A novel gastrin-processing pathway in mammalian antrum.

An antiserum, L221, has been developed that is specific for the C-terminal region of the N-terminal tridecapeptide (i.e., 1-13) fragment of the acid-stimulating hormone, G17. In contrast to N-terminal G17 antisera previously used to estimate 1-13 G17, L221 does not cross-react with other N-terminal gastrin fragments or with C-terminal extensions of G17. Using L221 in conjunction with conventional gastrin antisera, and reversed-phase HPLC, it has been possible to identify in addition to 1-13 G17 a further, formerly unrecognised gastrin fragment, 1-11 G17, in stomach extracts. The production of 1-13 G17, 1-11 G17 and other gastrin forms such as the biologically active hexapeptide G6 which is known to occur naturally cannot be explained by tryptic cleavage of progastrin. Instead, their biosynthesis could be explained by the actions of an enzyme with an endopeptidase 24.11-like specificity. In porcine antrum, unsulphated and sulphated G17 are present in similar amounts, but unsulphated 1-13 G17 was about twice as abundant as sulphate 1-13 G17. This is consistent with previous in vitro findings that endopeptidase 24.11 has a higher affinity for the Ala-11-Tyr-12 and Gly-13-Trp-14 bonds in unsulphated G17, than in sulphated G17. The results suggest a novel albeit minor, processing pathway for gastrin biosynthesis in pig antrum involving an enzyme resembling endopeptidase 24.11.

Deletion analogues of transportan.

Several shorter analogues of the cell penetrating peptide, transportan, have been synthesized in order to define the regions of the sequence, which are responsible for the membrane translocation property of the peptide. Penetration of the peptides into Bowes melanoma cells and the influence on GTPase activity in Rin m5F cellular membranes have been tested. The experimental data on cell penetration have been compared with molecular modeling of insertion of peptides into biological membranes. Omission of six amino acids from the N-terminus did not significantly impair the cell penetration of the peptide while deletions at the C-terminus or in the middle of the transportan sequence decreased or abolished the cellular uptake. Most transportan analogues exert an inhibitory effect on GTPase activity. Molecular modeling shows that insertion of the transportan analogues into the membrane differs for different peptides. Probably the length of the peptide as well as the location of aromatic and positively charged residues have major impact on the orientation of peptides in the membranes and thereby influence the cellular penetration. In summary, we have designed and characterized several novel short transportan analogues with similar cellular translocation properties to the parent peptide, but with reduced undesired cellular activity.

Synthesis and some pharmacological properties of deamino(4-threonine,8-D-arginine)vasopressin and deamino(8-D-arginine)vasopressin, highly potent and specific antidiuretic peptides, and (8-D-arginine)vasopressin and deamino-arginine-vasopressin.

Deamino[4-threonine,8-D-arginine]vasopressin (dTDAVP), deamino[8-D-arginine]vasopressin (dDAVP), [8-D-arginine[vasopressin (DAVP), and deamino-arginine-vasopressin (dAVP) were synthesized by the solid-phase method and tested for their biological activities. dTDAVP has an antidiuretic potency of 793+/-95 units/mg and undetectable vasporessor activity, less than 0.02unit/mg. The antidiuretic-pressor (A/P) ratio of dTDAVP is greater than 39 000. dDAVP has an antidiuretic potency of 1200+/-126 units/mg and a vasopressor potency of 0.39+/-0.02; its A/P ratio is thus 3000. DAVP has an antidiuretic potency of 253+/-44 units/mg, a vasopressor potency of 1.1+/-0.04 units/mg, and an A/P ratio of 240. The A/P ratios of dDAVP and DAVP are much higher than those originally reported. dAVP has an antidiuretic potency of 1745+/-385 units/mg, a vasopressor potency of 346+/-13, and an A/P ratio of 5; values are in general agreement with those in the literature. Threonine subsitution has thus brought about a significant enhancement in antidiuretic specificity, a finding entirely consistent with earlier observations that enhancement of lipophilicity at position 4 alone or in combination in arginine-vasopressin can lead to enhanced antidiuretic specificity.

Effects of a D-Cys6/L-Cys6 interchange in nonselective and selective vasopressin and oxytocin antagonists.

We report the solid-phase synthesis of the D-Cys6 analogues of arginine-vasopressin (AVP), peptide 1, of the selective AVP vasopressor (V1a receptor) antagonist [1-(beta-mercapto-beta,beta-pentamethylenepropionic acid),2-O-methyltyrosine]arginine-vasopressin (d(CH2)5[Tyr(Me)2]-AVP, (A)), peptide 2, of the three nonselective antidiuretic/vasopressor (V2/V1a receptor) AVP antagonists d(CH2)5[Tyr(Et)2]VAVP (B), d(CH2)5[D-Tyr(Et)2]VAVP (C), and d(CH2)5[D-Phe2]VAVP (D) (where V = Val4), peptides 3-5, of the nonselective oxytocin (OT) antagonists d(CH2)5-[Tyr(Me)2]OVT (E) and d(CH2)5[Tyr(Me)2,Thr4,Tyr-NH2(9)]OVT (F) (where OVT = ornithine-vasotocin), peptides 6 and 7, and of the selective OT antagonists desGly-NH2,d(CH2)5[Tyr(Me)2,Thr4]OVT (G) and d(CH2)5]D-Trp2,Thr4]OVT (H), peptides 8 and 9. We also present the repeat syntheses of the previously reported d(CH2)5[D-Trp2]AVT (peptide 10) and its D-Cys6 analogue (peptide 11) (where AVT = arginine-vasotocin). Peptides 1-11 were assayed for agonistic and antagonistic activities in in vivo V1a, V2, and oxytocic assays and in in vitro oxytocic assays without and with 0.5 mM Mg2+. With V2 and V1a agonistic potencies of 0.82 and 0.41 units/mg, [D-Cys6]AVP has retained less than 0.3% of the V2 and V1a potencies of AVP. It exhibits no oxytocic activity and is an in vitro OT antagonist. pA2 = 6.67 (no Mg2+); pA2 = 5.24 (0.5 mM Mg2+). By contrast, with one or two exceptions, a D-Cys6/L-Cys6 interchange in antagonists 2-9, although resulting in reductions of antagonistic potencies in all assays for virtually all peptides 2-9 relative to A-H, has been well tolerated. For peptides 2-5, the anti-V2 and anti-V1a pA2 values range from approximately 5.54 to 7.33 and from 7.19 to 8.06, respectively; the range of in vitro anti-OT pA2 values (no Mg2+) is 7.35-7.87; with 0.5 mM Mg2+, the range is 7.24-8.21. Peptides 2 and 4 have in vivo anti-OT pA2s = 6.60 and 7.16, respectively. For peptides 6-9, the range of in vitro anti-OT pA2 values (no Mg2+) is 7.65-7.96; with 0.5 mM Mg2+, the range is 7.41-7.65, and the in vivo anti-OT pA2 values range from 6.85 to 7.33. With an in vivo anti-OT pA2 = 7.33, peptide 6 is equipotent with its parent E. The in vivo anti-OT potencies of peptides 7-9 are significantly reduced relative to those of F-H. The in vitro anti-OT (0.5 mM Mg2+) pA2 values of 10 and 11 are 7.54 and 7.50, both significantly lower than those previously reported.(ABSTRACT TRUNCATED AT 400 WORDS)

The structural localization of galanin, and its function in modulating acetylcholine release in the olfactory bulb of adult rat.

The localization of galanin immunoreactivity was analyzed within the olfactory bulb of adult rats. Galanin-positive neurons were differentially distributed among the bulb layers. The density of stained neurons was highest in the glomerular and external plexiform layers. According to morphology, size, location and arrangement, a large proportion of galanin-immunoreactive neurons corresponds to external tufted cells and short-axon neurons in the superficial part of the external plexiform and glomerular layers. A smaller number were middle tufted cells and short-axons neurons while only a few short-axon neurons were labeled in the granule cell layer. Galanin-stained nerve fibers had different structures (thick fibers with or without varicosities, and thin fibers with or without varicosities). Among them were afferent immunoreactive nerve fibers entering the bulb through the olfactory nerve layer, but penetrating superficial layers. Correspondingly, a large number of galanin-positive axons (with or without varicosities) were observed in the olfactory nerve layer. A number of galanin-positive nerve fibers was also present in the glomerular and internal plexiform layers, while these fibers were scarce in the granule cell layer, their density was lowest in the external plexiform layer. These results suggest that galanin-positive axons present in the olfactory bulb originate from at least four different sources. From the periphery axon bundles enter the bulb together with olfactory nerve fibers from the rostral direction and with a fiber bundle from the ventral posterior surface, i.e. at the border between the olfactory tract and the main olfactory bulb along a large blood vessel. Central sources are local interneurons in the olfactory bulb and some extrabulbar brain regions. Double-labeling experiments combining acetylcholinesterase histochemistry with galanin immunocytochemistry did not show any co-localization of acetylcholinesterase and galanin in nerve cell perikarya or nerve fibers. Synthetic porcine galanin (1-29) promoted acetylcholine release in olfactory bulb tissue slices, suggesting that galanin can effectively modulate cholinergic transmission and perhaps other forms of neuronal transmission. It is concluded that galanin may be significantly involved in olfactory processing at cellular and synaptic levels.

Biological half-life and organ distribution of [3H]1-deamino-8-D-arginine-vasopressin in the rat.

The biological half-life of synthetic, radiochemically pure, biologically active [3H]1-deamino-8-D-arginine-vasopressin (dDAVP) in rats was found to be 5.33 +/- 0.28 (S.E.M.) min in the initial, transitional, fast phase and 56.28 +/- 3.27 min in the second, slow phase. The substance accumulated to the greatest extent in the kidney and small intestine and only slightly in the adenohypophysis. The results have suggested that the extended biological half-life may play a role in the more marked and longer antidiuretic effect of dDAVP. The explanation of the poor accumulation in the adenohypophysis may be that dDAVP does not possess an effect similar to that of corticotrophin releasing factor.

Dose-related effect of the oxytocin fragment (prolyl-leucyl-glycinamide) on ?-MPT-induced catecholamine disappearance and serotonin level in rat brain.

Graded doses of Pro-Leu-Gly-NH(2) (3.5 x 10(?12), 3.5 x 10(?11), 3.5 x 10(?10) or 3.5 x 10(?9) mol) were administered into the lateral cerebral ventricle of rats. The noradrenaline level of the dorsal hippocampus was increased 30 min after a dose of 3.5 x 10(?10) mol Pro-Leu-Gly-NH(2). The dopamine level was increased in the dorsal hippocampus and in the striatum. The serotonin level was increased in the hypothalamus, in the striatum and decreased in the dorsal hippocampus. The catecholamine disappearance following 350 mg/kg of ?-methyl-p-tyrosine indicated an accelerated dopamine disappearance in the striatum for each dose studied, while the hypothalamic noradrenaline disappearance was inhibited by a dose of 3.5 x 10(?11) mol of Pro-Leu-Gly-NH(2). The data indicate that Pro-Leu-Gly-NH(2) induces dose and region-dependent changes in the cerebral monoamine metabolism. The striatal dopamine and hypothalamic serotonin metabolism appeared to be the most sensitive for intraventricular Pro-Leu-Gly-NH(2).

Intracerebroventricularly administered atrial natriuretric peptide (ANP) antiserum attenuates fear-motivated learning behavior in rats.

Previous studies have demonstrated that rANP(1-28) administered into the lateral cerebroventricle facilitates consolidation of the passive avoidance response and delays extinction of the active avoidance response in fear-motivated learning in rats. To study the role of endogenous ANP in the same learning processes, the effects of different dilutions of ANP antiserum were investigated following their intracerebroventricular administration to rats. At dilutions of 1:40 and 1:60, the ANP antiserum attenuated consolidation of the passive avoidance response. It also facilitated extinction of the active avoidance response at a dilution of 1:2. The results suggest that endogenous ANP might be considered a modulating agent in the brain, and is involved in the learning processes and memory trace formation, since intracerebroventricularly administered antiserum against ANP attenuated fear-motivated learning behavior in the experimental animals.

Influence of dipeptides on precipitated morphine withdrawal in the mouse.

Effects of various dipeptides on naloxone-precipitated morphine withdrawal were studied in the mouse. Mice were rendered dependent on morphine by implantation of morphine pellets and the withdrawal syndrome was measured by the latency of the onset of stereotyped jumpings. In accordance with previous data, subcutaneous injection of Z-prolyl-D-leucine significantly delayed the onset of morphine withdrawal. The all-L enantiomer of the dipeptide (Z-L-prolyl-L-leucine) did not affect morphine withdrawal in the dose studied. Replacement of L-proline by L-glutamate or L-pyroglutamate (Z-L-glutamyl-L-leucine and L-pyroglutamyl-L-leucine) resulted in dipeptides which were more potent towards morphine withdrawal than Z-prolyl-D-leucine. Z-L-glycyl-L-proline attenuated the morphine withdrawal syndrome more effectively than Z-L-prolyl-D-leucine, but Z-L-leucyl-L-glycine was ineffective in this respect. The data reveal that certain dipeptides--which in their nonprotected forms are normal sequences of endogenous peptides--affect morphine withdrawal more potently than Z-prolyl-D-leucine, a synthetic dipeptide known to attenuate morphine dependence.

Interaction between synthetic amyloid-beta-peptide (1-40) and its aggregation inhibitors studied by electrospray ionization mass spectrometry.

It is generally postulated that amyloid-beta-peptides play a central role in the progressive neurodegeneration observed in Alzheimer's disease. Important pathological properties of these peptides, such as neurotoxicity and resistance to proteolytic degradation, depend on the ability of amyloid-beta-peptides to form beta-sheet structures and/or amyloid fibrils. Amyloid-beta-peptides are known to aggregate spontaneously in vitro with the formation of amyloid fibrils. The intervention on the amyloid-beta-peptides aggregation process can be envisaged as an approach to stopping or slowing the progression of Alzheimer's disease. In the last few years a number of small molecules have been reported to interfere with the in vitro aggregation of amyloid-beta-peptides. Melatonin, a hormone recently found to protect neurons against amyloid-beta-peptide toxicity, interacts with amyloid-beta-peptide (1-40) and amyloid-beta-peptide (1-42) and inhibits the progressive formation of beta-sheet and/or amyloid fibrils. These interactions between melatonin and the amyloid peptides have been demonstrated by circular dichroism (CD) and electron microscopy for amyloid-beta-peptide (1-40) and amyloid-beta-peptide (1-42) and by nuclear magnetic resonance (NMR) spectroscopy for amyloid-beta-peptide (1-40). Our electrospray ionization mass spectrometric (ESI-MS) studies also proved that there is a hydrophobic interaction between amyloid-beta-peptide (1-40) and melatonin and the proteolytic investigations suggested that the interaction took place on the 29-40 amyloid-beta-peptide segment. The wide-ranging application of these results would provide further information and help in biological research.

Inhibitory effect of galanin on dopamine induced increased oxytocin secretion in rat neurohypophyseal tissue cultures.

The regulation of oxytocin (OT) release by galanin (GAL) at the neurohypophyseal (NH) nerve terminal is not adequately understood. The effect of GAL on the secretion of OT was studied in 13- to 14-day cultures of isolated rat NH tissue. By this time, the hormone content of the medium had become constant. The OT content of the supernatant medium was determined by RIA after a 1- or 2-h incubation. A significantly decreased content of OT was found following incubation with 10(-6)-10(-8) M doses of GAL. Dopamine (DA) and the DA-active drugs apomorphine (APM) and Pro-Lys-Gly (PLG) (10(-6) M in each medium) increased the OT synthesis of NH tissue cultures. This elevation of OT secretion could be blocked by the administration of GAL together with DA, APM or PLG. The DA-blocking effect of GAL was prevented by previous treatment with the GAL receptor antagonist galantid (M15). The results indicate that OT release from the NH is directly influenced by the GAL-ergic system. The GAL-ergic control of OT secretion from NH tissue in rats can occur at the level of the posterior pituitary.

Inhibitory effect of galanin on dopamine-induced enhanced vasopressin secretion in rat neurohypophyseal tissue cultures.

The effect of galanin (GAL) on vasopressin (VP) secretion was studied in 13-14-day cultures of isolated rat neurohypophyseal (NH) tissue. The VP content of the supernatant was determined by radioimmunoassay (RIA) after a 1- or 2-h incubation. A significantly decreased content of VP was detected following the administration of 10(-6)-10(-9) M doses of GAL. Dopamine (DA) and the DA-active drugs apomorphine (APM) and Pro-Lys-Gly (PLG) (10(-6) M in each medium) increased the VP level of NH tissue cultures. This VP concentration elevation could be blocked by the administration of GAL together with DA, APM or PLG. The DA-blocking effect of GAL was prevented by previous treatment with the GAL receptor antagonist galantid (M15). The results indicate that VP release is directly influenced by the GAL-ergic system. The GAL-ergic control of VP secretion from NH tissue in rats can occur independently of the hypothalamus, at the level of the posterior pituitary.

Antiamnesic effects of D-pipecolic acid and analogues of Pro-Leu-Gly-NH2 in rats.

The antiamnesic effects of prolyl-leucyl-glycinamide (PLG) and analogues of this tripeptide were investigated in rats. Retrograde amnesia was induced by electroconvulsive shock treatment and the degree of amnesia was characterized by the attenuation of one-trial learning passive avoidance response. PLG resulted in dose-dependent attenuation of retrograde amnesia. Structural modifications included N-terminal protection, substitution of the C-terminal NH2 group, replacement of the N-terminal amino acid, and replacement of the second amino acid of the tripeptide. Some tripeptides, all of them containing D-pipecolic acid instead of the N-terminal proline, were more effective than PLG. Therefore, D-pipecolic acid, D-pipecolamide and their N-terminally protected analogues were also investigated, and were found to have powerful antiamnesic effects.

[A new synthesis of somatostatin]. / Eine neue Synthese von Somatostatin.

The synthesis of somatostatin by linking together Boc-Ala-Gly-Cys(Bzl)-Lys(Z)-Asn-Phe-NHNH2 and H-Phe-Trp-Lys[Z(pCl)]-Thr-Phe-Thr-Ser-Cys(Bzl)-OBzl is described. The two partial sequences were obtained from segments which in general are also used in preparing somatostatin analogues. The synthesis of the partial sequences was optimized. The intracerebroventricular application of the somatostatin thus obtained to rats produced an increase of the dopamine and serotonin contents in the striatum. The results achieved were identical with those realized with standard somatostatin (Serono).

[Synthesis of cyclo-[des(1,2-L-alanyl-glycine, 14-L-cysteine)-3-s-carboxymethyl-L-homocysteinamid, 8-D-tryptophan]-somatostatin]. / Synthese von cyclo-[Des(1,2-L-alanyl-glycin, 14-L-Cystein)-3-S-carboxymethyl-L-homocysteinamid, 8-D-Tryptophan]-somatostatin.

The authors describe the synthesis of a shorter-chained somatostatin analogue in which the ring size of the native molecule is largely preserved by incorporation of S-carboxymethyl-L-homocysteinamid and ring closure via its side-chain function. This synthesis involves the linkage of Boc-Phe-D-Trp-Lys[Z(pCl)]-Thr-Phe-Thr-Ser-OH to H-Hcy(CH2-CO-Lys[Z(oCl)]-Asn-Phe-OMe)-NH2 and subsequent azide cyclization. In contrast to somatostatin, the somatostatin analogue thus obtained produces, when applied intracerebroventricularly to rats, a decrease in the dopamine content of the striatum.

Amyloid-beta1-42 treatment does not have a specific effect on cholinergic neurons in in vitro basal forebrain neuronal cultures of rat.

The neurotoxic effect of amyloid-beta peptide (1-42) was investigated in cultures of neuronal tissue derived from the basal forebrain of embryonic rat. The axonal varicosities of the cholinergic cells were revealed by vesicular acetylcholine transporter staining, and the axonal varicosities in general by synaptophysin immunohistochemistry. The results demonstrate that the treatment of in vitro neuronal cultures with 20 microM amyloid-beta peptide (1-42) for 2 days on day 5, 12 or 15 exerted a neurotoxic effect on both the cholinergic and the non-cholinergic neurons. In the same cultures, the absolute number of synaptophysin-positive axon varicosities was reduced to greater extent (control: 203 +/- 37/field vs treated: 101 +/- 16/field) than the number of vesicular acetylcholine transporter-immunoreactive (control: 48 +/- 4/field vs treated: 0/field) structures. It is concluded that amyloid-beta peptide (1-42) does not have a specific effect only on the cholinergic neurons, but affects non-cholinergic neurons as well.