Hormone release from isolated nerve endings of the rat neurohypophysis.

Cazalis, Monique; Dayanithi, Govindan; Nordmann, J

doi:10.1113/jphysiol.1987.sp016686

Cited by 174 publications

(125 citation statements)

References 42 publications

(53 reference statements)

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“…The medium was then replaced with a buffer containing 270 mM sucrose, 10 mM Tris- HEPES, and 2 mM EGT A. The nerve endings were then isolated as described by Cazalis et al 9 Briefty, the neurohypophyses were homogenized in the buffered sucrose solution and centrifuged at 100 g for Imin. The supernatant was taken and centrifuged further at 2400 g for 5 min.…”

Section: Methodsmentioning

confidence: 99%

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The light chain of tetanus toxin inhibits calcium-dependent vasopressin release from permeabilized nerve endings

et al. 1992

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Abstract-The effects of tetanus toxin and its light and heavy chain subunits on vasopressin release were investigated in digitonin-permeabilized neurosecretory nerve terminals isolated from the neural lobe ofthe rat pituitary gland. Exocytosis was induced by chal1enging the permeabilized nerve endings with micromolar calcium concentrations. Tetanus toxin inhibited vasopressin release only in the presence of the reducing agent dithiothreitol. This effect was irreversible. The purified light chain of tetanus toxin strongly inhibited exocytosis in a dose-dependent manner with half-maximal effect at c. 10 nM. The action of the light chain was observed after only 2.5 min of preincubation. Separated heavy chain subunit had no effect on hormone secretion. Inhibition ofvasopressin release could be prevented by preincubating the light chain of tetanus toxin with an immune serum against tetanus toxin.The data c1early demonstrate that in mammalian neurosecretory nerve endings tetanus toxin acts at a step downstream from the activation by Ca2+ ofthe exocytotic machinery and that the functional domain of this toxin is confined to its light chain.In recent years, increasing use has been made of bacterial, plant and animal toxins in neurobiological research. A potent bacterial neurotoxin synthesized as a single-chain protein by Clostridium telani is tetanus toxin (for reviews see Refs 13, 24). Subsequent proteolytic processing leads to a pharmacologically more active molecule 27 which consists of disulfide-Iinked heavy (100,000 mol. wt) and light (50,000 mol. wt) chains. Tetanus toxin is a potent inhibitor of neurotransmitter secretion from neuronal tissue. For example, neurotransmitter release at central inhibitory synapses,17 at neuromuscular junctions l8 or at nerve endings of the neurohypophysis l4 is strongly reduced in the presence of the toxin. It has been shown that the individual chains of tetanus toxin alone are not neurotoxic in vivo. 16 Application of tetanus toxin to intact cells in culture or intoxication in vivo requires hours before transmitter release is blocked. This delay refiects the time required for the binding of the toxin to the plasma membrane followed by intemalization and intracellular action (cf. Ref. 13). In vertebrates the domain binding to the cell surface appears to be localized at the C-terminal of the heavy chain (cf. Ref. 24). tPresent address:

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Section: Methodsmentioning

confidence: 99%

“…a The Ca< , -evoked hormone release was calculated by subtracting the mean basal release determined in the fractions preceding the onset ofthe Ca2+ stimulus from the amount of hormone found in each of the following eight fractions. 9 Free Ca 2 , concentrations were ca1culated and controlIed with a Ca 2 +-sensitive electrode. 3 .…”

Section: Methodsmentioning

confidence: 99%

The light chain of tetanus toxin inhibits calcium-dependent vasopressin release from permeabilized nerve endings

et al. 1992

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“…Neurohypophysial terminals were homogenized as previously described (Cazalis et al, 1987) in a solution containing 270 mM sucrose, 10 mM HEPES, and 1 mM EGTA, pH 7.3. The homogenate was then plated onto a poly-D-lysine-coated 35 mm polystyrene dish or a coverslip, and after attachment, terminals underwent electrophysiological measurements or immunocytochemistry.…”

Section: Nh Terminal Preparationmentioning

confidence: 99%

Alcohol Tolerance in Large-Conductance, Calcium-Activated Potassium Channels of CNS Terminals Is Intrinsic and Includes Two Components: Decreased Ethanol Potentiation and Decreased Channel Density

Pietrzykowski¹,

Martin²,

Puig³

et al. 2004

J. Neurosci.

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Tolerance is an important element of drug addiction and provides a model for understanding neuronal plasticity. The hypothalamicneurohypophysial system (HNS) is an established preparation in which to study the actions of alcohol. Acute application of alcohol to the rat neurohypophysis potentiates large-conductance calcium-sensitive potassium channels (BK), contributing to inhibition of hormone secretion. A cultured HNS explant from adult rat was used to explore the molecular mechanisms of BK tolerance after prolonged alcohol exposure. Ethanol tolerance was intrinsic to the HNS and consisted of: (1) decreased BK potentiation by ethanol, complete within 12 min of exposure, and (2) decreased current density, which was not complete until 24 hr after exposure, indicating that the two components of tolerance represent distinct processes. Single-channel properties were not affected by chronic exposure, suggesting that decreased current density resulted from downregulation of functional channels in the membrane. Indeed, we observed decreased immunolabeling against the BK ␣-subunit on the surface of tolerant terminals. Analysis using confocal microscopy revealed a reduction of BK channel clustering, likely associated with the internalization of the channel.

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“…The medium was then replaced by a buffer containing 270 mM sucrose, 10 mM Tris-HEPES, 2 mM EGTA instead of 0.01 mM EGTA as described previously. 8 The nerve endings were then isolated as described 8 with some minor modifications. Briefly, the neurohypophyses were homogenized in the buffered sucrose solution and centrifuged at 100 g for Imin.…”

Section: Preparation 0/ Isolated Neurosecretory Nerve Terminalsmentioning

confidence: 99%

“…1 The Ca2+ evoked hormone release was calcula ted by subtracting the mean basal release determined in the fractions preceding the onset of the Ca 2 + stimulus from the amount of hormone found in each of the following eight fractions. 8 Free Ca2+ concentrations were calculated using the stability constants listed 18 and measured with a Cal+-sensitive electrode 4 as described recently.11…”

Section: Superfusion Of the Isolated Nerve Endingsmentioning

confidence: 99%

Release of vasopressin from isolated permeabilized neurosecretory nerve terminals is blocked by the light chain of botulinum A toxin

et al. 1990

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Abstract-The intracellular action on exocytosis of botulinim A toxin and constituent chains was studied using permeabilized isolated nerve endings from the rat neural lobe. The release of the neuropeptide vasopressin was measured by radioimmunoassay. In the presence ofthe reducing agent dithiothreitol, the two-chain form of botulinum A toxin inhibited vasopressin release induced by 10 Jl M free calcium. Half maximal inhibition was obtained with 15 nM botulinum A toxin. In the absence of the heavy chain the light chain of the toxin strongly inhibited exocytosis with a half maximal effect of 2.5 nM. The inhibitory effects on secretion could be prevented by incubating the light chain with an immune serum against botulinum A toxin. The heavy chain of botulinum A toxin did not affect vasopressin release. However, it prevented the inhibitory effects of the light chain on stimulated exocytosis.It is concluded that botulinum A toxin inhibits the calcium-dependent step leading to exocytosis by interfering with a target present in the isolated and permeabilized nerve terminals. The functional domain of this neurotoxin, which is responsible for the inhibition of vasopressin release, is present in its light chain.Botulinum A toxin belongs to a family of cIosely reIated neurotoxins. The 150,000 mol. wt protein consists of a heavy chain (100,000 mol. wt) and a light chain (50,000 mol. wt) covalently linked by a disulfide bond. It inhibits neuro transmission at the peripheral cholinergic nerve endings by inhibiting neurotransmitter release. A three-step model is thought to produce the paralytic effects. It consists of (i) extracellular binding, (ii) internalization and (iii) intracellular poisoning (cf. Refs 12 and 19). The toxins' effects are restricted to the nervous tissue when given extracellularly. However, an extracellular application does not allow the differentiation of the intracellular processes involved in the final inhibition of transmitter release and the role played by the individual chains of the toxin.Recently, using cultured adrenal chromaffin cells permeabilized with streptolysin 0, it has been shown that intracellularly applied botulinum A toxin is more effective in the presence of a reducing agent. Indeed, the light chain of botulinum A toxin alone inhibited Ca 2 + -stimulated catecholamine release. 5 ,20,21 In contrast, both the heavy and the light chain must be tPresent address:

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Hormone release from isolated nerve endings of the rat neurohypophysis.

Cited by 174 publications

References 42 publications

The light chain of tetanus toxin inhibits calcium-dependent vasopressin release from permeabilized nerve endings

The light chain of tetanus toxin inhibits calcium-dependent vasopressin release from permeabilized nerve endings

Alcohol Tolerance in Large-Conductance, Calcium-Activated Potassium Channels of CNS Terminals Is Intrinsic and Includes Two Components: Decreased Ethanol Potentiation and Decreased Channel Density

Release of vasopressin from isolated permeabilized neurosecretory nerve terminals is blocked by the light chain of botulinum A toxin

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