Synthesis, transport, and release of posterior pituitary hormones.

Vasopressin and oxytocin are made and released by neurons of the hypothalamo-neurohypophysial system. Pulse labeling these neurons with radioactive amino acid indicates that the two hormones and their respective neurophysin carrier proteins are synthesized as parts of separate precursor proteins. The precursors seem to be processed into smaller, biologically active molecules while they are being transported along the axon.

Ca(2+) binding protein frequenin mediates GDNF-induced potentiation of Ca(2+) channels and transmitter release.

Molecular mechanisms underlying long-term neurotrophic regulation of synaptic transmission and plasticity are unknown. We report here that long-term treatment of neuromuscular synapses with glial cell line-derived neurotrophic factor (GDNF) potentiates spontaneous and evoked transmitter release, in ways very similar to presynaptic expression of the Ca(2+) binding protein frequenin. GDNF enhances the expression of frequenin in motoneurons, and inhibition of frequenin expression or activity prevents the synaptic action of GDNF. GDNF also facilitates Ca(2+) influx into the nerve terminals during evoked transmission by enhancing Ca(2+) currents. The effect of GDNF on Ca(2+) currents is blocked by inhibition of frequenin expression, occluded by overexpression of frequenin, and is selective to N-type Ca(2+) channels. These results identify an important molecular target that mediates the long-term, synaptic action of a neurotrophic factor.

ets-1 in astrocytes: expression and transmitter-evoked phosphorylation.

The ets-1 protein has been primarily studied as a sequence-specific transcriptional regulator that is predominately expressed in lymphoid cells. In this report, we show that ets-1 is also expressed in astrocytes and astrocytoma cells and is regulated during both signal transduction and differentiation. Both isoforms of ets-1, p51 and p42, were found in astrocytes and astrocytoma cells, but whereas expression of p51 was strong, p42, the alternate splice product previously shown to lack the phosphorylation domain, was difficult to detect and was present at a level 10- to 40-fold lower than that of p51. This differed by roughly an order of magnitude from the ratio generally observable in T cells and thymocytes. In two astrocytoma lines of human origin, CCF and 1321N1, ets-1 phosphorylation was stimulated by bradykinin and carbachol, respectively. Glutamate, norepinephrine, and bradykinin elicited phosphorylation of p51 in cultures of primary rat type 1 astrocytes. ets-1 phosphorylation was dramatically blocked by KT5926, an inhibitor of myosin light-chain kinase, suggesting that this kinase may be involved in phosphorylation of ets-1 in vivo. Investigations of retinoic acid-induced differentiation in P19 cells provided further support for a strong correlation of ets-1 with the pathway for astrocyte differentiation.

Sparks and puffs in oligodendrocyte progenitors: cross talk between ryanodine receptors and inositol trisphosphate receptors.

Investigating how calcium release from the endoplasmic reticulum (ER) is triggered and coordinated is crucial to our understanding of how oligodendrocyte progenitor cells (OPs) develop into myelinating cells. Sparks and puffs represent highly localized Ca(2+) release from the ER through ryanodine receptors (RyRs) and inositol trisphosphate receptors (IP(3)Rs), respectively. To study whether sparks or puffs trigger Ca(2+) waves in OPs, we performed rapid high-resolution line scan recordings in fluo-4-loaded OP processes. We found spontaneous and evoked sparks and puffs, and we have identified functional cross talk between IP(3)Rs and RyRs. Local events evoked using the IP(3)-linked agonist methacholine (MeCh) showed significantly different morphology compared with events evoked using the caffeine analog 3,7-dimethyl-1-propargylxanthine (DMPX). Pretreatment with MeCh potentiated DMPX-evoked events, whereas inhibition of RyRs potentiated events evoked by low concentrations of MeCh. Furthermore, activation of IP(3)Rs but not RyRs was critical for Ca(2+) wave initiation. Using immunocytochemistry, we show OPs express the specific Ca(2+) release channel subtypes RyR3 and IP(3)R2 in patches along OP processes. RyRs are coexpressed with IP(3)Rs in some patches, but IP(3)Rs are also found alone. This differential distribution pattern may underlie the differences in local and global Ca(2+) signals mediated by these two receptors. Thus, in OPs, interactions between IP(3)Rs and RyRs determine the spatial and temporal characteristics of calcium signaling, from microdomains to intracellular waves.

Isolation and purification of calcium-binding proteins from bovine neurohypophyses.

An acidic calcium-binding protein was isolated from the soluble fraction of the homogenate of ox neurohypophyses. The protein has a molecular weight of 35 000 and a subunit weight of 15 000. The purification procedure involved ammonium sulphate fractionation, DEAE-cellulose chromatography and gel filtration on Sephadex G-100 and Sephadex G-50. Conventional and sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated it to be a protein distinct from the S-100 protein and the soluble hormone-binding proteins (neurophysins) abundant in the neurohypophysis. This appears to be the only Ca2+-binding protein in the soluble part of the homogenate, with an apparent Kdiss for Ca2+ of 1.1 X 10(-5) M (at 22 degrees C) and a binding capacity of 2 mol of calcium per mol of protein. Two different Ca2+-binding proteins of molecular weights 16 500 and 68 000, respectively, were identified in the sodium-deoxycholate-soluble proteins from an ox neurohypophysial microsome fraction. One of them (the former) has been isolated in high purity by DEAE-cellulose chromatography and gel filtration on Sephadex G-200. This protein binds 4 mol of calcium per mol of protein with an apparent Kdiss of 1.0 X 10(-5) M (at 22 degrees C). The sodium-deoxycholate-insoluble proteins from the microsomal fraction also have Ca2+-binding components. The soluble Ca2+-binding protein has properties similar to and may be identical to Ca2+-binding proteins which have been isolated from bovine brain and have been demonstrated to be modulators of brain cyclic nucleotide phosphodiesterase and of actinomyosin ATPase. It also resembles Ca2+-binding proteins isolated from bovine adrenals and the electroplax from electrophorus electricus.

The influence of pH on the absorption spectrum of arsenazo III.

The absorption spectrum of arsenazo III in media containing K+, Mg2+ and Ca2+ is sharply influenced by pH in the range of 7.5--5.0. The effect of pH is particularly pronounced in the wavelength range 532--602 nm due to the large pH dependence of the dissociation constant of Mg-arsenazo III complex. Therefore absorption changes at these wavelengths during muscle contraction cannot be used as reliable indicators of free ionized Ca2+ concentration in the cell. The effect of pH is less pronounced, but still noticeable at the wavelength pairs 575--650 or 660--685 nm. Multiple layers of muscle cells grown on polystyrene coils permit measurement of absorption changes of arsenazo III, introduced into the cells, by equilibration with 0.5 mM arsenazo III under routine culture conditions. The absorbance changes recorded at 660--685 nm are probably related to changes in intracellular free Ca2+ concentration.

Fusion of neurohypophyseal membranes in vitro.

Freeze cleaving electron microscopy has shown that fusion of isolated secretory vesicles from bovine neurohypophyses was induced by Ca2+ in micromolar concentrations. Mg2+ and Sr2+ were ineffective. Mg2+ inhibited Ca2+-induced fusion. In suspensions containing secretory vesicles as well as sheets of cell membrane, release of vasopressin parallel to intervesicular fusion and fusion of secretory vesicles with sheets of cell membrane was observed after exposure to Ca2+. Mg2+ and Sr2+ were ineffective in replacing Ca2+ as trigger for fusion or vasopressin release. Intervesicular fusion and exocytotic profiles were observed when isolated neurohypophyses or neurosecretosomes were exposed to cold.

Bax translocation to mitochondria subsequent to a rapid loss of mitochondrial membrane potential.

Bax, a pro-apoptotic member of the Bcl-2 family, is a cytosolic protein that inserts into mitochondrial membranes upon induction of cell death. Using the green fluorescent protein fused to Bax (GFP-Bax) to quantitate mitochondrial binding in living cells we have investigated the cause of Bax association with mitochondria and the time course relative to endogenous and induced changes in mitochondrial membrane potential (DeltaPsi(m)). We have found that staurosporine (STS) induces a loss in DeltaPsi(m) before GFP-Bax translocation can be measured. The onset of the DeltaPsi(m) loss is followed by a rapid and complete collapse of DeltaPsi(m) which is followed by Bax association with mitochondria. The mitochondria uncoupler FCCP, in the presence of the F(1)-F(0) ATPase inhibitor oligomycin, can trigger Bax translocation to mitochondria suggesting that when ATP levels are maintained a collapse of DeltaPsi(m) induces Bax translocation. Neither FCCP nor oligomycin alone alters Bax location. Bax association with mitochondria is also triggered by inhibitors of the electron transport chain, antimycin and rotenone, compounds that collapse DeltaPsi(m) without inducing rapid ATP hydrolysis that typically occurs with uncouplers such as FCCP. Taken together, our results suggest that alterations in mitochondrial energization associated with apoptosis can initiate Bax docking to mitochondria.

Permeability transition pore regulates both mitochondrial membrane potential and agonist-evoked Ca2+ signals in oligodendrocyte progenitors.

In this study, we investigated the importance of mitochondrial permeability transition pore (PTP) in agonist-evoked cytosolic Ca2+ ([Ca2+]c) signals in oligodendrocyte progenitor cells (OP cells). We measured transmembrane potential across the mitochondrial inner membrane (delta psi m) and [Ca2+]c in the immediate vicinity simultaneously using tetramethylrhodamine ethyl ester (TMRE) and calcium green respectively. Stimulation of OP cells with methacholine evoked robust [Ca2+]c signals in approximately 80% of cells which were either oscillatory or showed a peak followed by a plateau. Elevations in [Ca2+]c induced by supramaximal concentrations of the agonist (> 200 microM) were accompanied by changes in delta psi m in 33-42% of the total mitochondria investigated. The mitochondria that responded either depolarized (26-29%), hyperpolarized (7-13%) or showed no change (58-67%). Thus, of the responsive mitochondria, most (70%) depolarized during agonist-evoked [Ca2+]c signals. Blockade of PTP with cyclosporin A (CSA) reduced the number of mitochondria that depolarized with a corresponding increase in the number that hyperpolarized. In addition, CSA or its analogue methyl valine-4- CSA (MeVal-CSA), reduced the frequency of agonist-evoked global [Ca2+]c oscillations. In resting cells, CSA (63%) and MeVal-CSA (77%) hyperpolarized a majority of the mitochondria suggesting that PTP is constitutively active and may show flickering openings. Such hyperpolarizations were not mimicked by either cyclosporine H or verapamil and were inhibited by Ru360, which blocks the mitochondrial uniporter. This observation suggested that in resting cells, Ca2+ ions might redistribute between cytosol and mitochondrial matrix through the uniporter and the PTP. Taken together, these data suggest that PTP may play an important role in regulating delta psi m and local [Ca2+]c signals during agonist stimulation in OP cells.

A mathematical model of agonist-induced propagation of calcium waves in astrocytes.

In astrocytes, calcium signals evoked by neurotransmitters appear as waves within single cells, which spread to other cells in the network. Recent analysis has shown that waves are initiated at a single invariant site in the cell and propagated within the cell in a nonlinear and saltatory manner by regenerative amplification at specific predestined cellular sites. In order to gain insight into local cellular waves and wave collisions we have developed a mathematical model of cellular wave amplification loci. This model is in good agreement with experimental data which includes: ambient calcium gradients in resting cells, wave origination and local amplification and generation of local waves. As observed in experiments, the model also predicts that different locations in the cell can have different frequencies of oscillation. The amplification loci are thought to be specialized areas of the endoplasmic reticulum membrane containing a higher density or higher sensitivity of IP3 receptors. Our analysis suggests that the cellular loci act as weakly coupled oscillators each with its intrinsic latency and frequency of oscillation. Thus the appearance of the propagated calcium wave may be a reflection of these differences rather than an actual diffusional wave propagation.

Images of Ca2+ flux in astrocytes: evidence for spatially distinct sites of Ca2+ release and uptake.

In this study, we have developed a mathematical method to derive the Ca2+ fluxes underlying agonist-evoked Ca2+ waves in cultured rat cortical astrocytes. Astrocytes were stimulated with norepinephrine (100 nM) to evoke Ca2+ waves, which were recorded by measuring Fluo-3 fluorescence changes with high spatial and temporal resolution. Normalized fluorescence (delta F/F) was analyzed in discrete cellular spaces in a series of successive slices along the length of the cell. From these data, Ca2+ flux was then calculated using a one dimensional reaction-diffusion equation which utilizes the temporal and spatial derivatives of the fluorescence data and the diffusion coefficient of Ca2+ in the cytosol. This method identified distinct sites of positive flux (Ca2+ release into the cytosol) and of negative flux (Ca2+ removal from cytosol) and showed that in astrocytes, sites of Ca2+ release from stores regularly alternate with sites of Ca2+ removal from the cytosol. Cross correlation analysis of the two distribution patterns gave positive correlation at 2 microns out of phase and a negative correlation in phase. Thapsigargin-induced Ca2+ waves were analyzed to determine if the negative flux was due to Ca2+ uptake via thapsigargin-sensitive Ca2+ pumps. Negative flux sites were still found under these conditions, suggesting that multiple mechanisms of Ca2+ removal from the cytosol may contribute to negative flux sites. This method of calculation of flux may serve as a means to describe the distribution of functional ion channels and pumps participating in cellular Ca2+ signalling.

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.

Biosynthesis of vasopressin, oxytocin, and neurophysins: isolation and characterization of two common precursors (propressophysin and prooxyphysin).

[35S]Cysteine-labeled putative precursors for vasopressin-associated neurophysin (NP-VP) and oxytocin-associated neurophysin (NP-OT) were isolated from the supraoptic nuclei (SONs) of normal rats. Homozygous Brattleboro rats were deficient in one of these precursors, the NP-VP precursor. Direct support for the hypothesis that OT and its NP- and VP and its NP are synthesized from two separate macromolecular common precursors was obtained by limited proteolysis of the precursors with trypsin and identification of the fragments as NPs, VP, or OT by a number of criteria (14). In this paper, these common precursors designated propressophysin (precursor for NP-VP and arginine VP) and prooxyphysin (precursor for NP-OT and OT) were further characterized. Propressophysin was specifically bound by Concanavalin-A-Sepharose and was eluted by 0.2 M alpha-methyl mannoside, showing that it is glycosylated. In contrast, prooxyphysin does not appear to be glycosylated. Using [3H]fucose as label injected near the SONs, two proteins (mol wt, approximately 10,000) were identified, which appear to be derived from propressophysin, that are rapidly transported to the posterior pituitary of normal rats. These two proteins were absent in homozygous Brattleboro animals. Both propressophysin and prooxyphysin were bound by a NP-Sepharose affinity support. The binding was different from that of arginine VP or OT to NP, since it was independent of pH and was hydrophobic in nature. In addition to prooxyphysin, the SONs of homozygous Brattleboro rats also contained other [35S]cysteine-labeled protein (mol wt, 20,000) composed of a NP-like protein and NP-binding peptides. The tryptic peptides derived from these proteins were very different in their chromatographic (high performance liquid chromatography) properties than the tryptic peptides derived from propressophysin and prooxyphysin. This protein ('X') was not bound by a Concanavalin-A-Sepharose affinity column, although it had chromatographic properties similar to propressophysin on Sephadex G-75.

Proopiocortin-converting enzyme activity in bovine neurosecretory granules.

Neurosecretory granules (NSGs) from neural lobes of bovine pituitary glands were isolated in a highly purified form by metrizamide-sucrose gradient centrifugation. The purified NSGs were lysed and centrifuged, and the supernatants were further fractionated by gel filtration on Sephadex G-75. Proopiocortin-converting enzyme activity was assayed by incubation of [3H]arginine- or [3H]phenylalanine-labeled toad proopiocortin with NSG supernatant fractions. The processed products were identified by immunoprecipitation with ACTH and beta-endorphin antisera, followed by acid-urea gel electrophoresis. The optimum pH for the enzyme-mediated conversion was around pH 5.0. Conversion of toad proopiocortin by NSG converting enzyme activity was inhibited by leupeptin, antipain, p-chloromercuribenzoate, and pepstatin A, but not by diisopropyl fluorophosphate, EDTA, or N-alpha-p-tosyl-L-lysine-chloromethyl ketone HCl. The results suggest that the proopiocortin-converting enzyme activity in bovine neurosecretory granules is due to an acid-thiol protease which may contain secondary hydrophobic binding sites that are involved in substrate recognition.

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.

Differential cellular expression of isoforms of inositol 1,4,5-triphosphate receptors in neurons and glia in brain.

Inositol 1,4,5-trisphosphate receptors (IP3R) are mediators of second messenger-induced intracellular calcium release. Three isoforms are known to be expressed in brain, but their regional distributions and cellular localizations are little known. In order to better understand the roles of IP3 receptor isoforms in brain function, a first step is to define their distributions. We have used affinity-purified antibodies directed against peptides unique to each isoform to determine their sites of expression in rat brain. Type 1 IP3R (IP3R1) is dramatically enriched in Purkinje neurons in cerebellum and neurons in other regions, consistent with previous studies. By contrast, IP3R2 is only detected in glia, whereas IP3R3 is predominantly neuronal, with little detected in glia. IP3R3 is enriched in neuropil, especially in neuronal terminals (which often contain large dense core vesicles) in limbic and basal forebrain regions including olfactory tubercle, central nucleus of the amygdala, and bed nucleus of the stria terminalis. In addition, IP3R1 and IP3R3 have clearly distinct time courses of expression in developing brains. These data suggest separate roles for inositol 1,4,5-trisphosphate receptor isoforms in development, and for glial and neuronal function. The IP3R3 may be involved in regulation of neurotransmitter or neuropeptide release in terminals within specific nuclei of the basal forebrain and limbic system.

Evidence for anion channels in secretory vesicles.

Secretory vesicles from bovine neurohypophysis were reconstituted into lipid bilayers. Electrical measurements on the lipid bilayers under voltage clamp demonstrated the presence of channels that are permeable to chloride ions, are blocked by 4,4'-diisothiocyanostilbene-2,2'-disulfonate, and are slightly voltage dependent. When several different membrane fractions were used for the reconstitution, the probability of finding channels correlated with the fraction of secretory vesicle membrane in the membrane fraction, indicating that the secretory vesicles are the source of the channels. The observed anion channel can provide a pathway for the anion transport that has previously been described for secretory vesicles. The secretory vesicle anion channel may play a role in calcium-induced secretion.

Vasopressin administration to neonatal rats reduces antidiuretic response in adult kidneys.

Neonatal rats who had been given injections of vasopressin on days 1-7 after birth exhibited polyuria as adults. In vivo antidiuresis bioassays demonstrated that their kidneys were deficient in their ability to concentrate urine in response to stimulation with vasopressin. The kidneys also showed a reduction in vasopressin-induced cyclic AMP production, although parathyroid hormone- and calcitonin-induced levels were normal. This suggests a specific deficit in vasopressin receptor-adenylate cyclase function. In contrast, the neonatal treatment had no effect on the sensitivity of the adult vasculature to the hypertensive effects of vasopressin. These results show that short exposures to high levels of vasopressin early in development can produce a long-term defect in vasopressin responsiveness that is specific to the kidney.

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.

Dendrotoxin and 4-aminopyridine potentiate neurohypophysial hormone secretion during low frequency electrical stimulation.

The K+ channel blockers dendrotoxin (10(-9) M) and 4-aminopyridine (10(-6) M) produce similar patterns of augmentation of electrically evoked vasopressin and oxytocin secretion from isolated rat neurointermediate lobes. Both channel blockers produced a greater than two-fold increase in vasopressin release. Oxytocin release, however, was comparably increased only when endogenous opioid inhibition was abolished by the presence of naloxone. The potentiation of hormone release was dependent on the frequency of the applied stimulus. A large effect was seen with 1000 pulses at 4 Hz but not when the same number of pulses was delivered at 12 Hz. These findings suggest that a specific type of K+-channel, sensitive to dendrotoxin and low micromolar 4-aminopyridine, may play a significant role in the frequency-dependent facilitation of secretion from neurohypophysial nerve endings.