Time-Resolved In Vivo Measurement of Neuropeptide Dynamics by Capacitive Immunoprobe in Porcine Heart.

The ability to measure biomarkers in vivo relevant to the assessment of disease progression is of great interest to the scientific and medical communities. The resolution of results obtained from current methods of measuring certain biomarkers can take several days or weeks to obtain, as they can be limited in resolution both spatially and temporally (e.g., fluid compartment microdialysis of interstitial fluid analyzed by enzyme-linked immunosorbent assay [ELISA], high-performance liquid chromatography [HPLC], or mass spectrometry); thus, their guidance of timely diagnosis and treatment is disrupted. In the present study, a unique technique for detecting and measuring peptide transmitters in vivo through the use of a capacitive immunoprobe biosensor (CI probe) is reported. The fabrication protocol and in vitro characterization of these probes are described. Measurements of sympathetic stimulation-evoked neuropeptide Y (NPY) release in vivo are provided. NPY release is correlated to the sympathetic release of norepinephrine for reference. The data demonstrate an approach for the fast and localized measurement of neuropeptides in vivo. Future applications include intraoperative real-time assessment of disease progression and minimally invasive catheter-based deployment of these probes.

Rapid measurement of cardiac neuropeptide dynamics by capacitive immunoprobe in the porcine heart.

Sympathetic control of regional cardiac function occurs through postganglionic innervation from stellate ganglia and thoracic sympathetic chain. Whereas norepinephrine (NE) is their primary neurotransmitter, neuropeptide Y (NPY) is an abundant cardiac cotransmitter. NPY plays a vital role in homeostatic processes including angiogenesis, vasoconstriction, and cardiac remodeling. Elevated sympathetic stress, resulting in increased NE and NPY release, has been implicated in the pathogenesis of several cardiovascular disorders including hypertension, myocardial infarction, heart failure, and arrhythmias, which may result in sudden cardiac death. Current methods for the detection of NPY in myocardium are limited in their spatial and temporal resolution and take days to weeks to provide results [e.g., interstitial microdialysis with subsequent analysis by enzyme-linked immunosorbent assay (ELISA), high performance liquid chromatography (HPLC), or mass spectrometry]. In this study, we report a novel approach for measurement of interstitial and intravascular NPY using a minimally invasive capacitive immunoprobe (C.I. probe). The first high-spatial and temporal resolution, multichannel measurements of NPY release in vivo are provided in both myocardium and transcardiac vascular space in a beating porcine heart. We provide NPY responses evoked by sympathetic stimulation and ectopic ventricular pacing and compare these to NE release and hemodynamic responses. We extend this approach to measure both NPY and vasoactive intestinal peptide (VIP) and show differential release profiles under sympathetic stimulation. Our data demonstrate rapid and local changes in neurotransmitter profiles in response to sympathetic cardiac stressors. Future implementations include real-time intraoperative determination of cardiac neuropeptides and deployment as a minimally invasive catheter.NEW & NOTEWORTHY The sympathetic nervous system regulates cardiac function through release of neurotransmitters and neuropeptides within the myocardium. Neuropeptide Y (NPY) acts as an acute cardiac vasoconstrictor and chronically to regulate angiogenesis and cardiac remodeling. Current methodologies for the measure of NPY are not capable of providing rapid readouts on a single-sample basis. Here we provide the first in vivo methodology to report dynamic, localized NPY levels within both myocardium and vascular compartments in a beating heart.

Fast in vivo detection of myocardial norepinephrine levels in the beating porcine heart.

The sympathetic nervous system modulates cardiac function by controlling key parameters such as chronotropy and inotropy. Sympathetic control of ventricular function occurs through extrinsic innervation arising from the stellate ganglia and thoracic sympathetic chain. In the healthy heart, sympathetic release of norepinephrine (NE) results in positive modulation of chronotropy, inotropy, and dromotropy, significantly increasing cardiac output. However, in the setting of myocardial infarction or injury, sympathetic activation persists, contributing to heart failure and increasing the risk of arrhythmias, including sudden cardiac death. Methodologies for detection of norepinephrine in cardiac tissue are limited. Present techniques rely on microdialysis for analysis by high-performance liquid chromatography coupled to electrochemical detection (HPLC-ED), radioimmunoassay, or other immunoassays, such as enzyme-linked immunosorbent assay (ELISA). Although significant information about the release and action of norepinephrine has been obtained with these methodologies, they are limited in temporal resolution, require large sample volumes, and provide results with a significant delay after sample collection (hours to weeks). In this study, we report a novel approach for measurement of interstitial cardiac norepinephrine, using minimally invasive, electrode-based, fast-scanning cyclic voltammetry (FSCV) applied in a beating porcine heart. The first multispatial and high temporal resolution, multichannel measurements of NE release in vivo are provided. Our data demonstrate rapid changes in interstitial NE profiles with regional differences in response to coronary ischemia, sympathetic nerve stimulation, and alterations in preload/afterload.NEW & NOTEWORTHY Pharmacological, electrical, or surgical regulation of sympathetic neuronal control can be used to modulate cardiac function and treat arrhythmias. However, present methods for monitoring sympathetic release of norepinephrine in the heart are limited in spatial and temporal resolution. Here, we provide for the first time a methodology and demonstration of practice and rapid measures of individualized regional autonomic neurotransmitter levels in a beating heart. We show dynamic, spatially resolved release profiles under normal and pathological conditions.

Spatial and activity-dependent catecholamine release in rat adrenal medulla under native neuronal stimulation.

Neuroendocrine chromaffin cells of the adrenal medulla in rat receive excitatory synaptic input through anterior and posterior divisions of the sympathetic splanchnic nerve. Upon synaptic stimulation, the adrenal medulla releases the catecholamines, epinephrine, and norepinephrine into the suprarenal vein for circulation throughout the body. Under sympathetic tone, catecholamine release is modest. However, upon activation of the sympathoadrenal stress reflex, and increased splanchnic firing, adrenal catecholamine output increases dramatically. Moreover, specific stressors can preferentially increase release of either epinephrine (i.e., hypoglycemia) or norepinephrine (i.e., cold stress). The mechanism for this stressor-dependent segregated release of catecholamine species is not yet fully understood. We tested the hypothesis that stimulation of either division of the splanchnic selects for epinephrine over norepinephrine release. We introduce an ex vivo rat preparation that maintains native splanchnic innervation of the adrenal gland and we document experimental advantages and limitations of this preparation. We utilize fast scanning cyclic voltammetry to detect release of both epinephrine and norepinephrine from the adrenal medulla, and report that epinephrine and norepinephrine release are regulated spatially and in a frequency-dependent manner. We provide data to show that epinephrine is secreted preferentially from the periphery of the medulla and exhibits a higher threshold and steeper stimulus-secretion function than norepinephrine. Elevated stimulation of the whole nerve specifically enhances epinephrine release from the peripheral medulla. Our data further show that elimination of either division from stimulation greatly attenuated epinephrine release under elevated stimulation, while either division alone can largely support norepinephrine release.

Syndapin 3 modulates fusion pore expansion in mouse neuroendocrine chromaffin cells.

Adrenal neuroendocrine chromaffin cells receive excitatory synaptic input from the sympathetic nervous system and secrete hormones into the peripheral circulation. Under basal sympathetic tone, modest amounts of freely soluble catecholamine are selectively released through a restricted fusion pore formed between the secretory granule and the plasma membrane. Upon activation of the sympathoadrenal stress reflex, elevated stimulation drives fusion pore expansion, resulting in increased catecholamine secretion and facilitating release of copackaged peptide hormones. Thus regulated expansion of the secretory fusion pore is a control point for differential hormone release of the sympathoadrenal stress response. Previous work has shown that syndapin 1 deletion alters transmitter release and that the dynamin 1-syndapin 1 interaction is necessary for coupled endocytosis in neurons. Dynamin has also been shown to be involved in regulation of fusion pore expansion in neuroendocrine chromaffin cells through an activity-dependent association with syndapin. However, it is not known which syndapin isoform(s) contributes to pore dynamics in neuroendocrine cells. Nor is it known at what stage of the secretion process dynamin and syndapin associate to modulate pore expansion. Here we investigate the expression and localization of syndapin isoforms and determine which are involved in mediating fusion pore expansion. We show that all syndapin isoforms are expressed in the adrenal medulla. Mutation of the SH3 dynamin-binding domain of all syndapin isoforms shows that fusion pore expansion and catecholamine release are limited specifically by mutation of syndapin 3. The mutation also disrupts targeting of syndapin 3 to the cell periphery. Syndapin 3 exists in a persistent colocalized state with dynamin 1.

Activity-dependent fusion pore expansion regulated by a calcineurin-dependent dynamin-syndapin pathway in mouse adrenal chromaffin cells.

Neuroendocrine chromaffin cells selectively secrete a variety of transmitter molecules into the circulation as a function of sympathetic activation. Activity-dependent release of transmitter species is controlled through regulation of the secretory fusion pore. Under sympathetic tone, basal synaptic excitation drives chromaffin cells to selectively secrete modest levels of catecholamine through a restricted secretory fusion pore. In contrast, elevated sympathetic activity, experienced under stress, results in fusion pore expansion to evoke maximal catecholamine release and to facilitate release of copackaged peptide transmitters. Therefore, fusion pore expansion is a key control point for the activation of the sympatho-adrenal stress response. Despite the physiological importance of this process, the molecular mechanism by which it is regulated remains unclear. Here we employ fluorescence imaging with electrophysiological and electrochemical-based approaches to investigate the role of dynamin I in the regulation of activity-mediated fusion pore expansion in mouse adrenal chromaffin cells. We show that under elevated stimulation, dynamin I is dephosphorylated at Ser-774 by calcineurin. We also demonstrate that disruption of dynamin I-syndapin binding, an association regulated by calcineurin-dependent dynamin dephosphorylation, limits fusion pore expansion. Last, we show that perturbation of N-WASP function (a syndapin substrate) limits activity-mediated fusion pore expansion. Our results suggest that fusion pore expansion is regulated by a calcineurin-dependent dephosphorylation of dynamin I. Dephosphorylated dynamin I acts via a syndapin/N-WASP signaling cascade to mediate pore expansion.

Reduced calcium current density in female versus male mouse adrenal chromaffin cells in situ.

Neuroendocrine adrenal medullary chromaffin cells are a main output of the sympathetic nervous system. Acute stress activates the sympatho-adrenal stress reflex, excites adrenal chromaffin cells, and elicits catecholamine secretion into the circulation. Previous studies have demonstrated that stress-evoked serum catecholamine levels are greater in males. We investigated potential mechanistic bases for this gender dimorphism at the level of the adrenal medulla. We utilized in situ single-cell perforated patch voltage clamp to measure basic electrophysiological parameters that affect cell excitability. We found that chromaffin cells from male and female mice exhibit statistically identical depolarization-evoked calcium currents. However, the resting capacitance, an index of cell surface area, was significantly higher in cells from female mice. Thus the current density in female cells was significantly lower. We found that inhibition of protein kinase C, an enzyme shown to regulate both exocytosis and endocytosis, eliminates the cell surface area gender dimorphism. Finally, we performed kinetic simulations of the secretion process and report a predicted elevated secretory capacity in male cells. Thus, regulation of cell size may act to decrease cell excitability in female cells and may in-part represent the mechanistic basis for increased stress-evoked catecholamine secretion described in males.

Pituitary adenylate cyclase-activating peptide (PACAP) recruits low voltage-activated T-type calcium influx under acute sympathetic stimulation in mouse adrenal chromaffin cells.

Low voltage-activated T-type Ca(v)3.2 calcium channels are expressed in neurosecretory chromaffin cells of the adrenal medulla. Previous studies have shown that naïve adrenal chromaffin cells express a nominal Ca(v)3.2-dependent conductance. However, Ca(v)3.2 conductance is up-regulated following chronic hypoxia or long term exposure to cAMP analogs. Thus, although a link between chronic stressors and up-regulation of Ca(v)3.2 exists, there are no reports testing the specific role of Ca(v)3.2 channels in the acute sympathoadrenal stress response. In this study, we examined the effects of acute sympathetic stress on T-type Ca(v)3.2 calcium influx in mouse chromaffin cells in situ. Pituitary adenylate cyclase-activating peptide (PACAP) is an excitatory neuroactive peptide transmitter released by the splanchnic nerve under elevated sympathetic activity to stimulate the adrenal medulla. PACAP stimulation did not evoke action potential firing in chromaffin cells but did cause a persistent subthreshold membrane depolarization that resulted in an immediate and robust Ca(2+)-dependent catecholamine secretion. Moreover, PACAP-evoked secretion was sensitive to block by nickel chloride and was acutely inhibited by protein kinase C blockers. We utilized perforated patch electrophysiological recordings conducted in adrenal tissue slices to investigate the mechanism of PACAP-evoked calcium entry. We provide evidence that stimulation with exogenous PACAP and native neuronal stress stimulation both lead to a protein kinase C-mediated phosphodependent recruitment of a T-type Ca(v)3.2 Ca(2+) influx. This in turn evokes catecholamine release during the acute sympathetic stress response.

Dynamin and myosin regulate differential exocytosis from mouse adrenal chromaffin cells.

Neuroendocrine chromaffin cells of the adrenal medulla represent a primary output for the sympathetic nervous system. Chromaffin cells release catecholamine as well as vaso- and neuro-active peptide transmitters into the circulation through exocytic fusion of large dense-core secretory granules. Under basal sympathetic activity, chromaffin cells selectively release modest levels of catecholamines, helping to set the "rest and digest" status of energy storage. Under stress activation, elevated sympathetic firing leads to increased catecholamine as well as peptide transmitter release to set the "fight or flight" status of energy expenditure. While the mechanism for catecholamine release has been widely investigated, relatively little is known of how peptide transmitter release is regulated to occur selectively under elevated stimulation. Recent studies have shown selective catecholamine release under basal stimulation is accomplished through a transient, restricted exocytic fusion pore between granule and plasma membrane, releasing a soluble fraction of the small, diffusible molecules. Elevated cell firing leads to the active dilation of the fusion pore, leading to the release of both catecholamine and the less diffusible peptide transmitters. Here we propose a molecular mechanism regulating the activity-dependent dilation of the fusion pore. We review the immediate literature and provide new data to formulate a working mechanistic hypothesis whereby calcium-mediated dephosphorylation of dynamin I at Ser-774 leads to the recruitment of the molecular motor myosin II to actively dilate the fusion pore to facilitate release of peptide transmitters. Thus, activity-dependent dephosphorylation of dynamin is hypothesized to represent a key molecular step in the sympatho-adrenal stress response.

Enhanced dense core granule function and adrenal hypersecretion in a mouse model of Rett syndrome.

Rett syndrome (RTT) is a progressive developmental disorder resulting from loss-of-function mutations in the gene encoding methyl-CpG-binding protein 2 (MeCP2), a transcription regulatory protein. The RTT phenotype is complex and includes severe cardiorespiratory abnormalities, dysautonomia and behavioral symptoms of elevated stress. These findings have been attributed to an apparent hyperactivity of the sympathetic nervous system due to defects in brainstem development; however, the possibility that the peripheral sympathoadrenal axis itself is abnormal has not been explored. The present study demonstrates that the adrenal medulla and sympathetic ganglia of Mecp2 null mice exhibit markedly reduced catecholamine content compared with wild-type controls. Despite this, null animals exhibit significantly higher plasma epinephrine levels, suggesting enhanced secretory granule function in adrenal chromaffin cells. Indeed, we find that Mecp2 null chromaffin cells exhibit a cell autonomous hypersecretory phenotype characterized by significant increases in the speed and size of individual secretory granule fusion events in response to electrical stimulation. These findings appear to indicate accelerated formation and enhanced dilation of the secretory granule fusion pore, resulting in elevated catecholamine release. Our data therefore highlight abnormal catecholamine function in the sympathoadrenal axis as a potential source of autonomic dysfunction in RTT. These findings may help to explain the apparent 'overactivity' of the sympathetic nervous system reported in patients with RTT.

PACAP regulates immediate catecholamine release from adrenal chromaffin cells in an activity-dependent manner through a protein kinase C-dependent pathway.

Adrenal medullary chromaffin cells are a major peripheral output of the sympathetic nervous system. Catecholamine release from these cells is driven by synaptic excitation from the innervating splanchnic nerve. Acetylcholine has long been shown to be the primary transmitter at the splanchnic-chromaffin synapse, acting through ionotropic nicotinic acetylcholine receptors to elicit action potential-dependent secretion from the chromaffin cells. This cholinergic stimulation has been shown to desensitize under sustained stimulation, yet catecholamine release persists under this same condition. Recent evidence supports synaptic chromaffin cell stimulation through alternate transmitters. One candidate is pituitary adenylate cyclase activating peptide (PACAP), a peptide transmitter present in the adrenal medulla shown to have an excitatory effect on chromaffin cell secretion. In this study we utilize native neuronal stimulation of adrenal chromaffin cells in situ and amperometric catecholamine detection to demonstrate that PACAP specifically elicits catecholamine release under elevated splanchnic firing. Further data reveal that the immediate PACAP-evoked stimulation involves a phospholipase C and protein kinase C-dependent pathway to facilitate calcium influx through a Ni2+ and mibefradil-sensitive calcium conductance that results in catecholamine release. These data demonstrate that PACAP acts as a primary secretagogue at the sympatho-adrenal synapse under the stress response.

Increased secretory capacity of mouse adrenal chromaffin cells by chronic intermittent hypoxia: involvement of protein kinase C.

Previous studies have shown that catecholamine secretion from the adrenal medulla plays a critical role in chronic intermittent hypoxia (CIH)-induced alterations in cardiovascular function. In the present study we examined the cellular mechanisms associated with the effects of CIH on adrenal chromaffin cell catecholamine secretion. Experiments were performed on adult male mice (C57/BL6) that were exposed to 1-4 days of CIH or to normoxia. Perforated patch electrical capacitance recordings were performed on freshly prepared adrenal medullary slices that permit separating the chromaffin cell secretion from sympathetic input. CIH resulted in a significant increase in the readily releasable pool (RRP) of secretory granules, and decreased stimulus-evoked Ca(2+) influx. Continuous hypoxia (CH) either for 2.5 h (equivalent to hypoxic duration accumulated over 4 days of CIH) or for 4 days were ineffective in evoking changes in the RRP and Ca(2+) influx. CIH activated PKC in adrenal medullae as evidenced by increased phosphorylation of PKC at Thr(514) and PKC inhibitors prevented CIH-induced increases in the RRP and restored stimulus-evoked attenuation of Ca(2+) influx. CIH resulted in elevated thio-barbituric acid reactive substances (TBARSs, an index of oxidized proteins) and an antioxidant prevented CIH-induced changes in the RRP, suggesting the involvement of reactive oxygen species (ROS). These results demonstrate that CIH increases the RRP in adrenal chromaffin cells via ROS-mediated activation of PKC and suggest that CIH can directly affect the secretory capacity of chromaffin cells and contribute, in part, to elevated catecholamine levels.

Dysregulation of brain-derived neurotrophic factor expression and neurosecretory function in Mecp2 null mice.

Disruptions in brain-derived neurotrophic factor (BDNF) expression are proposed to contribute to the molecular pathogenesis of Rett syndrome (RTT), a severe neurological disorder caused by loss-of-function mutations in methyl-CpG-binding protein-2 (MeCP2). Although MeCP2 is a transcriptional regulator of BDNF, it is unknown how MeCP2 mutations affect transsynaptic BDNF signaling. Our findings demonstrate an early, abnormal neurosecretory phenotype in MeCP2-deficient neurons characterized by significant increases in the percentage of cellular BDNF content available for release. However, loss of MeCP2 also results in deficits in total cell BDNF content that are developmentally regulated in a cell-type-specific manner. Thus, the net effect of MeCP2 loss on absolute BDNF secretion changes with age and is determined by both the amount of BDNF available for release and progressive declines in total cellular BDNF. We propose, therefore, that loss of MeCP2 function disrupts transsynaptic BDNF signaling by perturbing the normal balance between BDNF protein levels and secretion. However, mutant neurons are capable of secreting wild-type levels of BDNF in response to high-frequency electrical stimulation. In addition, we found elevated exocytic function in Mecp2(-/y) adrenal chromaffin cells, indicating that the Mecp2 null mutation is associated with alterations of neurosecretion that are not restricted to BDNF. These findings are the first examples of abnormal neuropeptide and catecholamine secretion in a mouse model of RTT.

Adrenal chromaffin cells exhibit impaired granule trafficking in NCAM knockout mice.

Neural cell adhesion molecule (NCAM) plays several critical roles in neuron path-finding and intercellular communication during development. In the clinical setting, serum NCAM levels are altered in both schizophrenic and autistic patients. NCAM knockout mice have been shown to exhibit deficits in neuronal functions including impaired hippocampal long term potentiation and motor coordination. Recent studies in NCAM null mice have indicated that synaptic vesicle trafficking and active zone targeting are impaired, resulting in periodic synaptic transmission failure under repetitive physiological stimulation. In this study, we tested whether NCAM plays a role in vesicle trafficking that is limited to the neuromuscular junction or whether it may also play a more general role in transmitter release from other cell systems. We tested catecholamine release from neuroendocrine chromaffin cells in the mouse adrenal tissue slice preparation. We utilize electrophysiological and electrochemical measures to assay granule recruitment and targeting in wild-type and NCAM -/- mice. Our data show that NCAM -/- mice exhibit deficits in normal granule trafficking between the readily releasable pool and the highly release-competent immediately releasable pool. This defect results in a decreased rate of granule fusion and thus catecholamine release under physiological stimulation. Our data indicate that NCAM plays a basic role in the transmitter release mechanism in neuroendocrine cells through mediation of granule recruitment and is not limited to the neuromuscular junction and central synapse active zones.

Action potential stimulation reveals an increased role for P/Q-calcium channel-dependent exocytosis in mouse adrenal tissue slices.

Chromaffin cells of the adrenal medulla receive cholinergic input from the splanchnic nerve. Upon sympathetic activity, chromaffin cells fire action potentials that open voltage-gated calcium channels and evoke the exocytic release of catecholamines. Catecholamines then regulate homeostatic processes such as cardiac output and vascular tone. Thus control of the Ca(2+) influx in chromaffin cells represents a target for the regulation of multiple physiological functions. Previous reports utilized square pulse depolarizations to quantify the proportional exocytic response as a function of Ca(2+) channel subtype. In this study, we use perforated patch voltage clamp and action potential waveforms to depolarize cells in situ. We analyze Ca(2+) current components under conditions that match the dynamic native cell behavior. This approach revealed a greater role for P/Q-type calcium channels in evoked exocytosis than previously reported. Thus, the P/Q-type channels represent a more important control point for the regulation of catecholamine-dependent processes than previously believed.

Low frequency stimulation of mouse adrenal slices reveals a clathrin-independent, protein kinase C-mediated endocytic mechanism.

Evidence suggests that chromaffin cells employ separate mechanisms for evoked endocytosis and granule recycling when stimulated at basal (approximately 0.5 Hz) and stress-activated (approximately 15 Hz) rates. Previous studies have focused mainly on elucidating the cellular mechanisms responsible for membrane recycling under conditions similar to the stress-activated state and indicate a clathrin/dephosphin-mediated retrieval via coated pits. However, the mechanism for membrane internalisation at basal stimulus intensity remains largely unexplored. We electrically stimulated chromaffin cells in adrenal tissue slices at the sympathetic basal firing rate and measured cell capacitance in the perforated voltage clamp configuration. A new method for the separation of non-secretory from secretory cell capacitance signals is presented. Simultaneous catecholamine release was measured electrochemically to isolate the exocytic from endocytic components of the capacitance responses. Using this approach we demonstrate that firing patterns that mimic basal sympathetic input results in rapid and graded membrane retrieval. We show that block of the calcium-mediated protein phosphatase 2B, a common step in clathrin-mediated processes, did not alter endocytosis elicited at basal firing levels. We further blocked clathrin-mediated retrieval with a clathrin/dephosphin-disrupting peptide (PP-19) and found endocytosis to be blocked at 15 Hz stimulation but complete and indistinguishable from control cells at 0.5 Hz stimulation. Lastly, pharmacological treatments show that conventional isoforms of protein kinase C (cPKC) are required for the 0.5 Hz-evoked retrieval mechanism. From these data we conclude that unlike endocytosis evoked under stress conditions, basal firing activity results in a clathrin-independent rapid membrane retrieval mediated through conventional isoforms of PKC.

Calcium dependence of action potential-induced endocytosis in chromaffin cells.

Exocytosis occurs via fusion of transmitter-containing granules with the cell membrane, whereupon the granule contents are released and the cell membrane surface area increases. Exocytosis is followed by endocytosis to maintain proper cell membrane surface area and composition. We have shown that adrenal chromaffin cells internalize membrane in a biphasic manner following action potential stimulation. At basal firing rates (single - 0.5 Hz trains) endocytosis occurs by a rapid retrieval of membrane (termed Phase I) that is independent of the activity of the protein phosphatase calcineurin and wanes in efficiency with cell activity. At intermediate firing frequencies (>6 Hz) a second, calcineurin-sensitive, form of activity-enhanced endocytosis emerges (Phase II). In this study, we employ electrophysiological, electrochemical, and computational techniques to estimate intracellular Ca(2+) at the site of endocytosis by measuring secretion rates. The measured rates of secretion yield estimates of [Ca(2+)](i) based on a kinetic scheme for exocytosis calibrated under highly controlled [Ca(2+)](i). Based on this analysis, we propose that Phase I endocytosis is inhibited by cytosolic Ca(2+) with a K(inh)=605 nM, while Phase II endocytosis is activated by Ca(2+) with a K(act)=1.46 micro M. Molecular processes that may be consistent with the measured behaviors are discussed.

Ischemia-induced changes in monocarboxylate transporter 1 reactive cells in rat hippocampus.

This study aims to demonstrate the responses of monocarboxylate transporter 1 (MCT1) immunoreactive cells to transient global ischemia in rat hippocampus using confocal and electron microscopy. The MCT1 staining in CA1 pyramidal cells of the sham-operated controls appeared evenly distributed. Most of the MCT1 immunoreactive products were associated with the cell surface; however, some intracellular reaction products are also found. This pattern of stain was not altered in the first three days after an ischemic episode. As the neuronal demise progressed, the MCT1 immunoreactive cells became patchy in the 21-day post-ischemic rats. Besides the neuronal labeling, MCT1 immunoreactivity was found in astroglia, in endothelial cells and in the adjacent ependymal lining. The latter exhibited intense labeling both in the acute and long-term surviving rats. These data suggest that MCT1 plays a role in the initial and long-term neuronal survival in the hippocampus.