Characterization of MSH/ACTH-like immunoreactivity in sciatic nerves of Xenopus laevis by immunocytochemistry,Western blotting and radioimmunoassay

Summary Previous studies have indicated that rat neurofilament protein may contain an endogenous MSH-like epitope with neuroregenerative properties. The presence of such an epitope has now been studied in nerve tissue from Xenopus laevis. Western blot analyses of sciatic nerve tissue using an assortment of sequence-specific MSH/ACTH antisera revealed the presence of two major immunoreactive protein bands of 52 and 50 kDa, which contained a mid-region MSH-like epitope. Weaker staining occurred in another protein band at 135 kDa. Immunocytochemistry revealed the immunoreactivity to reside in the axis cylinders of the nerve fibers. Other antisera, recognizing other regions of MSH/ACTH produced strong staining of Xenopus intermediate lobes, but failed to stain sciatic nerves. Thus, the proteins detected have no clear relation to either Xenopus neurofilament proteins or proopiomelanocortin.     

Specificity of the glial fibrillary acidic protein for astroglia.

Glial fibrillary acidic protein (GFA) is the main constituent of glial filaments and the close similarity of GFA and neurofilament protein has been recently reported. However, the immunofluorescence staining of peripheral nerve which may be observed with GFA antisera is not due to cross-reaction between GFA and neurofilament protein. Staining of peripheral axons was also observed with control sera obtained by injecting the rabbits with nonimmunogenic GFA preparations isolated with the same procedure. Immune GFA antisera and control sera reacted with sodium dodecyl sulfate extracts of sciatic nerve. However, the precipitin line formed with peripheral nerve crossed the line against GFA protein, thus indicating nonidentity between the two antigens. Buffer extract of sciatic nerves that had been incubated with spinal cord reacted by immunodiffusion with GFA antisera, thus indicating that redistribution of GFA occurred under these conditions.     

Cytoskeletal proteins at the cholinergic synapse: distribution of desmin, actin, fodrin, neurofilaments, and tubulin in Torpedo electric organ

Treatment of the electric organ of Torpedo marmorata with Triton X-100 in the presence of 2 mM MgCl2 generated a cytoskeletal fraction in which a 54 kDa polypeptide is a major constituent. This 54 kDa polypeptide accounted for about 8% of the cellular protein when total electric organ tissue was analyzed by two-dimensional gel electrophoresis. Immunoblotting experiments showed that this protein reacts with monoclonal antibodies to desmin, the major intermediate filament protein of avian and mammalian muscle tissue. Negative stain analysis revealed that filaments of about 10 nm diameter are the major structural elements of the electric organ cytoskeleton. In the presence of Ca2+ there was a rapid degradation of the desmin-like protein and intermediate filaments due to a Ca2+-activated protease. Some of the resulting fragments retained antigenic activity against the desmin antibodies. Immunoblotting of membrane fractions enriched in acetylcholine receptor revealed desmin in addition to some actin. A further cytoskeletal component was identified from biochemical and immunological properties as a homologue of the mammalian neurofilament L-polypeptide. Thus Torpedo expresses proteins homologous to the mammalian desmin and neurofilament L-protein which can be detected using immunological approaches. Immunofluorescence microscopy was used to map the location of various cytoskeletal proteins of the cholinergic synapse on paraffin sections and on en face preparations of membranes. Desmin staining was restricted to electrocytes and in en face preparations was seen associated with both the ventral receptor-containing membrane and with the non-innervated dorsal membrane. Antibodies to neurofilament L-protein stained only the axons and not the electrocytes. Staining for fodrin, a non-erythrocyte spectrin, resulted in submembraneous decoration of both the axons and the electrocytes. Axonal staining for neurofilaments and microtubules did not extend into the ends of the nerve terminal arborizations.     

The utility of antisera to different synthetic adrenocorticotrophins (ACTH) and melanotrophins (MSH) for immunocytochemical staining of the dog pituitary gland

Using the immunoperoxidase technique and specific antisera to synthetic ACTH beta (1-24), ACTH beta (17-39) and bMSHbeta1, selective immunocytochemical staining was localized in a distinctive cell type in the pars distalis and pars tuberalis of the dog pituitary gland. Except for a rare cell, the pars distalis and pars tuberalis did not stain with an anti-bMSH alpha serum. In the pars intermedia immunoreactive cells containing ACTH beta(1-24), ACTHbetap(17-39), bMSHbeta and/or bMSH alpha were observed. The specificity and validity of the antisera were demonstrated by elimination of their immunostaining capacity after prior absorption with their respective antigens, while absorption with other antigens failed to decrease staining intensity. The cytoplasm of the ACTH/MSH cells showed a positive reaction to periodic-acid-Schiff and assumed a pale aniline blue colour, whilst the granules were stained with carmoisine L and acid alizarine blue. These ACTH/MSH cells were further differentiated from other functional cell types of the pars distalis on the basis of their typical cytological features, intraglandular distribution and by immunochemical double staining. It is concluded that ACTH and MSH beta were present and most probably produced by the corticomelanotrophs of the pars distalis and pars tuberalis. In addition to corticomelanotrophs analogous to those of the pars distalis and pars tuberalis, the pars intermedia showed many cells which contain MSH alpha alone or together with MSH beta and/or ACTH.     

Melanocortins and fast axonal transport in intact and regenerating sciatic nerve

The effect of ACTH/MSH peptides on fast axonal transport along intact or regenerating sciatic nerve was examined following injection of tritiated leucine into the rat lumbar spinal cord. The rate of fast axonal transport was not significantly changed by treatment with ACTH/MSH(4-10), the ACTH(4-9) analog ORG 2766, hypophysectomy, or adrenalectomy. Fast axonal transport was unchanged in regenerating nerves and in regenerating, ACTH(4-10)-treated nerves. However, treatment with ORG 2766 in dosages of either 1 or 10 micrograms/kg/day IP for seven days significantly reduced (62% and 64%, respectively) the crest height of the fast axonal transport curve of intact sciatic nerve. The results suggest that the reported peptide-induced enhancement of nerve regeneration is not due to changes in the rate of fast axonal transport.     

Intracellular acetylation of desacetyl alpha MSH in the Xenopus laevis neurointermediate lobe

High performance liquid chromatography (HPLC) followed by radioimmunoassay (RIA) of the chromatographic fractions were used to separate and quantify, respectively, the alpha MSH-like peptides stored in the neurointermediate lobe (NIL) of the Xenopus laevis (X. laevis) pituitary gland and released from the X. laevis NIL, in vitro. Immunoreactive (IR) material eluting with a similar HPLC retention time as desacetyl alpha MSH was the major IR peptide in the NIL. Material with a retention time similar to alpha MSH and immunological properties equivalent to alpha MSH was also present in the NIL. However, the retention times of the X. laevis and mammalian alpha MSH-like peptides were not identical, suggesting species difference in these peptides. Following incubation of NILs in the presence of [3H]-acetyl CoA, the X. laevis variant of alpha MSH was the major [3H]-labeled, immunoprecipitable material present. Following an incubation of NILs in the presence of [3H]-amino acids for 21 hours, immunoprecipitable [3H]-alpha MSH was detected in the NILs and the ratio of [3H]-desacetyl alpha MSH to [3H]-alpha MSH was similar to the ratio of IR-desacetyl alpha MSH to IR-alpha MSH. The X. laevis variant of alpha MSH was the major alpha MSH-like peptide released from the NILs into the incubation medium. Dopamine (50 microM) significantly inhibited the release of IR-alpha MSH but not IR-desacetyl alpha MSH. No net increase in total alpha MSH (sum of release and NIL content) was observed in the actively secreting (control) NIL group versus the dopamine-treated group. These results indicate that acetylation of desacetyl alpha MSH occurs intracellularly.     

Appearance and phosphorylation of the 210 kDalton neurofilament protein in newborn rat brain,spinal cord,and sciatic nerve

The appearance and in vivo phosphorylation of the 210 kDalton (kD) neurofilament protein (NF210K) in newborn rat brain, spinal cord, and sciatic nerve were invetigated. Electron microscopic examination of neurofilaments isolated from newborn rat brain and spinal cord demonstrated morphologically distinct filaments which contained cross-bridging side arms. Neurofilament proteins, phosphorylated in vivo, were separated by sodium dodecyl sulfate slab gel electrophoresis and were transferred from acrylamide gels to nitrocellulose sheets. The nitrocellulose sheets were treated with antiserum to the 70 kD, 145 kD and 210 kD neurofilament proteins by the immunoblot technique. The three neurofilament proteins were found to be present in newborn brain, spinal cord and sciatic nerve. The presence of NF210K in newborn rat brain was further confirmed by 2-dimensional gel electrophoresis followed by indentification of this protein by the immunoblot technique. Exposure of the immunostained nitrocellulose sheets to x-ray film revealed that the NF210K, NF145K, and NF70K proteins were phosphorylated in filaments prepared from newborn rat central and peripheral nervous systems. These results suggest that the synthesis and posttranslational modification of the neurofilament proteins may be synchronized or developmentally regulated. It is feasible that phosphorylation of the NF210K subunit may be a prerequisite for the formation of neurofilament cross-bridging elements which are necessary for radial growth of axons.     

Identification of the cDNA for xlcaax-1, a membrane associated Xenopus maternal protein.

xlcaax-1 is a cDNA coding for a CAAX box containing protein in Xenopus laevis that undergoes isoprenylation and palmitoylation. Here we report on the confirmation that this clone (formerly xlgv7) codes for a 110 kDa membrane associated protein and not an 80 kDa nuclear protein as originally believed (1). The reason for the misidentification was the presence of a common epitope on these two proteins recognized by the monoclonal antibody 37-1A9. We clarified the discrepancy by raising polyclonal antibodies against the xlcaax-1 protein produced in a bacterial expression system and demonstrating that these antibodies only recognize the 110 kDa protein on western blots of oocyte extracts. During early development xlcaax-1 protein starts reaccumulating from the neurula stage. In the adult frog both the xlcaax-1 protein and its cognate mRNA are highly enriched in the kidney. Consistent with the presence of CAAX box at the C-terminus this protein is associated with the membranes in Xenopus tissue culture cells (XTC).     

Identification of a lipid-anchored heparan sulfate proteoglycan in Schwann cells

Schwann cells synthesize both hydrophobic and peripheral cell surface heparan sulfate proteoglycans (HSPGs). Previous analysis of the kinetics of radiolabeling suggested the peripheral HSPGs are derived from the membrane-anchored forms (Carey, D., and D. Evans. 1989. J. Cell Biol. 108:1891-1897). Peripheral cell surface HSPGs were purified from phytic acid extracts of cultured neonatal rat sciatic nerve Schwann cells by anion exchange, gel filtration, and laminin-affinity chromatography. Approximately 250 micrograms of HSPG protein was obtained from 2 X 10(9) cells with an estimated recovery of 23% and an overall purification of approximately 2000-fold. SDS-PAGE analysis indicated the absence of non-HSPG proteins in the purified material. Analysis of heparinase digestion products revealed the presence of at least six core protein species ranging in molecular weight from 57,000 to 185,000. The purified HSPGs were used to produce polyclonal antisera in rabbits. The antisera immunoprecipitated a subpopulation of 35SO4- labeled HSPGs that were released from Schwann cells by incubation in medium containing phosphatidylinositol-specific phospholipase C (PI- PLC); smaller amounts of immunoprecipated HSPGs were also present in phytic acid extracts. In the presence of excess unlabeled PI-PLC- released proteins, immunoprecipitation of phytic acid-solubilized HSPGs was inhibited. SDS-PAGE analysis of proteins immunoprecipitated from extracts of [35S]methionine labeled Schwann cells demonstrated that the antisera precipitated an HSPG species that was present in the pool of proteins released by PI-PLC, with smaller amounts present in phytic acid extracts. Nitrous acid degradation of the immunoprecipitated proteins produced a single 67,000-Mr core protein. When used for indirect immunofluorescence labeling, the antisera stained the external surface of cultured Schwann cells. Preincubation of the cultures in medium containing PI-PLC but not phytic acid significantly reduced the cell surface staining. The antisera stained the outer ring of Schwann cell membrane in sections of adult rat sciatic nerve but did not stain myelin or axonal membranes. This localization suggests the HSPG may play a role in binding the Schwann cell plasma membrane to the adjacent basement membrane surrounding the individual axon-Schwann cell units.     

Protein composition and synthesis in the adult mouse spinal cord

Propepties of spinal cord proteins were studied in adult mice subjected to unilateral crush or electrical stimulation of sciatic nerve. The protein composition of spinal tissue was determined using SDS-polyacrylamide gel electrophoresis coupled with subcellular fractionation. Comparisons of mouse spinal cord and brain revealed similarities in the types but differences in the concentrations of myelin associated proteins, nuclear histones and other proteins. Comparisons with sciatic nerve proteins demonstrated differences in types of proteins but similarities in the concentration of myelin proteins and nuclear histones. The short term (<2 hrs.) incorporation of radioactive amino acids into spinal cord proteins revealed heterogeneous rates of incorporation. Neither nerve crush six days prior to testing nor sciatic nerve stimulation had a significant effect on the protein composition or amino acid incorporation rates of spinal cord tissue. These observations suggest that known differences in spinal cord function following alterations in nerve input may be dependent upon different mechanisms than have been found in the brain.     

Examination of a nerve injury-induced, 37 kDa protein: Purification and characterization

Following traumatic injury to the adult rat sciatic nerve the synthesis and accumulation of soluble, extra-cellular, 37 kDa protein is increased. This protein, which accumulates in the extracellular space of the injured nerve, accounts for nearly 5% of the total soluble pool of protein in an injured nerve 3 weeks after injury. 8 weeks after injury, when regeneration is nearly complete, this accumulated pool returns to control levels, yet if regeneration is blocked synthesis of the 37 kDa protein remains high. Recently this 37 kDa protein has been shown to be nearly identical to apolipoprotein E, the protein component of various lipoprotein particles. This finding suggests a role for the 37 kDa protein in cholesterol and lipid transport and metabolism during nerve repair within the nervous system, functions that have been ascribed to apo E in serum. Results are presented here describing the purification of the nerve injury induced 37 kDa protein and the subsequent production of specific rabbit antisera directed against it. By centrifugation analysis in a sucrose gradient, a native mass of 37 kDa was determined, revealing the 37 kDa protein's monomeric, native structure. Additionally injections of [³⁵S]methionine directly into the injured nerve allowed 1) a comparison of 37 kDa synthesis in vivo versus in vitro and 2) an examination of the presence or absence of retrogradely transported 37 kDa protein. The in vitro and in vivo collected material were found to share identical 2-dimensional electrophoretic mobilities, and no appreciable amount of transported 37 kDa protein was found in proximal regions of the injured nerve.Special Issue dedicated to Dr. E. M. Shooter and Dr. S. Varon.     

Microsequencing evidence for the maturation of human proopiomelanocortin into an 18 amino acid beta-melanocyte stimulating hormone [h beta MSH(5-22)] in nonpituitary tissue

Sixty pmoles of a material with molecular size, immunological, and RP-HPLC characteristics identical to that of h beta MSH(5-22) were purified from a bronchial carcinoid tumor responsible for the ectopic ACTH syndrome. The first 16 cycles of microsequencing revealed the following sequence: Asp-Glu-Gly-Pro-Tyr-Arg-Met-Glu-X-Phe-Arg-Trp-Gly-X-Pro- Pro-, identical to the first 16 amino acids of h beta MSH(5-22). Since this material was recognized by an antibody which requires the free COOH-terminal Asp22 residue, it can be assumed that it is indeed h beta MSH(5-22). We also show that neither the 5 N acetic acid nor the 1 N HCl extraction procedure artefactually generated h beta MSH-like material in normal or tumoral human pituitaries and in nonpituitary tumors. We conclude that h beta MSH(5-22) is a normal maturation product of proopiomelanocortin in the human nonpituitary tissues which express its gene, including the hypothalamus and ACTH-secreting tumors.     

Localization of multiple forms of ACTH- and β-endorphin-related substances in the pituitary of the reptile, Anolis carolinensis

Immunohistochemical studies on the pituitary of Anolis carolinensis detected ACTH-like, β-endorphin-like, and 16K fragment-like immunoreactivity in distinct clusters of cells in the anterior lobe; ACTH-like, αMSH-like, β-endorphin-like, and 16K fragment-like immunoreactivity was detected in all the cells of the intermediate lobe. Crude acid extracts of both lobes, when alayzed by radioimmunoassay, gave displacement curves in ACTH and β-endorphin assays which were parallel to the appropriate synthetic standard. Only extracts of the intermediate lobe gave parallel displacement curves in an αMSH radioimmunoassay. Extracts of both lobes crossreacted with antiserum to 16K fragment, but the displacement curves were not parallel to that of mouse 16K fragment standard. The levels of immunoreactive ACTH and β-endorphin in the intermediate lobe were approximately 8-fold higher than in the anterior lobe. Fractionation of anterior lobe and intermediate lobe extracts by either gel filtration on Sephadex G-75 in 10% formic acid or sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed multiple forms of ACTH-related and β-endorphin-related substances in both lobes. In the anterior lobe the major forms of immunoreactivity were, respectively, ACTH-sized and β-endorphin-sized. In the intermediate lobe the major forms of immunoreactivity were αMSH-sized, CLIP-sized, and β-endorphin-sized. In both lobes, antisera directed against ACTH and β-endorphin detected high molecular weight material with an apparent molecular weight slightly less than that of mouse pro-ACTH/endorphin; this material probably represents the putative common precursor for ACTH and β-endorphin in this species.     

Arsenic-induced neurotoxicity in relation to toxicokinetics: effects on sciatic nerve proteins

In our previous study in rats acutely exposed to As, we observed an effect of As on neurofilaments in the sciatic nerve. This study deals with the effects of inorganic As in Wistar rats on the cytoskeletal protein composition of the sciatic nerve after subchronic intoxication. Sodium meta-arsenite (NaAsO2) dissolved in phosphate-buffered saline (PBS) was administered daily in doses of 0, 3 and 10 mg/kg body weight/day (n=9 rats/group) by intragastric route for 4, 8 and 12 week periods. Toxicokinetic measurements revealed a saturation of blood As in the 3- and 10-mg/kg dose groups at approximately 14 microg/ml, with an increase in renal clearance of As at increasing doses. After exsanguination, sciatic nerves were excised and the protein composition was analyzed. Analysis of the sciatic nerves showed compositional changes in their proteins. Protein expression of neurofilament Medium (NF-M) and High (NF-H) was unchanged. Neurofilament protein Low (NF-L) expression was reduced, while mu- and m-calpain protein expression was increased, both in a dose/time pattern. Furthermore, NF-H protein was hypophosphorylated, while NF-L and microtubule-associated protein tau (MAP-tau) proteins were (hyper)-phosphorylated. In conclusion, we show that expression of mu- and m-calpain protein is increased by exposure to As, possibly leading to increased NF-L degradation. In addition, hyperphosphorylation of NF-L and MAP-tau by As also contribute to destabilization and disruption of the cytoskeletal framework, which eventually may lead to axonal degeneration.     

αMSH-like peptides in rat brain: Identification and changes in level during development

MSH-like peptides were extracted from rat brains and separated by high pressure liquid chromatography. Using C and N terminally directed antibodies, and a bioassay for melanotropic activity, two major melanotropic peptides were detected. One peptide was identified as αMSH and the other as des-acetyl αMSH, a form which has not been previously reported in brain. Analysis of the level of αMSH-like peptides in brain and pituitary gland during development, showed steady increases of pituitary αMSH from day 18 fetuses to adults, whereas in brain, significant increases were not observed until one day post partum. This difference in the time of onset of αMSH production in the two tissues suggests the presence of biosynthetically independant pools of αMSH-like peptides in pituitary and brain.     

The influence of peripheral nerve graft's predegeneration stage on the regrowth of hippocampal injured neurites and concomitant changes in submicrosomal fraction proteins of grafts.

The present work has a twofold aim: 1. To ascertain whether the stimulative influence of peripheral nerve grafts on injured hippocampal neurons depends on the time lapse after transection and; 2. To examine whether the mentioned effect runs parallel to the time-dependent changes of proteins contents and composition in the submicrosomal fraction from transected rat sciatic nerves. Fluorescence microscope examination revealed that FITC-HRP labeled cells extending their neurites into the implanted peripheral nerve segments were particularly numerous among the hippocampal neurons when 7- and 35-day-old predegenegated distal stumps were used as grafts. Discontinuous SDS-slab polyacrylamide gel electrophoresis of submicrosomal fraction proteins obtained from distal stumps of rat sciatic nerves was performed at the 7, 14, 21 and 35 days after transection. Among the obtained protein fractions the most interesting seem to be the ones of 47 and 54 kDa, which reached maximal levels at the 7th day and the 50 kDa fraction with a maximum at the 35th experimental day. It is possible that the growth promoting power of the employed grafts depends on the presence of proper proteins.     

Quantitative and Qualitative Alterations of Neuronal and Glial Intermediate Filaments in Rat Nervous System After Exposure to 2, 5-Hexanedione

The precise mechanism for the neurotoxicity of 2,5-hexanedione is not known, but cross-linking of neurofilament proteins has been suggested as one possibility. In this study the effects of long-term exposure to 2,5-hexanedione were studied in the rat nervous system with special reference to regional changes in the quantities of neuronal and glial intermediate filaments. Using enzyme-linked immunosorbent assays the concentrations of 68- and 200-kDa neurofilament polypeptides were shown to be reduced in all brain regions studied. Similar results were obtained in the sciatic nerve. The concentration of glial fibrillary acidic protein was decreased in the cerebellar vermis and the dorsal cerebral cortex, whereas it was increased in the spinal cord, a result suggesting a regional variation in glial sensitivity. The intermediate filaments of the exposed animals were also immunoblotted using polyclonal antisera against the various neurofilament polypeptides and glial fibrillary acidic protein. In all tissues studied, several aggregates with molecular weights higher than those of the monomeric polypeptides were demonstrated. Contrary to clinical observations, these data indicate pronounced effects in both CNS and PNS and call for further studies on CNS effects in humans.     

Binding of γ-Aminobutyric AcidA Receptors to Tubulin

Abstract: The rate of axonal transport of tubulin, actin, and the neurofilament proteins was measured in the peripheral and central projections of the rat L5 dorsal root ganglion (DRG). [³⁵S]Methionine was injected into the DRG, and the "front" of the radiolabeled protein was located 7, 14, and 20 days postinjection. Transport rates calculated for the neurofilament triplet proteins, tubulin, and actin in the peripheral nerve were ∼ 1.5-fold faster than those in the dorsal root. A progressive decrease in the rate of transport was observed from 7 to 20 days after radiolabeling in both the central and peripheral directions (neurofilaments, ∼ 1.7-fold; tubulin/actin, 2.1-fold). A surgical preparation, leaving the peripheral sciatic nerve with predominantly sensory fibers, was the basis for ELISAs for phosphorylation-dependent immunoreactivity of the high-molecular-weight neurofilament protein. In both dorsal roots and peripheral sensory axons the degree of phosphorylation was greater in nerve segments further away from the cell bodies. The degree of phosphorylation-related immunoreactivity correlates with the slowing of transport of radiolabeled cytoskeletal protein.     

Intracellular acetylation of desacetyl αMSH in the xenopus laevis neurointermediate lobe

High performance liquid chromatography (HPLC) followed by radioimmunoassay (RIA) of the chromatographic fractions were used to separate and quantify, respectively, the αMSH-like peptides stored in the neurointermediate lobe (NIL) of the Xenopus laevis (X. laevis) pituitary gland and released from the X. laevis NIL, in vitro. Immunoreactive (IR) material eluting with a similar HPLC retention time as desacetyl αMSH was the major IR peptide in the NIL. Material with a retention time similar to αMSH and immunological properties equivalent to αMSH was also present in the NIL. However, the retention times of the X. laevis and mammalian αMSH-like peptides were not identical, suggesting species difference in these peptides. Following incubation of NILs in the presence of [³H]-acetyl CoA, the X. laevis variant of αMSH was the major [³H]-labeled, immunoprecipitable material present. Following an incubation of NILs in the presence of [³H]-amino acids for 21 hours, immunoprecipitable [³H]-αMSH was detected in the NILs and the ratio of [³H]-desacetyl αMSH to [³H]-αMSH was similar to the ratio of IR-desacetyl αMSH to IR-αMSH. The X. laevis variant of αMSH was the major αMSH-like peptide released from the NILs into the incubation medium. Dopamine (50 μM) significantly inhibited the release of IR-αMSH but not IR-desacetyl αMSH. No net increase in total αMSH (sum of release and NIL content) was observed in the actively secreting (control) NIL group versus the dopaminetreated group. These results indicate that acetylation of desacetyl αMSH occurs intracellularly.     

Electron microscopic-immunocytochemical localization of adrenocorticotropin and melanocyte stimulating hormone in the pars intermedia cells of rats and mice

Summary Electron microscopic localization of adrenocorticotropin (ACTH) and melanocyte stimulating hormone (MSH) in light, dark and ACTH cells in the pars intermedia (PI) of rats and mice is attempted by using antisera to _p 1–24, _p 17–39 ACTH and _b MSH with the immunoglobulin-peroxidase bridge technique. All of the PI parenchymatous cells (light, dark and ACTH cells), except the marginal cuboidal and the ependymal like cells, in rats and mice show very good localization of ACTH and MSH staining. In the light and dark cells, stain of varying intensity is seen on the secretory granules, vesicles and also in many places on the surface of the rough endoplasmic reticulum. There is no staining on the mitochondria, in the nuclei or in the granules inside and around the cisternae of the Golgi complex. Dark stained dense core granules become larger and larger as they appear farther and farther away from the Golgi complex. On the other hand, in the ACTH cells of the PI, ACTH antisera show stronger stained granules in the Golgi complex including the cisternae, similar to the pars distalis (PD) ACTH cells. From these observations it is concluded that the corticotropin in light and dark cells, is not packaged or condensed in the Golgi complex like that in the ACTH cells. MSH synthesis in light and dark cells also seems to be similar to that of ACTH synthesis. It is likely that the granules accumulate ACTH and MSH secretions after they are liberated from the Golgi cisternae, and thus become bigger and bigger in size. In case of ACTH cells of PI and PD, corticotropin may be packaged in Golgi cisternae and the size of the granule does not change much. This shows that there are distinct immunocytochemical differences between the light, dark and ACTH cells of the PI. At the moment, it is difficult to say whether ACTH and MSH are present in the same granule or not.The present and previous studies show that the ACTH and MSH secretion in the PI of rats and mice depends on the hypothalamic neural control.This study was supported by MRC of Canada Grant nos. MA-3759, and MA-5160.The author gratefully wishes to thank Drs. P. Desaulles and W. Rittel (CIBA, Basle, Switzerland) for the synthetic _p 1–24 ACTH and _b MSH, Dr. R. F. Phifer for _p 17–39 ACTH, and Dr. S. S. Spicer for providing samples of rabbit anti-porcine 17–39 ACTH and anti-human ACTH sera, Drs. George Sétáló and Paul Nakane for their valuable advice. He also acknowledge the help of Mr. Shankar Nayak to prepare the antisera and the skilful technical assistance of Miss. Elise Poiré. He wishes to acknowledge Mr. Gatson Lambert for his photography.