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H Gainer

Publications and source records attributed to H Gainer.

At least 91 records · Page 5Linked to original sources
PubMed1986

Neurohypophysial peptide potencies in cultured anuran epithelia (A6).

To characterize the V2 receptor (for antidiuretic hormone), we have studied the effect of a number of neurohypophysial hormone analogues on cyclic AMP (cAMP) accumulation and short-circuit current in cultured epithelia formed by A6 cells. A6 is the designation of a continuous cell line derived from the kidney of Xenopus laevis. The order of potency for stimulating cAMP accumulation and short-circuit current in A6 epithelia is like that for stimulating water permeability in toad urinary bladder. As anticipated, arginine vasotocin (AVT), the antidiuretic hormone of Amphibia, is more potent than arginine vasopressin (AVP), the antidiuretic hormone of most mammals. The two hormones differ only in the third amino acid (Phe-3 in AVP is a substitution for Ile-3 in AVT). However, there are a number of striking differences in the responsiveness of these amphibian V2 receptors and mammalian V2 receptors to changes in the 7th, 8th, and 9th amino acids where AVT and AVP are identical. 1) Substitution of Lys-8 for Arg-8 in AVP results in marked loss of potency in Amphibia, whereas there is only modest loss of potency in mammals. 2) Desglycinamide AVP is nearly as potent as AVP in Amphibia, whereas it is inactive in mammals. 2) Tocinoic acid, lacking amino acids 7, 8, and 9, has activity in Amphibia, but pressinoic acid, lacking the same three amino acids, is inactive.

Amino Acids↗
PubMed1986

Action potentials and frequency-dependent secretion in the mouse neurohypophysis.

The frequency-dependence of secretion of arginine vasopressin (AVP) from the mouse neural lobe in vitro was studied and found to be comparable to that reported for the rat neural lobe in vitro. For a stimulus train of 600 pulses, the secretion of AVP per pulse (i.e., facilitation) increased to a maximum at 20 Hz. Compound intracellular action potentials were recorded from the mouse neural lobe using optical recording methods and potentiometric dyes. These extrinsic optical signals reflect the true time courses of transmembrane potential changes (e.g., action potentials), and the action potentials recorded from mouse neural lobes had a duration of 5 ms; at half-maximum peak height. Optical recordings during repetitive stimulation showed that significant spike broadening occurred in each subsequent spike at 10 and 16 Hz stimulation. These data are consistent with a spike broadening hypothesis of frequency-dependent facilitation in the neural lobe. However, 4-aminopyridine, a drug which causes spike broadening in neural tissues by blocking potassium channels, did not produce an increase in secretion of AVP per stimulus from the mouse neural lobe.

4-Aminopyridine↗
PubMed1986

Opioid receptors of bovine posterior pituitary neurosecretosomes are exclusively kappa.

Intact neurosecretosomes (NSS) from bovine posterior pituitary were prepared and characterized. Ligand binding studies were performed, using 3H-[D-Ala2-D-Leu5]enkephalin (DADL), 3H-etorphine and 3H-ethylketocyclazocine (EKC). The absence of specific binding of 3H-DADL and the inability of DADL to displace 3H-etorphine, implies the absence of mu, delta, or DADL-suppressible benzomorphan ("kappa-2") sites. Self- and cross displacement studies for etorphine and EKC suggested receptor heterogeneity. EKC fails to displace - 20% of specific binding of etorphine. Mathematical modelling indicates the presence of three classes of sites. The present findings imply that bovine posterior pituitary opioid receptors are exclusively of the kappa type.

Analgesics, Opioid↗
PubMed1985

Ultrastructural immunolocalization of vasopressin and neurophysin in neurosecretory cells of dehydrated rats.

It has been proposed that neurosecretory material may be transported in a non-vesicular compartment in magnocellular neurons in the hypothalamus of osmotically stimulated rats. We have reexamined this issue using postembedding electron microscopic immunoperoxidase labeling of the secreted peptides (vasopressin and neurophysin) found in these neurons. Reaction product was found exclusively over neurosecretory vesicles in cell bodies, and in axons in the median eminence and neurohypophysis. The findings are consistent with an exclusive secretory vesicle location of the neuropeptides in the hypothalamo-neurohypophysial system of both normal and dehydrated rats.

Animals↗
PubMed1985

Active calcium responses recorded optically from nerve terminals of the frog neurohypophysis.

Voltage-sensitive dyes were used to record by optical means membrane potential changes from nerve terminals in the isolated frog neurohypophysis. Following the block of voltage-sensitive Na+ channels by tetrodotoxin (TTX) and K+ channels by tetraethylammonium (TEA), direct electric field stimulation of the nerve terminals still evoked large active responses. These responses were reversibly blocked by the addition of 0.5 mM CdCl2. At both normal and low [Na+]o, the regenerative response appeared to increase with increasing [Ca++]o (0.1-10 mM). There was a marked decrease in the size of the response, as well as in its rate of rise, at low [Ca++]o (0.2 mM) when [Na+]o was reduced from 120 to 8 mM (replaced by sucrose), but little if any effect of this reduction of [Na+]o at normal [Ca++]o. In normal [Ca++]o, these local responses most probably arise from an inward Ca++ current associated with hormone release from these nerve terminals. At low [Ca++]o, Na+ appears to contribute to the TTX-insensitive inward current.

Action Potentials↗
PubMed1985

Large and rapid changes in light scattering accompany secretion by nerve terminals in the mammalian neurohypophysis.

Large changes in the opacity of the unstained mouse neurohypophysis follow membrane potential changes known to trigger the release of peptide hormones. These intrinsic optical signals, arising in neurosecretory terminals, reflect variations in light scattering and depend upon both the frequency of stimulation and [Ca2+]o. Their magnitude is decreased in the presence of Ca2+ antagonists and by the replacement of H2O in the medium by D2O. These observations suggest a correspondence between the intrinsic optical changes and secretory activity in these nerve terminals.

Animals↗
PubMed1985

The enzymology and intracellular organization of peptide precursor processing: the secretory vesicle hypothesis.

The 'secretory vesicle hypothesis of precursor processing' states that the initial endopeptidase cleavages which excise the nascent, biologically active peptides from their protein precursors occur primarily in secretory vesicles (or granules). Hence, all the processing steps subsequent to these cleavages must also occur within these organelles. Two types of evidence are presented in support of this view: (1) cell biological studies which implicate the secretory vesicle as the site of precursor conversion to peptides, and (2) enzymological studies which locate and characterize putative processing enzymes in secretory vesicles. The processing enzymes reviewed include the 'prohormone-converting enzymes' which cleave at pairs of basic amino acids, other endopeptidases, carboxypeptidase-B-like enzymes and aminopeptidase, and N-acetylation and alpha-amidation enzymes. The properties of these enzymes in relation to the nature of the processing micro-environment in the secretory vesicles is discussed.

Adrenal Medulla↗
PubMed1985

Ultrastructural localization of immunoreactive neurophysins using monoclonal antibodies and protein A-gold.

Using three different monoclonal antibodies against rat neurophysins (5), with protein A-gold as immunocytochemical marker (27), the murid hypothalamoneurohy-pophysial system was studied at the ultrastructural level. Postembedding staining was done on epoxy-embedded sections of supraoptic nuclei and posterior pituitaries. Specific immunolabeling of vasopressinergic and oxytocinergic neurosecretory granules was observed in tissues fixed with glutaraldehyde or glutaraldehyde mixtures (containing paraformaldehyde and picric acid), with or without osmium tetroxide postfixation and with or without sodium metaperiodate oxidation. Some autophagic vacuoles containing lysed neurosecretory granules were also neurophysin immunoreactive. Nonspecific background staining was extremely low. An attempt was made to appraise labeling intensities semiquantitatively by counting gold particles in relation to number of secretory granules per axonal varicosity. Immunoreactivity was measurably influenced by the mode of fixation, sodium metaperiodate oxidation, and titer and affinity of the antibody. The protein A-gold technique using monoclonal antibodies against neurophysins provides a superior means of ultrastructural analysis of the hypothalamoneurohypophysial system, both visually and morphometrically.

Animals↗
PubMed1985

Neurophysin in the hypothalamo-neurohypophysial system. I. Production and characterization of monoclonal antibodies.

Seven mouse monoclonal antibodies (IgGs) were produced against rat neurophysins (NPs). Three were specifically directed against vasopressin-associated NP (NP-AVP), and four were specific for oxytocin-associated NP (NP-OT). These specificities were observed in liquid phase assays, immunoblot, and immunoprecipitation experiments. Homozygous Brattleboro rat tissues and extracts, which do not contain vasopressin or NP-AVP, did not react with the anti-NP-AVP antibodies but reacted with high affinity to the anti-NP-OT antibodies. In immunoprecipitation assays the antibodies brought down the appropriate NPs as well as their precursor molecules synthesized in vivo with no detectable cross-reactivity. In solid phase assays where the antigens were presented in a different manner, there was a significant cross-reactivity of the anti-NP-AVP antibodies with NP-OT. The extent of this cross-reactivity in solid phase correlated with the cross-reactivities of the antibodies observed in immunocytochemical studies. These solid phase (and immunocytochemical) data demonstrated that liquid phase specificities and absorption controls of antibodies are inadequate to assess their immunocytochemical (solid phase) specificities. Posterior pituitary extracts from the mouse and frog, as well as purified NPs from the rat, cow, and human were studied for their cross-reactivities to two of the antibodies, PS 36 and PS 45. In liquid phase assays the anti-rat NP-OT antibody, PS 36, reacted only with rat and mouse NPs and did not cross-react with NPs from any of the other species. In contrast, the anti-rat NP-AVP antibody, PS 45, was cross-reactive across species lines including an NP-like antigen extracted from frog posterior pituitaries. Immunoblot staining with these antibodies showed heterogeneity of NP-AVP and NP-OT in the rat posterior pituitary. Analysis of the epitopes for PS 36 and PS 45 indicated the antigenic determinants were located near amino acid positions 80 to 81 in NP-OT and 75 to 86 in NP-AVP, respectively.

Animals↗
PubMed1985

Neurophysin in the hypothalamo-neurohypophysial system. II. Immunocytochemical studies of the ontogeny of oxytocinergic and vasopressinergic neurons.

Two anti-neurophysin monoclonal antibodies (MABs), PS 36 and PS 41, described in the preceding paper (Ben-Barak, Y, J.T. Russell, M.H. Whitnall, K. Ozato, and H. Gainer (1985) J. Neurosci. 5:000-000), allowed us to specifically stain for oxytocin-associated neurophysin (NP-OT) or vasopressin-associated neurophysin (NP-AVP) in the hypothalamus of developing rats. Staining with these MABs specific for NP-OT or NP-AVP showed that both types of neurophysin appeared in cells in the developing hypothalamus as early as embryonic day (E16) and continued to increase in immunoreactivity throughout fetal life. The literature indicated that oxytocin appears in the system between E20 and E22, much later than vasopressin (E16 to E17), which we confirmed in immunocytochemical experiments using affinity-purified antisera to these hormones. Since the MABs recognize the specific prohormones as well as the specific mature neurophysins (Ben-Barak, Y., J. T. Russell, M.H. Whitnall, K. Ozato, and H. Gainer (1985) J. Neurosci. 5: 81-97), we conclude that there is a developmental delay between the synthesis of the oxytocin prohormone (pro-oxyphysin) and its processing to form oxytocin and NP-OT. The delay in prohormone processing in the oxytocin cells was correlated with a delay in immunocytochemically detectable neurites as compared to the vasopressin cells. This reduced level of axonal and dendritic immunoreactivity was still obvious in the oxytocin cells at 9 days after birth. In contrast, the clustering of cells to form adult-like hypothalamic nuclei appeared to follow similar time courses for the two types of cells. Adult-like distributions of cells staining for NP-OT and NP-AVP were already apparent in the supraoptic and paraventricular nuclei by E17.

Animals↗
PubMed1984

An aminopeptidase activity in bovine pituitary secretory vesicles that cleaves the N-terminal arginine from beta-lipotropin60-65.

Secretory vesicles isolated from the neural and intermediate lobes of the bovine pituitary contained a membrane-bound aminopeptidase activity which cleaved arginine from beta-LPH60-65 (Arg-Tyr-Gly-Gly-Phe-Met) and Arg-MCA. Neither methionine enkephalin (Tyr-Gly-Gly-Phe-Met) nor Substance P, which has an N-terminal arginine followed by a proline, could serve as substrates for this aminopeptidase activity; nor could cathepsin B-like or chymotrypsin-like enzyme activities be detected in the vesicle preparations. Maximal enzyme activity was at pH 6.0, and the activity was inhibited by EDTA, stimulated by Co2+ and Zn2+, but was unaffected by leupeptin, pepstatin A, phenylmethylsulfonyl fluoride and p-chloromercuribenzenesulfonate, suggesting that the enzyme is a metalloaminopeptidase. The presence of this aminopeptidase activity in secretory vesicles suggests that it may be involved in peptide prohormone processing.

Aminopeptidases↗
PubMed1984

Phylogenetic cross-reactivities of monoclonal antibodies produced against rat neurophysin.

Previously described mouse monoclonal antibodies against rat neurophysins [Ben-Barak, Y., et al. (1985); Whitnall, M. H., et al. (1985)] were studied here for their cross-reactivities to neurophysins (NPs) from other vertebrate species. Posterior pituitary extracts from various mammals (rat, mouse, cow, human) and lower vertebrates (frog, ratfish) were studied. The monoclonal antibodies displayed several distinct patterns of cross-reactivity to the various species, indicating that the epitopes which they recognized were different. PS 67 bound strongly to rat pituitary extract in solid-phase radioimmunoassay (RIA) but showed no cross-reactivity with extracts from any of the other species tested, including the mouse. PS 36 cross-reacted with mouse and frog extracts but showed almost no cross-reactivity with cow and none to ratfish extracts. PS 41 cross-reacted with mouse, cow, and frog extracts. PS 45 was the most cross-reactive antibody and recognized an antigen in extracts from mouse, cow, frog, and ratfish pituitaries. Electrophoresis of proteins extracted from posterior pituitaries, followed by immunoblot staining with either PS 36 or PS 45, demonstrated that the NP-like molecules within each species are heterogeneous, i.e., more than two bands stained in each species. The frog NP stained by PS 45 was about twice the molecular weight of the mammalian NPs. The possible valve of the PS 45 antibody for future molecular cloning experiments on the arginine vasotocin precursor in lower vertebrates is discussed.

Animals↗
PubMed1984

Angiotensin I-generating acid endopeptidase activity in neurosecretory vesicles isolated from bovine pituitary.

Secretory vesicles purified from the neural and intermediate lobes of the bovine pituitary contain acidic endopeptidases which are capable of converting renin tetradecapeptide (RTD) substrate to Angiotensin I (AI). Preliminary characterization of the neurosecretory vesicle (NSV) endopeptidase showed that it had a pH optimum of 4.0, and unlike renin was inactive at pHs greater than 6.0. It is inhibited by 10(-6) M pepstatin A, but not by PMSF, leupeptin, PMBS, or the specific renin inhibitor H-142. This NSV endopeptidase differed from cathepsin D in that it was unable to degrade alpha-casein, but was quite active in generating AI from RTD (Vmax = 5 moles/g protein/hour). No enzyme activity that could convert AI to Angiotensin II could be detected in the NSVs suggesting that the acidic endopeptidase is involved in processing neurosecretory vesicle proteins other than those associated with the renin angiotensin system in the brain.

Angiotensin I↗
PubMed1984

Distribution of acid protease activity in the squid nervous system.

Acid protease activity was measured in homogenized stellate ganglion, axoplasm extruded from the squid giant axon, homogenized fin nerves, and in lysed synaptosomes prepared from the optic lobe of the squid. At least two different acid protease classes were distinguished on the bases of their inhibitor profiles. Acid protease activity was present in each of the above tissues except extruded axoplasm. This result suggests that the acid protease activity found in our homogenized fin nerves might be located not within the axons but rather in glial cells or extracellular tissue. The absence of acid protease activity in extruded axoplasm indicates that acid proteases are unlikely to play a significant role in the catabolism of intracellular proteins along the length of the axon.

Animals↗
PubMed1983

Onset of neurophysin self-association upon neurophysin/neuropeptide hormone precursor biosynthesis.

The potential of the common biosynthetic precursor of neurophysin and neuropeptide hormones to self-associate has been assessed by quantitative affinity chromatographic analysis. The precursor form, with the hormone sequence in the amino terminal region and assumed able to interact intramolecularly with the hormone binding site of the neurophysin domain of the folded precursor, exhibits an affinity for neurophysin-agarose which is intermediate between those of unliganded neurophysin and non-covalently hormone-liganded neurophysin. The results lead to a prediction that neurophysin self-association is established upon precursor synthesis and prior to limited proteolysis of the precursor to release mature neurophysin and hormone components. Such self-association could play a role in packaging of the precursor into secretory granules and in regulating subsequent precursor processing events within the granules.

Animals↗