Immunoreactive methionine-enkephalin secretion by porcine uterus.

The current study examined the presence of immunoreactive methionine-enkephalin (ir-MENK) in porcine uterine fluid and endometrial extracts, characterized ir-MENK biochemically, and investigated the effect of ovarian steroids on uterine secretion of ir-MENK. Porcine uterine fluid was collected by flushing the uterine lumen with saline. Endometrial tissues were extracted with acetic acid. Both uterine fluid and endometrial extracts exhibited inhibition curves parallel to that of authentic MENK in the MENK RIA system. Sephadex G-15 gel filtration chromatographic profiles indicated that both concentrated uterine fluid and endometrial extracts contained two peaks of ir-MENK, a major peak which coeluted with standard MENK, and a minor peak eluting near the void volume (Vo). Reverse phase-HPLC chromatographic profiles also demonstrated two peaks of ir-MENK for concentrated uterine fluid and endometrial extracts, a major peak which coincided with standard MENK, plus a highly hydrophilic peak. The effect of ovarian steroids on the uterine secretion of ir-MENK was examined by measuring ir-MENK in uterine fluids from cyclic and pregnant gilts as well as ovariectomized, ovarian steroid-treated gilts. Day effects (P less than 0.01) were detected for cyclic and pregnant gilts, since values for ir-MENK increased between days 8 and 14 after onset of estrus. In ovariectomized gilts, treatment with progesterone (P4) increased the uterine secretion of ir-MENK (202 +/- 9 vs. 65 +/- 4 pg/ml for control, P less than 0.05). The combined treatment of P4 and estradiol did not further enhance secretion of ir-MENK, while treatment with estradiol did not alter ir-MENK levels relative to values for control gilts. These results indicate the presence of ir-MENK in porcine uterine fluid and endometrium, and suggest that uterine secretion of ir-MENK is regulated primarily by P4.

Methionine-enkephalin and thyrotropin-stimulating hormone are intimately related in the human anterior pituitary.

The tissue distribution and function of opioid peptides in humans is incompletely defined. We report here that, unlike that in other species, the human anterior pituitary gland contains high concentrations of methionine-enkephalin (met-enkephalin). The met-enkephalin immunoreactive material was isolated and identified as authentic met-enkephalin by fast atom bombardment-mass spectrometry and Edman degradation sequencing. The met-enkephalin was localized in a large subpopulation of TSH immunoreactive cells (thyrotrophs). No other proenkephalin-derived opioid peptides were found in the pituitary, and there was no overlap between proopiomelanocortin and met-enkephalin immunoreactive cells. These results suggest that the human anterior pituitary gland contains a novel met-enkephalin precursor and a possible role for met-enkephalin in regulating human thyroid function.

The presence of antibacterial and opioid peptides in human plasma during coronary artery bypass surgery.

Antibacterial peptides, found in both invertebrates and vertebrates, represent a potential innate defense mechanism against microbial infections. However, it is unknown whether this process occurs in humans during surgery. We looked for evidence of release of antibacterial peptides during coronary artery bypass grafting (CABG). We used immunological techniques and antibacterial assays combined with high-performance gel-permeation chromatography, reverse-phase HPLC, N-terminal sequencing and comparison with synthetic standards to characterize the peptide B/enkelytin. We show the presence of anionic antibacterial peptide, the peptide B/enkelytin which correspond to the C-terminal part of proenkephalin A, from the plasma of patients undergoing CABG. Our studies show that peptide B/enkelytin is initially present at low levels in plasma and is then released in increased amounts just after skin incision. Antibacterial assays confirmed that the peptides specifically target gram-positive bacteria. We also demonstrate that peptide B/enkelytin is metabolized in vivo to the opioid peptides methionine-enkephalin-Arg-Phe and methionine-enkephalin, peptides that we show have granulocyte chemotactic activity. These findings suggest that in humans, surgical incision leads to the release of antibacterial peptides. Furthermore, these antibacterial peptides can be metabolized into compounds that have immune-activating properties.

Methionine-enkephalin-Arg-Phe immunoreactivity in heart tissue.

Previous findings of enkephalins in cardiac tissue led us to investigate enkephalin distribution in animal models used for cardiovascular research. Canine cardiac methionine-enkephalin (ME) concentrations are low and evenly distributed between atria (4.2 +/- 0.6 fmol/mg protein, n = 30) and ventricles (4.4 +/- 0.5). In contrast, methionine-enkephalyl-arginyl-phenylalanine (MEAP) immunoreactivity (IR) is higher and preferentially concentrated in the ventricle (112 +/- 12) vs. the atria (23.2 +/- 2.6 fmol/mg protein). HPLC analysis suggests the atrial/ventricular difference is partly due to altered posttranslational processing. Nearly 90% of ventricular IR is comprised of MEAP (46%) and peptide B (40%) whereas these peptides represent less than half of the atrial content. A nonneuronal localization is indicated because the peptide distribution does not correspond to the catecholamine distribution. Canine left ventricular tissue sections were processed for immunohistochemistry with the MEAP antibody. Fluorescence was distributed throughout the myocytes and concentrated in ordered lines perpendicular to the myocyte longitudinal axis corresponding to the area of the intercalated disc. This suggests opioids may be important in communication between cardiomyocytes, and possibly the presence of a unique peptide secretory mechanism utilizing the intercalated disc. The relative peptide content in cat and pig hearts was similar to the dog; however, the distribution was different. Feline cardiac ME content was distributed 2:1 in favor of the ventricles and corresponded with a preferential ventricular norepinephrine distribution. The MEAP-IR pattern gave a ventricular/atrial ratio lower (3.5:1) in cat heart vs. dog (5:1). In contrast, pig heart ME and MEAP-IR ventricular/atrial ratios were reversed for both ME (1:10) and MEAP (1:2). HPLC of pig left ventricle showed that MEAP and peptide B represented 33% and 39% of the MEAP-IR, respectively. These species variations may correlate to the differences observed in cardiac function.

Detection of Met-enkephalin and Leu-enkephalin in the posterior pituitary of the holostean fish, Amia calva.

Immunohistochemical analysis of the pituitary of the holostean fish, Amia calva, indicated that enkephalin-related immunoreactivity was restricted to the pars nervosa, and was not detected in other regions of the pituitary. Fractionation of acid extracts of posterior pituitaries by reverse phase HPLC followed by RIA analysis indicated the presence of immunoreactive Met-enkephalin and Leu-enkephalin. No immunoreactive forms were detected with RIAs specific for either Met-enkephalin-RF or Met-enkephalin-RGL. The molar ratio of Met- to Leu-enkephalin in this terminal field was 3:1 (n = 4). HPLC fractions were also digested with trypsin and carboxypeptidase B to test for C-terminally extended forms of Met-enkephalin. A novel modified form of Met-enkephalin was detected. Extracts of the posterior pituitary, forebrain, midbrain, hypothalamus and hindbrain were screened with RIAs specific for the Pro-dynorphin end products, alpha-neo-endorphin, dynorphin A(1-17), dynorphin A(1-8) and dynorphin B(1-13). The results of these analyses were negative. Collectively, these data suggest that a Pro-enkephalin-like molecule is present in holostean fish. The holostean enkephalin precursor contains at least Met-enkephalin and Leu-enkephalin. However, Pro-dynorphin-related end products with antigenic determinants similar to mammalian dynorphin A(1-17), dynorphin A(1-8), dynorphin B(1-13) and alpha-neo-endorphin could not be detected in the brain or pituitary of this species.

Isolation of immunoreactive beta-endorphin-related and Met-enkephalin-related peptides from the posterior pituitary of the amphibian, Xenopus laevis.

Acid extracts of the posterior pituitary of the amphibian, Xenopus laevis, were analyzed with two heterologous region specific beta-endorphin RIAs. Following gel filtration chromatography and cation exchange chromatography four peaks of immunoreactivity were detected. All four peaks were detected with a N-acetyl specific beta-endorphin RIA. Peak I represented 92% of the total immunoreactivity isolated following cation exchange chromatography. This peak had a net positive charge at pH 2.5 of +1 and an apparent molecular weight of 1.4 Kd. Following reverse phase HPLC, Peak I fractionated into two peaks: Peak Ia and Peak Ib. Both peaks were detected with the N-acetyl specific beta-endorphin RIA and a Met-enkephalin RIA, however, neither peak co-migrated with either Met-enkephalin or N-acetyl-beta-endorphin(1-16). At present it is not clear whether Peak I is derived from pro-opiomelanocortin or one of the other opioid polyproteins. Peaks II, III, and IV represented 8% of the total immunoreactivity recovered following cation exchange chromatography. These peaks had net positive charges of +3, +4, and +5, respectively, and apparent molecular weights of 2.8, 3.2, and 3.5 Kd, respectively. These apparently N-acetylated beta-endorphin-sized forms are minor end products of the pro-opiomelanocortin biosynthetic pathway.

Subgroups of parkinsonian patients differentiated by peptidergic immunostaining of caudate nucleus biopsies.

In this study, Met-enkephalin (Met-enk), substance P (SP) and tyrosine hydroxylase (TH) immunostaining was assessed in caudate nucleus biopsies from 15 Parkinson's disease patients who were treated surgically. According to the combination of changes in Met-enk, SP and TH immunostaining, several subgroups of parkinsonian patients were disclosed. Group I: Patients showing low SP and normal Met-enk immunostaining, and variably reduced TH immunoreactivity. Group II: both SP and Met-enk immunostaining were apparently of normal intensity in these PD patients, but they showed the greatest decrease in TH labeling. Group III: PD patients that showed normal SP, very low Met-enk and variably reduced TH immunostaining. Low Met-enk immunostaining tended to correlate with the severity of the disease as judged by higher Unified Parkinson's disease Rating Scale and gait scores. These results suggest that different neurochemical phenotypes may exist among Parkinson's disease patients. Peptidergic deficits should be taken into account for therapeutic intervention.

Production of bioactive enkephalin from the nonendocrine cell lines COS-7, NIH3T3, Ltk-, and C2C12.

Enkephalin is synthesized from proenkephalin in neuroendocrine cells. For the attempt to induce nonneuroendocrine origin cells to produce enkephalin, we used a mammalian expression vector for fusion peptides, pMEproCT beta, in which a fused peptide is designed to be cleaved by a yeast Kex2-like endoprotease furin. Met-Enkephalin was expressed in four nonneuroendocrine cell lines: COS-7, C2C12, Ltk-, and NIH3T3. The four cell lines produced a marked amount of Met-enkephalin, which appeared as a single peak on reverse-phase HPLC. Because transplantation of adrenal medullary cells to the subarachnoid space has been used to alleviate terminal cancer pain, and enkephalin appears to play a central role in relieving pain, this enkephalin expression vector may be useful for direct enkephalin expression in pericancerous tissues.

Effects of Met-enkephalin on the mechanical activity and distribution of Met-enkephalin-like immunoreactivity in the cat large intestine.

The effects of Met-enkephalin on the spontaneous and electrically evoked activity were investigated in longitudinal and circular strips isolated from different regions of the large intestine, i.e., proximal colon, distal colon and rectum. Met-enkephalin induced dose-dependent contractile responses which were reversibly blocked by naloxone (10(-6) M). In all longitudinal strips and in the circular strips of the rectum, the effects of Met-enkephalin were prevented by TTX (10(-7) M), demonstrating their neurogenic nature. In the circular strips from the colon, Met-enkephalin induced contractile responses after TTX, proving the existence of smooth muscle opioid receptors. The comparison between the EC50 values of Met-enkephalin showed that the opioid receptors in the different regions have different sensitivity to Met-enkephalin, while the opioid receptors in the longitudinal and circular layers of the same region have equal affinity. Atropine (10(-6) M) and guanethidine (10(-6) M) did not alter significantly the EC50 values, showing that the neurogenic effects of Met-enkephalin on the spontaneous activity involve mainly nonadrenergic, noncholinergic (NANC) neurotransmitter mechanisms. When the preparations were stimulated electrically, Met-enkephalin (10(-9) M) suppressed the cholinergic components of the responses. Met-enkephalin-containing nerve fibers were found in the myenteric plexus of the three intestinal regions. In the colon, where direct smooth muscle effects were observed, fibers containing Met-enkephalin-like immunoreactivity were found to go deep into the circular layer, suggesting that they could supply Met-enkephalin input to the smooth muscle cells.

Characterization of neuropeptides extracted from canine intestine.

Quantitative determination of neuropeptides in biologic tissues by radioimmunoassay requires both an efficient extraction of neuropeptides as well as maintenance of immunochemical reactivity. Vasoactive intestinal peptide, substance P, and met5-enkephalin were chosen for this study because they are neuropeptides which appear to be involved in multiple physiologic systems. Since all three neuropeptides have a methionine residue within their amino acid sequence, oxidation of methionine to methionine-sulfoxide during the extraction process could diminish their immunochemical reactivity. Multiple factors that might be important in extracting these neuropeptides from canine intestine, including pH of the solvent, tissue homogenization, heating, and addition of enzyme inhibitors, were examined. Concentrations of vasoactive intestinal peptide-like immunoreactivity and substance P-like immunoreactivity were significantly higher in acidic solvents, and tissue homogenization appeared to increase the concentrations of these two neuropeptides. Substance P-like immunoreactivity was increased by heating after tissue homogenization, suggesting heat-induced denaturation of tissue enzymes liberated by homogenization. Separation of acidic tissue extracts by high performance liquid chromatography followed by radioimmunoassay for all three neuropeptides revealed minor acid-induced oxidation of substance P. These results should be useful for planning the extraction of these three neuropeptides from other tissues.

Purification and assay of opioid activity of low molecular weight enkephalin-immunoreactive peptides generated by peptic digestion of rat plasma proteins.

A low molecular weight Methionine enkephalin-immunoreactive peptide (MEIP) and two Leucine-enkephalin immunoreactive peptides (LEIP) generated by peptic digestion of rat plasma were purified through gel filtration followed by five sequential reverse phase HPLC gradients in different solvent systems. Binding experiments of these peptides to opioid receptors of rat brains were performed. The two LEIPs were able to inhibit binding of [3H]naloxone to opioid receptors in rat brain membranes. No inhibition was found with the MEIP (which represented the only MEIP present in the low molecular weight fraction of pepsin-digested rat plasma). Sequencing revealed that the MEIP is a six residue peptide with the following sequence: Gly-Glu-Tyr-Gly-Phe-Gln. This sequence corresponds to that of residues 422-427 of rat serum albumin.

Enkephalin-containing polypeptides in human cerebrospinal fluid.

The chemical nature of peptides in human CSF with the enkephalin core sequence from proenkephalin A and proenkephalin B, was investigated. Direct measurements with radioimmunoassay (RIA) were run on enkephalin, enkephalin hexapeptides, dynorphin A, dynorphin A1-8 and dynorphin B. The hexapeptides occurred in about 3 times higher concentration than the corresponding enkephalins. The only dynorphin RIA which gave positive results was the one for dynorphin A. However, most dynorphin A immunoactive material showed higher apparent molecular weight (MW; 3 and 5 kdalton) than the standard (2 kdalton). To identify and quantitate every possible proenkephalin derived peptide with the enkephalin sequence, chromatographic fractions were treated with trypsin. The products, Leu-enkephalin-Arg6 (from proenkephalin B) and Met-enkephalin-Lys6/Arg6 (from proenkephalin A) were measured by specific RIAs and identified by HPLC. In the higher (greater than 5 kdalton) MW interval, there was about 10-fold higher yield of Met-enkephalyl than Leu-enkephalyl hexapeptides. In the intermediate 1-3 kdalton MW interval, most activity derived from proenkephalin B. Finally, from the low MW region, there was about 5 times more proenkephalin A peptides. The main dynorphin A peak at 5 kdalton was transferred to a major Leu-enkephalin-Arg6 peak by trypsin degradation. The data indicate the presence of a whole family of peptides from the two proenkephalin genes in human CSF. Precursors to the peptides supposed to be the active members in the proenkephalin families occur in relatively high concentrations and may provide good markers for activity in these peptide systems.

Theoretical interpretation of extraction (in brain) of peptides including concentration variations.

The transport properties of several peptides across blood-brain barrier (BBB) have been investigated theoretically in terms of simple diffusion and facilitated diffusion processes. Comparison of the calculated results from the simple diffusion and the experimental data reveals the presence of the facilitated diffusion of these substances which we have conceived of as a carrier-mediated process. The values of the partition coefficients f for these peptides were in the range 7 X 10(-4) less than or equal to f less than or equal to 200 X 10(-4). The calculated f values gave permeabilities, Ps, in lipids between 10(-7) less than or equal to Ps less than or equal to 14 X 10(-7) cm/s. These values were then used to estimate the extraction for peptides from simple diffusion alone which vary from 0.3 to 3.5% compared with the experimental extraction (0.4-12%) indicating the inadequacy of the simple diffusion alone to explain the experimental data. As for the carrier-mediated facilitated diffusion process we have used the activated-complex theory. The extraction in this case depends on the maximal rate of transport (Tmax)f and the reciprocal of the affinity constant Kt for the transport of peptides through BBB. We have deduced that (Tmax)f approximately 0.46 X 10(-3) pmol/g X s and Kt approximately 0.35 nM for Met-enkephalin (Met-ENK), Leu-enkephalin (Leu-ENK), glutathione, carnosine, alpha-MSH and MIF and (Tmax)f approximately 10 X 10(-3) pmol/g X s and Kt approximately 7 nM for AVP, beta LT, beta E and alpha E to explain the observed results. We have also obtained the quantitative variation of extraction with concentration of peptides in the brain-capillary and have established that the extraction decreases with increasing concentration of peptides, tending to a small constant value at high concentrations. It has been inferred that carrier-mediated facilitated diffusion is important for the transport of peptides across BBB.

Purification and quantification of opioid peptides in bone and joint tissues--a methodological study in the rat.

The occurrence of methionine-enkephalin-Arg(6)-Phe(7) (MEAP) and dynorphin B (DYNB) representing two main precursors of opioids was analyzed in specimens from rat cortical bone, periosteum, bone marrow and joint tissue by radioimmunoassay (RIA). MEAP and DYNB were extracted in a solution of 4% EDTA in 2 M acetic acid previously proven suitable for extraction of sensory and autonomic neuropeptides in bone and joints. In crude extracts of cortical bone, the immunoreactive (ir) levels of both opioids were under the detection limit of RIA. As for DYNB this also applied to crude extracts of joints and periosteum. Therefore, two purification methods were tested and compared, i.e. reverse phase C 18 and ion exchange chromatography. RIA of the elution fraction disclosed a significant difference between the two methods in terms of recovery, i.e. <5% and 50%, respectively. Thus, purification by ion exchange chromatography prior to RIA appeared to be the most suitable by providing measurable levels of both MEAP and DYNB in all tissues analyzed (highest in bone marrow, lowest in cortical bone). The described method offers a means of quantifying opioid peptides in bone and joints, which may be utilized in the analysis of regulatory mechanisms of nociception, growth and immune responses in different conditions.

Isolation of molluscan opioid peptides.

An acid extract of neural tissues of the mollusc, Mytilus edulis, was fractionated by high-pressure chromatography. Peak fractions with retention times of that of Met-enkephalin, Leu-enkephalin and Met-enkephalin-Arg-Phe were subjected to competitive displacement assays in the same neural tissues. The results showed that these fractions have the same binding activities as the authentic neuropeptides. The peptides from these fractions were purified by high-pressure liquid chromatography under isocractic conditions. Sequential amino acid analyses showed these peptides to have the same primary structures as Met-enkephalin, Leu-enkephalin and Met-enkephalin-Arg-Phe.

Met-enkephalin-Arg6-Gly7-Leu8 exists together with Met-enkephalin-Arg6-Phe7, Met-enkephalin and Leu-enkephalin in human stomach.

Studies on the nucleotide sequence of cloned DNA complementary to mRNA for preproenkephalin A from adrenal medulla and human pheochromocytoma have revealed that this precursor contains 4 copies of methionine-enkephalin(Met-Enk) and one copy each of leu-enkephalin (Leu-Enk), methionine-enkephalin-Arg6-Gly7-Leu8(Met-Enk-Arg6-Gly7-Leu8) and methionine-enkephalin-Arg6-Phe7(Met-Enk-Arg6-Phe7). We have demonstrated the existence of Met-Enk-Arg6-Gly7-Leu8 together with Met-Enk, Leu-Enk and Met-Enk-Arg6-Phe7 in human gastric antrum, using high performance liquid chromatography(HPLC) coupled with radioimmunoassays for these opioid peptides. The ratio of molar concentrations of these peptides in human gastric antrum is almost equal to the ratio of these peptides contained in preproenkephalin A. Furthermore, gel filtration studies on Sephadex G-50 showed that most of immunoreactivities of these peptides were eluted at the elution position of each synthetic peptide without any detectable immunoreactivities at high molecular weight positions. In addition, most of immunoreactivities of these four opioid peptides were detected in the muscular layer of the gastric antrum. These results indicate the presence of Met-Enk-Arg6-Gly7-Leu8 together with Met-Enk, Leu-Enk, and Met-Enk-Arg6-Phe7 in human gastric antrum and further suggest that these opioid peptides are derived from the same precursor as preproenkephalin A in the adrenal medulla and the processing of preproenkephalin A is almost completed in the human stomach.

Methionine-enkephalin, leucine-enkephalin methionine-enkephalin-Arg6-Phe7 and methionine-enkephalin-Arg6-Gly7-Leu8 in human pheochromocytoma.

Methionine-enkephalin(met-enkephalin)-, leucine-enkephalin(leu-enkephalin)-, methionine-enkephalin-Arg6-Phe7(met-enkephalin-Arg-Phe)- and methionine-enkephalin-Arg6-Gly7-Leu8(met-enkephalin-Arg-Gly-Leu)-like immunoreactivities(-LI) were studied in 16 pheochromocytomas by radioimmunoassays (RIAs) for these four opioid peptides. Met-enkephalin-Arg-Phe-LI and met-enkephalin-Arg-Gly-Leu-LI existed together with met-enkephalin-LI and leu-enkephalin-LI in 16 pheochromocytomas. Significant positive correlations were observed among contents of these four opioid peptides in 16 pheochromocytomas. The concentrations of these four opioid peptides in epinephrine producing pheochromocytomas were much higher than those in norepinephrine producing tumors. HPLC and gel exclusion chromatography followed by the RIAs showed the presence of met-enkephalin, leu-enkephalin, met-enkephalin-Arg-Phe and met-enkephalin-Arg-Gly-Leu together with their high molecular weight forms. These results indicate the co-existence of met-enkephalin, leu-enkephalin, met-enkephalin-Arg-Phe, met-enkephalin-Arg-Gly-Leu and their high molecular weight forms derived from preproenkephalin A in human pheochromocytomas and suggest the association of preproenkephalin A synthesis with epinephrine production in human pheochromocytomas.

Simultaneous extraction of beta-endorphin and leu- and met-enkephalins from human and rat plasma.

A simple, rapid and reliable procedure is described to simultaneously concentrate and purify beta-endorphin, leu- and met-enkephalins from small volumes of human and rat plasma before radioimmunoassay is performed. It uses C18 Sep-Pak reverse phase cartridges. The effectiveness of different protease inhibitors in preventing degradation of opiates by plasma and different solvent systems for eluting opiates is also evaluated.

Plasma levels and biochemical characterisation of circulating met-enkephalin in canine endotoxin shock.

Endogenous opioid peptides have been implicated in the pathophysiology of shock (1-5). In anaesthetised mongrel dogs, administration of E coli endotoxin caused a rise in plasma met-enkephalin-like immunoreactivity (MLI). Biochemical characterisation of MLI by gel filtration chromatography revealed various molecular forms: 31K, 8K, 3-5K and the native pentapeptide in approximately equal amounts. After enzymatic treatment of column fractions the 31K form predominated (90.7%). This is the first demonstration of elevated MLI in endotoxin shock.

Effect of 2,2,2-trifluoroethanol on capillary zone electrophoretic peptide separations.

The use of 2,2,2-trifluoroethanol-water mixtures for peptide separations by capillary zone electrophoresis (CZE) displays some advantages over aqueous solutions. First, the increase in viscosity reduces and stabilizes the running current and facilitates heat dispersion, with a consequent improvement in the number of theoretical plates. Second, the decrease in the dielectric constant leads to a modification of the dissociation constants of the ionizable groups. The consequence is a change in selectivity that, for several favourable peptide pairs, provides an increase in resolution. Third, the interaction trifluoroethanol with the peptide modifies the Stokes radius in a manner strongly dependent on the peptide sequence. This can also be utilized for an increase in CZE performance. Fourth, the structural properties of 2,2,2-trifluoroethanol are particularly useful for an improvement in the separation of large apolar peptides. Finally, the use of trifluoroethanol strongly stabilizes the capillary coating.