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J K Angleson

Publications and source records attributed to J K Angleson.

13 recordsLinked to original sources
PubMed2001

Sustained stimulation of exocytosis triggers continuous membrane retrieval in rat pituitary somatotrophs.

We studied the relationship between exocytosis and endocytosis in rat pituitary somatotrophs using patch-clamp capacitance, FM1-43 fluorescence imaging and amperometry. Stimulation of exocytosis through voltage-dependent Ca2+ channels by depolarizations (1-5 s) increased the capacitance by 4.3 +/- 0.9 % and the fluorescence by 6.6 +/- 1.1 % (10 cells). The correlation between the capacitance and fluorescence changes indicated that the cell membrane and granule membrane added via exocytosis were stained with the membrane-bound fluorescent dye FM1-43 in a quantitatively similar manner. Intracellular dialysis (0.5-4.5 min) with elevated Ca2+ (1.5-100 microM) evoked continuous exocytosis that was detected with a carbon fibre electrode from dopamine-loaded cells (10 cells) or as an increase in FM1-43 fluorescence (56 +/- 10 %; 21 cells). Interestingly during Ca2+ dialysis the capacitance did not significantly change (2 +/- 1 %; 31 cells), indicating that endocytosis efficiently retrieved increased cell membrane. Sustained endocytosis was not blocked when the intracellular GTP (300 microM) was replaced with GTP[gamma]S. Replacing intracellular Ca2+ (100 microM) with Ba2+ (300 microM) or Sr2+ (200 microM), or reducing the pH of the intracellular solution from 7.2 to 6.2 did not block sustained endocytosis. Our results suggest that pituitary somatotrophs have the ability to undergo continuous exocytosis and membrane retrieval that persist in whole-cell recordings.

Animals↗
PubMed2001

Intraterminal Ca(2+) and spontaneous transmitter release at the frog neuromuscular junction.

We investigated the relationship between intraterminal Ca(2+) concentration ([Ca(2+)](i)) and the frequency of miniature end plate potentials (MEPPs) at the frog neuromuscular junction by use of ratiometric imaging of fura-2-loaded nerve terminals and intracellular recording of MEPPs. Elevation of extracellular [KCl] over the range of 2-20 mM resulted in increases in [Ca(2+)](i) and MEPP frequency. Loading terminals with the fast and slow Ca(2+)-buffers bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid-acetoxymethyl (BAPTA-AM) and EGTA-AM resulted in equivalent reductions in the KCl-dependent increases in MEPP frequency. The [Ca(2+)](i) dependence of MEPP frequency determined by elevation of [Ca(2+)](i) due to application of 0.1-10 microM ionomycin was similar to that determined when [Ca(2+)](i) was raised by increasing extracellular KCl. Measurements in 10 mM extracellular KCl revealed that application of the phorbol ester phorbol 12 myristate 13-acetetate (PMA) caused an increase in MEPP frequency while the inactive analogue, 4 alpha-PMA, did not. PMA application also caused an increase in [Ca(2+)](i). The relationship between [Ca(2+)](i) and MEPP frequency in PMA was the same as was determined by the other methods of raising [Ca(2+)](i). Under all conditions tested, our data revealed a low [Ca(2+)](i) threshold for activation of transmitter release and are consistent with a K(d) for [Ca(2+)](i) on the order of 1 microM.

Animals↗
PubMed2000

Differential regulation of granule-to-granule and granule-to-plasma membrane fusion during secretion from rat pituitary lactotrophs.

We used fluorescence imaging of individual exocytic events together with electron microscopy to study the regulation of dense core granule-to-plasma membrane fusion and granule-to-granule fusion events that occur during secretion from rat pituitary lactotrophs. Stimulating secretion with elevated extracellular potassium, with the calcium ionophore ionomycin, or with thyrotropin releasing hormone or vasoactive intestinal polypeptide resulted in abundant exocytic structures. Approximately 67% of these structures consisted of multiple granules fused together sharing a single exocytic opening with the plasma membrane, i.e., compound exocytosis. For all of these stimulation conditions there appeared to be a finite number of plasma membrane fusion sites, approximately 11 sites around each cellular equator. However, a granule could fuse directly with another granule that had already fused with the plasma membrane even before all plasma membrane sites were occupied. Granule-to-plasma membrane and granule-to-granule fusion events were subject to different regulations. Forskolin, which can elevate cAMP, increased the number of granule-to-granule fusion events without altering the number of granule-to-plasma membrane fusion events. In contrast, the phorbol ester PMA, which activates protein kinase C increased both granule-to-granule and granule-to-plasma membrane fusion events. These results provide a cellular mechanism that can account for the previously demonstrated potentiation of secretion from lactotrophs by cAMP- and PKC-dependent pathways.

Animals↗
PubMed1999

Regulation of dense core release from neuroendocrine cells revealed by imaging single exocytic events.

Using FM1-43 fluorescence, we have optically detected single exocytic and endocytic events in rat pituitary lactotrophs. About fifty discrete fluorescent spots abruptly appear around the entire surface of a cell bathed in FM1-43 and high-potassium saline. The spots, which also immunostain for prolactin, reflect the labeling of dense cores as well as membranes of exocytosed secretory granules. Stained cores are not released, but remain attached to the cell and are eventually endocytosed. However, in cells exposed to dopamine (or an analog, bromocriptine), the cores dissolve and are secreted after several seconds. Solubilization of dense cores is mediated through a reduction in cytoplasmic cyclic AMP. Thus, the composition of secretions from individual secretory granules is regulated.

Animals↗
PubMed1999

Monitoring secretory membrane with FM1-43 fluorescence.

FM1-43 and similar styryl dyes have proven useful as probes for membrane trafficking because they reversibly stain membranes, are impermeable to membranes, and are more fluorescent when bound to membranes than when in solution. Because these dyes stain membranes in an activity-dependent manner, they are ideal for studies of neurotransmitter release mechanisms such as synaptic vesicle recycling, exocytosis, and endocytosis. FM dyes have been used in conjunction with other techniques such as fluorescent calcium indicator dyes and electrophysiological techniques to elucidate mechanisms of presynaptic calcium homeostasis and modulation of neurotransmitter release. Presynaptic membranes have been marked by FM dyes in studies of synaptogenesis and reinnervation. As a probe for endocytosed membranes, these dyes have been used to examine vacuole formation in yeast. These versatile membrane dyes are useful in a variety of applications.

Animals↗
PubMed1998

The synaptic vesicle cycle.

The ins and outs of the synaptic vesicle cycle are being examined in increasing detail with diverse investigative tools in a variety of cell types, particularly those with large granules. The cycle begins with the opening of a fusion pore that connects the vesicle lumen to the extracellular fluid. Sensitive electrophysiological techniques reveal the often-stuttering behavior of single pores in non-neuronal cells, through which small molecules trickle until the fusion pore expands and the remaining contents erupt from the vesicle. The granule membranes are then retrieved by multiple processes that appear to act in parallel and that are distinguished from each other kinetically and ultrastructurally. Following endocytosis, synaptic vesicles are then shuttled back into the vesicle pool, where they briefly mix with other vesicles, become immobilized, and remain gelled with their neighbors, even while moving en masse again to the presynaptic membrane as a prelude for another round of exocytosis.

Animals↗
PubMed1997

A comparison of the efficiency of G protein activation by ligand-free and light-activated forms of rhodopsin.

Activation of the photoreceptor G protein transducin (Gt) by opsin, the ligand-free form of rhodopsin, was measured using rod outer segment membranes with densities of opsin and Gt similar to those found in rod cells. When GTPgammaS was used as the activating nucleotide, opsin catalyzed transducin activation with an exponential time course with a rate constant k(act) on the order of 2 x 10(-3)s(-1). Comparison under these conditions to activation by flash-generated metarhodopsin II (MII) revealed that opsin- and R*-catalyzed activation showed similar kinetics when MII was present at a surface density approximately 10(-6) lower than that of opsin. Thus, in contrast to some previous reports, we find that the catalytic potency of opsin is only approximately 10(-6) that of MII. In the presence of residual retinaldehyde-derived species present in membranes treated with hydroxylamine after bleaching, the apparent k(act) observed was much higher than that for opsin, suggesting a possible explanation for previous reports of more efficient activation by opsin. These results are important for considering the possible role of opsin in the diverse phenomena in which it has been suggested to play a key role, such as bleaching desensitization and retinal degeneration induced by continuous light or vitamin A deprivation.

Animals↗
PubMed1997

Monitoring secretion in real time: capacitance, amperometry and fluorescence compared.

Techniques for measuring exocytosis, endocytosis and vesicle cycling in living cells in real time have resulted in a rapid expansion in the knowledge of these processes in neurons and other secretory cells. Several experimental approaches, developed during the past decade, have played key roles in this expansion. In this review we focus on three techniques: electrophysiological methods for monitoring membrane capacitance, electrochemical methods for detecting released secretory contents and optical methods for imaging membranes of endosomes and recycled vesicles that are stained with fluorescent dyes. Each technique has contributed unique and complementary information about the vesicle cycle, advancing our knowledge of the kinetics of membrane fusion and retrieval, the identity of the secretory contents and the spatial patterns and directional pathways involved in secretory membrane recycling. Naturally, each technique has inherent limitations; some of these shortcomings have recently been resolved by using more than one method simultaneously.

Animals↗
PubMed1996

High affinity interactions of GTPgammaS with the heterotrimeric G protein, transducin. Evidence at high and low protein concentrations.

A well known difference in nucleotide binding characteristics between heterotrimeric G proteins and small GTP binding proteins of the Ras superfamily is that the former bind GTP or guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) with a much lower affinity (Kd approximately 10(-8)-10(-7) M) than the latter (Kd approximately 10(-11)-10(-10)M). We report here that the alpha subunit of the heterotrimeric G protein transducin (Gt) binds GTPgammaS with an affinity comparable to that of Ras. High affinity binding was suggested by GTPgammaS titrations of rod outer segment samples with Gt concentrations in the range of 7 nM to 300 nM; the results were more consistent with a dissociation constant for GTPgammaS in the subnanomolar range, than with one in the 10(-8)-10(-7) M range typically reported for heterotrimeric G proteins. Equilibrium binding experiments with G protein concentrations in the subnanomolar to nanomolar range confirmed this conclusion and revealed a dissociation constant of 50 pM. Thus, transducin's affinity for GTPgammaS, and by inference, for GTP, appears to be approximately three orders of magnitude higher than previously reported. These results raise the possibility that some results obtained with high concentrations of nucleotide analogues may be due to minute traces of contaminants such as GTP, GTPgammaS, or GTPalphaS, that have high affinities for Gtalpha.

Guanosine 5'-O-(3-Thiotriphosphate)↗
PubMed1996

Low affinity interactions of GDPbetaS and ribose- or phosphoryl-substituted GTP analogues with the heterotrimeric G protein, transducin.

We have examined the effects of three commonly used classes of guanine nucleotide analogues on the retinal G protein, transducin (Gt), and found them to be quite different from those that might be expected from results with other GTP-binding proteins. The most surprising results were with guanosine 5'-O-(2-thiodiphosphate) (GDPbetaS); rather than inhibiting activation of Gt, GDPbetaS addition activated Gt as a result of a trace contaminant. Even when the contaminant levels were reduced 5-fold by chromatography, its effects dominated those of GDPbetaS, which binds Gt at least 1500-fold more weakly than guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS). The affinity of Gt for GDP was found to be at least 300-fold weaker than for GTPgammaS, while the affinities of GTP and GTPgammaS were similar. Ribose-modified GTP analogues, including 2'(3')-O-(N-methylanthraniloyl) GTP (mantGTP), 2'(3')-O-[(2-aminoethyl)carbamyl] GTP (edGTP), and adducts of fluorescein 5-isothiocyanate and rhodamine B-isothiocyanate with edGTP, interacted extremely weakly, if at all, with the GTP binding site of the alpha subunit of Gt. They were neither effective activators of Gt nor effective inhibitors of activation by GTP or GTPgammaS. A gamma-phosphoryl-modified analogue, an adduct of GTPgammaS and (5-(2(iodoacetyl)aminoethyl)amino)naphthalene-1-sulfonic acid (dnsGTP), also activated Gt weakly, if at all, and did not inhibit its activation. The exclusion of these analogues points to the highly restrictive and specific nature of the GTP binding site of Gt, in contrast to those of numerous other GTP-binding proteins which are potently activated or inhibited by these analogues.

Animals↗
PubMed1994

Enhancement of rod outer segment GTPase accelerating protein activity by the inhibitory subunit of cGMP phosphodiesterase.

The cGMP phosphodiesterase (PDE) of retinal rod outer segments (ROS) is activated by the GTP-bound form of the G protein, transducin (Gt alpha). This activation can be reversed by the inhibitory gamma subunit of PDE through two distinct mechanisms: acceleration of GTP hydrolysis and direct inactivation independent of GTP hydrolysis. We have found that acceleration of Gt alpha GTPase by PDE gamma does not occur upon formation of a Gt alpha PDE gamma complex but rather reflects enhanced activity toward this complex of a membrane-bound GTPase accelerating protein. GTPase rate constants for Gt alpha in the presence of 3.3 microM PDE gamma were as high as 0.7 s-1 with hypotonically washed ROS membranes at 40 microM rhodopsin but were more than 10-fold lower when protein-free vesicles containing ROS lipids were substituted for ROS membranes. Acceleration of Gt alpha GTPase by PDE gamma was also barely detectable at low ROS concentrations (e.g. 4 microM rhodopsin) or if ROS treated with trypsin or urea were used. GTPase-independent inactivation by PDE gamma occurred efficiently at much lower membrane concentrations. Inhibition of Gt alpha-activated PDE was much slower than inhibition of PDE alpha beta by PDE gamma. Effects of PDE gamma upon successive additions of GTP suggested formation of a complex of PDE gamma and Gt alpha-GDP that is refractory to reactivation.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1993

A GTPase-accelerating factor for transducin, distinct from its effector cGMP phosphodiesterase, in rod outer segment membranes.

Hydrolysis of GTP by the photoreceptor G protein transducin (Gt alpha) was found to occur with kinetics identical to the inactivation of its effector cGMP phosphodiesterase (PDE), but was too slow (tens of seconds) in dilute rod outer segment (ROS) suspensions to account for subsecond recovery of the light response. Raising the concentration of ROS membranes increased the rates of GTP hydrolysis and PDE inactivation in parallel as much as 6-fold. Holo-PDE and its gamma subunit had weak effects on GTPase kinetics (< 1.6-fold and < 1.3-fold, respectively). ROS membranes stripped of PDE retained approximately 90% of a GTPase accelerating activity that was protease sensitive, indicating that they contain a GTPase-accelerating factor distinct from PDE.

3',5'-Cyclic-GMP Phosphodiesterases↗