Transplacental transfer of the opioid growth factor, [Met]-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.     

Endogenous opioid systems regulate growth of neural tumor cells in culture

Endogenous opioid systems (i.e., opioids and opioid receptors) play a role in neural cancer. Using a tissue culture system of S20Y murine neuroblastoma to assess the effects of opioids on growth, [Met5]-enkephalin was the most potent compound to influence cell replication. With a median effective concentration of 10(-10) M, this peptide inhibited cell proliferation in a stereospecific and naloxone-reversible manner. [Met5]-Enkephalin depressed both DNA synthesis and mitosis. [Met5]-Enkephalin was detected in neuroblastoma cells by radioimmunoassay, and was found to increase in concentration in culture media over time, suggesting that these cells produced the peptide. Immunocytochemistry showed [Met5]-enkephalin-like activity in the cortical cytoplasm, but not the cell nucleus, of neuroblastoma cells. Binding of [3H]-[Met5]-enkephalin specific and saturable, and Scatchard analysis yielded a Kd of 1.2 +/- 0.1 nM and a binding capacity of 50.2 +/- 4.3 fmol/mg protein. [Met5]-Enkephalin also depressed the growth of N115 murine neuroblastoma, SK-N-MC human neuroblastoma, and HT-1080 human fibrosarcoma. These results indicate that [Met5]-enkephalin, a naturally occurring pentapeptide that is derived from proenkephalin A, is a potent inhibitor of cell growth. Since cancer cells produce [Met5]-enkephalin, and contain a binding site to this ligand, endogenous opioid systems appear to control cell proliferation by an autocrine mechanism.     

Endogenous opioids regulate cell proliferation in the retina of developing rat

The role of endogenous opioids and opioid receptors (endogenous opioid systems) in modulating cell proliferation in the developing mammalian retina was examined in 1-day-old rats. In contrast to a labeling index (LI) of 35.8% in control animals, administration of the opioid peptide [Met5]-enkephalin (100 micrograms/kg) significantly reduced (10.6%) the proportion of cells incorporating [3H]thymidine; concomitant injection of 1 mg/kg naloxone blocked the inhibitory effects of [Met5]-enkephalin on cell division. Naloxone (1 mg/kg) alone did not alter the LI. The interruption of endogenous opioid-opioid receptor interaction by naltrexone (50 mg/kg), a potent opioid antagonist, was accompanied by a significant increase (6.4%) in the LI relative to control levels. Immunocytochemical experiments revealed the presence of enkephalin-like immunoreactivity, with staining of the cortical cytoplasm of proliferating and differentiating retinal cells recorded; no immunoreactivity was noted in the adult retina. In vitro autoradiography using 125I-[Met5]-enkephalin indicated that [Met5]-enkephalin binding sites were localized to the developing retina; no binding of the radiolabeled ligand was recorded in the adult retina. These results demonstrate the presence of growth-related endogenous opioids and opioid receptors in the developing mammalian retina, but not in adult retina, and suggest that endogenous opioids serve as natural inhibitory trophic factors that tonically regulate cell proliferation.     

Opioid pathways in an avian retina. I. The content, biosynthesis, and release of Met5-enkephalin

By means of an enzyme-linked immunosorbent assay, the concentration of enkephalin-immunoreactive substances was estimated to be about 25 nM in the chicken retina. The biosynthesis of 3H-Met5-enkephalin in this retina was studied by a pulse-chase incubation technique. Isolated retinas were incubated with 0.2 ml of oxygenated Ringer's solution containing 40 microCi of [3H]methionine and trasylol, a peptidase inhibitor, for 30 min at room temperature. The tissue was then rinsed three times in large volumes of Ringer's solution and incubated in the same solution containing unlabeled methionine (100 micrograms/ml) and trasylol for at least another hour. The products synthesized were extracted in acetic acid and assayed by high performance liquid chromatography (HPLC) and immunoassay. A peak of radioactivity that comigrated with Met5-enkephalin on HPLC and cross-reacted with antibodies against enkephalins was detected. The level of 3H-Met5-enkephalin radioactivity increased approximately 10-fold as the chase-incubation period increased from 0 to 120 min, suggesting that, as in other tissues, Met5-enkephalin may be synthesized as part of a larger precursor. The newly synthesized Met5-enkephalin could be released by depolarization of the retina with high extracellular K+ concentration. Furthermore, this K+-stimulated release was greatly suppressed by 5 mM Co2+ in the medium, suggesting that this release is Ca2+-dependent and may be synaptically mediated.     

Detection of methionine-enkephalin at the 10(-16) mole level

A high performance radioimmunoassay for Met5-enkephalin has been developed, using two ways to circumvent the difficulties of obtaining such an assay for the opiate peptides. First, hemocyanin was used as the carrier for the antigen and secondly, a new iodination method for Met5-enkephalin was established, resulting in a [125I]probe with an intact methionyl residue. The Met5-enkephalin antibodies exhibited a very high affinity (Kd = 5 X 10(-12) M) and Met5-enkephalin could be detected at the 10(-16) mole level. The Met5-enkephalin content of individual cervical ganglia and individual cochlea was measured. We also measured the Met5-enkephalin content of brain from different areas of two rat strains: no significant differences were found.     

Localization of preproenkephalin A mRNA in the neonatal rat retina.

An opioid growth factor, [Met5]-enkephalin, is known to regulate developmental events in the neonatal rat retina. This growth factor interacts with the zeta (zeta) opioid receptor to modulate retinal ontogeny. Both peptide and receptor are present in developing retina, but not in adult retina. We have used in situ hybridization histochemistry to identify and localize preproenkephalin A mRNA in the neonatal rat retina. Preproenkephalin mRNA was localized to the ganglion cell layer, with some radiolabeling found in the neuroblast layer. This result indicates that 1) the mRNA to preproenkephalin A is present during the critical stage of development in the neonatal retina that coincides with the presence of the growth-regulating peptide, [Met5]-enkephalin, and 2) that the source of the opioid growth factor controlling the production of retinal cells appears to be autocrine (i.e., retinal neuroblasts) and paracrine (i.e., ganglion cells) in nature.     

Zeta (zeta), a growth-related opioid receptor in developing rat cerebellum: identification and characterization.

Endogenous opioids and opioid receptors (i.e. endogenous opioid systems) are expressed during neural ontogeny, and play a role in the development of the nervous system. Using [3H][Met5]-enkephalin, a potent ligand involved in neural growth, particularly cell proliferation, specific and saturable binding was detected in homogenates of 6-day-old rat cerebellum; the data were consistent with a single binding site. Scatchard analysis yielded a binding affinity (Kd) of 2.2 nM and a binding capacity (Bmax) of 22.3 fmol/mg protein. Binding was linear with protein concentration, dependent on time, temperature, and pH, and was sensitive to Na+, Mg2+, and guanyl nucleotides. Optimal binding required protease inhibitors, and pretreatment of the homogenates with trypsin markedly reduced binding, suggesting that the binding site was proteinaceous in character. The [Met5]-enkephalin binding site was an integral membrane protein located in the nuclear fraction. Competition experiments indicated that [Met5] enkephalin was the most potent displacer of [3H][Met5]-enkephalin, and that binding was stereospecific. In the adult rat cerebellum, non-opioid receptor binding of [3H][Met5]-enkephalin was recorded, mu and kappa receptors were also found in the developing rat cerebellum, while mu, delta, and kappa receptors were recorded in adult cerebellar tissue. The function, pharmacological and biochemical characteristics, subcellular distribution, and temporal expression of the [Met5]-enkephalin binding site suggest the presence of a unique opioid receptor, termed zeta (zeta), in the developing nervous system.     

The role of cytokines in mediating effects of prenatal infection on the fetus: implications for schizophrenia

Maternal infections with bacterial or viral agents during pregnancy are associated with an increased incidence of schizophrenia in the offspring at adulthood although little is known about the mechanism by which maternal infection might affect fetal neurodevelopment. Exposure of pregnant rodents to the bacterial endotoxin, lipopolysaccharide (LPS), results in behavioral deficits in the adult offspring that are relevant to schizophrenia. It is however unknown whether these effects are due to the direct action of the inflammatory stimulus on the developing fetus, or due to secondary immune mediators (cytokines) activated at maternal/fetal sites. In this study we sought to elucidate the site of action of LPS, following a single intraperitoneal (i.p.) injection, in pregnant rats at gestation day 18. Animals received 5 muCi of iodinated LPS ((125)I-LPS) and its distribution was assessed in maternal/fetal tissues (1-8 h). In addition, induction of the inflammatory cytokines, TNF-alpha, IL-1beta and IL-6, was measured in maternal/fetal tissues following maternal LPS challenge (0.05 mg/kg, i.p.) (2-8 h). (125)I-LPS was detected in maternal tissues and placenta, but not the fetus. This distribution was accompanied by significant increases in TNF-alpha, IL-1beta and IL-6 in maternal plasma and placenta, but not in fetal liver or brain. A significant increase in IL-1beta was however detected in fetal plasma, possibly due to transfer from the maternal circulation or placenta. Collectively, these data suggest that effects of maternal LPS exposure on the developing fetal brain are not mediated by the direct action of LPS, but via indirect actions at the level of the maternal circulation or placenta.     

Systemic administration of kainic acid differentially regulates the levels of prodynorphin and proenkephalin mRNA and peptides in the rat hippocampus.

The effects of systemic kainic acid (KA) administration on hippocampal levels of prodynorphin and proenkephalin mRNA, as well as opioid peptides derived from these precursors, were evaluated. A single subcutaneous injection of KA induced a range of seizure states, from mild wet dog shakes to generalized motor seizures. Northern blot analysis of hippocampal mRNA revealed an increase in both prodynorphin and proenkephalin mRNA levels which corresponded to the intensity of the convulsions. Conversely, hippocampal levels of immunoreactive dynorphin A (1-8) and [Met]5-enkephalin were decreased as a function of seizure frequency and intensity. The time course of KA-induced alterations in prodynorphin and proenkephalin mRNA and peptide levels was also investigated. Hippocampal prodynorphin mRNA levels rose at a dramatic rate. At 3 h following KA administration, mRNA levels were maximally elevated approximately 13-fold. The levels decreased over a 48 h period, eventually reaching control values. In contrast, proenkephalin mRNA levels increased more slowly. At 24 h, a maximal 24-fold increase was observed. At 72 h after injection, proenkephalin mRNA levels were still slightly elevated. In the same experiment, immunoreactive enkephalin peptide levels, although somewhat decreased at 3-12 h, began to increase between 12 and 24 h after injection, and were still rising at 72 h. In marked contrast, immunoreactive dynorphin peptide levels ranged from 40% to 80% of control values at all times tested.(ABSTRACT TRUNCATED AT 250 WORDS)     

Endogenous opioids and neural cancer: an immunoelectron microscopic study

[Met5]-enkephalin, an endogenous opioid peptide derived from proenkephalin A, participates in tumorigenic events by serving as a natural trophic factor that inhibits cell replication. In order to understand how endogenous opioids function in modulating neoplasia, the present study examined the fine structural association of enkephalin with the cellular components of a tumor cell. Immunoelectron microscopic studies were undertaken using antibodies recognizing [Met5]-enkephalin-like substances, and murine S20Y neuroblastoma cells that are known to be responsive to endogenous opioid modulation. Enkephalin was found throughout the cell body and process. Immunoreactivity was associated with the plasma membrane, outer nuclear envelope, and a variety of organelles. With the exception of aggregates of immunoreactivity subjacent to the inner nuclear envelope, the nucleus was not reactive. These results establish that growth-related enkephalins are localized discretely within neuroblastoma cells. Since neuroblastoma cells produce and secrete enkephalins, and enkephalins interact with receptors to mediate actions on cell replication, this study examined enkephalins involved in two different patterns of traffic; further work will be needed to examine each aspect.     

Distribution of Phenobarbital and Phenytoin in Pregnant Rats and Their Fetuses

Studies were conducted to determine the extent of fetal exposure to phenobarbital (PB) and phenytoin (PHT) in pregnant rats. Two groups of rats determined to be in the third week of pregnancy received 30 mg/kg as a single intraperitoneal (i.p.) dose of either PB or PHT. At various times (from 15 min to 48 h for PB and from 20 min to 16 h for PHT), the rats were killed and whole blood, plasma, maternal brain, fetal brain and liver, whole fetuses, and placentas were rapidly collected. Concentrations of PB, PHT, and its major metabolite, p-hydroxyphenytoin (HPPH), were measured by high-performance liquid chromatography (HPLC). PB and PHT appeared rapidly in plasma and all tissues; the maximum HPPH concentrations appeared much later. The highest PB concentrations were detected in plasma, followed by placenta, whole fetus, maternal brain, fetal liver, and fetal brain. In contrast, PHT concentrations were higher in fetal brain and liver than in whole fetuses. HPPH concentrations were much lower than PHT and were not detected in maternal brain. These results show that PB and PHT readily distribute to the rat fetus, that drug distribution within the fetus is regional, and that changes in maternal plasma concentrations of these antiepileptic drugs (AEDs) are accompanied by parallel changes in fetal tissue.     

Maternal thermal stimulation changes metabolic activity in fetal hypothalamus

To study whether the central nervous system in the perinatal fetal rat can operate during maternal cooling and warming, we examined the 2-deoxy-D-[14C]glucose ([14C]DG) uptake in the fetal brain. Full-term pregnant rats were placed at three different ambient temperatures of 35-37 degrees C, 24-25 degrees C and 0-10 degrees C. Saline containing 20 microCi/100 g of [14C]DG was injected into the superior caval vein in the pregnant rats. Forty-five min after the injection, the mother rats were decapitated and the fetal brains were taken out for autoradiography. The [14C]DG uptake was significantly influenced by maternal thermal stimulation in the hypothalamus and not in other brain regions examined such as the cerebral cortex, the basal ganglia and the limbic nuclei. Glucose utilization in the fetal anterior hypothalamus, paraventricular hypothalamus and dorsomedial hypothalamus significantly increased when the mother rat was exposed to heat compared to when the mother rat was in the thermoneutral condition. During maternal cooling, glucose utilization in the ventromedial hypothalamus and dorsomedial hypothalamus significantly decreased. There was no area activated by cooling and/or inhibited by warming. Compared to a similar study in adult rats (Am. J. Physiol., 248 (1985) R84-92), the present results suggest that although the perinatal fetal brain does not respond to thermal stimulation in terms of glucose utilization as fully as the adults, a few hypothalamic nuclei have already acquired thermal responses, which might be a possible neuronal basis for the thermoregulatory responses just after birth in rats.     

Endogenous opioid systems and neural cancer: Transmission and scanning electron microscopic studies of murine neuroblastoma in tissue culture

Endogenous opioid systems participate in carcinogenic events. To understand further the action of opioids on growth, S20Y neuroblastoma cells in tissue culture were exposed to i) [Met5]-enkephalin, a naturally occurring opioid pentapeptide, at a concentration (10(-6) M) that inhibits cell replication by 66% of control levels, ii) [Met5]-enkephalin (10(-6) M) and the opioid antagonist naloxone (10(-6) M) which blocks opioid agonist action, or iii) naltrexone (10(-6) M), a potent antagonist that disrupts endogenous opioid-opioid receptor interaction and increased cell number 76% above control values. The morphology of cells exposed to these agents for 2-4 days were similar to controls (i.e., exposed to sterile water) as determined by scanning and transmission electron microscopy. These results support the hypothesis that endogenous opioid systems act as trophic factors as they regulate growth; their effects on cell growth and survival, however, do not alter the basic ultrastructural morphology of the cells. Moreover, these data strengthen the validity of paradigms and therapeutic regimens that utilize opioid agonists and antagonists to modulate the relationship of endogenous opioid-opioid receptor interactions in neural cancer.     

Maternal-fetal distribution of manganese in the rat following inhalation exposure to manganese sulfate

Studies examining the pharmacokinetics of manganese during pregnancy have largely focused on the oral route of exposure and have shown that the amount of manganese that crosses the rodent placenta is low. However, limited information exists regarding the distribution of manganese in fetal tissues following inhalation. The objective of this study was to determine manganese body burden in CD rats and fetuses following inhalation of a MnSO4 aerosol during pregnancy. Animals were evaluated following pre-breeding (2 weeks), mating (up to 14 days) and gestational (from gestation day (GD) 0 though 20) exposure to air or MnSO4 (0.05, 0.5, or 1 mg Mn/m(3)) for 6h/day, 7 days/week. The following maternal samples were collected for manganese analysis: whole blood, lung, pancreas, liver, brain, femur, and placenta. Fetal tissues were examined on GD 20 and included whole blood, lung, liver, brain, and skull cap. Maternal lung manganese concentrations were increased following exposure to MnSO4 at >or=0.05 mg Mn/m(3). Maternal brain and placenta manganese concentrations were increased following exposure of pregnant rats to MnSO4 at >or=0.5 mg Mn/m(3). Increased fetal liver manganese concentrations were observed following in utero exposure to MnSO4 at >or=0.5 mg Mn/m(3). Manganese concentrations within all other fetal tissues were not different from air-exposed controls. The results of this study demonstrate that the placenta partially sequesters inhaled manganese, thereby limiting exposure to the fetus.     

Inhibition of release of neurohypophysial hormones by endogenous opioid peptides in pregnant and parturient rats

Naloxone, an opiate receptor antagonist, was used to determine whether opioid peptides modulate release of oxytocin (OT) or vasopressin (AVP) in the rat after expulsion of the fetus, i.e. parturition. We measured the concentrations of AVP and OT in plasma and in the neurointermediate lobe of the pituitary of pregnant rats given naloxone (5 mg/kg, s.c.) or saline on day 20 of gestation, and on day 21 either before or during the expulsive stage of labor. Non-pregnant rats in diestrus were given naloxone for comparison. On days 20 and 21 of gestation, before the onset of parturition, plasma [AVP] but not [OT] was elevated, compared to the non-pregnant controls. After delivery of the first two pups, plasma [OT] approximately doubled, whereas plasma [AVP] remained unchanged. Blocking the action of endogenous opioid peptides with naloxone caused an elevation of plasma [OT] in pregnant animals on days 20 and 21 of gestation and during parturition. Naloxone, however, did not alter plasma [AVP] in either parturient or preparturient animals. In contrast, [AVP], but not [OT], was increased in plasma of non-pregnant rats given naloxone. The content of OT in the neuro-intermediate lobe was similar in pregnant and non-pregnant rats and was unaffected by delivery of the first two pups. However, AVP content and the ratio of AVP/OT in the pituitary were lower in pregnant animals before and during delivery than in the non-pregnant controls. The content of neither hormone was altered by naloxone. Thus, AVP release apparently increases and pituitary stores of this peptide are decreased by day 20 of gestation, when labor has not yet begun.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Inhibition of release of neurohypophysial hormones by endogenous opioid peptides in pregnant and parurient rats

Naloxone, an opiate receptor antagonist, was used to determine whether opioid peptides modulate release of oxytocin (OT) or vasopressin (AVP) in the rat after expulsion of the fetus, i.e. parturition. We measured the concentrations of AVP and OT in plasma and in the neurointermediate lobe of the pituitary of pregnant rats given naloxone (5 mg/kg, s.c.) or saline on day 20 of gestation, and on day 21 either before or during the expulsive stage of labor. Non-pregnant rats in diestrus were giben naloxone for comparison. On days 20 and 21 of gestation, before the onset of parturition, plasma [AVP] but not [OT] was elevated, compared to the non-pregnant controls. After delivery of the first two pups, plasma [OT] approximatelyy doubled, whereas plasma [AVP] remained unchanged. Blocking the action of endogenous opioid peptides with naloxone caused an elevation of plasma [OT] in pregnant animals on days 20 and 21 of gestation and during parturition. Naloxone, however, did not alter plasma [AVP] in either parturient or preparturient animals. In contrast, [AVP], but not [OT], was increased in plasma of non-pregnant rats given naloxone. The content of OT in the neuro-intermediate lobe was similar in pregnant and non-pregnant rats and was unaffected delivery of the first two pups. However, AVP content and the ratio of AVP/OT in the pituitary were lower in pregnant animals before during delivery than in the non-pregnant controls. The content of neither hormone was altered by naloxone. Thus, AVP release apparently increase and pituitary stores of this peptide are decreased by day 20 gestation, when labor has not yet begun. In contrast, OT secretion becomes elevated only during delivery. Inhibition of OT release by opioid peptides may: (1) allow preferential release of AVP during pregnancy; and (2) prevent depletion of pituitary stores of OT and neuronal fatigue during the 1–2 h period of parturition in the rat.     

Evidence for the presence of mu- and kappa- but not of delta-opioid sites in the human fetal brain

The presence of multiple opioid binding sites in human fetal brain at 20 weeks gestational age was determined using the following selective tritiated ligands: D-Ala2, N-MePhe4, Gly-ol5-enkephalin (DAGO) for the mu-type, D-Pen2, D-Pen5-enkephalin (DPDP) for the delta-type and U-69,593 for the kappa-type. [3H]DAGO and [3H]U-69,593 each bind to a single class of high-affinity sites in membrane preparations with respective Kd values of 0.96 +/- 0.33 nM and 0.57 +/- 0.20 nM. The binding capacity for [3H]DAGO is 1.38 +/- 0.21 pmol/g tissue whereas [3H]U-69,593 has a binding capacity of 1.04 +/- 0.22 pmol/g tissue. Using DPDP we did not detect any specific high-affinity binding sites in human fetal brain. The non-selective tritiated opioid ethylketazocine (EKC) labels a homogenous class of sites with a Kd of 0.17 +/- 0.01 nM and a binding capacity of 2.15 +/- 0.15 pmol/g tissue, a value that is not statistically different from the total capacity obtained with [3H]DAGO and [3H]U-69,593. Competition studies using selective unlabeled opioids against the binding of [3H]-DAGO, [3H]U-69,593 or [3H]EKC indicate that opioid sites present in human fetal brain possess similar pharmacological characteristics to those found in human and mammalian adult brain. The results of this study present the first evidence for the presence of mu- and kappa-type opioid sites in human fetal brain and this may support the postulated role of the opioid system in the neurobiological development.     

Decreased levels of [Met]enkephalin, neuropeptide Y, substance P, and vasoactive intestinal peptide in parkinsonian adrenal medulla

Adrenal medullary tissue was collected from parkinsonian patients at autopsy and at the time of autologous transplantation of the adrenal medulla to the caudate nucleus, and from nonparkinsonian patients at autopsy and during nephrectomy. Levels of the following neuropeptides were measured by radioimmunoassay in samples of the medullary tissue: neuropeptide Y (NPY), substance P (SP), [Met]enkephalin ([Met]ENK), vasoactive intestinal peptide (VIP), peptide YY, and bombesin-like immunoreactivity. Regression analysis was used to establish a relationship between patient age, time to organ harvest, and peptide levels in nonparkinsonian tissue. Levels of [Met]ENK, VIP, NPY, and SP were significantly lower in parkinsonian adrenal medullae than that predicted from the control group. These results suggest that the adrenal medulla of a parkinsonian patient is severely compromised, either by the disease process itself or by the antiparkinsonian medications used to treat the symptoms of the disease.     

Proenkephalin-A derived peptides do not modulate cardiovascular effects of epinephrine on the isolated rat atrial preparations

The catecholamines and the opioid peptides are found to be co-localized in the adrenomedullary chromaffin cells. They are co-secreted from the chromaffin granules in response to various stimuli. The stress-induced released of epinephrine is known to exert its effect on the cardiovascular system resulting in the changes in heart rate and blood pressure. However, the role of the co-released proenkephalin-A derived peptides has not been extensively characterized. Previous work from several investigators suggested that the peptides modulate cardiac functions of the catecholamines. There is considerable conflicting results among these reports. Results from the isolated rat atrial preparation indicated that enkephalins attenuated the increase in atrial rate induced by norepinephrine through restriction of the calcium fluxes. Nonetheless, others reported insensitivity of the enkephalins in similar or different test systems. We further re-examined these discrepancies using the isolated rat atrial preparation to investigate the opioid peptide modulatory effect on the cardiovascular changes induced by exogenous epinephrine. Alterations in rate and force of contraction resulting from epinephrine and the peptides were both studied in parallel. The opioid peptides used in this study were [Met5]-enkephalin (ME), [Leu5]-enkephalin (LE), FMRFamide, [Met5]-enkephalyl-Arg6-Phe7 (MEAP), peptide E, and the non-selective opioid agonist, etorphine. We report here that none of the opioid peptides were effective in alleviating or attenuating the increase in heart rate and developed tension caused by epinephrine. The peptides did not affect the basal beating rate nor the force of contraction. Thus, the present results clearly demonstrate the insensitivity of the enkephalins in modulating the cardiac effects of epinephrine. They further indirectly support the prejunctional synaptic nerve endings as the potential peripheral site of action of the peptides.