scholarly journals Hypotonicity and peptide discharge from a single vesicle

2008 ◽  
Vol 295 (3) ◽  
pp. C624-C631 ◽  
Author(s):  
Jernej Jorgačevski ◽  
Matjaž Stenovec ◽  
Marko Kreft ◽  
Aleksandar Bajić ◽  
Boštjan Rituper ◽  
...  

Neuroendocrine secretory vesicles discharge their cargo in response to a stimulus, but the nature of this event is poorly understood. We studied the release of the pituitary hormone prolactin by hypotonicity, because this hormone also contributes to osmoregulation. In perfused rat lactotrophs, hypotonicity resulted in a transient increase followed by a sustained depression of prolactin release, as monitored by radioimmunoassay. In single cells imaged by confocal microscopy, hypotonicity elicited discharge of the fluorescently labeled atrial natriuretic peptide cargo from ∼2% of vesicles/cell. In contrast, KCl-induced depolarization resulted in a response of ∼10% of vesicles/cell, with different unloading/loading time course of the two fluorescent probes. In cell-attached studies, discrete changes in membrane capacitance were recorded in both unstimulated and stimulated conditions, reflecting single vesicle fusion/fissions with the plasma membrane. In stimulated cells, the probability of occurrence of full fusion events was low and unchanged, whereas over 95% of fusion events were transient, with the open fusion pore probability, the average pore dwell-time, the frequency of occurrence, and the fusion pore conductance increased. Hypotonicity only rarely elicited new fusion events in silent membrane patches. The results indicate that, in hypotonicity-stimulated lactotrophs, transient vesicle fusion mediates hormone release.

1997 ◽  
Vol 153 (1) ◽  
pp. R5-R10 ◽  
Author(s):  
N Masumoto ◽  
Y Ikebuchi ◽  
T Matsuoka ◽  
K Tasaka ◽  
A Miyake ◽  
...  

Abstract The synaptic membrane protein synaptosomal-associated protein (SNAP-25) has recently been implicated as one of the key proteins involved in exocytotic membrane fusion in neurons. However, the role of SNAP-25 in pituitary hormone release is not known. In this study, we determined that SNAP-25 is involved in regulated exocytosis in the clonal pituitary cell line GH4C1. SNAP-25 messenger RNA and protein were detected in GH4C1 cells by RT-PCR and immunoblot analysis, respectively. Immunofluorescence analysis indicated that SNAP-25 protein was localized in the plasma membrane. Next, to determine the function of SNAP-25 in GH4C1 cells, specific inhibitors of SNAP-25, botulinum neurotoxin (BoNT) /A or /E, and antisense SNAP-25 oligonucleotide were used. Neither BoNT/A nor BoNT/E affected thyrotropin-releasing hormone (TRH)-induced cytosolic Ca2+ increase, but both inhibited TRH-induced exocytosis. Moreover, they dose-dependently inhibited TRH-induced prolactin release. The introduction of antisense oligonucleotide into the cells also inhibited TRH-induced prolactin release. These results suggest that SNAP-25 is involved in regulated exocytosis in GH4C1 cells.


1999 ◽  
pp. 17-22 ◽  
Author(s):  
G Van den Berghe ◽  
P Wouters ◽  
CY Bowers ◽  
F de Zegher ◽  
R Bouillon ◽  
...  

OBJECTIVE: During prolonged critical illness, nocturnal pulsatile secretion of GH, TSH and prolactin (PRL) is uniformly reduced but remains responsive to the continuous infusion of GH secretagogues and TRH. Whether such (pertinent) secretagogues would synchronize pituitary secretion of GH, TSH and/or PRL is not known. DESIGN AND METHODS: We explored temporal coupling among GH, TSH and PRL release by calculating cross-correlation among GH, TSH and PRL serum concentration profiles in 86 time series obtained from prolonged critically ill patients by nocturnal blood sampling every 20 min for 9 h during 21-h infusions of either placebo (n=22), GHRH (1 microg/kg/h; n=10), GH-releasing peptide-2 (GHRP-2; 1 microg/kg/h; n=28), TRH (1 microg/kg/h; n=8) or combinations of these agonists (n=8). RESULTS: The normal synchrony among GH, TSH and PRL was absent during placebo delivery. Infusion of GHRP-2, but not GHRH or TRH, markedly synchronized serum profiles of GH, TSH and PRL (all P< or =0.007). After addition of GHRH and TRH to the infusion of GHRP-2, only the synchrony between GH and PRL was maintained (P=0.003 for GHRH + GHRP-2 and P=0.006 for TRH + GHRH + GHRP-2), and was more marked than with GHRP-2 infusion alone (P=0.0006 by ANOVA). CONCLUSIONS: The nocturnal GH, TSH and PRL secretory patterns during prolonged critical illness are herewith further characterized to include loss of synchrony among GH, TSH and PRL release. The synchronizing effect of an exogenous GHRP-2 drive, but not of GHRH or TRH, suggests that the presumed endogenous GHRP-like ligand may participate in the orchestration of coordinated anterior pituitary hormone release.


1992 ◽  
Vol 134 (1) ◽  
pp. 33-41 ◽  
Author(s):  
G. R. Hart ◽  
H. Gowing ◽  
J. M. Burrin

ABSTRACT We have demonstrated that the novel hypothalamic peptide pituitary adenylate cyclase-activating poly-peptide (PACAP-38; 0·1–100 nmol/l) caused an increase in the release of GH, ACTH, LH and α-subunit and accumulation of intracellular cyclic AMP from dispersed rat anterior pituitary cells in static culture for 24 h. There were no significant effects on TSH or prolactin release over the same time-period. PACAP-38 (10 nmol/l) increased the release of GH by 1·3-fold (P<0·05), ACTH by 1·9-fold (P<0·05), LH by 3·5-fold (P<0·001) and α-subunit by 2·0-fold (P< 0·005) and the accumulation of intracellular cyclic AMP by >2-fold (P<0·001) after 24 h. However, the time-course for the effect of PACAP-38 (1 mmol/l) on hormone release and intracellular cyclic AMP levels showed a temporal dissociation. The effect of PACAP-38 on GH and ACTH levels did not reach significance until 24 h whereas the effect of PACAP-38 on LH and α-subunit release reached significance after 4 h implying a different mechanism of action for their release. To investigate the PACAP-induced secretion of LH and α-subunit further, we examined the effects of PACAP after down-regulation of protein kinase C (PKC). PACAP-38 at a dose maximal for the stimulation of LH and α-subunit release (10 nmol/l) added together with the PKC activator, 12-0-tetradecanoyl-phorbol-13-acetate (TPA; 0·1 μmol/l) had no greater effect on LH and α-subunit release than TPA alone over a 4 h incubation period. Increasing the pretreatment time with TPA (0–5 h) at a dose (0·1 μmol/l) known to deplete PKC activity substantially, reduced the ability of PACAP-38 to stimulate LH and α-subunit release and intracellular cyclic AMP levels significantly. We conclude that the stimulatory actions of PACAP on LH and α-subunit relies in part on PKC activity. Journal of Endocrinology (1992) 134, 33–41


2013 ◽  
Vol 456 (1) ◽  
pp. 25-33 ◽  
Author(s):  
Ying Lai ◽  
Xiaochu Lou ◽  
Yongseok Jho ◽  
Tae-Young Yoon ◽  
Yeon-Kyun Shin

We used the single-vesicle-fusion assay to dissect the function of the Syt1 linker region, and our results suggest that the Syt1 linker region might have some capacity to extend for docking and fold to facilitate pore opening.


1991 ◽  
Vol 54 (3) ◽  
pp. 262-266 ◽  
Author(s):  
Krzysztof Lyson ◽  
Samuel M. McCann

1986 ◽  
Vol 87 (6) ◽  
pp. 857-884 ◽  
Author(s):  
J R Hume ◽  
A Uehara

The objective of these experiments was to test the hypothesis that the "creep currents" induced by Na loading of single frog atrial cells (Hume, J. R., and A. Uehara. 1986. Journal of General Physiology. 87:833) may be generated by an electrogenic Na/Ca exchanger. Creep currents induced by Na loading were examined over a wide range of membrane potentials. During depolarizing voltage-clamp pulses, outward creep currents were observed, followed by inward creep currents upon the return to the holding potential. During hyperpolarizing voltage-clamp pulses, creep currents of the opposite polarity were observed: inward creep currents were observed during the pulses, followed by outward creep currents upon the return to the holding potential. The current-voltage relations for inward and outward creep currents in response to depolarizing or hyperpolarizing voltage displacements away from the holding potential all intersect the voltage axis at a common potential, which indicates that inward and outward creep currents may have a common reversal potential under equilibrium conditions and may therefore be generated by a common mechanism. Measurements of inward creep currents confirm that voltage displacements away from the holding potential rapidly alter equilibrium conditions. Current-voltage relationships of inward creep currents after depolarizing voltage-clamp pulses are extremely labile and depend critically upon the amplitude and duration of outward creep currents elicited during preceding voltage-clamp pulses. An optical monitor of mechanical activity in single cells revealed (a) a similar voltage dependence for the outward creep currents induced by Na loading and tonic contraction, and (b) a close correlation between the time course of the decay of the inward creep current and the time course of mechanical relaxation. A mathematical model of electrogenic Na/Ca exchange (Mullins, L.J. 1979. Federation Proceedings. 35:2583; Noble, D. 1986. Cardiac Muscle. 171-200) can adequately account for many of the properties of creep currents. It is concluded that creep currents in single frog atrial cells may be attributed to the operation of an electrogenic Na/Ca exchange mechanism.


2012 ◽  
Vol 8 (1) ◽  
pp. 1-16 ◽  
Author(s):  
Minjoung Kyoung ◽  
Yunxiang Zhang ◽  
Jiajie Diao ◽  
Steven Chu ◽  
Axel T Brunger

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