Purification and sequencing of a rat intestinal 22 amino acid C-terminal CCK fragment

Fractionation on Sephadex G50 gel of methanol extracts of rat intestine revealed two molecular forms of cholecystokinin (CCK) of about equal immunopotency: one form has an elution volume between CCK33 and CCK12; the other elutes in the salt region as does authentic CCK8. Purification and sequencing have demonstrated that the smaller molecular form is CCK8 with a sequence identical to the pork and sheep CCK8's that had previously been sequenced. Purification and sequencing of the larger molecular form reveals that it is a 22 amino acid C-terminal CCK fragment identical with pig CCK22 except that glycine instead of serine is present at the nineteenth residue from the C-terminus. This sequence is consistent with that predicted by cloned cDNA encoding preprocholecystokinin from a rat medullary thyroid carcinoma. CCK22 has not previously been reported to be a prominent molecular form in either pig or dog intestines.     

Cholecystokinin octapeptide analogues stable to brain proteolysis

Based on recent findings identifying the initial degradative cleavage of CCK-8 at the Met3-Gly4 bond by a metalloendopeptidase, two analogues of CCK-8 with D-Ala and D-Trp substitutions at the Gly4 position were synthesized as stable analogues. Their stability to proteolysis by brain membranes and their binding potency at central CCK receptors were quantified. Both peptides are stable to degradation by peptidases in cortical synaptic membrane preparations. The analogues are nearly equipotent to CCK-8 in their affinities for inhibition of 125I-CCK-33 binding to guinea pig cortical membranes. L-Ala and L-Trp substituted peptides were synthesized for comparison. Both these peptides are degraded by synaptic membranes and the L-Trp substituted peptide possesses a greatly reduced affinity for central CCK receptors. Therefore, the structure of CCK due to the D conformation of Gly is more capable of interacting with brain CCK receptors. Further conformational analysis will establish whether the stabilized structure is a beta-bend or a beta-turn. Since these peptides are highly potent and stable to brain proteolysis they may be useful as stable CCK analogues for in vivo application.     

Crucial role of position 40 for interactions of CCK-58 revealed by sequence of cat CCK-58

Evidence suggests that amino terminal extensions of CCK-8 affect the carboxyl terminal bioactive region of CCK. Cat CCK-58 was purified by low pressure reverse phase and ion-exchange chromatography steps and several reverse phase HPLC steps. The purified peptide and its tryptic fragments were characterized by mass spectral analysis and microsequence analysis. The structure of cat CCK-58 is: AVQKVDGEPRAHLGALLARYIQQARKAPSGRMSVIKNLQSLDPSHRISDRDY(SO3) MGWMDF-amide. Cat and dog CCK-58 are identical except for position 40 which is serine in cat and asparagine in dog. Radioimmunoassay detected cat CCK-58 about 1/10th as well as dog CCK-58, indicating a marked effect on C-terminal immunoreactivity. Cat CCK-58 with a serine at position 40, the same residue found in pig, mouse, cow and rabbit CCK-58, can be used as a unique bioprobe for defining how amino terminal amino acids influence the structure and bioactivity of the carboxyl terminal region of CCK.     

A synthetic peptide derivative that is a cholecystokinin receptor antagonist

So far, there are no known peptidic effective receptor antagonists of both peripheral and central effects of cholecystokinin (CCK). Here, we describe a synthetic peptide derivative of CCK, t-butyloxycarbonyl-Tyr(SO3-)-Met-Gly-D-Trp-Nle-Asp 2-phenylethyl ester 1 (where Nle is norleucine), which is a potent CCK receptor antagonist. In rat and guinea pig dispersed pancreatic acini, this peptide derivative did not alter amylase secretion, but was able to antagonize the stimulation caused by cholecystokinin-related agonists. It caused a parallel rightward shift in the dose-response curve for the stimulation of amylase secretion with half-maximal inhibition of CCK-8-stimulated amylase release at a concentration of about 0.1 microM. Compound 1 was able to inhibit the binding of labeled CCK-9 (the C-terminal nonapeptide of CCK) to rat and guinea pig pancreatic acini (IC50 = 5 X 10(-8) M) as well as to guinea pig cerebral cortical membranes (IC50 = 5 X 10(-7) M). These results indicate that Compound 1 is a potent competitive CCK receptor antagonist.     

Biological effects of newly synthesized cholecystokinin analogs

Cholecystokinin (CCK) is a gut hormone that regulates pancreatic endocrine functions via CCK(A) receptors. CCK(4) (Trp-Met-Asp-Phe-NH(2)) has an insulinotropic effect, but is 1,000-fold less potent than CCK(8) in rodents. The in vitro potencies with respect to binding, the biological effects and the selectivity of newly synthesized CCK(4) analogs constructed by computer modelling experiments were investigated in vitro in rat pancreas and brain, INS-1 cells, and guinea pig ileum. Exchanging various amino acids, e.g. Met by either Pro or Nle, and modifying Phe by adding various substituents in different positions led to compounds which were more effective as insulin secretagogues than CCK(4) itself and even show insulinotropic effects comparable with those of CCK(8) (e. g. compounds M1 and M2 being substituted at Phe). Some compounds which possess electron withdrawing groups on the C-terminal Phe and possess a Pro instead of a Met were especially effective. The CCK(A) receptor antagonist L-364,718, but not by the CCK(B) receptor antagonist L-365,260, inhibited the insulinotropic effects. The synthetic CCK(4) compounds were not selective for the endocrine pancreas: e.g. M1 and M2 had binding activity with respect to rat brain homogenates but no activity with respect to contraction of the guinea pig ileum. The data indicate that some of the newly synthesized CCK tetrapeptides exhibit a high affinity for the CCK receptor of beta-cells and have an insulinotropic effect much higher than CCK(4).     

Identification and characterization of a novel CCK-like immunoreactive peptide in pig CNS

Antibodies directed against the C-terminus of cholecystokinin octapeptide (CCK8) and caerulein were used to study immunoreactive peptides in pig brain. One antibody, a mouse monoclonal raised to caerulein (c.MAb), reacts equally with heptadecapeptide gastrin (G17), CCK8 and caerulein, the other raised to CCK8 (L48) shows 10 times lower immunoreactivity with caerulein compared with G17 and CCK8. Extracts were purified by adsorption to alginic acid, gel filtration chromatography and reversed phase HPLC. In addition to material with the expected properties of CCK33, 39 and 58 a novel peptide was identified that reacted 10 times better with c.MAb compared with L48. This material emerged in a similar position to CCK58 on Sephadex G50 but had a greater retention time on reversed phase HPLC. It had CCK-like bioactivity and digestion with trypsin gave a fragment showing a pattern of immunoreactivity similar to that of the parent compound. This pattern of activity is distinct from other known mammalian CCKs; the material may represent an addition to the gastrin-CCK family in mammals.     

Neuropeptides in the gut: quantification and characterization of cholecystokinin octapeptide-, bombesin- and vasoactive intestinal polypeptide-like immunoreactivities in the myenteric plexus of the guinea-pig small institute

The localization of CCK8-, bombesin- and VIP-like immunoreactivities in the myenteric plexus of the guinea pig small intestine has been studied by radioimmunoassay of extracts of longitudinal muscle strips obtained with and without adherent myenteric plexus; concentrations were compared with those in other regions of the gut. In innervated strips of longitudinal muscle of ileum there was approximately 14 pmol/g CCK8-, 32 pmol/g bombesin- and 135 pmol/g VIP-like immunoreactivity; concentrations were reduced by over 70% in denervated strips. Gel filtration and ion exchange chromatography indicated that over 80% of CCK immunoreactivity was due to CCK8; no evidence was found of significant amounts of smaller COOH-terminal fragments. Bombesin immunoreactivity occurred in two forms, the major one resembling the amphibian tetradecapeptide in its elution from gel filtration columns. Immunoreactive VIP differed markedly from porcine VIP in immunochemical and chromatographic properties; the data suggest that guinea pig VIP is less basic than porcine VIP and that the two peptides differ in structure in their NH2-terminal regions. Some functional implications of these findings are discussed.     

Cholecystokinin octapeptide purified from brains of Australian marsupials

Cholecystokinin octapeptides (CCK8s) have been purified from methanol extracts of two brains from each of two Australian marsupials, Tammar Wallaby and Eastern Quoll, containing 3 nmol and 2 nmol of the peptides, respectively. Immunoreactive CCK was concentrated on QMA SepPak cartridges and purified by two successive HPLC steps on Nova C18 radial-pak cartridges. The sequence of each of the peptides is identical with that previously reported for Old World mammals (DYMGWMDF). This is in contrast to the previously reported sequence for CCK8 from the South American hystricomorphs, guinea pig and chinchilla, which differs in a substitution of valine for methionine in position 3 from the NH2-terminus. Although evolutionary history suggests that marsupials migrated from South America into Australia before the two continents separated, this peptide resembles that found in Old World mammals rather than that of South American hystricomorphs. Such molecular data are useful in assessing phylogenetic relationships among taxa.     

Effect of cholecystokinin octapeptide and somatostatin on the motility of guinea pig and canine gallbladder

Species differences have been observed in the effect of cholecystokinin octapeptide (CCK OP) on the canine and guinea pig gallbladder smooth muscle motility. 1. CCK OP was more potent stimulant in canine than in guinea pig gallbladder smooth muscles. Its pD2 values were 10 and 9.2, respectively. 2. The acetylcholine (10(-4) M)-induced maximum contractions in canine gallbladder muscle strips were by 50% lower as compared to the CCK OP (10(-8) M) maximum responses while in guinea pig gallbladder muscle strips the acetylcholine (ACh) maximum responses were by 20% lower than the CCK OP maximum responses. 3. CCK OP increased [3H]ACh release by 27% in canine gallbladder and by 40% in guinea pig gallbladder. 4. Somatostatin (SOM) had not any direct myogenic effect in guinea pig and canine gallbladder but it decreased [3H]ACh release from gallbladder intrinsic cholinergic neurons.     

Occurrence of two cholecystokinin binding sites in guinea-pig brain cortex

Saturation experiments of the highly potent cholecystokinin analogue [3H]Boc(diNle28,31)CCK27-33 ([3H]BNDL-CCK7, 100 Ci/mmol) with guinea pig brain cortex in a large concentration range (0.05 nM to 30 nM) show the presence of two different binding sites (A site: KD = 0.13 nM, Bmax = 35 fmol/mg; B site: KD = 6.4 nM, Bmax = 92 fmol/mg). Both sites exhibit different sensitivity to sodium ions and therefore can be selectively investigated at [3H]BDNL-CCK7 concentration lower than 1 nM for the A site in Tris buffer and in Krebs buffer for the B site. The selectivity factors KIB/KIA of various CCK related peptides vary from 58 for CCK4 to 26 for CCK8 and 4 for the antagonist (Nle28,31) CCK27-32-NH2. The occurrence of two different CCK binding sites in the brain could explain biphasic pharmacological effects of CCK8.     

Species differences in structure-activity relationships of adenosine agonists and xanthine antagonists at brain A1 adenosine receptors

A series of 28 adenosine analogs and 17 xanthines has been assessed as inhibitors of binding of N6-R-[3H]phenylisopropyladenosine binding to A1 adenosine receptors in membranes from rat, calf, and guinea pig brain. Potencies of N6-alkyl- and N6-cycloalkyladenosines are similar in the different species. However, the presence of an aryl or heteroaryl moiety in the N6 substituent results in marked species differences with certain such analogs being about 30-fold more potent at receptors in calf than in guinea pig brain. Potencies at receptors in rat brain are intermediate. Conversely, 2-chloroadenosine and 5'-N-ethylcarboxamidoadenosine are about 10-fold less potent at receptors in calf brain than in guinea pig brain. Potencies of xanthines, such as theophylline, caffeine and 1,3-dipropylxanthine are similar in the different species. However, the presence of an 8-phenyl or 8-cycloalkyl substituent results in marked species differences. For example, a xanthine amine conjugate of 1,3-dipropyl-8-phenylxanthine is 9-fold more potent at receptors in calf than in rat brain and 110-fold more potent in calf than in guinea pig brain. Such differences indicate that brain A1 adenosine receptors are not identical in recognition sites for either agonists or antagonists in different mammalian species.     

Synthesis of cholecystokinin-related peptides and their biological properties

Two tyrosine-O-sulfate containing cholecystokinin-related peptides were synthetized, i.e. the undeca- and the dodecapeptide amide corresponding to the C-terminus of this gut hormone; in the undecapeptide the N-terminal serine was O-substituted with the 2-deoxy-alpha-D-galactosyl group. Within the limits of error of the biological assay systems the two CCK peptides exhibited potencies in stimulating amylase secretion from dispersed rat pancreatic acini comparable to those of the CCK deca-, nona- and octapeptide.     

Characterization of neuromedin U like immunoreactivity in rat, porcine, guinea-pig and human tissue extracts using a specific radioimmunoassay

Two novel bioactive peptides termed neuromedin U-8 and neuromedin U-25 have recently been isolated from porcine spinal cord but nothing is known of their occurrence and molecular forms in other species. Following gel permeation chromatography, a specific radioimmunoassay detected only a single molecular form of neuromedin U-like immunoreactivity (NmU-LI) in rat, porcine and human central nervous system and gastrointestinal tract. Only guinea pig tissue extracts revealed two molecular forms of NmU-Li. Reverse phase high performance liquid chromatographic (HPLC) analysis demonstrated that porcine NmU-LI co-eluted with synthetic neuromedin U-25 standard. Human and rat NmU-LI however, was more hydrophobic on HPLC thus indicating species differences.     

Differences in Action of PACAP-27 and PACAP-38 on Guinea Pig Gallbladder Smooth Muscle Using Synthetic C-terminally Modified PACAP Peptides

Pituitary adenylate cyclase activating polypeptide (PACAP) occurs in two bioactive forms, PACAP-38 and PACAP-27 that have identical N-terminal sequences but differ by the presence of a C-terminal 11 residue elongation in the former. Although VIP and PACAP have several similar biological actions due to their amino acid sequence similarity, we have found that they evoke opposite responses in the guinea pig gallbladder smooth muscle, where PACAP induces contraction while VIP causes relaxation. In addition the response to PACAP-38 is four times lower than that of PACAP-27. In a previous study we have reported the role of the N-terminal α-helical regions of PACAP-27 which play a key role in gallbladder contraction. In the present study the biological action on the guinea pig gallbladder was investigated using a synthetic mini-library of C-terminally deleted peptides related to PACAP-38. The effects caused by residues within the C-terminus are not a result of a response via the M-receptor or Na⁺ channel, but most likely arise from a delicate balance between the differential effects of PACAP-38 on specific PAC1 and VPACs receptors.     

Clonotypic heterogeneity of Lewis rat T cells specific for the encephalitogenic 68-86 region of myelin basic protein

Experimental autoimmune encephalomyelitis was induced in a Lewis rat by sensitization with synthetic peptide GP68-86, representing the 68-86 sequence of guinea pig myelin basic protein (GPMBP). To delineate T cell determinants of GP68-86, lymph node cells from this rat were activated in culture with GP68-86 and were fused with cells of the mouse thymoma BW5147. The resultant hybrids were cloned by limiting dilution and screened for GP68-86-evoked secretion of IL2 in the presence of rat splenocytes. Twelve T cell hybrids derived in this manner were tested for reactivity to different heterologous species of MBP as well as to substituted or truncated analogs of GP68-86. The hybrids generally exhibited potent reactivity to GPMBP but differed markedly in their reactivity to autologous rat MBP (RMBP). A few exceptional hybrids exhibited crossreactivity with peptides in which native serine75 or serine80 residues of GPMBP were substituted with either alanine75 (A75) or proline80 (P80) residues. These cross-reactive hybrids also possessed high levels of anti-RMBP reactivity. The remaining hybrids were unresponsive to the A75 and P80 substituted peptides and, with one exception, had relatively low levels of anti-RMBP reactivity. Unique reactivity patterns were also revealed by hybrid responses to peptides having modified C-terminal 84-86 residues. In summary, the contrasting fine specificities of different hybrids indicated that several distinct clones of T cells mediate the immune response of Lewis rats against the 68-86 region of GPMBP. Furthermore, heterogeneity in the hybrid response to "self" RMBP may reflect substantial differences in encephalitogenic potency of the T cell clones from which these hybrids were derived.     

A pseudopeptide that is a potent cholecystokinin agonist in the peripheral system is able to inhibit the dopamine-like effects of cholecystokinin in the striatum

There are no known specific effective cholecystokinin (CCK) receptor antagonists of both peripheral and central nervous systems. Here, we describe experiments which demonstrate that a synthetic pseudopeptide analogue of CCK-7 is a potent agonist in the peripheral system and behaves as a selective and highly potent inhibitor of the dopamine-like effects of CCK in the striatum. This compound, t-butyloxycarbonyl-Tyr (SO3H)-Nle psi (COCH2)Gly-Trp-Nle-Asp-Phe-NH2, is able to stimulate enzyme secretion from rat pancreatic acini, with high efficacy and potency. It is also very potent in inhibiting the binding of labeled CCK-8 to rat pancreatic acini (IC50 = 5 nM) and to guinea pig and mouse brain membranes (IC50 = 0.7 nM). However, this compound is able to antagonize the effects of intrastriatally injected t-butyloxycarbonyl-[Nle28,31] CCK-8 in mice, with high potency.     

Characterization of cholecystokinin receptors in bullfrog (Rana catesbeiana) brain and pancreas

The binding of biologically active 125I-Bolton-Hunter-CCK-33 to bullfrog brain and pancreatic membrane particles was characterized. Both tissues exhibited time-dependent, saturable, reversible, and high affinity binding without evidence for cooperative interaction. Both bullfrog CCK receptors resembled their mammalian counterparts in having acidic pH optima for tracer binding and a Kd of about 0.5 nM. However, the receptors differed from their mammalian counterparts in that (1) the bullfrog brain membranes bound more tracer per mg protein than did the pancreatic membranes, (2) both bullfrog CCK receptors were relatively insensitive to dibutyryl cGMP, and (3) both bullfrog brain and pancreatic CCK receptors exhibited the same general specificity toward a variety of CCK and gastrin peptides. For both tissues, the relative order of receptor binding potency was CCK-8 greater than caerulein = CCK-33 greater than gastrin-17-II greater than CCK-8-ns = gastrin-17-I greater than caerulein-ns greater than gastrin-4 with the sulfated CCK peptides being 1000-fold more potent than their nonsulfated analogs. Sulfated gastrin was also relatively potent, being only 10-fold weaker than CCK-8. Gastrin-4 was 20 000-fold weaker than CCK-8 in interacting with the brain CCK receptor. The latter finding is in sharp contrast to the mammalian brain CCK receptor. We conclude that the bullfrog brain and pancreas contain similar CCK receptors of probable physiological significance and may represent an ancestral condition from which the two distinct CCK receptors present in mammalian brain and pancreas have evolved.     

Porcine VIP is more potent than guinea pig VIP in relaxing the guinea pig uterine artery

The vasodilator potency of guinea pig VIP (gp VIP) on the guinea pig uterine artery was compared with the potency of porcine VIP (p VIP), which differs in amino acid sequence at four locations. When antioxidants were not used, the two peptides were approximately equipotent in causing relaxation of precontracted vessel segments. Use of the antioxidants ascorbic acid and dithiothreitol resulted in significantly increased potency of both peptides. Porcine VIP was 15 times more potent than gp VIP synthesized by the same method (tBoc), and gp VIP synthesized by tBoc methodology was 2 times more potent than gp VIP synthesized by Fmoc methodology. Therefore, care should be taken in the choice and handling of synthetic peptides when aiming to mimic actions of endogenous peptides.     

Signal peptides having standard and nonstandard cleavage sites can be processed by Imp1p of the mitochondrial inner membrane protease

We have performed a site-directed mutagenesis study showing that residues comprising the type I signal peptidase signature in the two catalytic subunits of the yeast inner membrane protease, Imp1p and Imp2p, are functionally important, consistent with the idea that these subunits contain a serine/lysine catalytic dyad. Previous studies have shown that Imp1p cleaves signal peptides having asparagine at the -1 position, which deviates from the typical signal peptide possessing a small uncharged amino acid at this position. To determine whether asparagine is responsible for the nonoverlapping substrate specificities exhibited by the inner membrane protease subunits, we have substituted asparagine with 19 amino acids in the Imp1p substrate i-cytochrome (cyt) b(2). The resulting signal peptides containing alanine, serine, cysteine, leucine, and methionine can be cleaved efficiently by Imp1p. The remaining mutant signal peptides are cleaved inefficiently or not at all. Surprisingly, none of the amino acid changes results in the recognition of i-cyt b(2) by Imp2p, whose natural substrate, i-cyt c(1), has alanine at the -1 position. The data demonstrate that (i) although the -1 residue is important in substrates recognized by Imp1p, signal peptides having standard and nonstandard cleavage sites can be processed by Imp1p, and (ii) a -1 asparagine does not govern the substrate specificity of the inner membrane protease subunits.     

Parallel bioassay of litorin and phyllolitorins on smooth muscle preparations

Phyllolitorin and Leu8-phyllolitorin, two nonapeptide amides from the skin of the South American frog Phyllomedusa sauvagei, are representatives of a novel bombesin subfamily, characterized by the occurrence in their molecule of a serine residue substituting the usual histidine residue at position 3 from the C-terminus. In parallel bioassay on ten different smooth muscle preparations and rat blood pressure, phyllolitorin and Leu8-phyllolitorin were virtually equiactive, but the two peptides appeared remarkably less potent that litorin in all test preparations, except the rat urinary bladder. The shape of contractions produced by the phyllolitorins and promptness of cessation of their action upon washing seem to indicate a looser binding of these peptides to their receptors and/or a more rapid inactivation, in comparison to litorin.