Methionine Enkephalin-like Immunoreactivity Research Articles

Segregation of met–enkephalin from vesicular acetylcholine transporter and choline acetyltransferase in sympathetic preganglionic varicosities mostly lacking synaptophysin and synaptotagmin

Sympathetic preganglionic neurons (SPN) coexpress the acetylcholine (ACh)-synthesizing enzyme choline acetyltransferase and different peptides in their cell bodies, but can express them independently in separate varicosities, indicating that SPN segregate transmitters to different synapses. Consequently, there are populations of preganglionic varicosities (peptidergic and noncholinergic) that store peptides but not ACh. We studied in the cell bodies and axon processes of the rat SPN the expression and the proportional coexpression of the vesicular ACh transporter-like immunoreactivity (VAChT), a specific marker of cholinergic synaptic vesicles or ChAT-like immunoreactivity (ChAT), and the peptide methionine enkephalin-like immunoreactivity (mENK), and confirmed the presence of a population of SPN peptidergic, noncholinergic varicosities. We characterized these varicosities by exploring the occurrence of synaptophysin-like immunoreactivity (Syn), a marker of small clear vesicles, and synaptotagmin-like immunoreactivity (Syt), a preferential marker of large dense core vesicles. We found that (i) VAChT and mENK, like ChAT–mENK, were coexpressed in only 59% of the mENK-containing varicosities, although they colocalized in the SPN cell bodies; and (ii) almost 60% of the population of mENK-containing varicosities did not express Syn or Syt, and over 80% of the mENK-containing varicosities negative for VAChT also lacked Syn. These data prove that SPN segregate mENK from VAChT and ChAT, and show that most of the subset of mENKergic varicosities negative for VAChT also does not express Syn, suggesting the presence of a different vesicular pattern in these sympathetic preganglionic varicosities.

Coexistence of enkephalin‐ and tyrosine hydroxylase‐like immunoreactivities in nerve fibers of the temporomandibular joint capsule of the guinea pig

Background The innervation of joints has been suggested to play an important role for their normal function as well as for the pathogenesis of chronic arthritic conditions. It is therefore important to elucidate the functional anatomy of this innervation. Methods The expression of methionine enkephalin-like immunoreactivity (ENK-LI) and tyrosine hydroxylase (TH)-LI as well as their possible colocalization were examined in the temporomandibular joint of the guinea pig. Results Nerve fibers with ENK-LI were demonstrated in the synovium of the guinea pig temporomandibular joint capsule with the use of indirect immunofluorescence. The ENK+ fibers were found close to the surface of the synovial membrane, deeper in the synovium, and at the walls of blood vessels. Coexistence of ENK- and TH-LI was observed frequently in the synovial nerve fibers. After removal of the superior cervical ganglion (SCG), the ENK-containing nerve fibers were reduced substantially in number on the operated side or were completely absent if the cervical sympathetic trunk was also excised. Conclusions The findings indicate that the majority of fibers with ENK-LI derive from the SCG. ENK may act as a neuromodulator on the catecholaminergic nerves and may also have an analgesic effect in the joint. Anat. Rec. 251:226–229, 1998. © 1998 Wiley-Liss, Inc.

Coexistence of enkephalin- and tyrosine hydroxylase-like immunoreactivities in nerve fibers of the temporomandibular joint capsule of the guinea pig.

The innervation of joints has been suggested to play an important role for their normal function as well as for the pathogenesis of chronic arthritic conditions. It is therefore important to elucidate the functional anatomy of this innervation. The expression of methionine enkephalin-like immunoreactivity (ENK-LI) and tyrosine hydroxylase (TH)-LI as well as their possible colocalization were examined in the temporomandibular joint of the guinea pig: Nerve fibers with ENK-LI were demonstrated in the synovium of the guinea pig temporomandibular joint capsule with the use of indirect immunofluorescence. The ENK+ fibers were found close to the surface of the synovial membrane, deeper in the synovium, and at the walls of blood vessels. Coexistence of ENK- and TH-LI was observed frequently in the synovial nerve fibers. After removal of the superior cervical ganglion (SCG), the ENK-containing nerve fibers were reduced substantially in number on the operated side or were completely absent if the cervical sympathetic trunk was also excised. The findings indicate that the majority of fibers with ENK-LI derive from the SCG ENK may act as a neuromodulator on the catecholaminergic nerves and may also have an analgesic effect in the joint.

Plasma methionine enkephalin-like immunoreactivity in patients with methamphetamine psychosis.

Plasma methionine enkephalin-like immunoreactivity in patients with methamphetamine psychosis.

HPLC/RIA analysis of bioactive methionine enkephalin content in the seizure-susceptible El mouse brain

We previously reported a deficit of methionine enkephalin-like immunoreactivity (ME-LI), in the cerebral cortex, septal area, hippocampus, and striatum and the abnormal metabolism of opioid peptides in the hippocampus and striatum of seizure-susceptible El mice, which are involved in the pathogenesis of seizures. However, these findings suggest that the ME-LI does not necessarily reflect the bioactive methionine enkephalin (ME). Herein, we measured the biologically active peptide, ME excluding cross-reactive substances by using HPLC coupled with radioimmunoassay to clarify the abnormal function of enkephalinergic neurons in the El mouse brain. The ME content in 25-day-old El mice that had no seizures was significantly decreased in the hippocampus and septal area, as compared with corresponding regions in ddY mice (seizure-nonsusceptible; the mother strain of El). At the age of 50 days when El mice displayed abortive seizures, this content in both stimulated El[s] and nonstimulated El[ns] was significantly reduced in the septal area and cerebral cortex. At the age of 150 days when El mice exhibit tonic-clonic seizures, this content in both El[s] and El[ns] was significantly reduced in the septal area, cerebral cortex and striatum. These findings were generally compatible with our previous findings. This study further supports our hypothesis that a deficit of anticonvulsant endogenous ME, in the cerebral cortex, septal area, and hippocampus of seizure-susceptible El mice play an important role in the pathogenesis of seizures.

Metabolic profile of opioid peptides differs in the hippocampus and striatum of seizure-susceptible E1 mice

We previously suggested that a deficit of anticonvulsant endogenous methionine enkephalin, in the cerebral cortex, septal area, hippocampus, and striatum of seizure-susceptible El mice plays a role in the pathogenesis of seizures. To determine whether a hypofunction of enkephalinergic neuron may be due to metabolic abnormalities of opioid peptides in the El mouse brain, we measured methionine enkephalin-like immunoreactivity (ME-LI) of 50 fractions eluted by high performance liquid chromatography obtained from those four regions of the brain of El and seizure-nonsusceptible ddY mice (the mother strain of El mice). We observed the same ME-LI patterns of 50 fractions in the cerebral cortex and septal area in El and ddY mice, whereas exhibited differing ME-LI patterns in the hippocampus and striatum in the two stains. Different ME-LI patterns may imply the difference in the metabolic profile of opioid peptides. Thus, an abnormal metabolism of opioid peptides in the hippocampus and striatum of the El mouse may be involved in the pathogenesis of seizures.

Differential effects of isoflurane, halothane, and ketamine on the regional methionine-enkephalinlike immunoreactivity in the mouse brain.

The widely used measurement index for anesthetic potency, minimum alveolar concentration (MAC), is hypothesized to be the sum of the effects on multiple neural systems whose contribution to anesthesia differs depending on the agents used. The present study, which compared the effects of halothane, isoflurane, and ketamine, at equipotent level of anesthesia, on the methionine-enkephalinergic neurons in 9 brain regions, showed a significant difference in the methionine-enkephalin-like immunoreactivity (Met-ENK-like IR) among the anesthetics in each region. The order of the Met-ENK-like IR was: halothane > ketamine > isoflurane in the caudatus putamen; halothane > isoflurane ≊ketamine in the nucleus accumbens and the ventral pallidum; halothane ≊isoflurane > ketamine in the globus pallidus, the nucleus dorsomedialis hypothalami, and the nucleus ventromedialis hypothalami; and halothane > isoflurane > ketamine in the arcuate nucleus, the periaqueductal gray, and the nucleus reticularis parvocellularis. These findings indicate that these three anesthetics affect the methionine-enkephalinergic neurons in the motor and pain controlling pathways in different fashions.

Effects of space flight stress on proopiomelanocortin, proenkephalin A, and tachykinin neuropeptidergic systems in the rat posterior pituitary

β-endorphin-like immunoreactivity (BE-li), methionine enkephalin-like immunoreactivity (ME-li), and substance P-like immunoreactivity (SP-li) were measured in the posterior pituitary of rats that experienced a 5-day space flight in a Space Shuttle. ME-li and SP-li were both significantly lower compared to the control rats. However, there was no difference in BE-li between flight and control rats. These data suggest that the space flight stress diminished the methionine enkephalin (ME) and substance P (SP) concentrations in the posterior pituitary without affecting the β-endorphin (BE) concentration. Thus, the proenkephalin A and tachykinin, but not proopiomelanocortin, neuropeptidergic systems in the posterior pituitary may respond to this type of unique stress.

Assay of Preproenkephalin A Processing Enzymes in Human Lumbar Cerebrospinal Fluid

Vmax and Km measurements have been obtained for endogenous peptidases that are important for methionine enkephalin (ME) homeostasis in humans. Those peptidases in human lumbar cerebrospinal fluid (CSF) act upon several synthetic biologically significant peptides that are also contained within the preproenkephalin Ahuman, 1-267 molecule. The amount of endogenous methionine enkephalin-like immunoreactivity (ME-li) in human lumbar CSF is 74.1 ± 5.7 fmol ME-li/ml CSF (n = 56; x ± SE). The kinetic parameters of the various enzymes that inactivate exogenous, synthetic methionine enkephalin (ME, YGGFM) and that also produce ME from two different portions of the preproenkephalin Ahuman, 1-267 precursor molecule were determined. The enzyme that inactivates synthetic ME to FM, and that correlates to the rate of decrease of ME, has a Vmax = 560 ± 43.3 nmol/ml/min and a Km = 4514 ± 373 μM (n = 56; x ± SE). Preproenkephalin Ahuman, 186-193 (PA = YGGFMRGL) was added to CSF samples to characterize those processing and converting enzymes that produce the ME pentapeptide. Vmax, as measured by the rate of the decrease of PA to produce YGGFMR, was 0.192 ± 0.038 nmol/ml/min and a Km of 513 ± 121 μM (n = 10; x ± SE). Similarly, a bovine analog to preproenkephalin Ahuman, 128-140 (PPEhuman, GSEILAKRYGGFM; PPEbovine, 125-137, GGEVLGKRYGGFM) was used to characterize that enzyme system that produces ME from an N-terminally extended ME peptide. That endopeptidase had a Vmax of 0.120 ± 0.048 nmol/ml/min with a Km of 734 ± 296 μM (n = 10). Those endogenous enzymes in human CSF may relate to the proopiomelanocortin convertase enzymes that contain the subtilisin-like catalytic domain.

Methionine enkephalin-like immunoreactivity, substance P-like immunoreactivity and β-endorphin-like immunoreactivity post∼mortem stability in rat pituitary

Significant post-mortem changes in peptide concentration occur within the previously unstudied timeframe, i.e. within 1 h, for the proenkephalin A, proopiomelanoccortin, and tachykinin neuropeptidergic systems in the pituitary. These data differ from data obtained in other studies that concluded that peptides are stable for up to 72 h post-mortem. The post-mortem stability of the three neuropeptides, methionine enkephalin, substance P, and β-endorphin, was studied in the rat pituitary to test the hypothesis that significant post-mortem concentration changes of those three neuropeptides occur in the immediate post-mortem time period.

Methionine enkephalin-like, substance P-like, and β-endorphin-like immunoreactivity in human parotid saliva

These three neuropeptides were measured at daily baseline values by radioimmunoassay. Stimulated parotid saliva was collected from 31 subjects using a modified Carlson-Crittenden device affixed over Stenson's duct. Methionine enkephalin-like immunoreactivity ranged from 6.6 to 11.7 fmol/ml, with a mean of 9.3 fmol/ml. Substance P-like immunoreactivity ranged from 6.1 to 12.6 fmol/ml, with a mean of 9.3 fmol/ml. β-Endorphin-like immunoreactivity ranged from 1.2 to 3.6 fmol/ml, with a mean of 2.6 fmol/ml. This is believed to be the first documentation of methionine enkephelin- and substance P-like activities in human parotid saliva and the first demonstration of β-endorphin-like activity in any type of human saliva. Substance P-like activity was significantly higher in morning than evening samples; β-endorphin-like activity also tended to be higher in the morning samples. Substance P and β-endorphinlike immunoreactivities covaried in a significant positive manner, suggesting either common control mechanisms or similar responses to physiological variables.

Opioids in cerebrospinal fluid in hypotensive newborn pigs.

This study was designed to determine if opioids were detectable in cerebrospinal fluid (CSF) and if these concentrations were altered by hemorrhagic hypotension. This study was further designed to determine the effects of topically administered opioids on pial arteriolar diameter during normotension and hypotension. Closed cranial windows were used to determine pial arteriolar diameter. Periarachnoid cortical and cisterna magna CSF was collected from piglets during normotension and hypotension (systemic arterial pressure decreased from 63 +/- 1 to 33 +/- 1 mm Hg). Opioid profiles were assessed qualitatively by radioreceptor assay, and individual opioids were measured quantitatively by radioimmunoassay. Periarachnoid cortical and cisterna magna CSF methionine enkephalin-, leucine enkephalin-, dynorphin-, and beta-endorphin-like receptor active values all were increased by hypotension. When quantified by radioimmunoassay, periarachnoid cortical CSF values for methionine enkephalin-like immunoreactivity were 1,167 +/- 58 and 2,975 +/- 139 pg/ml for normotension and hypotension, respectively. Periarachnoid cortical CSF radioimmunoassay values for dynorphin-like immunoreactivity were 15 +/- 2 and 28 +/- 2 pg/ml for normotension and hypotension, respectively. When applied topically to the cortical surface, synthetic methionine enkephalin increased pial arteriolar diameter (134 +/- 4, 158 +/- 4, and 163 +/- 4 microns for control, 574 pg/ml [10(-10) M], and 5,740 pg/ml [10(-9) M], respectively). Similarly, topical synthetic leucine enkephalin and dynorphin elicited pial arteriolar dilation. However, beta-endorphin produced arteriolar constriction. Hypotension attenuated methionine and leucine enkephalin-induced dilation and reversed dynorphin-induced dilation to concentration-dependent constriction. beta-Endorphin-induced constriction was not changed by hypotension. Therefore, opioids could contribute to the control of the cerebral circulation during hypotension.

Developmental and regional alteration of methionine enkephalin-like immunoreactivity in seizure-susceptible E1 mouse brain

Methionine enkephalin-like immunoreactivity (ME-LI) in the brain of El mice (seizure-susceptible strain) was measured by radioimmunoassay (RIA) to elucidate the relation between seizures and the opioid system. The lyophilized supernatants of tissue extracts were subjected to ME RIA. The concentration of ME-LI in 25-day-old El mice that had no seizures was significantly decreased in the hippocampus. At the age of 50 days when El mice displayed abortive seizures, the levels of ME-LI in both El(+) and nonstimulated El(o) mice were also significantly reduced in the hippocampus and septal area. It was further shown that the ME-LI concentrations in both 150-day-old adult El(+) during interictal periods and El(o) mice were markedly decreased in the cerebral cortex, septal area, and striatum, as compared with the corresponding regions in ddY mice (seizure-nonsusceptible strain; the mother strain of El). The decrease of ME-LI in the El mouse brain was generally compatible with our previous findings concerning the up-regulation of opioid delta receptors in this species. These results suggest that the reduction of ME-LI in the El mouse brain is not due to convulsions, but could be associated with the pathogenesis of seizure diathesis and seizure manifestations in the El mouse.

Distribution of substance P and methionine-enkephalin in salivary glands and effect of chronic morphine treatment on levels of these peptides.

Substance P-like immunoreactivity (SPLI) and methionine-enkephalin-like immunoreactivity (MELI) were determined in salivary glands from rats by radioimmunoassay. In all salivary glands investigated (submandibular gland, sublingual gland and parotid gland), SPLI and MELI were detected. The amount of both peptides is comparable to or relatively higher than those found in any other peripheral tissue. The level of SPLI showed a tendency to increase following chronic treatment with morphine: the enhancement in the submandibular gland and the sublingual gland was especially remarkable. The level of MELI was decreased, particularly in the submandibular gland.

Distribution of Substance P and Methionine-Enkephalin in Salivary Glands and Effect of Chronic Morphine Treatment on Levels of These Peptides

Substance P-like immunereactivity (SPLI) and methionine-enkephalinlike immunoreactivity (MELI) were determined in salivary glands from rats by radioimmunoassay. In all salivary glands investigated (submandibular gland, sublingual gland and parotid gland). SPLI and MELI were detected. The amount of both peptides is comparable to or relatively higher than those found in any other peripheral tissue. The level of SPLI showed a tendency to increase following chronic treatment with morphine; the enhancement in the submandibular gland and the sublingual gland was especially remarkable. The level of MELI was decreased, particularly in the submandibular gland.

Postnatal sequential development of dopaminergic and enkephalinergic perineuronal formations in the lateral septal nucleus of the rat correlated with local neuronal maturation

Tyrosine-hydroxylase (TH-IR) and methionine-enkephalin like immunoreactivity (MetE-IR) were analyzed in the lateral septal nucleus (LSN) of the rat from birth (PO) to adulthood. TH-IR labeled specifically the dopaminergic (DA) pericellular arrangements of the LSN, as checked by negative dopamine-beta-hydroxylase and phenylethanolamine-N-methyl transferase-IR. TH-IR and Met-IR processes were present at birth in the medial LSN and extended lateralwards and caudalwards from P0 to P6 to constitute two main DA terminal fields (medial and lateral) surrounding a MetE one. Within these fields, the development of perineuronal baskets followed a similar medial to lateral sequence: DA axons first surrounded a few neuronal cell bodies at P3 in the medial part of the intermediate LSN; at P6, Met-IR axons encircled more laterally located perikarya, and only at P9, some neurons located along the ventricle in the lateral DA field became surrounded. The initial aspect of TH-IR baskets consisting of few axons surrounding the cell body rapidly evolved in a positive network encapsulating the perikaryon and long segments of the proximal dendrites, whereas MetE-IR varicosities remained restricted around the perikaryon and the initial dendritic segments. Ultrastructural study at P14 revealed numerous TH-IR and MetE-IR axosomatic and axodendritic profiles. TH-IR axosomatic varicosities exhibited asymmetrical synapses, whereas MetE-IR ones displayed rare symmetrical contacts. The medio-lateral gradient of development of the perineuronal baskets was parallel to the postnatal neuronal development of the LSN as evaluated by cytological criteria: neuronal density, cell size and Nissl staining. Therefore, the formation of DA and MetE perineuronal arrangements in the LSN does not seem to be subordinate to the nature of the neurotransmitter they contain but related to the level of differentiation of their target neurons. A similar sequential set-up in the development of afferences paralleling the neuronal differentiation is discussed.

Catecholamines and opioid peptides in human phaeochromocytomas.

The tissue contents of catecholamines (CAs) and methionine-enkephalin-like immunoreactivity (Met-enk) were examined in 8 patients with phaeochromocytomas (Phs). In 6, Ph cells were cultured, and the modes of secretion of CAs and Met-enk were examined. Tissue contents of CAs and Met-enk varied from patient to patient, but larger amounts of Met-enk were found in medullary Phs than extramedullary ones, regardless of the secreting CA type. Three patients with extramedullary Phs had clinically showed a sustained hypertension, whereas five with medullary Phs were normotensive or had occasional paroxysmal hypertension. Nicotine (10(-7) M to 10(-4) M) stimulated simultaneous secretion of CAs and Met-enk from the cultured human Ph cells. Met-enk, however, was not secreted in proportion to either epinephrine (E), norepinephrine (NE) or total CAs (E + NE). Met-enk, FK33-824 (FK) (Met-enkephalin analogue), and dynorphin 1-13 (Dyn) significantly suppressed the secretion of CA evoked by 10(-5) M nicotine. The 50% inhibitory concentrations (IC50) of Met-enk, FK, and Dyn were 5 X 10(-6) M, 9.9 X 10(-5) M, and 9.0 X 10(-8) M, respectively, in one patient and 9.0 X 10(-6) M, 1.5 X 10(-7) M, and 1.4 X 10(-7) M in another. In one patient, 10(-5) M naloxone inhibited the CA secretion evoked by 10(-5) M nicotine and did not reverse the 10(-5) M FK-induced suppression of CA secretion in the presence of 10(-5) M nicotine. These results suggest that human Phs may be heterogeneous with regard to storage and secretion of CAs and opioid peptides.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of nitrous oxide upon regional brain levels of methionine-enkephalin-like immunoreactivity in rats

The influence of a 60-min exposure to 75% nitrous oxide/25% oxygen upon concentrations of methionine-enkephalin-like immunoreactivity (MELI) in discrete brain regions was investigated in conscious rats. Compared to room air-exposed control animals, nitrous oxide-exposed rats had 12–18% increases in MELI in the brainstem, spinal cord, hypothalamus and corpus striatum. There were no significant differences between control and nitrous oxide-exposed rats in levels of MELI in the cerebral cortex or diencephalon.

Ontogeny of dynorphin-like immunoreactivity in the hippocampal formation of the rat

Light microscopic immunocytochemical techniques were used to analyze the ontogeny of dynorphin (A)-like immunoreactivity (DLI) in the hippocampal formation of the Sprague-Dawley rat. For comparison purposes, alternate sections of the same brains were processed for the localization of methionine enkephalin-like immunoreactivity (ELI). DLI was first detectable in CA3a stratum lucidum and the suprapyramidal hilus on postnatal day (P) 6. On P7, DLI was evenly present throughout the full extent of the mossy fiber system. From P8 to P19, DLI progressively increased in intensity and could be localized in the fine axons and spherical swellings. The mossy fiber system and occasional perikarya superficial to stratum granulosum were the only hippocampal elements that exhibited DLI. In corroboration with earlier results, stratum lucidum ELI was first detected within large spherical bouton-like swellings on P13. From these data it is concluded that DLI appears in morphologically immature mossy fibers soon after they reach their target fields. In contrast, enkephalin is first detected within morphologically elaborated mossy fiber boutons well after the establishment of functional synapses.

Acute and chronic morphine modifies the in vivo release of methionine enkephalin-like immunoreactivity from the cat spinal cord and brain

The effect of acute and chronic morphine treatment was investigated on the spontaneous and evoked release of methionine enkephalin-like immunoreactivity (MELI) in vivo using the technique of cat spinal superfusion and ventriculocisternal perfusion. Stimulation of sciatic nerve, at intensities known to activate small-diameter nerve fibers, resulted in a consistent release of MELI from the spinal cord and the brain. Local application of morphine (5 × 10 −4M) to the spinal cord resulted in a significant decrease in the evoked release of MELI. Naloxone (2 mg/kg i.v.), administered during morphine treatment, produced an increase in the spontaneous and greatly augmented the evoked release of spinal and ventricular MELI. In morphine-naive animals, naloxone did not affect the spontaneous or evoked MELI release. In cats chronically exposed to parenternal morphine by implantation of morphine pellets (2 × 75mg), the spontaneous release of spinal and ventricular MELI was significantly greater than this release in control animals. Stimulation of sciatic nerves evoked a normal release of MELI in morphine-pelleted animals. Administration of naloxone to these animals resulted in a large and sustained increase in the spontaneous release of MELI from the spinal cord and brain. The material released by stimulation was identified as methionine enkephalin-like on the basis of similar results with two different antisera, parallel displacement curves with serial dilutions of spinal and ventricular perfusates and comigration with methionine enkephalin on a Sephadex G-25 column. These results suggest that if there is a tonic suppression of enkephalin release mediated by opiate receptors, these receptors display a tolerance development also. The facilitated release by naloxone in the chronic morphine-treated animals may indicate that reversal of the ongoing opioid inhibition results either in an excessive drive of the enkephalinergic neuron by other excitatory systems or the loss of a tonic auto-inhibition, which is not present in the non-tolerant animal.