Topical application of naltrexone facilitates reepithelialization of the cornea in diabetic rabbits.

Delayed corneal reepithelialization is a complication of diabetes, and may lead to ulcers and erosions, which cause ocular morbidity and visual loss. This study examined the efficacy of naltrexone (NTX), a long-acting, potent opioid antagonist, applied topically, to facilitate the repair of standardized corneal abrasions in diabetic (alloxan-induced) New Zealand White rabbits (glucose levels>450 mg/dL). NTX at a concentration of 10(-4)M, or sterile vehicle (SV), was administered topically 4 times per day for 7 days to the abraded eye of uncontrolled Type 1 diabetic (DB), insulin-controlled Type 1 diabetic (DB-IN), or non-diabetic (Normal) rabbits. Wound healing was monitored, and non-invasive (tonopen, pachymeter, hand-held slit lamp, and retinal camera) and invasive (histopathology) measurements evaluated. Corneal reepithelialization in the uncontrolled DB rabbits was significantly enhanced (up to a 47% reduction in wound area) following treatment with NTX relative to both Normal SV and DB SV rabbits at 24, 48, and 56 h following surgery. At 72 h, DB NTX rabbits had residual defects that were 64-82% smaller than Normal and DB SV animals. NTX treated DB-IN rabbits had residual defects that were 9-37% smaller than DB-IN rabbits receiving SV, and 6-40% smaller than Normal rabbits. No signs of toxicity from topical applications were noted. These data confirm and extend those documented in rats that demonstrated a lack of toxicity of NTX at a wide range of dosages, as well as efficacy for enhanced corneal epithelialization. These studies set the stage for clinical trials using NTX as a therapy for diabetic keratopathy.

Transplacental transfer of the opioid growth factor, [Met(5)]-enkephalin, in rats.

Placental transfer of the pentapeptide [Met5]-enkephalin, known to function as a growth regulating factor and neuromodulatory agent, was studied in pregnant Sprague-Dawley rats. Using separation by reversed phase high-performance liquid chromatography, and analysis by derivative spectroscopy, [Met5]-enkephalin was detected in 20-day-old fetal tissue including brain, heart, lung, and kidney. Fetal tissues from pregnant rats given an injection of 40 mg/kg [Met5]-enkephalin on gestation day 20 had markedly elevated levels of peptide within 1 h, indicating the transplacental transfer of this opioid. [Met5]-enkephalin levels were increased from control samples at 1, 2, 4, and 14 h post-injection of peptide, but not at 24 h. Evaluation of breakdown products of [Met5]-enkephalin, along with the related peptide [Leu5]-enkephalin, revealed that elution times differed substantially from [Met5]-enkephalin. These data indicate that [Met5]-enkephalin is present in fetal organs, crosses the placenta, does not appear to be restrictive in organ specificity, and is sustained in fetal tissues at detectable levels for at least 14 h. Given that [Met5]-enkephalin tonically inhibits DNA synthesis in the fetus, these results raise the question of whether an elevated level of this peptide (either maternally or from the fetus) may be detrimental to cellular ontogeny in the fetus, and perhaps have long-term implications for postnatal development.

Epithelial adhesion complexes and organ culture of the human cornea.

The effects of extended organ culture of human cornea on the structural integrity, particularly adhesion complexes, of the epithelium were determined. Human corneas were placed in organ culture using an immersion method. The structure of the cornea prior to culture (0 h) and at 7, 14, and 18 days in culture was evaluated by staining with hematoxylin/eosin, and by ultrastructural analysis that included a morphometric study of the type and number of adhesion complexes. Human corneas prepared immediately (0 h) and those in culture after 7 days showed similar structural organization and anatomical features. In contrast to 0 h specimens, the corneal epithelium at 14 days in culture exhibited signs of deterioration, with increases in cellular contraction, extracellular space, electron density of the cytoplasm, nuclear invaginations, and nucleoplasmic opacity, as well as aggregations of junctional complexes between cells. At 18 days in culture, the ocular surface epithelium was markedly reduced in thickness and consisted of no more than 2-3 cell layers; a distinct basal layer was not detected, and the morphology of the suprabasal and basal layers were similar. The basement membrane was disorganized, and anchoring complexes composed of hemidesmosomes were often absent. The number and type of the anchoring complexes associated with the basal epithelium and Bowman's membrane were comparable until 14 days of age, although the total number of hemidesmosomes per microm of epithelial plasmalemma was subnormal. After 2 weeks in culture, there were 38-72% fewer anchoring complexes and a decrease of 44% in the number of hemidesmosomes/microm of membrane from samples prepared immediately and after 7 days in culture. These results indicate that the structural integrity of human corneal epithelium in organ culture is compromised after 14 days in vitro using an immersion system of tissue culture. Thus, long-term use of cultures to define homeostasis and wound healing of the ocular surface epithelium, which necessitates normal architecture including anchoring complexes between epithelium and Bowman's membrane, may not be appropriate and requires careful monitoring both qualitatively and quantitatively at the electron microscopic level of resolution.

Molecular characterization and distribution of the opioid growth factor receptor (OGFr) in mouse.

The native opioid growth factor (OGF), [Met(5)]-enkephalin, is a tonic inhibitory peptide that modulates cell proliferation and tissue organization during development, cancer, cellular renewal, wound healing, and angiogenesis. OGF action is mediated by a receptor mechanism. The receptor for OGF, OGFr, has been cloned and sequenced in humans and rats. Using primers based on the rat OGFr cDNA, and a mouse embryo expressed sequence tag, the full-length 2.1 kb mouse OGFr cDNA was sequenced. The open reading frame was found to encode a protein of 634 amino acids, and 14 imperfect repeats of 9 amino acids each were a prominent feature. The molecular weight of OGFr was calculated as 70679, and the isoelectric point was 4.5. Northern blot analysis revealed a 2.1 kb OGFr mRNA transcript in adult mouse brain, heart, lung, liver, kidney, and triceps surae muscle. The amino acids for mouse and rat OGFr were 93% similar and 91% identical, but the mouse and human shared only a 70% similarity and a 58% identity. These results emphasize the molecular validity of OGFr, and explain the interaction of OGF with respect to normal and abnormal growth in mouse cells and tissues.

Cellular dynamics of corneal wound re-epithelialization in the rat. III. Mitotic activity.

The number and distribution of mitotic epithelial cells in the ocular surface during homeostasis and in response to abrasion of the mammalian cornea were determined. Normal rats and those receiving a 3 mm diameter centrally located epithelial defect, received an intraperitoneal injection of colchicine 6 h prior to sacrifice. Mitosis in the basal epithelium during homeostasis was comparable in magnitude across the ocular surface epithelium, with the exception of a few mitotic figures in the midline. Thirty percent of the mitotic figures were in the basal layer (layer 1), and 70% were in layer 2; the cells in layer 2 were often noted to retain connection to the basal lamina by cytoplasmic stalks. Mitosis was rarely noted in the regenerating epithelium. However, summation of M phase cells in both the basal and suprabasal epithelium adjacent to the wound showed increases of 3- and 5-fold at 30 and 36 h after abrasion, respectively, from levels at homeostasis and the time of injury. In striking contrast to homeostatic epithelium, 80% of the mitotic cells were located in layer 1 of the corneal epithelium, with normal distribution observed by 72 h. Mitosis in the limbus and conjunctiva was increased 3-fold at 30 h and 24 h, respectively, from values at homeostasis and the time of debridement. These results, using rigorous statistical analysis and precise topographic assessment, showed that mitosis is not impeded - but rather often accelerated - following denuding of the corneal epithelium and that the spatial distribution of mitotic cells is correlated with wounding. The data revealed that re-epithelialization of the corneal epithelium is not dependent on mitosis in the regenerating epithelium, but rather in the adjacent unwounded epithelium of the cornea, with most cells being located in the basal layer until re-epithelialization is completed. Mitotic cells in the limbus and conjunctiva may be related to replenishment of ocular surface epithelial cells used in the repair process rather than directly supplying the abraded surface.

Opioid growth factor regulates the cell cycle of human neoplasias.

The native opioid growth factor (OGF), [Met5]-enkephalin, is a tonic inhibitory peptide that modulates cell proliferation and migration, as well as tissue organization, during development, cancer, homeostatic cellular renewal, wound healing, and angiogenesis. OGF action is mediated by the OGF receptor (OGFr). To investigate the target of OGF as to cell proliferation, the effects of excess OGF, and a deprivation of OGF-OGFr interaction by an opioid antagonist, naltrexone (NTX), were examined in 3 human cancer cell lines: pancreatic (BxPC-3), colon (HT-29), and head and neck (CAL-27). OGF exposure decreased growth, DNA synthesis, and mitosis, and increased the doubling time from control levels. FACS analysis revealed a marked increase in cells in the G0/G1 phase and compensatory reduction in cells in S and G2/M phases. Consistent with this observation, the percentage of labeled mitosis (PLM) analysis showed a notable increase in the time of the G0/G1 phase. Receptor blockade with NTX increased the rate of growth, length of DNA synthesis and mitotic phases, and decreased doubling time from control values. FACS analysis indicated an increase in the proportion of cells in S and G2/M phases, and a decrease in the number of cells in the G0/G1 phase. PLM evaluation demonstrated a shortening of the length of the S and G2 phases in the 3 cell lines, and decreases in the M and G0/G1 phases in some cancers. These results indicate that OGF action is directed at the G0/G1 phase, but interruption of OGF-OGFr interfacing has widespread repercussions on the cell cycle. The data on blockade of OGF-OGFr during log phase growth suggest a requisite escorting of the growth peptide and its receptor through the cell cycle.

Elevated levels of opioid growth factor in the plasma of patients with pancreatic cancer.

Opioid growth factor (OGF, [Met5]-enkephalin) is an endogenous peptide that regulates the growth of human pancreatic cancer. To evaluate whether human subjects with pancreatic cancer have alterations in plasma levels of OGF, fasting blood samples were obtained from 15 patients with histologically confirmed pancreatic adenocarcinoma. Forty-five subjects with other malignancies, 20 patients with acute pancreatitis, and 30 aged-matched patients without cancer served as control populations. Individuals with pancreatic cancer had OGF values, as determined by radioimmunoassay, that were up to 7.3-fold greater than control subjects. No differences were found between OGF values obtained from patients with other malignancies, acute pancreatitis, or subjects without cancer. The sensitivity and specificity of OGF for pancreatic cancer were greater than either CA 19-9 or CEA. These data indicate that pancreatic cancer is associated with a marked increase in plasma OGF levels and suggest that this peptide may serve as a useful diagnostic tool in the screening for this disease.

Opioid growth factor modulates angiogenesis.

OBJECTIVE: Induced angiogenesis has recently been attempted as a therapeutic modality in patients with occlusive arterial atherosclerotic disease. We investigated the possible role of endogenous opioids in the modulation of angiogenesis. METHODS: Chick chorioallantoic membrane was used as an in vivo model to study angiogenesis. Fertilized chick eggs were incubated for 3 days, explanted, and incubated for an additional 2 days. Three-millimeter methylcellulose disks were placed on the surface of the chorioallantoic membrane; each disk contained opioid growth factor ([Met(5)]-enkephalin; 5 microgram), the short-acting opioid receptor antagonist naloxone (5 microgram), opioid growth factor and naloxone together (5 microgram of each), the long-acting opioid antagonist naltrexone (5 microgram), or distilled water (control). A second series of experiments was performed with distilled water, the angiogenic inhibitor retinoic acid (1 microgram), and vascular endothelial growth factor (1 microgram) to further evaluate our model. The developing vasculature was imaged 2 days later with a digital camera and exported to a computer for image analysis. Total number of blood vessels, total vessel length, and mean vessel length were measured within a 100-mm(2) region surrounding each applied disk. Immunocytochemical analysis was performed with antibodies directed against opioid growth factor and its receptor (OGFr). RESULTS: Opioid growth factor had a significant inhibitory effect on angiogenesis, both the number of blood vessels and the total vessel length being decreased (by 35% and 20%, respectively) in comparison with control levels (P <.005). The simultaneous addition of naloxone and opioid growth factor had no effect on blood vessel growth, nor did naloxone alone. Chorioallantoic membranes exposed to naltrexone displayed increases of 51% and 24% in blood vessel number and length, respectively, in comparison with control specimens (P <.005). These results indicate that the opioid growth factor effects are receptor mediated and tonically active. Immunocytochemistry demonstrated the presence of both opioid growth factor and OGFr within the endothelial cells and mesenchymal cells of the developing chorioallantoic membrane vessel wall. Retinoic acid significantly reduced the number and the total length of blood vessels, whereas vascular endothelial growth factor increased both the number and the length of blood vessels in comparison with the controls (P <.0001). The magnitude of opioid growth factor's effects were comparable to those seen with retinoic acid, whereas inhibition of opioid growth factor with naltrexone induced an increase in total vessel length comparable to that for vascular endothelial growth factor. CONCLUSIONS: These results demonstrate for the first time that endogenous opioids modulate in vivo angiogenesis. Opioid growth factor is a tonically active peptide that has a receptor-mediated action in regulating angiogenesis in developing endothelial and mesenchymal vascular cells.

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.

In vivo effects of chronic treatment with [MET5]-enkephalin on hematological values and natural killer cell activity in athymic mice.

The role of endogenous opioids in immunological mechanisms was examined by subjecting athymic (nu/nu) mice to chronic injections of the opioid agonist [Met5]-enkephalin (MET) or continuous opioid receptor blockade with naltrexone (NTX). After 8 days of treatment, neither excess peptide nor deprivation of opioids from receptors had any effect on body weight, spleen index (spleen to body weight ratio), total and differential white blood cell counts, and natural killer (NK) cell activity in peripheral blood or splenic lymphocytes. At 28 days, chronic treatment with MET or NTX had no effect on any of these parameters with the exception of an elevation from controls in NK cell activity in peripheral blood in mice receiving NTX, and subnormal NK cell activity related to splenic lymphocytes in the MET group. These results suggest that chronic exposure to an opioid agonist, or persistent opioid receptor blockade, have little influence on a variety of immunological properties in athymic mice, suggesting that native opioids such as MET do not play a marked role in defense mechanisms in the athymic mouse.

Cloning, sequencing, chromosomal location, and function of cDNAs encoding an opioid growth factor receptor (OGFr) in humans.

The native opioid growth factor (OGF), [Met(5)]-enkephalin, is a tonic inhibitory peptide that modulates cell proliferation and tissue organization during development, cancer, cellular renewal, wound healing, and angiogenesis. OGF action is mediated by a receptor mechanism. We have cloned and sequenced cDNAs encoding multiple spliced forms of a human OGF receptor. The open reading frame in the longest cDNA was found to encode a protein of 697 amino acids, and 8 imperfect repeats of 20 amino acids each were a prominent feature. Altogether, five alternatively spliced forms were observed. The cDNA hybridized to mRNA from a variety of normal and neoplastic cells and tissues. Functional studies using antisense oligonucleotides to OGFr demonstrated an enhancement in cell growth. Fluorescent in situ hybridization (FISH) experiments showed the chromosomal location to be 20q13.3. This OGF receptor has no homology to classical opioid receptors. These results provide molecular validity for the interaction of OGF and OGF receptor in the regulation of growth processes in humans.

The opioid growth factor receptor in human head and neck squamous cell carcinoma.

Carcinoma of the head and neck is the sixth leading cause of cancer in the world, and the third most common neoplasia in developing countries. More than 90% of head and neck cancers are squamous cell carcinomas (SCCHN). Approximately half of the patients afflicted die within 5 years of diagnosis and survival rates for cancer of the upper aero-digestive tract have not changed in 25 years. The opioid growth factor (OGF), ¿Met5-enkephalin, inhibits the growth of SCCHN in vitro and in vivo, and acts in a receptor-mediated fashion. Receptor binding assays using CAL-27 human SCCHN cells in culture and ¿3H-¿Met5-enkephalin were employed to identify and characterize the receptor responsible for the growth-regulatory effects of OGF. Specific and saturable binding was recorded, and Scatchard analysis showed that the data were consistent for a single binding site with a binding affinity (Kd) of 5.0+/-0.9 nM and maximal binding capacity (Bmax) of 47.5+/-1.7 fmol/mg protein. Subcellular fractionation studies determined that the optimal binding occurred with the nuclear fraction. Competition experiments demonstrated that cold ¿Met5-enkephalin was at least 7-fold greater than ligands selective for classical opioid receptors. Binding was detected in 4 other SCCHN cell lines. Receptor number in xenografts of CAL-27 was decreased almost 5-fold compared to the same cells grown in vitro. Binding to radiolabeled ¿Met5-enkephalin was recorded in SCCHN obtained from surgical resections. The function, pharmacological and biochemical characteristics, distribution and subcellular location of OGF binding in human SCCHN were consonant with the OGF receptor (OGFr).

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.

Identification and characterization of opioid growth factor receptor in human pancreatic adenocarcinoma.

Pancreatic cancer is the fourth most common cancer-related mortality in the United States, and the ninth most common cause of death from cancer worldwide. The opioid growth factor (OGF), [Met5]-enkephalin, inhibits the growth of human pancreatic adenocarcinoma in vitro and in vivo, and acts in a receptor-mediated fashion. Ligand binding assays using PANC-1 human pancreatic tumor cells and [3H]-[Met5]-enkephalin were performed to identify and characterize the receptor responsible for the growth-regulatory effects of OGF in pancreatic cancer. Specific and saturable binding was detected, and a Scatchard analysis revealed that the data were consistent for a single binding site with a binding affinity of 1.2+/-0.3 nM and a binding capacity of 36.4+/-4.1 fmol/mg protein. Subcellular fractionation studies showed that binding was restricted to the nuclear fraction. Competition experiments revealed that cold [Met5]-enkephalin was the most effective ligand at displacing [3H]-[Met5]-enkephalin; ligands for mu, delta, and kappa opioid receptors exhibited little or no competition. Binding was detected in 3 other human pancreatic tumor cell lines. Receptor number in xenografts of Capan-1 was decreased 8.6-fold compared to the same cells grown in tissue culture. Binding to radiolabeled [Met5]-enkephalin was detected in pancreatic cancers obtained from surgical resections. Binding capacity, but not binding affinity, was 7.1-fold greater in normal pancreatic tissues than in pancreatic neoplasia. The function, pharmacological and biochemical characteristics, distribution, and subcellular location of OGF binding in human pancreatic cancer were consistent with the OGF receptor (OGFr). In addition, human pancreatic cancer appears to have a low number of receptors for OGF, having the net effect of diminishing control of cellular replicative events.

Cloning, sequencing, expression and function of a cDNA encoding a receptor for the opioid growth factor, [Met(5)]enkephalin.

The native opioid growth factor (OGF), [Met(5)]enkephalin, is a tonic inhibitory peptide that modulates cell proliferation and tissue organization during development, cancer, cellular renewal, wound healing and angiogenesis. OGF action is mediated by a receptor mechanism. We have cloned and sequenced a 2.1-kilobase (kb) cDNA for a receptor to OGF (OGFr). The open reading frame was found to encode a protein of 580 amino acids, and eight imperfect repeats of nine amino acids each were a prominent feature. The protein encoded by this cDNA exhibited the pharmacological, temporal and spatial characteristics of the OGFr. Functional studies using antisense technology demonstrated an enhancement in cell growth. The molecular organization of the OGFr has no homology to classical opioid receptors. These results provide molecular validity for the interaction of OGF and OGFr in the regulation of growth processes.

Human renal cell cancer proliferation in tissue culture is tonically inhibited by opioid growth factor.

PURPOSE: Peptide growth factors alter cellular events by binding to specific receptors. One group of peptides, the endogenous opioids, is important in the growth of normal and neoplastic tissue. [Met5]enkephalin, also termed opioid growth factor (OGF), is a tonically active inhibitory factor that interacts with the OGF receptor, OGFr, formerly identified as Greek zeta (zeta) and appears to be autocrine produced by human cancer cells. This study examined the hypothesis that OGF directly inhibits proliferation of renal cell carcinoma in tissue culture. MATERIALS AND METHODS: Human renal cancer cells (Caki-2) were grown using routine tissue culture techniques. A variety of natural and synthetic opioids including OGF, opioid antagonists, and opioid antibodies were added to renal cancer cell cultures to determine role of these peptides in renal cell carcinoma. The experiments were repeated in serum-free media, and with 4 other human renal cancer cell lines: Caki-2, A498, SN12C, and ACHN. Immunocytochemistry was performed to examine the presence of OGF and its receptor. RESULTS: OGF was the most potent opioid peptide to influence human renal cell carcinoma. OGF depressed growth within 12 hours of treatment, with cell numbers subnormal by up to 48% of control levels. OGF action was receptor mediated, reversible, not cytotoxic, neutralized by antibodies to the peptide, and detected in the human renal cell carcinoma lines examined. OGF appeared to be autocrine produced and secreted, and was constitutively expressed. Both OGF and its receptor were detected in these cells. CONCLUSION: OGF tonically inhibits renal cancer cell proliferation in tissue culture, and may play a role in the pathogenesis and management of human renal cell cancer.

Cellular dynamics of corneal wound re-epithelialization in the rat. II. DNA synthesis of the ocular surface epithelium following wounding.

To determine the fate of ocular surface epithelial cells in response to corneal injury, epithelial cells undergoing DNA synthesis were labeled at discrete time points following abrasion. A 3-mm diameter epithelial defect was made in the center of the rat cornea. One hour prior to sacrifice, animals received an intraperitoneal injection of [3H]-thymidine. DNA synthesis in the regenerating epithelium was of low abundance (< 2%). The undamaged corneal epithelium had a labeling index (LI) that was markedly elevated from unwounded specimens in the first 48 h after abrasion. The LIs of suprabasal cells were increased at 24 h and 36 h; these cells were believed to be displaced basal epithelial cells. Basal and suprabasal cells of the limbus and conjunctiva exhibited increases in LIs from unwounded subjects at singular timepoints (within 24 h of injury). These results, using rigorous statistical analysis, show that DNA synthesis is not impeded--but rather often accelerated--following denuding of the corneal epithelium. Re-epithelialization is not dependent on DNA synthesis in the regenerating region, but appears to be related to an increase in DNA synthesis (along with centripetal migration) in the adjacent, undamaged epithelium. The increase in DNA synthesis occurring in the limbus and conjunctiva does not directly supply cells to the regenerating region within the 72 h required for epithelial restitution, but is conjectured to serve in replenishing ocular surface epithelial cells used in the repair process.

Opioid growth factor and organ development in rat and human embryos.

In addition to neurotransmission, the native opioid peptide, [Met5]enkephalin, is a tonically active inhibitory growth molecule that is termed opioid growth factor (OGF). OGF interacts with the zeta (zeta) opioid receptor to influence cell proliferation and tissue organization. We now identify OGF and the zeta receptor in embryonic derivatives including ectoderm, mesoderm, and endoderm of the rat on gestation day 20. Messenger RNA for preproenkephalin (PPE), the precursor of OGF, was detected in the developing cells, suggesting an autocrine production of this peptide. Acute exposure of the pregnant female to OGF resulted in a decrease in DNA synthesis in cells of organs representing all three germ layers, and did so in a receptor-mediated fashion. The influence of OGF was direct, as evidenced in organ culture studies. Blockade of endogenous opioid interaction using naltrexone (NTX) produced an increase in DNA synthesis, indicating the constitutive and functional nature of opioid activity on growth during prenatal life. Human fetal cells contained OGF and the zeta receptor. These data support the hypothesis that endogenous opioid modulation of organ development is a fundamental principle of mammalian embryogenesis, and that OGF has a profound influence on ontogeny. Irregularities in the role of opioids as growth regulators in relationship to the more than 500,000 newborns suffering from birth defects each year in the US needs to be examined.

A rapid sample preparation method for the HPLC determination of the opioid antagonist naltrexone in serum.

HPLC with UV and electrochemical detection has routinely been employed for the determination of the opioid antagonist naltrexone in serum. Sample preparation protocols range from liquid/liquid to solid phase extraction. The sample preparation described in this communication uses ultrafiltration as the mode of sample preparation prior to HPLC analysis. The method is accurate, precise and saves considerable time compared to previously published techniques.

Regulation of human head and neck squamous cell carcinoma growth in tissue culture by opioid growth factor.

Squamous cell carcinoma of the head and neck (SCCHN) is the sixth most common malignancy worldwide. Approximately half of the patients afflicted die within 5 years of diagnosis, and surviving patients may be left with severe esthetic and functional compromise. In this study, we discovered that an endogenous opioid peptide, [Met5]-enkephalin, inhibited the growth of human SCCHN in vitro; in view of this pentapeptide's action it has been termed opioid growth factor (OGF). OGF was found to be a constitutively expressed, receptor-mediated growth inhibitory agent that appears to be autocrine produced and secreted. Growth regulation was dose-related, reversible, cytostatic, and independent of serum. All 6 human SCCHN cell lines examined exhibited growth modulation by OGF. Blockade of peptide-receptor interaction by opioid antagonists (naltrexone), or addition of antibody to OGF, resulted in substantial increases in cell number compared to control levels, showing the tonic nature of OGF-zeta activity. Immunocytochemical studies detected both OGF and its related receptor, zeta, in these cells, correlating with earlier findings of peptide and receptor in specimens of SCCHN obtained at surgery. These data suggest that a native opioid peptide, OGF, interacts with a novel opioid receptor, zeta, to tonically arrest the growth of human SCCHN.