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We performed 3D reconstruction of the microscopic structure of the adrenal medulla of the adult rats using serial histological sections with histochemical differentiation of adrenaline-storing (A) and noradrenaline-storing (NA) cells. Medulla volume is 1.18 ± 0.17 mm3 . Chromaffin tissue consists of 82.9 ± 2.6% of A and 17.1 ± 2.6% of NA cells. Cords of the chromaffinocytes run along the nerves in the adrenal cortex and form cones when merging with medulla bulk. There is no unambiguously greater prevalence of A cells over NA in the areas of the medulla bordering on the cortex as compared to deep layers of medulla. NA cells form a network of beams. Their concentration increases with distance from the entry site of the nerves and is maximal on the opposite side. This testifies to the fallacy of the point of view about the disordered distribution of NA cells in the medulla. Based on the polar asymmetric arrangement of the adrenal chromaffin tissue, if it is necessary to completely remove the medulla with the keeping or reimplantation of the cortex, the subcapsular cortex zone located on the pole opposite to the entrance of nerves should be chosen. In addition, comparable results in the stereological examination of the medulla can be obtained only if taking its areas similar in location. The pronounced relationship in the arrangement of A and NA cells with nerves clearly indicates that in vivo nerve factors play a key role in differentiation and stabilization of the A and NA cells phenotypes.

Using Nav1.3 and FGF14 KO mice, Martinez-Espinosa et al. provide new findings on how intracellular FGF14 proteins interfere with the endogenous fast inactivation gating and regulate the “long-term inactivation” of Nav1.3 channels that sets Nav channel availability and spike adaptation during sustained stimulation in adrenal chromaffin cells.

Emerging evidence suggests that gaseous molecules, carbon monoxide (CO), and hydrogen sulfide (H(2)S) generated by heme oxygenase (HO)-2 and cystathionine γ-lyase (CSE), respectively, function as transmitters in the nervous system. Present study examined the roles of CO and H(2)S in hypoxia-induced catecholamine (CA) release from adrenal medullary chromaffin cells (AMCs). Studies were performed on AMCs from adult (≥6 wk of age) wild-type (WT), HO-2 null, CSE null, and HO-2/CSE double null mice of either gender. CA secretion was determined by carbon fiber amperometry and [Ca(2+)](i) by microflurometry using Fura-2. HO-2- and CSE immunoreactivities were seen in WT AMC, which were absent in HO-2 and CSE null mice. Hypoxia (medium Po(2) 30–38 mmHg) evoked CA release and elevated [Ca(2+)](i). The magnitude of hypoxic response was greater in HO-2 null mice and in HO inhibitor-treated WT AMC compared with controls. H(2)S levels were elevated in HO-2 null AMC. Either pharmacological inhibition or genetic deletion of CSE prevented the augmented hypoxic responses of HO-2 null AMC and H(2)S donor rescued AMC responses to hypoxia in HO-2/CSE double null mice. CORM3, a CO donor, prevented the augmented hypoxic responses in WT and HO-2 null AMC. CO donor reduced H(2)S levels in WT AMC. The effects of CO donor were blocked by either ODQ or 8pCT, inhibitors of soluble guanylyl cyclase (SGC) or protein kinase G, respectively. These results suggest that HO-2-derived CO inhibits hypoxia-evoked CA secretion from adult murine AMC involving soluble guanylyl cyclase (SGC)-protein kinase G (PKG)-dependent regulation of CSE-derived H(2)S. NEW & NOTEWORTHY Catecholamine secretion from adrenal chromaffin cells is an important physiological mechanism for maintaining homeostasis during hypoxia. Here, we delineate carbon monoxide (CO)-sensitive hydrogen sulfide (H(2)S) signaling as an important mediator of hypoxia-induced catecholamine secretion from murine adrenal chromaffin cells. Heme oxygenase-2 derived CO is a physiological inhibitor of catcholamince secretion by hypoxia and the effects of CO involve inhibition of cystathionine γ-lyase-derived H(2)S production through soluble guanylyl cyclase-protein kinase G signaling cascade.

Martinez-Espinosa et al. show that FGF14 and the voltage-dependent Na+ channel Nav1.3 are the main regulators of rapidly inactivating currents and Nav channel availability in mouse adrenal chromaffin cells. Their results uncover a role for FGF14 in regulating Nav channel function., Adrenal chromaffin cells (CCs) in rodents express rapidly inactivating, tetrodotoxin (TTX)-sensitive sodium channels. The resulting current has generally been attributed to Nav1.7, although a possible role for Nav1.3 has also been suggested. Nav channels in rat CCs rapidly inactivate via two independent pathways which differ in their time course of recovery. One subpopulation recovers with time constants similar to traditional fast inactivation and the other ∼10-fold slower, but both pathways can act within a single homogenous population of channels. Here, we use Nav1.3 KO mice to probe the properties and molecular components of Nav current in CCs. We find that the absence of Nav1.3 abolishes all Nav current in about half of CCs examined, while a small, fast inactivating Nav current is still observed in the rest. To probe possible molecular components underlying slow recovery from inactivation, we used mice null for fibroblast growth factor homology factor 14 (FGF14). In these cells, the slow component of recovery from fast inactivation is completely absent in most CCs, with no change in the time constant of fast recovery. The use dependence of Nav current reduction during trains of stimuli in WT cells is completely abolished in FGF14 KO mice, directly demonstrating a role for slow recovery from inactivation in determining Nav current availability. Our results indicate that FGF14-mediated inactivation is the major determinant defining use-dependent changes in Nav availability in CCs. These results establish that Nav1.3, like other Nav isoforms, can also partner with FGF subunits, strongly regulating Nav channel function.

Martinez-Espinosa et al. show that fast and slow recovery of voltage-dependent Na+ (Nav) channels in rat adrenal chromaffin cells depends on two independent inactivation pathways. Competition between these pathways regulates changes in Nav channel availability., Voltage-dependent sodium (Nav) current in adrenal chromaffin cells (CCs) is rapidly inactivating and tetrodotoxin (TTX)–sensitive. The fractional availability of CC Nav current has been implicated in regulation of action potential (AP) frequency and the occurrence of slow-wave burst firing. Here, through recordings of Nav current in rat CCs, primarily in adrenal medullary slices, we describe unique inactivation properties of CC Nav inactivation that help define AP firing rates in CCs. The key feature of CC Nav current is that recovery from inactivation, even following brief (5 ms) inactivation steps, exhibits two exponential components of similar amplitude. Various paired pulse protocols show that entry into the fast and slower recovery processes result from largely independent competing inactivation pathways, each of which occurs with similar onset times at depolarizing potentials. Over voltages from −120 to −80 mV, faster recovery varies from ∼3 to 30 ms, while slower recovery varies from ∼50 to 400 ms. With strong depolarization (above −10 mV), the relative entry into slow or fast recovery pathways is similar and independent of voltage. Trains of short depolarizations favor recovery from fast recovery pathways and result in cumulative increases in the slow recovery fraction. Dual-pathway fast inactivation, by promoting use-dependent accumulation in slow recovery pathways, dynamically regulates Nav availability. Consistent with this finding, repetitive AP clamp waveforms at 1–10 Hz frequencies reduce Nav availability 80–90%, depending on holding potential. These results indicate that there are two distinct pathways of fast inactivation, one leading to conventional fast recovery and the other to slower recovery, which together are well-suited to mediate use-dependent changes in Nav availability.

This protocol is about culturing of adrenal chromaffin cells from rats. For rat-derived cultures, adrenal glands from 7- to 12-day-old Sprague Dawley rats are dissected in ice-cold Hanks Balanced Salt Solution (HBSS).

Voltage-dependent sodium (Nav) current in adrenal chromaffin cells (CCs) is rapidly inactivating and TTX-sensitive. The fractional availability of CC Nav current has been implicated in regulation of action potential (AP) frequency and the occurrence of slow-wave burst firing. To ascertain whether features of CC Nav inactivation might influence AP firing, we recorded Nav current in rat CCs, primarily from adrenal medullary slices. A key feature of CC Nav current is that recovery from inactivation, even following brief (5 ms) inactivation steps, exhibits two exponential components of generally similar amplitude. Variations of standard paired pulse protocols support the view that entry into the fast and slower recovery processes result from largely independent, competing inactivation pathways, both of which occur with similar onset times at depolarizing potentials. Over voltages from −120 to −80 mV, faster recovery varies from ~3 to 30 ms, while slower recovery from about 50-400 ms. At strong activation voltages (+0 mV and more positive), the relative entry into slow or fast recovery pathways is similar and independent of voltage. Trains of brief inactivating steps result in cumulative increases in the slower recovery fraction. This supports idea that brief recovery intervals preferentially allow recovery of channels from fast recovery pathways, thereby increasing the fraction of channels in the slow recovery pathway with each subsequent inactivation step. This provides a mechanism whereby differential rates of recovery produce use-dependent accumulation in slower recovery pathways. Consistent with use-dependent accumulation of channels in slow recovery pathways, repetitive AP clamp waveforms at 1-10 Hz frequencies reduce Nav availability to 10-20% of initial amplitude dependent on holding potential. The results indicate that there are two distinct pathways of fast inactivation, one that leads to normal fast recovery and the other with a slower time course, which together are well-suited to mediate use-dependent changes in Nav availability.

Adrenal chromaffin cells (CCs) in rodents express a rapidly inactivating, TTX-sensitive sodium current. The current has generally been attributed to Nav1.7, although a possible role for Nav1.3 has also been suggested. Nav channels in rat CCs rapidly inactivate into two separable pathways, which differ in their time course of recovery from inactivation. One population recovers with time constants similar to traditional fast inactivation and the other about 10-fold slower. Inactivation properties suggest that the two pathways result from a single homogenous population of channels. Here we probe the properties and molecular components of the Nav current present in mouse CCs. We first confirm that functional properties of Nav current in rat and mouse cells are generally similar in terms of activation range, steady-state inactivation, and dual pathway fast inactivation. The results then show that all inward Nav current is absent in CCs from Nav1.3 KO mice. Subsequently, in a mouse with KO of fibroblast growth factor homology factor 14 (FGF14), we find that the slow component of recovery from fast inactivation is completely absent in most CCs, with no change in the time constant of fast recovery. Experiments probing the use-dependence of Nav current diminution between WT and FGF14 KO mice directly demonstrate a role of slow recovery from inactivation in determination of Nav current availability. Overall, the results indicate that the FGF14-mediated inactivation is the major determinant in defining use-dependent changes in Nav availability in CCs. We also consider the potential impact that inactivating FGF’s with different recovery kinetics can exert on differential use-dependent changes in Nav availability.

Developmental exposure to low doses of endocrine disrupter DDT results in negative changes in fine structure and impaired functional activity of adrenal chromaffin cells.

Summary The protein α-synuclein has a central role in the pathogenesis of Parkinson’s disease (PD). Similar to other proteins that accumulate in neurodegenerative disease, however, the function of α-synuclein remains unknown. Localization to the nerve terminal suggests a role in neurotransmitter release and over-expression inhibits regulated exocytosis, but previous work has failed to identify a clear physiological defect in mice lacking all three synuclein isoforms. Using adrenal chromaffin cells and neurons, we now find that both over-expressed and endogenous synuclein serve to accelerate the kinetics of individual exocytotic events, promoting cargo discharge and reducing pore closure (‘kiss-and-run’). Thus, synuclein exerts dose-dependent effects on dilation of the exocytotic fusion pore. Remarkably, mutations that cause PD abrogate this property of α-synuclein without impairing its ability to inhibit exocytosis when over-expressed, indicating a selective defect in normal function.

Good insulation at the glass/fiber interfaces is the most critical factor for successfully manufacturing high-quality cylindrical glass carbon fiber electrodes (CgCFEs). In the conventional process of fabricating CgCFEs, poor insulation is unavoidable when using epoxy as the insulator. In this study, we were the first to use silicone rubber as a new type of insulating material to make silicone rubber-insulated CgCFEs (SCgCFEs) which detected dopamine release in the striatum in vivo after medial forebrain bundle stimulation and recorded quantal catecholamine release from rat adrenal chromaffin cells. Our results indicate that silicone rubber can form perfect insulation in SCgCFEs which are stable, easily manufactured, and can be used in the measurement of easily oxidized monoamine neurotransmitters.

Ca2+ currents ( ICa) recorded from adrenal chromaffin cells (CCs) of spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats are similar to one another, but different from those recorded in other rodent species. ICa in WKY/SHR CCs comprises an early, transient ( ICae) and a late, sustained component ( ICas). In Wistar CCs, ICae is absent, and ICas is of greater amplitude. Activation and steady-state inactivation of ICae and ICas in WKY/SHR CCs suggest the recruitment of at least two populations of Ca2+ channels with different voltage dependence and kinetics. In WKY/SHR CCs, ICae is inhibited by nifedipine, enhanced by BAY K 8644, is not blocked by the mibefradil analog NNC 55–0396, and displays Ca2+-dependent inactivation and fast deactivation kinetics, suggesting that it results from the opening of L-type rather than T-type Ca2+ channels. ICae properties suggest that it originates from the opening of Ca2+ channels formed with the short splice variant (CaV1.342A). RT-PCR showed that expression of CaV1.342A mRNA is similar in both Wistar and WKY/SHR, but that the long variant (CaV1.342) is virtually absent in WKY/SHR. Thus ICae corresponds to the recruitment of CaV1.342A channels, unmasked by the absence of CaV1.342 channels. Studies in WKY CCs do not report major functional alterations, despite the unusual expression pattern of CaV1.3 splice variants. It remains to be established if more subtle functional alterations exist, and if the atypical splicing pattern of CaV1.3 could be related to the functional and behavioral alterations reported in WKY/SHR rats, including their susceptibility to develop hypertension.

We present the electrochemical response to single adrenal chromaffin vesicles filled with catecholamine hormones as they are adsorbed and rupture on a 33 μm diameter disk-shaped carbon electrode. The vesicles adsorb onto the electrode surface and sequentially spread out over the electrode surface, trapping their contents against the electrode. These contents are then oxidized, and a current (or amperometric) peak results from each vesicle that bursts. A large number of current transients associated with rupture of single vesicles (86%) are observed under the experimental conditions used, allowing us to quantify the vesicular catecholamine content.

1 The effects of the immunosuppressants, tacrolimus (FK506) and cyclosporin A (CsA), on catecholamine (CA) release were examined in cultured bovine adrenal chromaffin cells. 2 In intact cells, FK506 (1-30 microM) inhibited CA release stimulated by acetylcholine (ACh; 100 microM), 1,1-dimethyl-4-phenyl-piperazinium (DMPP, 10 microM) or high K+ (40 mM). CsA (1-30 microM) had a little inhibitory effect on the ACh- or DMPP-stimulated CA release, whereas it enhanced the high K(+)-stimulated CA release. 3 In beta-escin-permeabilized cells, FK506 inhibited CA release stimulated by Ca2+ (1 and 10 microM) in the presence and absence of MgATP (2 mM). CsA induced CA release under Ca(2+)-free condition and enhanced the Ca(2+)-stimulated CA release in the presence and absence of MgATP. 4 It is known that the Ca(2+)-dependent exocytosis involves at least two distinct steps, ATP-requiring priming stage and ATP-independent fusion step in adrenal chromaffin cells. Therefore, it is suggested that FK506 inhibits the Ca(2+)-dependent exocytosis probably at the fusion step whereas CsA induces CA release from bovine adrenal chromaffin cells.

Although adrenal glucocorticoids cortisol and corticosterone (CORT) have numerous “genomic” effects on adrenomedullary chromaffin cells, acute modulatory actions remain largely unknown, despite rapid stress-related changes in secretion. We report that 1 μM glucocorticoids rapidly modulate gating of chromaffin cell BK channels and action potential firing. In general, CORT, or the analog dexamethasone (DEX), increased channel activity in inside-out bovine patches, an effect not blocked by the glucocorticoid receptor (GR) antagonist RU38486. By contrast, these steroids could profoundly inhibit BK activation in many rat patches, while facilitating activation in others. We show that BK inhibition arises from a negative shift in the voltage dependence of BK inactivation paralleling that for activation. We report that rat cells characteristically exhibit greater repetitive firing ability than bovine cells in the absence of glucocorticoids. In both species, steroid application typically increased firing responses to smaller current injections, attributable to BK-enhanced repolarization and Na+ channel deinactivation. However, in rat cells, where BK inactivation is generally faster and more complete, glucocorticoids tended to dampen responses to stronger stimuli. Thus, in the context of natural variation in BK gating, glucocorticoids can either promote or limit firing responses. We suggest that steroids exploit BK gating variety to tailor catecholamine output in a species- and context-specific fashion.

Activation of protein kinase C (PKC) seems to promote vesicle recruitment to the release-ready state prior to Ca2+ -triggered fusion in chromaffin cells. To understand spatio-temporal regulation of vesicle recruitment by PKC, we studied the effects of a phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), on the vesicle movements in living chromaffin cells by imaging with a fluorescence microscope-cooled CCD system. About 60 approximately 80% of the chromaffin vesicles showed a rapid movement, about 20% showed a moderate movement, and the rest showed slow/no movement in resting and post-stimulation. The vesicles with slow/no movement increased to 40% upon a depolarizing stimulation, and TPA increased this population to about 70%. TPA treatment, in addition, increased the number of visible chromaffin vesicles beneath the plasma membrane, suggesting that the potentiation of vesicle recruitment by PKC involves a substantial increase in the subplasmalemmal distribution of vesicles.

At early life, the adrenal chromaffin cells respond with a catecholamine surge under hypoxic conditions. This response depends on Ca2+ entry through voltage-activated calcium channels (VACCs). We have investigated here three unresolved questions that concern this response in rat embryo chromaffin cells (ECCs): 1) the relative contribution of L (α1D, Cav1.3), N (α1B, Cav2.2), and PQ (α1A, Cav2.1) to the whole cell Ca2+ current ( ICa); 2) the relative contribution of L and N/PQ channels to the cytosolic Ca2+ elevations triggered by hypoxia (Δ[Ca2+]c); and 3) the role of L and non-L high-VACCs in the regulation of the catecholamine surge occurring during prolonged (1 min) hypoxia exposure of ECCs. Nimodipine halved peak ICa and blocked 60% the total Ca2+ entry during a 50-ms depolarizing pulse to 0 mV (QCa). Combined ω-agatoxin IVA plus ω-conotoxin GVIA (Aga/GVIA) blocked 30% of both ICa peak and QCa. This relative proportion of L- and non-L VACCs was corroborated by Western blot that indicated 55, 23, and 25% relative expression of L, N, and PQ VACCs. Exposure of ECCs to hypoxia elicited a mild but sustained Δ[Ca2+]c; the area of Δ[Ca2+]c was blocked 50% by nifedipine and 10% by Aga/GVIA. Exposure of ECCs to 1-min hypoxia elicited an initial transient burst of amperometric secretory spikes followed by scattered spikes along the time of cell exposure to hypoxia. This bulk response was blocked 85% by nimodipine and 35% by Aga/GVIA. Histograms on secretory spike frequency vs. time indicated a faster initial inactivation when Ca2+ entry took place through N/PQ channels; more sustained secretion but at a lower rate was associated to Ca2+ entry through L channels. The results suggest that the HIS response may initially be controlled by L and P/Q channels, but later on, N/PQ channels inactivate and the delayed HIS response is maintained at lower rate by slow-inactivating L channels.

Both bovine and rat adrenal chromaffin cells have served as pioneering model systems in cellular neurophysiology, including in the study of large conductance calcium- and voltage-dependent K+(BK) channels. We now report that while BK channels dominate the outward current profile of both species, specific gating properties vary widely across cell populations, and the distributions of these properties differ dramatically between species. Although BK channels were first described in bovine chromaffin cells, rapidly inactivating ones were discovered in rat chromaffin cells. We report that bovine cells can also exhibit inactivating BK channels with varying properties similar to those in rat cells. However, a much smaller proportion of bovine cells exhibit inactivating BK current, the proportion of the total current that inactivates is usually smaller, and the rate of inactivation is often much slower. Other gating features differ as well; the voltage dependence of channel activation is much more positive for bovine cells, and their rates of activation and deactivation are faster and slower, respectively. Modeling studies suggest that channel heterogeneity is consistent with varying tetrameric combinations of inactivation-competent versus -incompetent subunits. The results suggest that chromaffin BK channel functional nuances represent an important level for evolutionary tailoring of autonomic stress responses.

The effect of hypoxia on small-conductance Ca(2+)-activated K+ current was investigated in a study of adult rat adrenomedullary chromaffin cells (AMCs), which were maintained in short-term culture. The nystatin-perforated, whole-cell patchclamp technique was used to study the effect of hypoxia with minimum perturbation of the intracellular milieu. Under voltage-clamp conditions, acute hypoxia (P(O2) approximately equal to 25 mmHg) suppressed the whole-cell outward currents of more than half the AMCs (24/46). This suppression was eliminated after application of apamin (400 nM), a selective inhibitor of small-conductance Ca(2+)-activated K+ current (I(SK)(Ca)) (n=5), suggesting that an apamin-sensitive component of whole-cell currents is suppressed during hypoxia. In contrast to I(SK)(Ca), Ca2+ current (I(Ca)) (n=10) was not affected by hypoxia. Finally, under current-clamp conditions, hypoxia reversibly depolarized the resting membrane potential of adult AMCs (34/40). Apamin, however, eliminated the hypoxia-induced depolarization (400 nM) (7/8), suggesting that hypoxic depolarization is related to the suppression of I(SK(Ca). From the above results, we conclude that adult AMCs are sensitive to hypoxia, and that I(SK)(Ca) contributes to the hypoxia-induced suppression of whole-cell outward current and depolarization of the resting membrane potential in adult AMCs.

In digitonin-permeabilized adrenal chromaffin cells, Ca2+-induced catecholamine release can be resolved into at least two sequential steps: a MgATP-dependent priming step and a MgATP-independent Ca2+-triggered step. Botulinum neurotoxins types A and E cleaved SNAP-25, and blocked MgATP-independent Ca2+-induced catecholamine release from the permeabilized chromaffin cells. When the permeabilized cells were primed by pretreatment with MgATP, the amount of SNAP-25 associated with VAMP-2 decreased, and the fraction of SNAP-25 proteolyzed by the neurotoxins increased. These results suggest that dissociation of SNAP-25 and VAMP-2 occurs during the MgATP-dependent priming step, and SNAP-25 plays some important roles in the subsequent MgATP-independent step.

1. Intracellular Ca2+ clearance mechanisms were studied in rat adrenal chromaffin cells, by measuring slow tail currents through small-conductance Ca(2+)-activated K+ channels and using indo-1 photometry following depolarization-induced Ca2+ loading. 2. Following several-hundred millisecond depolarizations, [Ca2+]i decayed in three phases. An initial fast decay was followed by a long-lasting, low plateau, then [Ca2+]i returned to the resting level slowly. 3. Replacement of external Na+ moderately slowed [Ca2+]i decay, indicating a contribution of plasma membrane Na(+)-Ca2+ exchange. 4. Raising external pH or application of extracellular Eosin of La3+ prolonged slow tail currents, indicating a contribution of plasma membrane Ca(2+)-ATPase to Ca2+ clearance. 5. Ca(2+)-induced Ca2+ release from caffeine-sensitive stores occurred during depolarization. 6. Inhibition of endoplasmic reticulum Ca(2+)-ATPase had little effect on Ca2+ clearance. 7. Slow tail currents and [Ca2+]i decay following 0.2 - 2 s depolarizations were much prolonged by mitochondrial inhibition with carbonyl cyanide m-chlorophenylhydrazone (CCCP) or Ruthenium Red, which abolished the initial rapid decay and plateau of [Ca2+]i. 8. In conclusion, mitochondrial Ca2+ uptake plays a major role in Ca2+ clearance by rapidly and reversibly sequestering Ca2+ during depolarization-evoked Ca2+ loads.

Cytosolic Ca 2+ (Ca 2+ c ) clearance from adrenal chromaffin cells was studied by whole-cell patch clamp and indo-1 Ca 2+ photometry after influx of Ca 2+ through voltage-dependent Ca 2+ channels. We isolated the rates of Ca 2+ c clearance by several mechanisms using combinations of the following agents (with their expected targets): Li + or TEA substituted for Na + (Na + –Ca 2+ exchange), 1 mM La 3+ applied after the depolarization (Na + –Ca 2+ exchange and plasma membrane Ca 2+ -ATPase), 1 μM thapsigargin (pumping into reticular stores), and 2 μM carbonyl cyanide m-chlorophenylhydrazone (uptake into mitochondria). Remarkably, whenever [Ca 2+ ] c rose above ∼500 nM, Ca 2+ c clearance by mitochondria exceeded clearance by either Na + –Ca 2+ exchange or the Ca 2+ pumps of the plasma and reticular membranes. As [Ca 2+ ] c fell again, Ca 2+ reemerged from mitochondria, prolonging the final return to basal levels.

Using high resolution capacitance (Cm) measurement techniques, we mapped the kinetics of exocytosis evoked by brief (5-100 ms) depolarizations that activate voltage-dependent Ca2+ channels in rat adrenal chromaffin cells. After correcting the Cm signal for contributions from Na+ channel-gating charge movements, the initial kinetics of exocytosis were consistent with depolarization-triggered release occurring initially from an immediately releasable pool of only approximately 17 secretory vesicles. In contrast, repetitive application of longer depolarizations evoked release from a distinct, larger readily releasable pool of approximately 170 vesicles. Our results suggest that the secretory response of a chromaffin cell to an action potential is limited by the size of the immediately releasable pool rather than by a fusion mechanism that is slower than that at synapses.

Many pathological conditions involve alterations in expression of nicotinic acetylcholine receptors (nAChRs). The following studies were designed to investigate cellular mechanisms involved with expression and turnover of alpha3beta4* nAChRs. These studies support constitutive turnover of adrenal alpha3beta4* nAChRs and the use of cultured adrenal chromaffin cells to study nAChR regulation.

Intense stimuli, such as trains of depolarizing pulses or the caffeine-induced release of calcium from intracellular stores, readily depress the secretory response in neuroendocrine cells. Secretory responses are restored by rest periods of minutes in duration. This recovery was accelerated when the concentration of cytosolic calcium was moderately increased and probably resulted from calcium-dependent replenishment of a pool of release-ready granules. Continuously increased concentrations of calcium led the over-filling of such a pool. Subsequently, secretory responses to stronger calcium stimuli were augmented. Hormone-induced calcium transients with a plateau phase of increased concentration of calcium may enhance the secretory response in this way.

Dendritic formation of mastoparan molecule by a carboxyl-terminal linkage to a lysine branch increased the original mastoparan activity for hemolysis and catecholamine release from adrenal chromaffin cells. The most potent dendrimer in hemolytic activity, approximately 8000 times as active as mastoparan, was octameric mastoparan.

We have determined that there are components to the time course of Ca(2+)-dependent secretion from digitonin-permeabilized bovine adrenal chromaffin cells that can be distinguished by Ca2+ sensitivity and ATP dependence. The effects of various Ca2+ concentrations are different on the initial rates and later rates of secretion. The earliest rates (5 s) are half-maximal between 30-100 microM Ca2+ and maximal by 300 microM Ca2+. Later rates of secretion are maximal by 10 microM and decline above 30 microM Ca2+. At low Ca2+ concentrations secretion begins after a lag of several seconds. The early rates of secretion (within 1 min) are dependent on the prior effects of MgATP. MgATP primes the cells to secrete. Later rates require the continuous presence of MgATP for optimal secretion. Incubation with low concentrations of Ca2+ increases the ability of MgATP to stimulate subsequent Ca(2+)-dependent secretion. Preincubation with Ca2+ has no effect on the rapid loss of ATP-independent secretion with time after permeabilization. The data indicate that: 1) as secretion progresses in digitonin-permeabilized cells, different events become rate-limiting; 2) maximal secretion at the early times requires at least 10-fold higher Ca2+ concentrations than at later times; 3) the rate at which Ca2+ initiates secretion is concentration-dependent; and 4) Ca2+ not only triggers the final events in secretion but enhances the ability of ATP to prime secretion.

Etched carbon fiber microelectrodes of different radii have been used for amperometric measurements of single exocytotic events occurring at adrenal chromaffin cells. Frequency, kinetic, and quantitative information on exocytosis provided by amperometric spikes were analyzed as a function of the surface area of the microelectrodes. Interestingly, the percentage of spikes with foot (as well as their own characteristics), a category revealing the existence of sufficient long-lasting fusion pores, was found to be constant whatever the microelectrode diameter was, whereas the probability of overlapping spikes decreased with the electrode size. This confirmed that the prespike foot could not feature accidental superimposition of separated events occurring at different places. Moreover, the features of amperometric spikes investigated here (charge, intensity and kinetics) were found constant for all microelectrode diameters. This demonstrated that the electrochemical measurement does not introduce significant bias onto the kinetics and thermodynamics of release during individual exocytotic events. All in all, this work evidences that information on exocytosis amperometrically recorded with the usual 7 microm diameter carbon fiber electrodes is biologically relevant, although the frequent overlap between spikes requires a censorship of the data during the analytical treatment.

Exploring the interactions between radiofrequency (RF) radiation and biological systems is essential for developing RF-based non-lethal stunning/immobilizing weaponry. To this end a research effort was initiated to identify RF parameters potentially capable of selectively altering exocytosis, the process underlying neurotransmitter release and hence nervous system functioning. Major accomplishments included designing, setting up, testing, characterizing and optimizing a waveguide-based RF exposure system for assessing effects on exocytosis, using neurosecretory adrenal chromaffin cells as an in vitro model, and performing experiments that lead to the novel observation of enhanced neurosecretion in response to modulated RF fields in the 0.75 -0.85 GHz frequency range. The research, which has been transitioned into the DoD EPSCoR program, has been presented at four international meetings, two local research conferences, and has culminated in one peer-reviewed publication, a manuscript in preparation and a Master's thesis.

Cytochrome b(561) in adrenal chromaffin vesicle membranes conveys electron equivalents from extravesicular ascorbate to the intravesicular monodehydroascorbate radical. We conducted a stopped-flow study on the reaction of ascorbate with purified cytochrome b(561) in the detergent-solubilized state for the first time. The time course of the reduction of oxidized cytochrome b(561) with ascorbate could not be fitted with a single exponential but with a linear combination of at least four exponential functions. This result is consistent with the notion that cytochrome b(561) contains two hemes b, each having a distinct redox potential and a function upon reactions with ascorbate and monodehydroascorbate radical. The fastest phase, which was assigned to the first one-electron donation from ascorbate to heme b on the extravesicular side, was further analyzed by transient phase kinetics employing a two-step bi-uni sequential ordered mechanism. The result showed K(s) = 2.2 mM for ascorbate at pH6.0. At a region below pH5.5, there was a significant lag before the reduction of hemes b occurred. This time lag was interpreted as due to a pH-dependent transient state before the first electron transfer to take place. The fastest phase was completely lost by N-carbethoxylation of heme-coordinating histidyl residues (His88 and His161) and Lys85 upon treatment with diethylpyrocarbonate. The presence of ascorbate during the treatment inhibited the N-carbethoxylation of the histidyl residues and, thereby, restored the final reduction level of hemes b. But the reduction rate was still only one-twentieth of the native form. This result suggested an important role of the conserved Lys85 for the interaction with ascorbate.