232 research outputs found
Matrix-free calcium in isolated chromaffin vesicles
No full textIsolated secretory vesicles from bovine adrenal medulla contain 80 nmol of Ca2+ and 25 nmol
of Mg2+ per milligram of protein. As determined with a Ca2+-selective electrode, a further accumulation
of about 160 nmol of Ca2+/mg of protein can be attained upon addition of the Ca2+ ionophore A23187.
During this process protons are released from the vesicles, in exchange for Ca2+ ions, as indicated by the
decrease of the pH in the incubation medium or the release of 9-aminoacridine previously taken up by the
vesicles. Intravesicular Mg2+ is not released from the vesicles by A23 187, as determined by atomic emission
spectroscopy. In the presence of N H Q , which causes the collapse of the secretory vesicle transmembrane
proton gradient (ApH), Ca2+ uptake decreases. Under these conditions A23 187-mediated influx of Ca2+
and efflux of H+ cease at Ca2+ concentrations of about 4 pM. Below this concentration Ca2+ is even released
from the vesicles. At the Ca2+ concentration at which no net flux of ions occurs the intravesicular matrix
free Ca2+ equals the extravesicular free Ca2+. In the absence of NH4C1 we determined an intravesicular
pH of 6.2. Under these conditions the Ca2+ influx ceases around 0.15 pM. From this value and the known
pH across the vesicular membrane an intravesicular matrix free Ca2+ concentration of about 24 pM was
calculated. This is within the same order of magnitude as the concentration of free Ca2+ in the vesicles
determined in the presence of NH4C1. Calculation of the total Ca2+ present in the secretory vesicles gives
an apparent intravesicular Ca2+ concentration of 40 mM, which is a factor of lo4 higher than the free
intravesicular concentration of Ca2+. It can be concluded, therefore, that the concentration gradient of free
Ca2+ across the secretory vesicle membrane in the intact chromaffin cells is probably small, which implies
that less energy is required to accumulate and maintain Ca2+ within the vesicles than was previously
anticipated
An autocrine role for pituitary GABA: Activation of GABA-B receptors and regulation of growth hormone levels
No full textThere is increasing evidence suggesting that the neurotransmitter gamma-aminobutyric acid (GABA) is a local factor involved in the regulation of endocrine organs. Examples of such functions are documented in the pancreas, but recent results suggest that GABA may act in a similar way in the pituitary, in which GABA receptors are expressed and pituitary growth hormone (GH) cells provide a source of GABA. We hypothesised that GABA secreted in somatotropes may act as an autoregulatory signaling molecule. To test this hypothesis we first examined the nature of GABA receptors expressed by GH cells. RT-PCR analysis demonstrated that GABA-B receptor subunits R1 and R2 are present in the whole rat pituitary. Laser microdissection of immunostained GH cells, followed by RT-PCR as well as immunoelectron microscopy, showed that GABA-B receptors are expressed on somatotropes. To investigate GABA-B receptor function in somatotropes, we used rat GH3 adenoma cells, which, like pituitary GH cells, express GABA-B R1 and R2 (as assessed by RT-PCR and immunoelectron microscopy) and produce GABA (checked by high performance liquid chromatography). After inhibition of endogenous GABA synthesis, GH production was stimulated by baclofen, a chromatography). After inhibition of endogenous GABA synthesis, GH production was stimulated by bactofen, a GABA-B receptor agonist. By contrast, blocking GABA-B receptors by an antagonist, phaclofen, decreased GH levels. We conclude that in GH-producing cells, GABA acts as an autocrine factor via GABA-B receptors to control GH levels. Copyright (C) 2002 S. KargerAG, Basel
Molecular Aspects of Secretory Granule Exocytosis by Neurons and Endocrine Cells
No full textNeuronal communication and endocrine signaling are fundamental for integrating
the function of tissues and cells in the body. Hormones released by endocrine
cells are transported to the target cells through the circulation. By contrast, transmitter
release from neurons occurs at specialized intercellular junctions, the synapses.
Nevertheless, the mechanisms by which signal molecules are synthesized,
stored, and eventually secreted by neurons and endocrine cells are very similar.
Neurons and endocrine cells have in common two different types of secretory
organelles, indicating the presence of two distinct secretory pathways. The synaptic
vesicles of neurons contain excitatory or inhibitory neurotransmitters, whereas the
secretory granules (also referred to as dense core vesicles, because of their electron
dense content) are filled with neuropeptides and amines. In endocrine cells, peptide
hormones and amines predominate in secretory granules. The function and content
of vesicles, which share antigens with synaptic vesicles, are unknown for most
endocrine cells. However, in B cells of the pancreatic islet, these vesicles contain
GABA, which may be involved in intrainsular signaling.'
Exocytosis of both synaptic vesicles and secretory granules is controlled by
cytoplasmic calcium. However, the precise mechanisms of the subsequent steps,
such as docking of vesicles and fusion of their membranes with the plasma membrane,
are still incompletely understood. This contribution summarizes recent observations
that elucidate components in neurons and endocrine cells involved in
exocytosis. Emphasis is put on the intracellular aspects of the release of secretory
granules that recently have been analyzed in detail
Expression of the neural cell adhesion NCAM in endocrine cells of the ovary
No full textIn the adult mammalian ovary morphogenesis and differentiation processes are under hormonal control and, thus, occur in a highly regulated way during the sexual cycle. Cell-cell interactions, such as cell adhesion and cell separation, are crucial during these events. Here we show that the ovarian endocrine cells, which are prototypes of steroid-producing cells, express neural cell adhesion molecules (NCAMs). The combined use of in situ hybridization histochemistry, immunocytochemistry at the light and electron microscope levels, S1 nuclease protection assays, and Western blotting revealed that in the ovary of the adult rat during the estrus cycle and pregnancy, NCAM mRNA and the 140-kDa isoform of this protein are expressed mainly in granulosa cells of growing preantral and antral follicles and in corpora lutea. Since the granulosa cells lining the forming antrum and the antral fluid were strongly immunoreactive, a role for NCAM in the formation of the follicular antrum is proposed. The expression of NCAM was also associated with luteal cells of the active corpus luteum, indicating a role for NCAM in the morphogenesis of this endocrine compartment. Moreover, thecal cells of large follicles and hypertrophic thecal cells of atretic follicles expressed NCAM, as did interstitial cells, which are derived from thecal cells of atretic follicles. We propose that the adhesion molecule, NCAM, is an important factor involved in the recognition and intercellular interaction of ovarian endocrine cells and, thus, participates in the regulation of the cyclic remodeling processes of the ovarian endocrine compartment
Ca2+-induced fusion of Golgi-derived secretory vesicles isolated from rat liver
No full textDuring the transport of plasma proteins from the
cytoplasma of hepatocytes to the extracellular fluid
srnall vesicles may act as shuttles between the Golgi
complex and the plasma membrane. This type of
intracellular transfer is weil established for various
secretory cells and may be adopted also for the
hepatocyte. Recent investigations have shown that
secretory vesicles fuse with each other during
secretion in mast cells [4] exocrine [5,6] and
endocrine pancreatic tissue [7]. The intervesicular
fusion provides a tool for studies on membrane fusion,
since Golgi-derived vesicles can be isolated from the
hepatocyte and their interaction with various agents,
suggested to trigger membrane fusion, can be
monitored by freeze-cleaving
Ca2+ binding to Chromaffin Vesicle Matrix Proteins
No full textRecently we found that Ca2+ within chromaffin vesicles is largely bound [Bulenda, D., & Gratzl,
M. (1985) Biochemistry 24, 7760-77651. In order to explore the nature of these bonds, we analyzed the
binding of Ca2+ to the vesicle matrix proteins as well as to ATP, the main nucleotide present in these vesicles.
The dissociation constant at pH 7 is 50 pM (number of binding sites, n = 180 nmol/mg of protein) for
Ca2+-protein bonds and 15 pM (n = 0.8 pmol/pmoi) for Ca2+-ATP bonds. When the pH is decreased
to more physiological values (pH 6), the number of binding sites remains the same. However, the affinity
of Ca2+ for the proteins decreases much less than its affinity for ATP (dissociation constant of 90 vs. 70
pM). At pH 6 monovalent cations (30-50 mM) as well as Mg2+ (0.1-0.5 mM), which are also present
within chromaffin vesicles, do not affect the number of binding sites for Ca2+ but cause a decrease in the
affinity of Ca2+ for both proteins and ATP. For Ca2+ binding to ATP in the presence of 0.5 mM Mg2+
we found a dissociation constant of 340 pM and after addition of 35 mM K+ a dissociation constant of 170
pM. Ca2+ binding to the chromaffin vesicle matrix proteins in the presence of 0.5 mM Mg2+ is characterized
by a Kd of 240 pM and after addition of 15 mM Na' by a Kd of 340 pM. The similar affinity of Ca2+
for protein and ATP, especially at pH 6, in media of increased ionic strength and after addition of Mg2+,
points to the possibility that the intravesicular medium determines whether Ca2+ is preferentially bound
to ATP or the chromaffin vesicle matrix proteins. Purified chromogranin A, after sodium dodecyl sulfate-
polyacrylamide gel electrophoresis, stains with a carbocyanine dye ("Stains-all") and, following blotting
onto nitrocellulose, binds to 45Ca2+. A spectrophotometric analysis of dye binding to chromaffin vesicle
matrix proteins revealed a strong absorption band at 615 nm for the dye-protein complex. Since the observed
spectral changes were unaffected by the presence of Ca2+ (100 pM free), the sites interacting with the dye
and Ca2+ must be regarded as different
Birch pollen rupture
No full textThe video shows birch (Betula pendula) pollen grains immersed in water. It can be seen that the pollen grains rupture and expel cytoplasmic material including starch granules. These materials are also referred to as subpollen particles.
The video is a supplement to the article "Isolation of sub-pollen particles (SPPs) of birch: SPPs are potential carriers of ice nucleating macromolecules" in Biogeosciences.
Full reference: Burkart, J., Gratzl, J., Seifried, T. M., Bieber, P., and Grothe, H.: Isolation of subpollen particles (SPPs) of birch: SPPs are potential carriers of ice nucleating macromolecules, Biogeosciences, 18, 1–15, 2021
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