Systemic injections of gastro-intestinal peptides alter behavior in rats

Twenty-four male albino rats were given daily intraperitoneal injections of vasoactive intestinal polypeptide (VIP), motilin, human gastrin I (1–17) or the diluent control vehicle at a dose of 100 μg/kg for four consecutive days and food intake, water intake, body weight, and running wheel activity were determined every 24 hours. Animals injected with motilin or human gastrin I (1–17) exhibited decreased food intake relative to those injected with VIP or diluent, which did not differ from each other, although food intake increased reliably over days. The mean water consumption followed the same pattern as that of food intake. As expected from the above results, VIP produced weight gains as compared with rats injected with motilin or gastrin but not reliably more than after diluent. A reliable effect of trials for weight gain was the greatest on day three. Running wheel activity was not affected by injections of human gastrin I (1–17), motilin, or diluent but was reliably decreased by VIP. No significant differences existed across days. Although the results indicate that GI peptides may affect behavior when injected systemically and that like other peptides they have multiple effects, caution is urged in the interpretation of behavioral results at this time.     

Normal mouse serum contains peptides which induce fibroblasts to grow in soft agar

The untransformed mouse fibroblast cells NIH/3T3, C3H/10T1/2, and rat NRK cells do not grow in soft agar in medium supplemented with 10% fetal calf serum. When fetal calf serum in the growth medium was supplemented with less than 1% of sera from mice or other vertebrates, however, these cells responded, forming large colonies. The morphology of soft agar colonies was a function of the treated cell type. In the presence of 10% serum from C57BL/6 mice, NRK cells grew to smooth-surfaced spherical colonies, while NIH/3T3 colonies showed individual round cells on their surface and C3H/10T1/2 cells grew as extended cells forming columns of end to end connected fibroblasts. Mus Musculus Castaneus-Epithelial (MMC- E) cells were not stimulated to grow in soft agar under these conditions. The major fibroblast colony-inducing factor (F-CIF) was partially purified from mouse serum by acid/ethanol-extraction, gel permeation chromatography, and reverse-phase high-pressure liquid chromatography. F-CIF is a polypeptide, which does not compete for binding to epidermal growth factor (EGF) receptors, but stimulates normal fibroblasts to form small colonies in semisolid medium and very large colonies in the presence of added EGF (2 ng/ml). In contrast to unfractionated mouse serum, purified F-CIF did not induce C3H/10T1/2 cells to grow in soft agar, suggesting that serum contains additional cell type-specific agar growth-stimulating activities.     

Mechanism of the cardiovascular response to systemic intravenous administration of leucine-enkephalin in the conscious dog

Leucine-enkephalin (Leu⁵-ENK) (35 μg/kg) increased heart rate and mean systemic arterial blood pressure following intravenous injection into chronically-instrumented, conscious dogs. Repeated injections at five-minute intervals were not associated with a diminished response. Naloxone (1 mg/kg) pre-treatment inhibited both heart rate and blood pressure increases. Prazosin (1 mg/kg) attenuated the increase in blood pressure but did not influence the heart rate response. Propranolol (1 mg/kg) attenuated the heart rate response but not the pressor response. Clonidine (30 μg/kg) attenuated the positive chronotropic effect of Leu⁵-ENK. Atropine (1 mg/kg) plus propranolol (1 mg/kg) blocked the heart rate response but the pressor effect was still present. The attenuation of the heart rate response by propranolol and the pressor response by prazosin suggests an adrenergic component to the enkephalin response; the reduction in the heart rate response by clonidine and atropine-propranolol indicates a role for cholinergic mechanisms in the chronotropic response. Hexamethonium (10 mg/kg) blocked the heart rate response and markedly inhibited the pressor response. Vagal interruption attenuated both heart rate and blood pressure responses. It is concluded that intravenous Leu⁵-ENK stimulates afferent pathways located in fibers which are contained in the vagosympathetic trunk to reflexly increase heart rate and blood pressure.     

Isolation and characterization of fluorescent Pseudomonas associated with the roots of rice and banana grown in Sri Lanka

Bacterial populations in different parts of the rhizosphere of rice and banana in Sri lanka were examined. On rice, the number of aerobic bacteria and the population of fluorescent bacteria were higher in the rhizoplane as compared to the exorhizosphere. However, the opposite was observed with banana. Percentage of fluorescent bacteria was significantly higher on banana (10.8%) than on rice from the wet and dry zones of Sri Lanka (4.3% and 2.7%, respectively). In the endorhizosphere fraction of rice, bacterial populations were very low. Fluorescent bacteria were absent.Based on 33 phenotypical tests, 89 fluorescent isolates were grouped into 5 clusters. The three major clusters covered the isolates belonging to the Pseudomonas fluorescens-putida group, whereas the remaining small clusters contained other UV-fluorescent bacteria. SDS-PAGE of total cell proteins enabled classification of the isolates into one of 12 different protein-polymorphic types. Only a partial correlation was found between the latter classification and the phenotypical one. Cyanogenesis was observed with strains of P. fluorescens only. Isolates P. fluorescens RW9S1 and P. cepacia RW5P1 displayed a potent antagonism against several fungi.     

Influence of spinal cord transection on the presence and axonal transport of CGRP-, chromogranin A-, VIP-, synapsin I-, and synaptophysin-like immunoreactivities in rat motor nerve

Using immunofluorescence and cytofluorimetric scanning (CFS), we investigated the short-term (1-7 days) influence of lower thoracic spinal cord transection on lumbar motor neurons. The content of calcitonin gene-related peptide- (CGRP) like immunoreactivity (LI), chromogranin A (Chr A) -LI, vasoactive intestinal polypeptide (VIP)-LI, Syn I-LI, and synaptophysin (p38)-LI in motor perikarya, and the anterograde and retrograde axonal transport of these substances in the sciatic nerve, were studied in nerve crush (6 h) experiments. During the week after transection, CGRP-LI in perikarya decreased, whereas Chr A-LI increased. VIP-LI, co-localized with Chr A-LI in motor perikarya, did not change after transection. The antero- and retrograde transport of CGRP-LI in the sciatic nerve, occurring in both motor and sensory axons, appeared unchanged in cytofluorimetric scanning (CFS) graphs, but the microscopical picture clearly showed that large motor axons had a decreased content of CGRP-LI at 3 and 7 days posttransection, whereas thinner axons were unchanged in fluorescence intensity. The anterograde transport of Chr A-LI, present in both motor and postganglionic adrenergic axons, was decreased 1 and 3 days after lesion, but returned to control by day 7. There was a marked decrease in anterograde transport of VIP-LI, present mainly in postganglionic sympathetic axons, at day 3, but at 7 days transport was normal. The amounts of transported p38, the synaptic vesicle marker, were in the normal range during the whole period. Syn I-LI accumulation anterogradely was somewhat decreased at 3 and 7 days posttransection, and at 1 day the retrograde accumulation was significantly increased. The results suggest that removal of supraspinal input to intact lower motor neurons causes alterations in metabolism and axonal transport of organelle-associated substances, partly probably related to the complex pattern of transmitter leakage from degenerating, descending nerve terminals. These alterations appear to take place also in postganglionic sympathetic neurons in the sciatic nerve, that originate in the lumbar sympathetic chain. © 1992 John Wiley & Sons, Inc.     

Evidence that Inhibition of Nicotine-Mediated Catecholamine Secretion from Adrenal Chromaffin Cells by Enkephalin, β-Endorphin, Dynorphin (1-13), and Opiates is not Mediated via Specific Opiate Receptors

Abstract: The opioid peptides Met- and Leu-enkephalin, dynorphin (1-13), and β-endorphin and the narcotic analgesics, morphine, levorphanol, and dextrorphan all produced a dose-dependent inhibition of nicotine (5 × 10^?6m )-mediated release of [³H]norepinephrine ([³H]NE) from bovine adrenal chromaffin cells in culture. None of these agents affected [³H]NE release induced by high K⁺ (56 mm ). Although the above results suggest that the opioid peptides and narcotic analgesics inhibit catecholamine release from adrenal chromaffin cells in culture, we suggest that these effects are not mediated by specific opiate binding sites, since (1) the inhibition was only produced with high concentrations of the agents—the threshold concentrations were 10^?7 to 10^?5m and higher; (2) the inhibition produced by the narcotic analgesics did not display stereospecificity, because the (d-isomer, dextrorphan, was slightly more active than the l-isomer, levorphanol; (3) the narcotic antagonists naloxone, naltrexone, and levallorphan did not reverse the inhibition produced by either the narcotic analgesics (e.g., morphine) or the opioid peptides (e.g., dynorphin). These three antagonists themselves inhibited the nicotine-mediated release of [³H]NE from the adrenal chromaffin cells in culture. Finally (4), the I₂-Tyr¹ substituted analogues of β-endorphin and dynorphin that are biologically less active than the parent compounds produced an inhibition of the nicotine-mediated [³H]NE release similar to that of their parent compounds. These results do not support the idea that high-affinity stereospecific opiate binding sites are involved in the inhibitory modulation of nicotinic evoked catecholamine release from bovine adrenal chromaffin cells in culture.     

Measurement of Methionine-Enkephalin [Arg6,Phe7] in Rat Brain by Specific Radioimmunoassay Directed at Methionine Sulphoxide Enkephalin[Arg6,Phe7]

Abstract: A specific and sensitive radioimmunoassay procedure for Metenkephalin[Arg⁶,Phe⁷] which allows its measurement in regions of the rat brain is described. The antiserum was raised against the methionine sulphoxide derivative of the peptide, and all samples and standards were oxidized with hydrogen peroxide prior to use in the assay with chloramine T-oxidized ¹²⁵I-labelled Met(O)-enkephalin[Arg⁶,Phe⁷]. The only significant cross-reactivity was 30% with the reduced heptapeptide Met-enkephalin[Arg⁶,Phe⁷]. The assay showed less than 0.15% cross-reactivity with fragments of the heptapeptide and with leucine-enkephalin-containing peptides. Acid acetone extraction of rat striatum followed by Sephadex G-50 chromatography and reverse-phase high pressure liquid chromatography showed that essentially all immunoreactivity co-chromatographed with Met-enkephalin[Arg⁶,Phe⁷]. This confirmed the specificity of the assay and showed that the striatum does not contain a high concentration of larger molecular weight forms with the heptapeptide at the COOH terminus. Distribution of the heptapeptide followed that of methionine enkephalin, with highest concentrations in the globus pallidus, intermediate levels in caudate-putamen and hypothalamus, and low levels in cortex and cerebellum.     

Hydrolysis of cyclosporin A: Identification of 1,11 seco-cyclosporin A and 4,5 secoisoCyclosporin A by FAB-MS/MS

We have previously reported that treatment of CsA with aqueous HCl gives rise to the formation of a number of water-soluble compounds. Two of these were identified from their FAB-MS/MS spectra as open-chain nona- and decapeptides. We describe here the identification of two other main compounds deriving from the same treatment. Identification was rendered possible from the comparison of their FAB-MS/MS spectra with those of methyl and acetyl derivatives. The two compounds are water-soluble, open-chain undecapeptides corresponding to 1,11 seco-CsA and of 4,5 seco-isoCsA, respectively.     

Dynorphin-Related Opioid Peptides in the Neurointermediate Pituitary of Rats Are Not α-N-Acetylated

Immunoreactive dynorphin in the neurointermediate pituitary of rats was found to consist of four different molecular weight forms. The three larger molecular weight forms, with apparent molecular weights of 4800, 3200, and 1700, constituted more than 80% of the total dynorphin immunoreactivity, and each liberated leucine-enkephalin but not alpha-N-acetyl-leucine-enkephalin upon enzymatic treatment with trypsin followed by carboxypeptidase B. Only a minor portion of the smallest dynorphin-related molecular weight form, dynorphin-(1-8), released alpha-N-acetyl-leucine-enkephalin upon enzymatic cleavage. This suggests that the vast majority of dynorphin-related peptides in the rat neurointermediate pituitary is not alpha-N-acetylated. The exceptionally high opiate-like activity of the molecular weight 1700 dynorphin suggests that this dynorphin-related opioid peptide may constitute the major part of opioid activity in the neurointermediate pituitary of rats.