Search PubMed⌕ Search

Biomedical subjects

H Gainer

Publications and source records attributed to H Gainer.

At least 55 records · Page 3Linked to original sources
PubMed1990

Characterization of the distinctive neurofilament subunits of the soma and axon initial segments in the squid stellate ganglion.

The stellate ganglion, which gives rise to the giant axons of the squid, was dissected into two parts, one containing primarily cell bodies and the other axon initial segments. A neurofilament protein-enriched extract of each was prepared and compared biochemically and immunochemically with an axoplasmic neurofilament preparation and with the glial sheath that surrounds the axons. Both parts of the ganglion lacked the 220 kDa subunit of axoplasmic neurofilaments (NFs). However, they did contain a protein of about 190 kDa that reacted with the Pruss anti-intermediate filament antibody (aIFA; Pruss et al.: Cell 27:419-428, 1981), but not with a phosphorylation-dependent NF antibody (Cohen et al.: J Neurosci 7: 2056-2074, 1987). Dephosphorylation of the axoplasmic NF220 yielded a product that comigrated on two-dimensional (2D) gel electrophoresis with the 190 kDa ganglion protein, suggesting that the latter represented the incompletely phosphorylated precursor of NF220. The major low molecular weight aIFA-reactive species in the ganglion preparations was a polypeptide of about 65 kDa. A relatively small quantity of that polypeptide was also found in axoplasm and it comigrated in 2D gels with an aIFA-reactive polypeptide from the glial sheath. These results indicate that the site of modification of the 190 kDa NF precursor to the 220 kDa axonal form is probably at the point where the axon initial segments leave the ganglion, which is several mm distal to its site of synthesis in the cell body. Furthermore, the filament network of the axoplasm and possibly the cell bodies includes a glial-like intermediate filament protein in addition to the NF protein subunits.

Animals↗
PubMed1990

Structure of mouse vasopressin and oxytocin genes.

Mouse vasopressin (VP) and oxytocin (OT) genes were isolated from a genomic library and the nucleotide sequences of the two genes were determined. The two genes have similar three exon structures and a high similarity in the part of exon 1 encoding vasopressin or oxytocin nonapeptide and in exon 2 encoding the central core of neurophysin. They are linked together in a tail to tail orientation separated by a short 3.5 kb intergenic sequence and are transcribed from opposite strands. Both genes have a single transcription initiation site downstream from a TATA-like sequence and a single polyadenylated transcript of about 760 bp for the vasopressin mRNA and about 700 bp for the oxytocin mRNA.

Amino Acid Sequence↗
PubMed1989

Spatial and temporal expression of phosphorylated and non-phosphorylated forms of neurofilament proteins in the developing nervous system of Xenopus laevis.

Immunocytochemical studies of developing Xenopus laevis embryos and tadpoles (stages 12 1/2 to 46) were performed using a panel of 11 monoclonal antibodies to phosphorylated and non-phosphorylated forms of the neurofilament proteins. These included nine antibodies to the middle molecular weight neurofilament protein (XNF-M, 175 kDa), and two additional antibodies to non-phosphorylated forms of the other two neurofilament proteins (XNF-L, 73 kDa; XNF-H, 205 kDa). The developmental expression of XNF-M, XNF-L and XNF-H, and the progressive phosphorylation of XNF-M in the rhombencephalon, spinal cord, and optic nerve were studied using these antibodies. In the spinal cord and rhombencephalon, non-phosphorylated forms of XNF-M were initially detected during neural tube stages (stages 22-26), one day before XNF-L and XNF-H at early tadpole stages (stage 35/36). In the eye, XNF-M was observed initially during tailbud stages (stage 29/30), but neither XNF-L nor XNF-H was seen even by stage 46 (swimming tadpole). The phosphorylation of XNF-M occurred over a protracted period of several days, both in the neural tube and visual system, and could be divided into four phases. (1) When initially expressed, XNF-M was hypophosphorylated. This was indicated by the early immunostaining of axons and cell bodies with antibodies to dephosphorylated epitopes on XNF-M and by the absence of staining with antibodies to phosphorylated epitopes. (2) After a short timelag (3-9 h) axons were stained by some, but not all antibodies to phosphorylated epitopes. (3) Approximately one day later, all antibodies to phosphorylated epitopes stained the relevant axons. However, XNF-M was not yet fully phosphorylated, as indicated by the continued staining of these axons with antibodies to dephosphorylated epitopes of XNF-M. (4) Two to 3 days after the initial expression of XNF-M, dephosphorylated epitopes disappeared from the axons, establishing the adult pattern. During development, the most heavily phosphorylated neurofilament proteins present at a given stage were found first in distal regions of the axons and progressed gradually toward the neuronal perikarya as development proceeded. This gradient of phosphorylation, established early within the axon, suggests that neurofilaments in the axons mature from their distal ends toward the cell body, a process which may be regulated by local factors within the axons themselves. The similarity of the basic features of NF-M phosphorylation in mammalian, avian, and amphibian axons underscores the importance of this phenomenon for the development of a mature axon.

Aging↗
PubMed1989

Coexisting peptides in hypothalamic neuroendocrine systems: some functional implications.

1. Coexisting with oxytocin or vasopressin in the cell bodies and nerve terminals of the hypothalamic-neurohypophysial system are smaller amounts of other peptides. For a number of these "copeptides" there is strong evidence of corelease with the major magnocellular hormones. Guided by the location of their specific receptors we have studied the effects of three copeptides, dynorphin, cholecystokinin (CCK), and corticotropin releasing hormone (CRH), on the secretion of oxytocin and vasopressin from isolated rat neural lobe or neurointermediate lobe preparations in vitro. 2. Dynorphin is coreleased with vasopressin from neural lobe nerve terminals and acts on neural lobe kappa-opiate receptors to inhibit the electrically stimulated secretion of oxytocin. Naloxone augments oxytocin release from the neural lobe in a manner directly proportional to the amount of vasopressin (and presumably dynorphin) released. 3. Cholecystokinin, coreleased with oxytocin by neural lobe terminals, has been shown to have high-affinity receptors located in the NL and to stimulate secretion of both oxytocin and vasopressin. CCK's secretagogue effect was independent of electrical stimulation and extracellular Ca2+ and was blocked by an inhibitor of protein kinase C. 4. CRH, coreleased with OT from the neural lobe, has receptors in the intermediate lobe of the pituitary, but not in the neural lobe itself. CRH stimulates the secretion of oxytocin and vasopressin from combined neurointermediate lobes but not from isolated neural lobes. Intermediate lobe peptides, alpha and gamma melanocyte stimulating hormone, induced secretion of oxytocin and vasopressin from isolated neural lobes. Their effect was, like that of CCK, independent of electrical stimulation and extracellular Ca2+ and blocked by an inhibitor of protein kinase C. 5. Among the CRH-producing parvocellular neurons of the paraventricular nucleus, in the normal rat, approximately half also produce and store vasopressin. After removal of glucocorticoid influence by adrenalectomy, virtually all of the CRH neurons contain vasopressin. 6. The two subtypes of CRH neurosecretory cells found in the normal rat possess different topographical distributions in the paraventricular nucleus, suggesting the possibility of differential innervation. Stress selectively activates the vasopressin containing subpopulation of CRH neurons, indicating that there are separate channels of regulatory input controlling the two components of the parvocellular CRH neurosecretory system.

Animals↗
PubMed1989

Cholecystokinin evokes secretion of oxytocin and vasopressin from rat neural lobe independent of external calcium.

Cholecystokinin (CCK) and its receptors are abundantly represented in the central nervous system. However, a specific role or mechanism of action for CCK in this context has not been established. CCK coexists with oxytocin in magnocellular neurons of the hypothalamic-neurohypophysial system, sharing common neurosecretory vesicles with oxytocin in the neural lobe of the pituitary. The neural lobe, which consists primarily of oxytocin- and vasopressin-containing axons and nerve terminals and their surrounding glia, provides a relatively simple model system allowing for the study of the regulation of neurosecretion at the nerve terminal level, free from the complex array of synaptic effects present throughout the rest of the central nervous system. In this paper, we demonstrate the presence of high-affinity CCK binding sites in the rat neural lobe and show that activation of these receptors by the sulfated octapeptide, CCK-8, and related peptides causes potent secretion of oxytocin and vasopressin from the isolated nerve terminals. The secretagogue action of CCK-8, which is blocked by a CCK receptor antagonist (L-364,718), is independent of electrical stimulation and extracellular calcium and is blocked by an inhibitor of protein kinase C. Thus, the action of CCK on the neural lobe provides an example of peptide ligand-induced neurosecretion apparently mediated by second messengers rather than depolarization-induced calcium influx.

Alkaloids↗
PubMed1989

Evidence that cells expressing luteinizing hormone-releasing hormone mRNA in the mouse are derived from progenitor cells in the olfactory placode.

In situ hybridization histochemistry and immunocytochemistry were used to study the prenatal expression of luteinizing hormone-releasing hormone (LHRH) cells in the mouse. Cells expressing LHRH mRNA and peptide product were first detected on embryonic day 11.5 (E11.5) in the olfactory pit. On E12.5, the majority of LHRH cells were located on "tracks" extending from the olfactory pit to the base of the telencephalon. From E12.5 to E15.5, LHRH cells were detected in a rostral-to-caudal gradient in forebrain areas. Prior to E12.5, cells expressing LHRH mRNA were not detected in forebrain areas known to contain LHRH cells in postnatal animals. Quantitation of cells expressing LHRH mRNA showed that the number of labeled cells on E12.5 (approximately 800) equaled the number of LHRH cells in postnatal animals, but more than 90% of these cells were located in nasal regions. Between E12.5 and E15.5, the location of LHRH cells shifted. The number of LHRH cells in the forebrain increased, while the number of LHRH cells in nasal regions decreased over this same period. These findings establish that cells first found in the olfactory pit and thereafter in forebrain areas express the LHRH gene and correspond to the position of LHRH immunopositive cells found at these developmental times. To further examine the ontogeny of the LHRH system, immunocytochemistry in combination with [3H]thymidine autoradiography was used to determine when LHRH cells left the mitotic cycle. We show that LHRH neurons exhibit a discrete time of birth, suggesting that they arise as a single neuronal population between E10.0 and E11.0. Postnatal LHRH neurons were "birth-dated" shortly after differentiation of the olfactory placode and before LHRH mRNA was expressed in cells in the olfactory pit. Taken together, these studies support the hypothesis that all LHRH cells in the central nervous system arise from a discrete group of progenitor cells in the olfactory placode and that a subpopulation of these cells migrate into forebrain areas where they subsequently establish an adult-like distribution.

Animals↗
PubMed1989

Differential effects of estrogen on luteinizing hormone-releasing hormone gene expression in slice explant cultures prepared from specific rat forebrain regions.

Five serially sectioned tissue slices (400 microns) from the preoptic area/hypothalamus of postnatal day 4 rats were cultured using a slice explant roller culture technique. After 18 days in culture, these slices thinned sufficiently to allow immunocytochemical and in situ hybridization histochemical assays for LHRH peptide and LHRH mRNA, respectively. Large numbers of neurons containing mRNA encoding LHRH were detected in these slices using in situ hybridization histochemistry (ISHH). These 35S-labeled cells were distributed in the cultured slices in a pattern similar to that found with LHRH immunocytochemistry and ISHH in vivo, indicating that LHRH neurons were maintained in these cultures in an organotypic manner. Densitometric single cell analyses after ISHH of the culture slices were performed using a Loats image analysis system, so as to provide a density value per cell (density/cell). Comparisons of these density values from the slice explants cultured in presence or absence of 10(-7) M estradiol found that: 1) under basal (control) culture conditions there were no consistent differences in the frequency distributions of the density/cell values between all the five slices derived from either male or female rats, 2) mean density/culture values under control conditions did not differ significantly between slices and sexes, 3) the presence of estradiol in the culture media resulted in an overall decrease in density/cell values, with the most significant decrease occurring in slice 3 which is comparable to the level of the organum vasculosum lamina terminalis/rostral preoptic area (OVLT/rPOA) in vivo, and 4) this decrease in density/cell values in slice 3 due to estradiol treatment, was greater in cultures derived from female vs. male tissues.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Immunocytochemical identification of non-neuronal intermediate filament proteins in the developing Xenopus laevis nervous system.

Intermediate filament proteins in the postmetamorphic Xenopus laevis nervous system were identified by their crossreactivities on Western blots with a pan-specific intermediate filament antibody (anti-IFA). These intermediate filament protein bands on Western blots were characterized as 3 cytokeratin-like proteins (49, 55, and 58 kDa), one vimentin-like protein (53 kDa), two distinct glial fibrillary acidic protein (GFAP)-like proteins (60 and 67 kDa), and 3 neurofilament proteins (73, 175, and 200 kDa) by evaluation of their crossreactivities with specific antibodies directed against the mammalian forms of these proteins. This panel of antibodies to mammalian proteins, and two additional antibodies directed against a Xenopus GFAP-like protein and a Xenopus neurofilament (NF-M) protein, were used in immunocytochemical studies to determine the developmental expression of these proteins in the Xenopus nervous system. The first antigen to be detected during development was cytokeratin immunoreactivity, which was located in the inner lining of the embryonic neural tube as early as stage 19, and which in immunocytochemical studies in postmetamorphic frogs was abundant in meninges and processes forming the ventricular lining of the ependymal zone. Vimentin immunoreactivity was found in numerous neuroepithelial cell processes in the rhombencephalon and anterior spinal cord by stage 22, in the prosencephalon by stage 33/34, and in the retina by stage 29/30. In the postmetamorphic frog, vimentin immunoreactivity was found to be abundant in radial processes throughout the brain and spinal cord. NF-M protein immunoreactivity was first detected in neurons in the developing neural tube between stages 22 and 24, in the retina by stages 29/30, and continued to increase throughout development. GFAP-like immunoreactivity was detected very early in radial cells in the neural tube (stage 24), and by stage 42 was found throughout the nervous system. This early appearance of GFAP-like immunoreactivity implies that the onset of glial cell differentiation is a relatively early event in Xenopus.

Animals↗
PubMed1988

Identities, antigenic determinants, and topographic distributions of neurofilament proteins in the nervous systems of adult frogs and tadpoles of Xenopus laevis.

Three proteins with nominal molecular weights of 73 kDa (XNF-L), 175 kDa (XNF-M), and 205 kDa (XNF-H) were identified as putative neurofilament proteins in the nervous system of the frog, Xenopus laevis. These conclusions were based on four criteria: (1) these proteins were enriched in cytoskeletal preparations; (2) they reacted with a monoclonal antibody (anti-IFA) that cross-reacts with an epitope found in all intermediate filament proteins; (3) they cross-reacted with monoclonal antibodies directed against specific mammalian neurofilaments; and (4) antibodies that reacted with these proteins on Western blots specifically stained neurons in immunohistochemical analyses. The neurofilament proteins in Xenopus were antigenically similar, but not identical to mammalian neurofilament proteins. The principal difference was that four antibodies that reacted on Western blots with rat NF-H reacted with XNF-M in Xenopus. However, similarly to mammals, antibodies against phosphorylated XNF-M specifically labeled axons, whereas an antibody that reacted only with dephosphorylated epitopes on XNF-M specifically labeled neuronal cell bodies in immunohistochemistry. Three other antibodies that reacted equally well with untreated or alkaline-phosphatase-treated XNF-M or XNF-H proteins also showed axonally restricted staining in the adult Xenopus nervous system. An XNF-L (XC5D10) antibody was produced which stained axons and cell bodies equivalently throughout the adult Xenopus nervous system. By 3 days of development (stage 42; Xenopus tadpoles), antibodies to all three molecular weight forms of the frog neurofilament proteins detected specific neurons in the brainstem and spinal cord; and antibodies to phosphorylated and dephosphorylated epitopes on XNF-M could discriminate between axons and cell bodies in the rhombencephalon. The immunoreactivities of four antibodies directed at XNF-L, -M, or -H, which were unaffected by alkaline phosphatase treatment, differed significantly in their immunohistochemical staining patterns in adult vs. premetamorphic frogs.

Aging↗
PubMed1988

Activators of protein kinase C potentiate electrically stimulated hormone secretion from the rat's isolated neurohypophysis.

Two activators of protein kinase C, phorbol 12,13-dibutyrate (PDBu) and 1-oleoyl-2-acetylglycerol (OAG), augment electrically stimulated vasopressin and oxytocin secretion from the nerve terminals of the isolated rat neurointermediate lobe. The increased hormone release produced by PDBu is specific to the beta-phorbol conformation, and is dependent upon electrical stimulation in the presence of calcium. Furthermore, the potentiation of release was evident during low frequency stimulation (4 Hz) but not when the same number of pulses were applied at 20 Hz. This occlusion of the phorbol ester's effect by high-frequency stimulation suggests that activation of protein kinase C may play a role in the normal process of frequency-dependent facilitation of secretion in the neurohypophysial system.

Animals↗
PubMed1988

In vitro polymorphism and phase transitions of the neurofilamentous network isolated from the giant axon of the squid (Loligo pealei L.).

Using electron microscopy (EM), optical diffraction and image reconstruction techniques, we have demonstrated polymorphism of neurofilamentous network (NFN) in vitro based on phase transitions of the protein assemblies. The specific polymorphic appearances depended upon a number of factors, such as K+, Mg2+, Ca2+ ions, as well as the charge and hydration state of the molecules. Furthermore, modifications initiated by the state of phosphorylation of the sidearm proteins played an important role, especially in determining the sidearm disposition of the NFN. The Ca2+-activated protease removed the sidearms. Other enzymes activated by Ca2+ may initiate new association patterns of the peptide remnants and the intercoiling of two smooth neurofilaments (NFs) into paired helical filament-like (PHF-like) strands. Prolonged storage of the isolated NFs in Rubinson-Baker solution resulted in autocrosslinking and intercoiling of modified NFN components. The in vitro polymorphism and phase transitions of squid NFN induced under controlled conditions have been compared to modifications of cytoskeleton observed by EM in frontal lobe biopsies of Alzheimer patients. We conclude that similar processes, as induced in vitro, do occur in neurons of Alzheimer patients.

Alzheimer Disease↗
PubMed1988

Major pro-vasopressin-expressing and pro-vasopressin-deficient subpopulations of corticotropin-releasing hormone neurons in normal rats. Differential distributions within the paraventricular nucleus.

Two approximately equal subpopulations of corticotropin-releasing hormone (CRH)-containing parvocellular axons can be identified in the external zone of the median eminence in normal (unadrenalectomized) rats: one that contains pro-vasopressin (AVP)-derived peptides (i.e. AVP, AVP-associated neurophysin and the carboxy terminal glycopeptide) copackaged with CRH in secretory vesicles, and another that contains no detectable pro-AVP-derived peptides. In this study, antibodies to pro-AVP-derived peptides were used to demonstrate for the first time that similar subpopulations of CRH-containing parvocellular perikarya exist in the paraventricular nucleus of the hypothalamus in normal rats treated with colchicine. Electron-microscopic immunocytochemistry was performed on serial ultrathin sections to identify neurosecretory cell perikarya containing CRH that also expressed pro-AVP peptides or pro-oxytocin-derived neurophysin. Of the CRH-positive neurons that were detected, more than half stained positively for two pro-AVP peptides: AVP-associated neurophysin and the carboxy-terminal glycopeptide. Many of these cells also stained for AVP, but staining was variable, making quantitation of AVP-positive cells difficult. The remaining CRH-positive neurons contained no detectable pro-AVP peptides, and less than 0.5% of these CRH perikarya contained oxytocin-associated neurophysin. In the neurons that stained positively for both CRH and the pro-AVP peptides, CRH and the pro-AVP peptides were localized in the same secretory vesicles. The pro-AVP expressing and pro-AVP-deficient CRH neurons were distributed differently within the paraventricular nucleus.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Dynorphin A inhibits and naloxone increases the electrically stimulated release of oxytocin but not vasopressin from the terminals of the neural lobe.

Oxytocin release from the rat neurohypophysis is under endogenous opioid inhibition. It has recently been established that dynorphin precursor-derived peptides are colocalized with vasopressin (VP) in the secretory granules in nerve terminals of the neural lobe, and that the opiate receptors in the neural lobe are restricted to the kappa-subtype. Therefore, we hypothesized that dynorphin, which is copackaged and thus coreleased with VP, is the endogenous opioid that inhibits release from neighboring oxytocin (OT) terminals. To test this hypothesis we examined the effects of dynorphin-(1-8), dynorphin-(1-17), and naloxone on the electrically stimulated release of OT and VP from isolated rat neurointermediate lobes throughout a range of stimulus frequencies. Both dynorphin-(1-8) and -(1-17) (2 microM) produced a substantial reduction in OT release during a 4-Hz stimulus, and this effect was abolished by naloxone (10 microM). Neither form of dynorphin, however, affected OT secretion at a stimulus frequency of 12 or 30 Hz at concentrations up to 10 microM. Naloxone (10 microM) by itself did not affect OT release during the 4-Hz stimulus, but it produced a substantial increase in OT release at a stimulus frequency of 12 Hz. In contrast, neither form of dynorphin produced inhibition, nor did naloxone augment VP secretion at any frequency tested. Frequency-dependent secretion curves (4, 8, 12, 20, and 30 Hz) for OT and VP in the presence and absence of naloxone indicated that the degree of naloxone augmentation of OT release at a given stimulus frequency was positively correlated with the amount of VP release at that frequency. These data support the hypothesis that dynorphin released in parallel with VP during in vitro stimulations of the rat neurohypophysis simultaneously inhibits stimulated OT release.

Animals↗
PubMed1988

Identification and quantification of calcium-binding proteins in squid axoplasm.

The identities and quantities of calcium-binding proteins were determined in axoplasm isolated from the squid giant axon. 45Ca-binding assays on nitrocellulose filters containing axoplasm proteins separated by SDS-polyacrylamide electrophoresis revealed 4 major calcium-binding bands. These included the high-molecular-weight (Mr greater than 330 and 220 X 10(3] neurofilament proteins, an unidentified protein band that migrated around Mr 55,000, and a diverse group of proteins that migrated together around Mr 17,000. The low-molecular-weight (Mr 17,000) calcium-binding proteins could be resolved into calmodulin (ca. 120 mumol/kg axoplasm), 2 other Mr 17,000 calcium-binding proteins, and a small amount of calcineurin B. It is estimated that these calcium-binding proteins in squid axoplasm could theoretically bind about 1 mmol Ca2+/kg axoplasm. 125I-Calmodulin overlay and Western blot analyses disclosed a number of calmodulin-binding proteins in axoplasm. These included fodrin, calcineurin A, and Ca2+/CaM protein kinase II subunits.

Animals↗
PubMed1988

Differential biosynthesis and posttranslational processing of vasopressin and oxytocin in rat brain during embryonic and postnatal development.

The biosynthesis and posttranslational processing of arginine vasopressin (AVP) and oxytocin (OT) peptides in the developing rat brain and pituitary were studied using antibodies and complementary separation methods that permitted a quantitative radioimmunoassay (RIA) analysis of precursor, intermediate, and completely processed forms of the peptides. Precursor forms of the peptides were first detected in rat brain as early as embryonic day (E) 15 for AVP and E17 for OT. Proteolytic cleavage products of the precursors were detected 1 d later for both peptides. AVP was present in a fully processed (amidated) from immediately (E16) and throughout fetal development. OT was cleaved from its precursor starting on E18 but remained in an intermediate (C-terminal extended) form until E21, when amidated OT was first detected in the pituitary. Hence, Pro-AVP processing in the fetus was immediate and complete, whereas Pro-OT processing in the fetus was much slower and incomplete, resulting in the generation of partially processed, nonamidated stable forms of the peptide (OT-Gly10, OT-Gly10-Lys11, and OT-Gly10-Lys11-Arg12). The presence of OT-Gly10-Lys11-Arg12 as a major, stable intermediate form, indicated that the in vivo pattern of endoproteolytic cleavage occurred principally at the C-terminus of the pair of basic amino acids at the tripeptide spacer sequence (Gly-Lys-Arg) in the precursor. Although both precursors were first expressed nearly simultaneously in the brain, the steady-state levels of the precursors were very different throughout fetal life. From E16-E21, the quantities of AVP precursors and peptides were 5- to 10-fold greater than those of OT, suggesting a much higher level of precursor biosynthesis in the AVP neurons. In addition to these differences in the regulation of biosynthesis and processing, AVP peptides were axonally transported to the pituitary 3 d earlier than OT peptides, and in far greater (20-fold) abundance. The early presence and abundance of amidated AVP in the brain and pituitary suggests a trophic function for this peptide during development.

Animals↗
PubMed1987

Immunocytochemical studies of vasotocin, mesotocin, and neurophysins in the Xenopus hypothalamo-neurohypophysial system.

Mesotocinergic and vasotocinergic neurons, which constitute the principal neurons in the hypothalamo-neurohypophysial system in Xenopus, were studied by immunocytochemical techniques. Antibodies that could unequivocally distinguish mesotocin, vasotocin, and their respective neurophysins were used in these studies. A monoclonal antibody directed at rat oxytocin-associated neurophysin (PS-36) detected an antigen that was colocalized with vasotocin, whereas a monoclonal antibody to rat vasopressin-associated neurophysin (PS-45) crossreacted with an antigen in mesotocinergic cells. As vasotocin is regarded as an evolutionary precursor of vasopressin, and as mesotocin is usually associated with oxytocin, we were surprised to see this apparent eptitope switch in the associated neurophysins. One interpretation of this epitope switch is that the final exons encoding for the carboxy-terminals of the mammalian neurophysins, which contain the PS-45 and PS-36 antibody epitopes, are in reversed positions in Xenopus. Approximately 4,000 mesotocinergic and vasotocinergic neurons and their fibers were topographically mapped in the Xenopus hypothalamus. The two types of neurons were intermingled and scattered throughout a large contiguous region including but not limited to the preoptic recess. Small, medium size, and large cells contained these antigens. Immunoreactive fibers were seen in the preoptic area, the neurohypophysial tract, the median eminence, and the neural lobe of the pituitary. The neurophysin-specific monoclonal antibodies have several advantages as phenotypic markers in development; i.e., high titer, low background, and affinity for the prohormone forms as well as for the fully processed neurophysin polypeptides. Their antigens are related gene products whose expression is central to the identity of the two cell types and whose expression is differentially controlled in development. This characterization of their adult distribution provides a basis for future studies of the development of peptidergic phenotype in the central nervous system of Xenopus.

Animals↗
PubMed1987

Characterization of kappa opioid receptors in neurosecretosomes from bovine posterior pituitary.

The binding properties of opioid receptors on isolated nerve terminals (neurosecretosomes) from bovine posterior pituitaries were characterized. Both [3H]etorphine and [3H]ethylketocyclazocine ([3H]EKC) showed high-affinity binding with complex binding isotherms, consistent with the presence of multiple classes of binding sites. [D-Ala2,D-Leu5]enkephalin showed no specific binding and failed to displace [3H]etorphine at high concentrations, indicating the absence of mu, delta, or benzomorphan (kappa 2) sites. Mathematical modelling of the data suggested the presence of three classes of binding sites. The first was of high affinity with Kd values of 0.9 and 2.0 nM for etorphine and EKC, respectively. The second class of sites appeared to bind etorphine with a KD of 150 nM, and EKC with extremely low affinity (unmeasurable binding). The third class of sites was characterized by KD values of 7 and 2 microM for etorphine and EKC, respectively. These results indicate that the nerve terminals of bovine posterior pituitary contain opioid binding sites of the kappa type. Furthermore, these binding sites appear heterogeneous, consisting of at least two and possibly more subtypes or states.

Animals↗