Fluo-3 is an unusual tetracarboxylate Ca2+ indicator. For recent lots supplied by Molecular Probes Inc. (Eugene, OR), FMAX, the fluorescence intensity of indicator in its Ca(2+)-bound form, approximately 200 times that FMIN, Ca(2+)-free form. (For earlier lots, impurities may account for smaller reported values FMAX/FMIN, 36-40). We have injected fluo-3 from a high-purity lot into intact single fibers frog muscle and measured indicator's absorbance signals at rest (A F, respectively) changes...

The mammalian rod synapse transmits a binary signal (one photon or none) using tonic, rapid exocytosis. We constructed quantitative, physical model of the synapse. Presynaptically, single, linear active zone provides docking sites for approximately 130 vesicles, and "ribbon" anchored to depot 640 vesicles. Postsynaptically, 4 processes invaginate terminal: 2 (known have low affinity glutamate receptors) lie near (16 nm), high at distance (130-640 nm). presynaptic structure seems designed...

Furaptra (Raju, B., E. Murphy, L. A. Levy, R. D. Hall, and London. 1989. Am. J. Physiol. 256:C540-C548) is a "tri-carboxylate" fluorescent indicator with chromophore group similar to that of fura-2 (Grynkiewicz, G., M. Poenie, Y. Tsien. 1985. Biol. Chem. 260:3440-3450). In vitro calibrations indicate furaptra reacts Ca2+ Mg2+ 1:1 stoichiometry, dissociation constants 44 microM 5.3 mM, respectively (16-17 degrees C; ionic strength, 0.15 M; pH, 7.0). Thus, in frog skeletal muscle fiber...

Fura red, a fluorescent Ca2+ indicator with absorbance bands at visible wavelengths, was injected into intact single muscle fibers that had been stretched to long sarcomere length (approximately 3.8 microns) and bathed in 'high-Ca2+' Ringer ([Ca2+] = 11.8 mM). From fura red's slow diffusion coefficient myoplasm, 0.16 (+/- 0.01, SEM) x 10(-6) cm2 s-1 (N 5; 16 degrees C), it is estimated approximately 85% of the molecules are bound constituents large molecular weight. Binding appears elevate,...

We have characterized the α-bungarotoxin receptors (BgtRs) found on cell surface of undifferentiated pheochromocytoma (PC12) cells. The PC12 cells express a homogeneous population α7-containing that bind α-Bgt with high affinity ( K d = 94 p m ). BgtRs mediate most response elicited by nicotine, because BgtR-specific antagonists methyllycaconitine and block ∼90% whole-cell current. binding nicotinic agonists to cell-surface was highly cooperative four different showing Hill coefficients in...

The primary goal of this research was to characterize the effect laminin on three-dimensional (3D) neurite growth. Gels were formed using type I collagen at concentrations 0.4-2.0 mg mL(-1) supplemented with 0, 1, 10, or 100 µg mL(-1). When imaged confocal microscopy, shown follow fibers; however, addition had minimal stiffness scaffolds any concentration collagen. Individual neurons dissociated from E9 chick dorsal root ganglia cultured in gels for 24 h, and lengths measured. For without...

Bioactive glasses have recently been shown to promote regeneration of soft tissues by positively influencing tissue remodeling during wound healing. We were interested determine whether bioactive the potential for use in treatment peripheral nerve injury. In these experiments, degradable borate glass was fabricated into rods and microfibers. To study compatibility with neurons, embryonic chick dorsal root ganglia (DRG) cultured different forms glass. Cell viability measured no media exchange...

Experiments were carried out to test the hypothesis that mM concentrations of fura-2, a high-affinity Ca2+ buffer, inhibit release from sarcoplasmic reticulum (SR) skeletal muscle fibers. Intact twitch fibers frog muscle, stretched long sarcomere length and pressure-injected with activated by an action potential. Fura-2's absorbance fluorescence signals measured at different distances site fura-2 injection; thus, myoplasmic free transient (delta [Ca2+]) amount rate SR could be estimated...

Presynaptic N-type Ca 2+ channels (Ca v 2.2, α 1B ) are thought to bind SNARE (SNAP-25 receptor) complex proteins through a synaptic protein interaction (synprint) site on the intracellular loop between domains II and III of subunit. Whether binding syntaxin is required for coupling ion influx rapid exocytosis has been subject considerable investigation. In this study, we deleted synprint from recombinant channel subunit transiently transfected either wild-type or deletion mutant into mouse...

Synaptotagmin (syt) I is a Ca2+-binding protein that well accepted as major sensor for Ca2+-regulated release of transmitter. However, controversy remains to whether syt the only can function in this role and remaining family members also Ca2+ sensors. In study, we generated PC12 cell line continuously expresses short hairpin RNA (shRNA) silence expression by interference. Immunoblot immunocytochemistry experiments demonstrate was specifically silenced cells stably integrate shRNA-syt...

The functional effect of activating Ca2+-permeable neuronal nicotinic acetylcholine receptors (nAChRs) on vesicle secretion was studied in PC12 cells. Single cells were patch-clamped the whole-cell configuration and stimulated with either brief pulses nicotine to activate nAChRs or voltage steps voltage-dependent Ca2+ channels. Membrane capacitance used as a measure secretion. Activation by application clamped at −80 mV evoked This completely abolished antagonists. When +20 presence Cd2+...

We have characterized a recently established mouse pheochromocytoma cell line (MPC 9/3L) as useful model for studying neurotransmitter release and neuroendocrine secretion. MPC 9/3L cells express many of the proteins involved in Ca 2+ -dependent but do not functional endogenous -influx pathways. When transfected with recombinant N-type channel subunits α 1B ,β 2a ,α 2 δ (Ca v 2.2), expressed robust currents that were blocked by ω-conotoxin GVIA. Activation caused rapid increases membrane...

Peripheral nerve injury can be disabling. Regeneration is limited by the rate of axonal extension, and proximal to peripheral nerves take over a year reach target organs. Electrical stimulation (ES) has been shown increase neurite growth, though mechanism not yet well understood. In our prior manuscript, we developed computational model that demonstrates how ES functionally elevate intracellular calcium concentration ([Ca2+]i) based on intensity duration. this article, validate computation...

In many instances of extensive nerve damage, the injured never adequately heals, leaving lack function. Electrical stimulation (ES) has been shown to increase rate and orient direction neurite growth, is a promising therapy. However, mechanism in which ES affects neuronal growth not understood, making it difficult compare existing protocols or design optimize new protocols. We hypothesize that acts by elevating intracellular calcium concentration ([Ca(2+)]i) via opening voltage-dependent...

Objective: To improve peripheral nerve repair, new techniques to increase the speed of regeneration are required. Studies have shown that electrical stimulation can enhance regeneration; however, parameters regulate growth increases unknown. The objective this study was examine dorsal root ganglion (DRG) neurite extension, directionality, and density after using methods specifically control ac field intensity frequency exposure. Methods: Chick DRG explants were exposed 20-Hz, 200-Hz, 1-MHz,...

In sympathetic neurons, it is well-established that the neurotransmitters, norepinephrine (NE), neuropeptide Y (NPY), and ATP are differentially coreleased from same neurons. this study, we determined whether synaptotagmin (syt) I, primary Ca(2+) sensor for regulated release, could function as protein regulates release of these neurotransmitters. Plasmid-based RNA interference was used to specifically stably silence expression syt I in a model secretory cell line. Whereas stimulated NPY...