 or SP-B 53-78. The magnitude of anisotropy increase with these peptides is similar to that observed with an equivalent molar ratio of native SP-B isolated from a bovine source. These observations suggest that these four synthetic peptides have the structural and compositional characteristics required for surface ordering of the membrane bilayer in a manner similar to that observed with native SP-B, thereby facilitating the surfactant-like properties of phospholipid mixtures.     

Plant peptide hormone phytosulfokine (PSK-alpha): synthesis of new analogues and their biological evaluation.

Phytosulfokine-alpha (PSK-alpha), a sulfated growth factor (H-Tyr(SO3H)-Ile-Tyr(SO3H)-Thr-Gln-OH) universally found in both monocotyledons and dicotyledons, strongly promotes proliferation of plant cells in culture. In our studies on structure/activity relationship in PSK-alpha the synthesis of a series of analogues was performed: [H-D-Tyr(SO3H)1]- (9), [H-Phe(4-SO3H)1]- (10), [H-D-Phe(4-SO3H)1]- (11), [H-Phg(4-SO3H)1]- (12), [H-D-Phg(4-SO3H)1]- (13), H-Phe(4-NHSO2CH3)1]- (14), [H-D-Phe(4-NHSO2CH3)1]- (15), [H-Phe(4-NO2)1]- (16), [H-D-Phe(4-NO2)1]- (17), [H-Phg(4-NO2)1]- (18), [H-D-Phg(4-NO2)1]- (19), [H-Hph(4-NO2)1]- (20), [H-Phg(4-OSO3H)1]- (21), [Phe(4-NO2)3]- (22), [Phg(4-NO2)3]- (23), [Hph(4-NO2)3]- (24), [H-Phe(4-SO3H)1, Phe(4-SO3H)3]- (25) [H-Phe(4-NO2)1, Phe(4-NO2)3]- (26), [H-Phg(4-NO2)1, Phg(4-NO2)3]- (27), [H-Hph(4-NO2)1, Hph(4-NO2)3]- (28) and [Val3]- PSK-alpha (29). For modification of the PSK-alpha peptide chain the novel amino acids and their derivatives were synthesized, such as: H-L-Phg(4-SO3H)-OH (1), H-D-Phg(4-SO3H)-OH (2), Fmoc-Phg(4-SO3H)-OH (3), Fmoc-D-Phg(4-SO3H)-OH (4), Boc-Phg(4-NHSO2CH3)-OH (5), Boc-D-Phg(4-NHSO2CH3)-OH (6) Boc-Phe(4-NHSO2CH3)-OH (7), and Boc-D-Phe(4-NHSO2CH3)-OH (8). Peptides were synthesized by a solid phase method according to the Fmoc procedure on a Wang-resin. Free peptides were released from the resin by 95% TFA in the presence of EDT. All peptides were tested by competitive binding assay to the carrot membrane using 3H-labelled PSK according to the Matsubayashi et al. test.     

Rational design,synthesis, and biological evaluation of novel growth hormone releasing factor analogues

Since its initial discovery in 1982, growth hormone-releasing factor (GRF) has been the subject of intense investigation. This interest was prompted by the potential application of GRF for Stimulating growth in dwarf humans and for performance enhancement in livestock. Substantial research has been focused upon the development of potent, long-acting analogs as therapeutics. Herein is described a summary of the cumulative efforts of various laboratories endeavoring in this quest. The rationale utilized in GRF analog development is discussed: (1) determination of bioactive core. (2) evaluation of secondary structure, and (3) elucidation of degradation pathways (chemical and enzymatic). Using this information, several series of linear (unnatural and natural sequence) and cyclic GRF analogs were designed, synthesized, and evaluated. Stimulated by the constraints of commercial production, innovative, alternative methods of synthesis were explored: solid-phase, solution-phase, enzymatic, and recombinant. To date, the most promising candidate for drug development is [His¹, Val², Gln⁸, Ala¹⁵, Leu²⁷]-hGRF(1-32)-OH. This natural sequence analog, consisting of rodent and human sequences, incorporates the bioactive core, preferred secondary structure, resistance to chemical and enzymatic degradation: with the added benefit of amenability to large-scale recombinant synthesis. © 1994 John Wiley & Sons, Inc.     

Production of antisera to growth hormone-releasing factor: usefulness in radioimmunoassay and passive immunization

We have produced two antisera (R-1 & R-2) to human growth hormone-releasing factor (GRF) [1-44] NH2. Both antisera can be used for human GRF radioimmunoassay (RIA) at a final dilution of 1:50000. The antiserum R-2 was specific for the C-terminal amidated sequence of human GRF-44 and selectively recognized GRF [1-44] NH2 but not GRF [1-44] OH or GRF [1-40] OH. The antiserum R-1 also significantly bound 125I-rat GRF [1-43] OH at a final dilution of 1:5000 and enabled us to establish RIA for rat GRF. In both RIA systems, intra- and inter-assay coefficients of variation at 50% inhibition were 8 and 12%, respectively. A median effective dose was 90-120 pg in human GRF RIA and 250-300 pg in rat GRF RIA. Utilizing the RIA, we demonstrated that the hypothalamic GRF content in rats which received monosodium glutamate during the neonatal period was less than 20% of that of controls. However, the hypothalamic GRF content was not altered in rats made hypothyroid by methimazole administration, another condition known to greatly impair GH secretion. An iv administration of the antiserum R-1 significantly suppressed GH release following the injection of antisomatostatin serum. Thus, these antisera can be a useful tool in examining the physiological and/or pathophysiological roles of GRF in human and rat.     

Expression of growth hormone-releasing factor analog Leu27GRF(1-44)OH in Escherichia coli: purification and characterization of the expressed protein

A recombinant plasmid has been constructed to direct the synthesis of Leu27GRF(1-44)OH in Escherichia coli as a fusion protein containing a hexa-His tail followed by amino acids 1-99 of interferon-gamma and a methionine residue at the N-terminal. The expression of the 18-kDa fusion protein (H6GAMGRF) was induced by isopropylthiogalactoside treatment and the protein accumulated as insoluble aggregates in inclusion bodies. The protein aggregates were solubilized in 6 M guanidine-HCl and purified directly by affinity chromatography on a Nichelate column. The growth hormone-releasing factor (GRF) moiety was released from the fusion protein by cyanogen bromide cleavage and purified to homogeneity by anion-exchange chromatography followed by reverse-phase chromatography. The identity of the GRF peak was determined by comparing its retention time with that of synthetic Leu27GRF(1-44)OH. The purified material was further characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, N-terminal sequencing, and amino-acid analysis. The recombinant-derived product and the synthetic compound showed identical reactivities toward anti-GRF polyclonal antibodies and were essentially equipotent as determined by an in vitro biological assay for growth hormone-releasing activity.     

Comparison of the insulinotropic activity of glucagon-superfamily peptides in rat pancreas perfusion.

Previous studies have demonstrated that glucagon-superfamily peptides stimulate insulin release from the pancreatic islets in a glucose dependent manner. In this study we have carried out a structure-activity study of their insulinotropic activity using a rat pancreas perfusion with 5.5 mM glucose concentration. The following peptides were examined: glucagon-like peptide-1(7-36)amide (tGLP-1), glucagon, gastric inhibitory peptide (GIP), peptide having an amino-terminal histidine and carboxy-terminal isoleucine amide (PHI), vasoactive intestinal polypeptide (VIP), growth hormone releasing factor(1-29)amide (GRF), GRF(1-27)amide and synthetic hybrid-peptides of PHI-GRF, PHI(1-11)-GRF(12-27) and PHI(1-20)-GRF(21-27). Their potencies were evaluated as: tGLP-1 = GIP > glucagon > PHI = VIP > PHI(1-20)-GRF(21-27) > PHI(1-11)-GRF(12-27) > GRF(1-29) = GRF(1-27). It is clear that 0.1 nM tGLP-1 stimulated insulin release, whereas 1 microM GRF(1-29) did not. These results indicate that 1) in addition to N-terminal amino acid (histidine or tyrosine), position 4 (glycine), position 9 (aspartic acid) and position 11 (serine) in the amino acid sequence are important for their insulinotropic activity, 2) not only the N-terminal portion but also the C-terminal portion of these peptides contribute to their insulinotropic activity.     

The effects of antagonists of vasoactive intestinal peptide on nonadrenergic noncholinergic inhibitory responses in feline airways

Relaxations of the feline intrapulmonary bronchus (IPB) induced by VIP or nonadrenergic noncholinergic (NANC) inhibitory nervous stimulation were unaffected by the VIP receptor antagonist [Ac-Tyr1,D-Phe2]-GRF (1-29) (30 microM). A second VIP antagonist, [pCl-D-Phe6,Leu17]-VIP (30 microM), also had no effect on NANC relaxation responses or IPB sensitivity to VIP. However, responses to three of the four highest VIP concentrations were inhibited by this antagonist. These results indicate that [Ac-Tyr1,D-Phe2]-GRF (1-29) and [pCl-D-Phe6,Leu17]-VIP are not effective competitive antagonists of VIP receptors in feline airways and, hence, have but limited applicability in determining the role of VIP in mediating airway NANC inhibitory responses in this tissue.     

Conformational studies on bombesin antagonists: CD and NMR characterization of [Thr6, Leu13 psi(CH2NH) Met14] bombesin (6-14).

The conformational flexibility of the [Thr6, Leu13 psi(CH2NH) Met14] bombesin (6-14) nonapeptide has been studied by CD and one- and two-dimensional (1D and 2D) nmr techniques. The CD and nmr parameters in different solvents and in a micellar environment (SDS) are compared with the data collected for the parent bombesin (BN) and [D-Phe12, Leu14]BN. A preliminary investigation on spantide is also reported. In particular, the results obtained from CD measurements indicate that there is a shift from random coil structures, in aqueous solutions, toward folded structures in apolar media (2,2,2-trifluoroethanol) and in a membrane-mimetic environment (40 mM SDS) for all three peptides, namely BN, [D-Phe12, Leu14]BN, and [Thr6, Leu13 psi(CH2NH) Met14]BN (6-14). Spantide, which also possesses some inhibitory activity against BN but very little sequence similarity, even in water, shows an ordered conformation. Nuclear magnetic resonance parameters such as backbone NH-alpha CH coupling constant values, amidic temperature coefficients, and the presence of only sequential nuclear Overhauser effects have not provided, so far, any clear evidence for a preferential ordered structure in the peptides studied, and this may be due to rapid exchange among different conformers in the nmr time scale.     

Leucine pools in normal and dystrophic chicken skeletal muscle cells in culture

The specific radioactivity of [3H]Leu in the extracellular, intracellular, and Leu-tRNA pools of normal (white leghorn) and dystrophic (line 307) embryonic chick breast muscle cultures was analyzed as a function of equilibration time and extracellular Leu concentration (0.05-5 mM). The primary results were the following 1) [3H]Leu equilibrated to a constant specific radioactivity in the intracellular and Leu-tRNA pools within 2 min after addition to both normal and dystrophic cultures. 2) After equilibration, the extracellular [3H] Leu specific radioactivity in dystrophic cell culture medium was lower than that of medium exposed to normal cells (especially at low Leu concentrations), probably because of increased release of unlabeled Leu from the dystrophic cells as a result of faster protein breakdown. Accordingly, the specific radioactivities in the intracellular and the Leu-tRNA pools were also lower in dystrophic cells. 3) At 5 mM extracellular Leu, the specific radioactivity in the Leu-tRNA pool was approximately 40% lower than the specific radioactivity in the intracellular pool in both normal and dystrophic cells. Thus, high concentrations of extracellular Leu cannot be used to "flood out" reutilization of unlabeled Leu (released by protein degradation) during protein synthesis. 4) At 5.0 mM extracellular Leu, the specific radioactivity of [3H]Leu in the intracellular pool was comparable to that in the extracellular pool in normal and dystrophic cells; however, the specific radioactivity of Leu-tRNA (i.e. the immediate precursor to protein synthesis) was only 55-65% of the extracellular specific radioactivity in normal and dystrophic cells. In conclusion, reutilization of Leu from protein degradation is higher in dystrophic muscle cell cultures than in normal muscle cell cultures, and accurate rates of protein synthesis in cell cultures can only be obtained if specific radioactivity of amino acid in tRNA is measured.     

Synthesis, purification and biological evaluation of porcine corticotropin-releasing factor

The 41-residue sequences of recently identified porcine corticotropin-releasing factors [Ile40]pCRF and [Asn40]pCRF were assembled on a benzhydrylamine resin support. Deprotection and cleavage from the resin were accomplished by HF treatment. The crude peptides were purified by HPLC. The homogeneity of the final materials, obtained in 0.2% and 0.4% overall yield for [Ile40]pCRF and [Asn40]pCRF respectively, was assessed after the isolation by HPLC and amino acid analysis. Both sequences of the synthetic 41-residue pCRF stimulated the release of corticotropin (ACTH) from superfused rat pituitary cells on a column, the responses being related to a log-dose of CRF in the range of 1-20 ng/ml. [Ile40]pCRF and [Asn40]pCRF also augmented the in vivo release of ACTH in rats pretreated with chlorpromazine, morphine and Nembutal. [Ile40]pCRF appeared to be equipotent to ovine CRF and about twice as active as [Asn40]pCRF. The data indicate that synthetic porcine [Ile40]pCRF and [Asn40]pCRF have high biological activity.