Brain spectrin(240/235) and brain spectrin(240/235E): two distinct spectrin subtypes with different locations within mammalian neural cells.

Adult mouse brain contains at least two distinct spectrin subtypes, both consisting of 240-kD and 235-kD subunits. Brain spectrin(240/235) is found in neuronal axons, but not dendrites, when immunohistochemistry is performed with antibody raised against brain spectrin isolated from enriched synaptic/axonal membranes. A second spectrin subtype, brain spectrin(240/235E), is exclusively recognized by red blood cell spectrin antibody. Brain spectrin(240/235E) is confined to neuronal cell bodies and dendrites, and some glial cells, but is not present in axons or presynaptic terminals.

Synapsin I-mediated interaction of brain spectrin with synaptic vesicles.

We have established a new binding assay in which 125I-labeled synaptic vesicles are incubated with brain spectrin covalently immobilized on cellulosic membranes in a microfiltration apparatus. We obtained saturable, high affinity, salt- (optimum at 50-70 mM NaCl) and pH- (optimum at pH 7.5-7.8) dependent binding. Nonlinear regression analysis of the binding isotherm indicated one site binding with a Kd = 59 micrograms/ml and a maximal binding capacity = 1.9 micrograms vesicle protein per microgram spectrin. The fact that the binding of spectrin was via synapsin was demonstrated in three ways. (a) Binding of synaptic vesicles to immobilized spectrin was eliminated by prior extraction with 1 M KCl. When the peripheral membrane proteins in the 1 M KCl extract were separated by SDS-PAGE, transferred to nitrocellulose paper and incubated with 125I-brain spectrin, 96% of the total radioactivity was associated with five polypeptides of 80, 75, 69, 64, and 40 kD. All five polypeptides reacted with an anti-synapsin I polyclonal antibody, and the 80- and 75-kD polypeptides comigrated with authentic synapsin Ia and synapsin Ib. The 69- and 64-kD polypeptides are either proteolytic fragments of synapsin I or represent synapsin IIa and synapsin IIb. (b) Pure synapsin I was capable of competitively inhibiting the binding of radioiodinated synaptic vesicles to immobilized brain spectrin with a Kl = 46 nM. (c) Fab fragments of anti-synapsin I were capable of inhibiting the binding of radioiodinated synaptic vesicles to immobilized brain spectrin. These three observations clearly establish that synapsin I is a primary receptor for brain spectrin on the cytoplasmic surface of the synaptic vesicle membrane.

Naltrexone modulates tumor response in mice with neuroblastoma.

Naltrexone, an opiate antagonist, had both stimulatory and inhibitory effects, depending on the dosage, on the growth of S20Y neuroblastoma in A/Jax mice. Daily injections of 0.1 milligram of naltrexone per kilogram of body weight, which blocked morphine-induced analgesia for 4 to 6 hours per day, resulted in a 33 percent tumor incidence, a 98 percent delay in the time before tumor appearance, and a 36 percent increase in survival time. Neuroblastoma-inoculated mice receiving 10 milligrams of naltrexone per kilogram, which blocked morphine-induced analgesia for 24 hours per day, had a 100 percent tumor incidence, a 27 percent reduction in the time before tumor appearance, and a 19 percent decrease in survival time. Inoculation of neuroblastoma cells in control subjects resulted in 100 percent tumor incidence within 29 days. These results show that naltrexone can modulate tumor response and suggest a role for the endorphin-opiate receptor system in neuro-oncogenic events.

Autoradiographic identification of ornithine decarboxylase in mouse kidney by means of alpha-[5-14C]difluoromethylornithine.

alpha-Difluoromethylornithine is an enzyme-activated irreversible inhibitor of ornithine decarboxylase that forms a covalent bond with the enzyme. When alpha-[5-14C]difluoromethylornithine was administered to androgen-treated mice, only ornithine decarboxylase became labeled. Autoradiographic examination of kidney, liver, and brain indicated much more extensive incorporation of labeled difluoromethylornithine into kidney protein than into the protein of the other tissues. Such incorporation was greatly reduced by prior treatment of the mice with cycloheximide. These results correlate with the presence of ornithine decarboxylase activity which is much higher in the kidney than in the other tissues and is lost rapidly when protein synthesis is inhibited. The binding of this drug in vivo, therefore, is useful for determining the distribution of ornithine decarboxylase. The enzyme was predominantly located in the proximal tubule cells of the kidney in androgen-treated mice.

Increased brain size and cellular content in infant rats treated with an opiate antagonist.

From birth to day 21, rat offspring received daily injections of naltrexone at a dosage that blocked morphine-induced analgesia 24 hours a day. At 21 days, body, brain, and cerebellar weights of naltrexone-injected animals were 18, 11, and 5 percent greater than corresponding control weights. In addition, morphometric analysis of the cerebrum revealed a somatosensory cortex that was 18 percent thicker than that of the controls. The cerebellum of naltrexone-treated rats was 41 percent larger in total area and contained at least 70 percent more glial cells and 30 percent more granule neurons. Neurons derived prenatally were unaffected by drug treatment. These results show that naltrexone can stimulate body and brain growth in rats and suggest a role for the endorphin and opiate receptor system in development.

Distribution of enkephalin immunoreactivity in germinative cells of developing rat cerebellum.

The cellular distribution of enkephalin, an endogenous opioid, in the developing rat cerebellum was determined by immunocytochemistry. Methionine and leucine enkephalin were concentrated in the external germinal layer, a matrix of proliferative cells; staining was confined to the cortical cytoplasm. Enkephalin was not detected by immunocytochemistry in differentiated neural cells. These results indicate that endogenous opioids are involved specifically in early phases of nervous system development, particularly cell proliferation and differentiation.

Identification and location of brain protein 4.1.

Protein 4.1 is a membrane skeletal protein that converts the low-affinity interaction between spectrin and actin into a high-affinity ternary complex of spectrin, protein 4.1, and actin that is essential to the structural stability of the erythrocyte. Pig brain was shown to contain an 87-kilodalton immunoreactive analog of protein 4.1 that has partial sequence homology with pig erythrocyte protein 4.1 and the same location as spectrin in the cortical cytoplasm of neuronal and glial cell types of the cerebellum.

Modulation of murine neuroblastoma in nude mice by opioid antagonists.

Naltrexone, an opioid antagonist, had an inhibitory effect on the growth of murine S20Y neuroblastoma in BALB/c nude mice. Daily injections of 0.1 mg naltrexone/kg, which invoked a receptor blockade for 6-8 hours/day, resulted in 31-92% delay in latency time prior to tumor expression and a 27-49% increase in mean survival time; the magnitude of antitumor response was governed by tumor burden. Inoculation of neuroblastoma (10(6)-2.5 X 10(4) cells) resulted in measurable tumors in 10-13 days and mean survival times of 30-34 days. Immunoreactive beta-endorphin was detected in tumor tissue (39.7 pg/mg protein). Receptor binding assays revealed specific saturable binding of ligands related to delta- and kappa-binding sites, but not for the mu-binding site. These results demonstrate that opioid antagonist modulation of neuro-oncogenesis is not dependent on the integrity of T-cell-mediated immunity and suggest the feasibility of utilizing the nude mouse model in exploring the role of endogenous opioids in human cancers.

Opioid receptors and endogenous opioids in diverse human and animal cancers.

Receptor binding studies demonstrated specific high-affinity, saturable binding of a number of opioid ligands to a wide variety of neural and nonneural human and animal tumors. Radioimmunoassays revealed the presence of beta-endorphin and methionine-enkephalin in these tumors. Both methionine- and leucine-enkephalin were detected in tumor tissue by immunocytochemistry, with immunoreactivity related to the cortical cytoplasm of tumor cells, but not to cell nuclei. Endogenous opioids and receptors were found in benign and malignant tumors representative of ectodermal, mesodermal, and endodermal origin. Receptors and endogenous opioid peptides were present in tumors from many different species, including those transplanted into nude mice. These results suggest that opioid receptors and endogenous opioids are fundamental features of human and animal cancers.

Specific labeling of O6-alkylguanine-DNA alkyltransferase by reaction with O6-(p-hydroxy[3H]methylbenzyl)guanine.

O6-Alkylguanine-DNA alkyltransferase (AGT) is an important DNA repair protein that removes adducts from the O6 position of guanine by transferring them to a cysteine residue within its amino acid sequence. Mammalian AGTs are readily inactivated by incubation with O6-benzyl-guanine (BG), which is an alternative substrate for the protein. To examine this inactivation in more detail and to develop a procedure for the specific labeling of human AGT, we synthesized a BG derivative, O6-(p-hydroxy[3H]methylbenzyl_guanine ([3H]HMBG) and examined its interaction with AGT in HT29 cell extracts and in HT29 cells. Incubation of human AGT with [3H]HMBG led to the incorporation of radioactivity in the protein in a time- and temperature-dependent manner. This reaction was specific, since neither AGT that was first inactivated by reaction with BG nor proteins other than AGT were labeled. Digestion of the labeled AGT with trypsin showed that a single peptide contained the label. Sequencing of this peptide indicated that the label was bound to cysteine-145. These results demonstrate that AGT accepts HMBG as a substrate and becomes inactivated by transfer of a p-hydroxymethylbenzyl residue to the cysteine-145 acceptor site. When [3H]HMBG was added to cultures of HT29 cells which are Mer+ and express active AGT, radioactivity was incorporated in a macromolecule and could be detected by autoradiography. No such labeling occurred with BE or CHO cells, which are Mer- and lack AGT. Examination of the interaction of [3H]HMBG with mutant AGT proteins that differ greatly in their abilities to react with BG showed that there was a strong correlation between the reaction with BG and the labeling with [3H]HMBG. These results indicate that [3H]HMBG is a potentially useful reagent for the detection and localization of AGT activity and for the investigation of its mechanism of action.

The opioid growth factor, [Met5]-enkephalin, and the zeta opioid receptor are present in human and mouse skin and tonically act to inhibit DNA synthesis in the epidermis.

Opioid peptides serve as tonically active negative growth factors in neural and non-neural cells, in addition to being neuromodulators. To investigate the involvement of opioids in homeostatic renewal of epithelial cells in the epidermis, mice were given systemic injections of the potent opioid antagonist, naltrexone (NTX) (20 mg/kg). Disruption of opioid-receptor interaction by NTX resulted in an elevation of 42 and 72% in DNA synthesis in skin from the dorsum and plantar surface of the hindfoot, respectively, within 2 h; response to NTX was dependent on the circadian rhythm in each region examined. Injection of the naturally occurring and potent opioid growth factor (OGF), [Met5]-enkephalin, at 1 mg/kg depressed DNA synthesis in the dorsum and plantar surface by 42 and 19%, respectively, within 2 h; the effects of OGF complied with the pattern of circadian rhythm in each area of skin. The decreases in labeling index evoked by OGF were blocked by concomitant administration of the opioid antagonist, naloxone (10 mg/kg); naloxone alone at the dosage utilized had no influence on cell replicative processes. In tissue culture studies, OGF and NTX respectively depressed and elevated DNA synthesis. Both OGF and its receptor, zeta, were detected in all but the cornified layer of the epidermis in murine skin from the dorsum, plantar surface, pinnae, and tail. In addition, both peptide and receptor were observed in basal and suprabasal cells of the human epidermis. These results lead to the suggestion that an endogenous opioid peptide and its receptor are present and govern cellular renewal processes in the skin in a direct manner, regulating DNA synthesis in a tonically inhibitory, circadian rhythm-dependent fashion.

Opiates, endorphins, and the developing organism: a comprehensive bibliography, 1982-1983.

A comprehensive bibliography of the literature concerned with opiates, endorphins, and the developing organism for 1982 and 1983 is presented. Utilized with a companion paper (Neurosci Biobehav Rev 6: 439-479, 1982) these articles cover clinical and laboratory references beginning in 1875. For the years 1982 and 1983, a total of 385 citations was recorded. A series of indexes accompanies the citations in order to make the literature more accessible. These indexes are divided into clinical and laboratory topics. The clinical section is subdivided into: age of subject examined, maternal aspects, the fetus, and the offspring. The laboratory section is subdivided into: type of opiate/endorphin studied, species utilized, and major subject areas explored.

Opioids and the developing organism: a comprehensive bibliography, 1984-1988.

A comprehensive bibliography of the literature concerned with opioids and the developing organism for 1984-1988 is presented. Utilized with companion papers (Neurosci. Biobehav. Rev. 6:439-479; 1982; 8:387-403; 1984), these articles cover the clinical and laboratory references beginning in 1875. For the years 1984, 1985, 1986, 1987, and 1988, a total of 877 citations were recorded. A series of indexes accompanies the citations in order to make the literature more accessible. These indexes are divided into clinical and laboratory topics, and subdivided into such topics as the type of opioid explored and the general area of biological interest (e.g., physiology).

Opiates, endorphins and the developing organism: a comprehensive bibliography.

A comprehensive bibliography of the literature concerned with opiates, endorphins, and the developing organism is presented. A total of 1378 clinical and laboratory references, with citations beginning in 1875, are recorded. A series of indexed accompanies the citations in order to make the literature more accessible. These indexes are divided into clinical and laboratory topics. The clinical section is subdivided into: age of subject examined; maternal aspects; effects on the fetus; pharmacology, physiology, and the withdrawal syndrome; and "other" effects on the offspring. The laboratory section is subdivided into: type of opiate/endorphin studied; species utilized; and major subject areas explored.

Temporal variation in cellular proliferation during recornification of mouse tail skin.

The influence of the time of injury on subsequent epidermal regeneration is unknown. Epidermal cell proliferation of tail skin in C57BL/6J mice in response to tape stripping was followed for 7 days by radiolabelled thymidine incorporation and autoradiography. The homeostatic labelling index (LI) of the basal epidermis of unmanipulated, unwounded (control) animals was 7.6% and did not vary depending on the time of day. Tape stripping increased the LI of epidermal basal cells 110% above control values 24 h after injury. Labelling indexes of epidermal basal cells in the skin adjacent to the wounded area were 7.0%. Basal cell DNA synthesis stimulated by wounding exhibited a distinct temporal variation at 24 h postinjury, with tail skin wounded at 12.00 h found to be 275% greater than control values and elevated 78% from LIs recorded at any other time point. This temporal spike was due to the time of day at which wounding occurred rather than the time point when the LI was determined. Mice wounded at 12.00 h and terminated 27 h later (15.00 h) had LIs that were 52% greater than wounds created at 09.00 h and examined at 12.00 h the following day. Higher levels of DNA synthesis in tail skin injured at 12.00 h compared to wounding at 09.00 h was detected 12-48 h after injury. Furthermore, DNA synthesis in wounds created at 12.00 h returned to baseline levels 1-2 days earlier than tail skin wounded at 09.00 h. Investigation of other strains of mice detected differences in radiolabelling of epidermal basal cells 24 h after tape stripping at 12.00 h or 09.00 h in CD-1 and BALB/cJ mice, but not in the C3H/HeJ strain. These results indicate: (a) there is no diurnal variation in the LI of mouse tail skin under normal homeostatic conditions (b) tape stripping is a potent stimulator of basal cell turnover in the epidermis (c) the time of wounding determines the magnitude of the increase in the LI of basal cells following injury, and (d) the proliferative response to wounding of the tail is dependent on the strain of mouse.

The opioid growth factor, [Met5]-enkephalin, inhibits DNA synthesis during recornification of mouse tail skin.

Opioid peptides serve as tonically active negative growth regulators in renewing and regenerating epithelia. To examine the involvement of opioids in renewal of the stratum corneum after tape stripping of tail skin, C57BL/6 J mice were given systemic injections of the potent opioid antagonist, naltrexone (NTX, 20 mg/kg i.p.) following injury. Blockade of opioid-receptor interaction by NTX for 4 h resulted in an elevation of 36-66% in basal cell DNA synthesis measured 24 h after injury. Injection of the endogenous opioid peptide, [Met5]-enkephalin (OGF, 10 mg/kg i.p.) 4 h before termination, suppressed radiolabelled thymidine incorporation in the basal cell layer by 37-46% at 24 h after wounding. The magnitude of the effects on DNA synthesis of OGF, but not NTX, depended on the timing of administration with respect to injury. OGF maximally depressed basal cell labelling (72%) when given 16 h after tape stripping. Concomitant administration of naloxone (10 mg/kg) with OGF blocked the inhibition of DNA synthesis; naloxone alone at the dosage utilized had no effect on cell labelling. Both OGF and its receptor, OGFr, were detected by immunocytochemistry in the basal and suprabasal cell layers, but not the cornified layer of tape stripped and uninjured tail skin. These results indicate: (a) a native opioid peptide and its receptor are expressed in epidermal cells of injured and uninjured mouse tail skin; (b) removal of the stratum corneum by tape stripping does not disrupt the function of the endogenous opioid growth system; (c) the proliferative response to wounding of the tail is tonically inhibited by the receptor-mediated action of an endogenous opioid peptide; and (d) DNA synthesis by basal cells can be elevated by disrupting opioid peptide receptor interactions.

Endogenous opioid systems and the regulation of dendritic growth and spine formation.

The role of endogenous opioid systems (endogenous opioids and opioid receptors) in neuronal development was examined in 10- and 21-day-old rats by utilizing an opioid antagonist (naltrexone) paradigm. Throughout the first 3 weeks of life, Sprague-Dawley rats were given daily subcutaneous injections of either 50 mg/kg naltrexone, a dosage that invoked a complete (24 hours/day) receptor blockade, or 1 mg/kg naltrexone, a dosage which intermittently blocked (4-6 hours/day) opioid receptors and exacerbated opioid action; animals injected with sterile water served as controls. Pyramidal cells from the frontoparietal cortex (layer III) and hippocampal field CA1, and cerebellar Purkinje cells, were impregnated by using the Golgi-Kopsch method; total and mean dendrite segment length, branch frequency, and spine concentration were analyzed morphometrically. Perturbations of endogenous opioid systems caused region-dependent alterations in dendrite complexity and/or spine concentration in all brain areas. Continuous opioid receptor blockade resulted in dramatic increases in dendrite and/or spine elaboration compared to controls at 10 days in all brain regions; however, these increases were only evident in the hippocampus at 21 days. With intermittent blockade, dendrite and/or spine growth were often subnormal, being predominant at day 21. Our results indicate that endogenous opioid systems are critical regulators of neuronal differentiation, and they control growth through an inhibitory mechanism. Considering previous findings demonstrating that neurobehavioral ontogeny is dependent on endogenous opioid-opioid receptor interactions, the present results suggest an opioid-dependent, structure-function relationship between neuronal and behavioral maturation.

Opioid antagonist (naltrexone) stimulation of cell proliferation in human and animal neuroblastoma and human fibrosarcoma cells in culture.

Endogenous opioids play a role in carcinogenic events by serving as inhibitory growth factors that alter cell proliferative events by interaction with opioid receptors. The present study addresses the question of whether endogenous opioid systems function tonically in tissue culture. Using S20Y neuroblastoma, a cell line that produces a growth-related opioid peptide (i.e.[Met5]enkephalin) and contains the zeta receptor known to be associated with growth, the effects of opioid receptor blockade by naltrexone, a potent opioid antagonist, was examined. Drug concentrations of 10(-4) to 10(-8) M naltrexone stimulated cell proliferation, with 32-86% more cells found in the naltrexone groups than control from 12 to 48 h after initiating drug exposure; drug concentrations of 10(-9) to 10(-13) M had no effect on growth. Evaluation of labeling and mitotic indices revealed that both DNA synthesis and mitosis were increased by naltrexone, as were the number of cells with process lengths greater than 40 microns. Naltrexone (10(-6) M) also stimulated the growth of N115 murine neuroblastoma, SK-N-MC human neuroblastoma, and HT-1080 human fibrosarcoma. These results indicate that endogenous opioids function in vitro to regulate growth by inhibitory mechanisms, and do so actively. This autocrine mechanism in tissue culture also occurs in other animal neuroblastoma cell lines, as well as for human neuroblastoma and fibrosarcoma cell lines.

Ultrastructural localization of enkephalin-like immunoreactivity in developing rat cerebellum.

Methionine enkephalin, an endogenous opioid peptide, participates in the regulation of growth in the developing brain. In the present study, enkephalin-like immunoreactivity was localized in the cerebellum of developing and adult rats by immunoelectron microscopy. In 10-day-old animals, enkephalin-like immunoreactivity was found in the somata of proliferating, migrating and differentiating neural cells, and was associated with the plasma membrane, microtubules, filaments, mitochondria, endoplasmic reticulum and nuclear envelope. Both neurons and glia in the cerebellum of the preweaning rat displayed a similar profile of immunoreactivity. Reaction product was also detected in the dendrites and dendritic spines of Purkinje cells where it was concentrated in postsynaptic densities. The majority of internal granule neurons in 10-day-old animals were not immunoreactive, nor were axons, glial processes and postsynaptic elements (with the exception of mossy fiber terminals). At weaning (Day 21), enkephalin-like immunoreactivity was confined primarily to the somata of Purkinje, basket and stellate neurons, and to Purkinje cell dendrites and synaptic spines. Adult rats (day 75) exhibited no enkephalin-like immunoreactivity. These results establish that enkephalin or an enkephalin-like substance can be detected during the ontogeny of both neurons and glia in the cerebellar cortex, and appears to be associated with certain structural elements.

Reepithelialization of the human cornea is regulated by endogenous opioids.

PURPOSE: To determine the influence of endogenous opioid modulation on reepithelialization of the human cornea. METHODS: Eight-millimeter-diameter epithelial defects were created with a trephine and mechanical scraping in the center of human corneas. Resurfacing was studied in organ culture. The size of the defect, the number of specimens with complete reepithelialization, and rate of closure were evaluated using topical fluorescein and morphometric analysis. The influence of opioid receptor blockade was studied using the potent and long-acting opioid antagonist, naltrexone (NTX; 10(-6) M), and the effects of excess (10(-6) M) opioid growth factor (OGF), [Met5]enkephalin, also were determined. The modulatory activity of NTX and OGF on DNA synthesis was evaluated by monitoring the labeling index (LI) using radioactive thymidine. The presence and location of OGF and its receptor (OGFr) were ascertained by immunocytochemistry 1 hour and 24 hours after abrasion. RESULTS: NTX accelerated the wound-healing process, with 21% to 89% less defect than controls observed from 24 to 96 hours. At 72 hours, 62% of the subjects in the NTX group had complete closure of the corneal defects, in contrast to only 19% of the control specimens. All epithelial abrasions were resurfaced in the NTX group between 96 and 120 hours, whereas all controls were not closed until 168 hours. The rate of healing in the NTX group was 1.06 mm2/h compared to a rate of 0.68 mm2/h in the control group. OGF delayed corneal wound healing, with 24% to 260% more defect recorded than in control specimens at day 7. The healing rate of the OGF group was 0.42 mm2/h compared to 0.82 mm2/h for control subjects. The corneal epithelium adjacent to the wound had an LI that was 152% greater than control specimens, whereas OGF decreased the LI of this region by 75%. OGF and OGFr were detected in the epithelium bordering the damaged region at 1 hour, and both peptide and receptor were noted in the regenerating epithelium at 24 hours. CONCLUSIONS: These results indicate that an endogenous opioid is present and functions as a tonically active, receptor-mediated, negative growth factor during reepithelialization of the abraded human cornea.