alpha-Latrotoxin alters spontaneous and depolarization-evoked quantal release from rat adrenal chromaffin cells: evidence for multiple modes of action.

alpha-Latrotoxin (alpha-LT) potently enhances both "spontaneous" and "depolarization-evoked" quantal secretion from neurons. Here we have used the patch-clamped rat adrenal chromaffin cell to examine simultaneously the effects of alpha-LT on membrane current or voltage, cytosolic Ca, and membrane capacitance, the latter used as an assay for exocytosis. In chromaffin cells exposed to toxin concentrations of >100 pM, the development of large conductance, Ca-permeable ion channels, accompanied by a rise in cytosolic Ca to levels near 1 microM, precedes the initiation of spontaneous exocytosis. These channels appear to be induced de novo, because they occur concurrently with massive reduction or pharmacological block of voltage-dependent Na and Ca currents. However, enhancement of depolarization-evoked release, seen in many cells at <50 pM toxin, often occurs in the absence of a rise in background cytosolic Ca or de novo channel activity. These results favor Ca entry through toxin-induced channels underlying initiation of spontaneous release and direct modulation of the secretory machinery by the toxin-bound receptor contributing to enhancement of depolarization-evoked secretion as well as spontaneous release.

alpha-Latrotoxin-induced quantal release of catecholamines from rat adrenal chromaffin cells.

alpha-Latrotoxin (alpha-LT) potently enhances quantal release of neurotransmitter from nerve terminals. To develop the adrenal chromaffin cell as a 'model' system for the study of mechanisms of toxin action, we used amperometry to examine secretion of catecholamines and spectrofluorometry to measure cytosolic Ca. Several key features of toxin action emerged. (1) Release occurs at concentrations of toxin >35 pM and the pattern of release changes from repeated brief bursts to more continuous discharges of varying duration as the toxin concentration increases. (2) Release requires extracellular calcium in the micromolar range, but not the activity of native voltage-dependent calcium channels. (3) Release is associated with a rise in cytosolic calcium to near micromolar range. (4) Provided calcium is later restored, release can be induced even though the toxin is applied and washed away in calcium-free saline. These features largely resemble those of alpha-LT action on nerve terminals. They suggest that in chromaffin cells, as in neurons, the Ca-dependence of toxin-enhanced quantal release is based on Ca entry through toxin-induced channels rather than a requirement of extracellular Ca for toxin binding.

Characterization of a Ca2+-activated K+ current in insulin-secreting murine betaTC-3 cells.

1. The whole-cell perforated-patch recording mode was used to record a Ca2+-dependent K+ current (IK(Ca)) in mouse betaTC-3 insulin-secreting cells. 2. Depolarizing voltage steps (to potentials where Ca2+ currents are activated) evoked a slowly activating, outward current, which exhibited a slow deactivation (in seconds) upon subsequent hyperpolarization. 3. This current was shown to increase with progressively longer depolarizing voltage steps. It could be reversibly abolished by the removal of Ca2+ from the external medium or by application of Ca2+ channel blockers, such as Cd2+ and nifedipine. It was concluded that the depolarization-evoked current was activated by Ca2+. 4. Variations in external K+ concentration led to shifts in the reversal potential of the Ca2+-dependent current as predicted by the Nernst equation for a K+-selective current. 5. The Ca2+-activated K+ current was insensitive to external TEA (10 mM), a concentration sufficient to block the large-conductance Ca2+-dependent (maxi-KCa) channel in beta-cells. It was also insensitive to apamin, tubocurarine and scyllatoxin (leiurotoxin I), specific blockers of small-conductance KCa channels. 6. The current was blocked by quinine, a non-specific KCa channel blocker and, surprisingly, by charybdotoxin (ChTX; 100 nM) but not iberiotoxin, a charybdotoxin analogue, which blocks the maxi-KCa channel. It was sensitive to block by clotrimazole and could be potently and reversibly potentiated by micromolar concentrations of niflumic acid. Thus, the current exhibited unique pharmacological characteristics, not conforming to the known KCa channel classes. 7. The ChTX-sensitive KCa channel was permeable to Tl+, K+, Rb+ and NH4+ but not Cs+ ions. 8. The ChTX-sensitive IK(Ca) could be activated by the muscarinic agonists in the presence or absence of external Ca2+, presumably by releasing Ca2+ from internal stores. 9. Acutely isolated porcine islet cells also exhibited a slow IK(Ca) resembling that described in betaTC-3 cells in kinetic properties, insensitivity to TEA (5 mM) and sensitivity to quinidine, an analogue of quinine. The porcine IK(Ca), however, was not sensitive to block by 100-200 nM ChTX. It is likely, that species differences account for pharmacological differences between the mouse and porcine slow IK(Ca).

An optimized approach to membrane capacitance estimation using dual-frequency excitation.

We present an optimized solution to the problem of membrane impedance estimation when a patch-clamped cell is stimulated by a dual-frequency, sinusoidal excitation. The complete data set of raw whole-cell current samples is typically reduced, via digital lock-in detection, to measurements of the complex cell model admittance at the two stimulus frequencies. We describe a statistical model of both data sets and demonstrate that the admittance data adequately represent the essential features obtained from the raw data. The parameter estimates obtained by a nonlinear weighted least-squares solution (NWLS), which under normal recording conditions is equivalent to the maximum likelihood solution, essentially obtain the theoretical lower bound on variance established by the Cramér-Rao bound. Our software implementation of the NWLS solution produces estimates of the cell model parameters that are less noisy than other dual-frequency systems. Our system can be used 1) to measure slow changes in membrane capacitance-in the face of large, slow changes in membrane resistance, 2) to detect with confidence capacitance changes expected from the exocytosis of moderate-sized dense core granules, and 3) to reduce the cross-talk between transient changes in membrane conductance and membrane capacitance.

Single-cell measurements of quantal secretion induced by alpha-latrotoxin from rat adrenal chromaffin cells: dependence on extracellular Ca2+.

alpha-Latrotoxin (alpha-LT), from black widow spider venom, is a potent enhancer of the spontaneous quantal release of neurotransmitter from a variety of nerve terminals and clonal neurosecretory cells. Using electrochemical amperometry and estimation of membrane impedance by phase detection, we present evidence that alpha-LT induces exocytosis of catecholamines from rat adrenal chromaffin cells beginning as rapidly as 30 s after close application of the toxin. This release is largely dependent on adequate levels of extracellular Ca2+ ([Ca2+]o). Lowering [Ca2+]o from 2 mM to

Rapid fluctuations in transmitter release from single vesicles in bovine adrenal chromaffin cells.

Single-vesicle release of catecholamines from chromaffin cells can be detected in real time as current spikes by the electrochemical method of amperometry. About 70% of spikes are preceded by a small "foot," the trickle of transmitter out of the early fusion pore. In addition, 20-50% of foot signals exhibit rapid fluctuations that we interpret as flickering of the fusion pore. There are also "stand-alone" foot signals, which may reflect transient fusions, in which the vesicles do not collapse completely into the plasma membrane. The number and frequency of the foot flickering are affected by intracellular Ca2+ concentration.

Amperometric detection of quantal secretion from patch-clamped rat pancreatic beta-cells.

Serotonin (5-HT) is taken up in insulin granules and co-released with insulin on stimulation of pancreatic islet beta-cells. Based on these observations, we have used microcarbon fiber amperometry to examine secretogogue-induced 5-HT release from rat beta-cells preloaded for 4-16 h with 5-HT and then exposed to a bath solution containing 10 microM forskolin. In response to local application of KCl (60 mM) or tolbutamide (50-200 microM), we recorded barrages of amperometric events. Each amperometric event consisted of a short pulse of current measurable at electrode voltages that catalyze 5-HT oxidation. With either secretogogue, release was calcium-dependent. On combining amperometry with perforated patch whole-cell recording, we found that barrages of such events were well coupled in time and graded in intensity with depolarization-induced Ca2+ currents and well correlated with increases in membrane capacitance. In cell-attached patch recording, amperometric events evoked by application of tolbutamide followed the closure of ATP-sensitive K+ channels and coincided with the onset of electrical activity. These experiments suggest that amperometry is a useful technique for studying, in real time, the dynamic aspects of stimulus-secretion coupling in beta-cells. Their performance was facilitated by the design of a new carbon fiber electrode (ProCFE) described within.

Voltage-dependent Na+ and Ca2+ currents in human pancreatic islet beta-cells: evidence for roles in the generation of action potentials and insulin secretion.

We describe three voltage-dependent inward currents in human pancreatic beta-cells. First, a rapidly inactivating Na+ current, blocked by tetrodotoxin (TTX) is seen upon brief depolarization to or beyond -40 mV. Second, a transient, low-voltage-activated (LVA), amiloride-blockable Ca2+ current is seen upon depolarization to or beyond -55 mV; it inactivates within less than 1s of sustained depolarization to -40 mV. Third, a more sustained, high-voltage-activated (HVA) Ca2+ current, which shows variable sensitivity to dihydropyridines is seen upon depolarization to or beyond -40 mV, and thereafter slowly inactivates over a time course of many seconds. Our pharmacological evidence suggests that all three currents contribute to action potential initiation and upstroke when the background membrane potential (Vm) is equal or negative to -45 to -40 mV, a situation often induced by glucose concentrations (5-6 mM) in the range of those seen post-prandially. Consistent with this, TTX drastically reduces both transient and sustained insulin secretion in the presence of 5-6 mM glucose, but has little effect in 10 mM glucose, at which concentration cells rapidly depolarize to approximately -35 mV, a Vm sufficient to rapidly inactivate Na+ and LVA Ca2+ currents.

Coupling of exocytosis to depolarization in rat pancreatic islet beta-cells: effects of Ca2+, Sr2+ and Ba(2+)-containing extracellular solutions.

Using rat beta-cells we present evidence that Sr2+ and Ba2+, like Ca2+, support depolarization-induced increases in membrane capacitance which reflect insulin granule exocytosis. Even with identical total charge entry, Sr2+ and Ba2+ are 3-5 and 20-fold less effective than Ca2+ in supporting release. While exocytosis supported by Sr2+ is graded with cation entry and complete within 250ms of depolarization, exocytosis supported by Ba2+ begins abruptly after a threshold of charge entry and continues for many seconds. Ba(2+)-supported release continues in the presence of greatly enhanced cytosolic Ca2+ buffering, arguing against release of Ca2+ from stores as its principal action. These results suggest that Sr2+ and Ba2+ support exocytosis largely by binding to Ca(2+)-dependent release-activating sites, though with less affinity than Ca2+.

Amperometric detection of stimulus-induced quantal release of catecholamines from cultured superior cervical ganglion neurons.

Amperometry has been used for real-time electrochemical detection of the quantal release of catecholamines and indolamines from secretory granules in chromaffin and mast cells. Using improved-sensitivity carbon fiber electrodes, we now report the detection of quantal catecholamine release at the surface of somas of neonatal superior cervical ganglion neurons that are studded with axon varicosities containing synaptic vesicles. Local application of a bath solution containing high K+ or black widow spider venom, each of which greatly enhances spontaneous quantal release of transmitter at synapses, evoked barrages of small-amplitude (2-20 pA), short-duration (0.5-2 ms) amperometric quantal "spikes". The median spike charge was calculated as 11.3 fC. This figure corresponds to 3.5 x 10(4) catecholamine molecules per quantum of release, or approximately 1% that evoked by the discharge of the contents of a chromaffin granule.

Pharmacology of volume regulation following hypotonicity-induced cell swelling in clonal N1E115 neuroblastoma cells.

When exposed to hypotonic solutions, clonal N1E115 neuroblastoma cells initially swell and later undergo a regulatory volume decrease (RVD). We studied the effects of a variety of transport inhibitors on the time course of cross-sectional area of N1E115 cells exposed to a solution of reduced osmolarity (pi = 186 mosm). Application to the bath of either: (i) blockers of net K efflux through K channels (e.g. isotonic KCl or 20 mM TEA); or (ii) blockers of net efflux through anion channels (e.g. isotonic methanesulfonate, 10 microM DIDS or 100 microM IAA-94) all prevent RVD. In contrast, ouabain (a Na+/K+ pump blocker), bumetanide (a Na+/K+/Cl- cotransporter blocker) and SITS (a HCO3-/Cl- exchange blocker) do not. These data support the involvement of these channels over pumps or exchangers in solute exit during RVD. Only variable block of RVD was achieved using blockers of stretch activated non-selective cation C+ (SA) channels (i.e., amiloride and gadolinium, Gd3+) or a membrane permeant Ca chelator (BAPTA-AM) suggesting that neither the opening of C+ (SA) channels nor a global rise in cytosolic Ca2+ is critical for triggering RVD.

Action potential-induced quantal secretion of catecholamines from rat adrenal chromaffin cells.

Using single rat adrenal chromaffin cells, we examined the coupling of action potential activity to quantal release of catecholamines by combining perforated patch current-clamp recording with electrochemical microcarbon fiber amperometry. Chromaffin cells display steeper dependence of quantal release on action potential frequency than many nerve terminals, as well as more desynchronized release following an action potential. Also in contrast to neurons, in chromaffin cells, a major chemical secretagogue (acetylcholine) triggers potent quantal release even in the absence of electrical activity. These findings are consistent with an hypothesis that a major component of exocytosis from chromaffin cells involves diffusion of Ca2+ to secretion sites which are less well co-localized with Ca2+ channels than those in nerve terminals.

cAMP-enhancing agents "permit" stimulus-secretion coupling in canine pancreatic islet beta-cells.

Isolated canine islets of Langerhans differ from isolated islets of other species (including rodents and man) in that elevated glucose concentrations are unable to stimulate insulin secretion. Here we demonstrate that addition to the perifusate of isobutylmethylxanthine (IBMX), forskolin or 8-CPT-cAMP, all of which enhance cytosolic cAMP, permits insulin secretion in response to glucose, leucine or tolbutamide. These cAMP enhancers increase secretogogue-induced electrical activity in beta-cells and restore depolarization-induced, Ca(2+)-dependent granule exocytosis measured as stepwise increases in membrane capacitance. We propose that the primary permissive action of cAMP is to tightly link Ca2+ entry to insulin granule release, while a secondary action is to tighten the link between glucose metabolism and cell depolarization.

Enhancers of cytosolic cAMP augment depolarization-induced exocytosis from pancreatic B-cells: evidence for effects distal to Ca2+ entry.

We have investigated the effects of cAMP-enhancing agents on depolarization-induced membrane capacitance increases (delta Cm) in single rat pancreatic B-cells. Concentrations of IBMX, 8-CPT cAMP and forskolin, which enhance cAMP and insulin release, all enhance depolarization-induced delta Cm's seen in response to single voltage-clamp pulses and reduce the depression of delta Cm responses often seen with trains of pulses. These effects often occur in the absence of changes in peak Ca2+ current or the total Ca2+ charge entry during the depolarizing pulse. These data suggest that cAMP-modulating maneuvers may directly affect the mechanism of insulin granule mobilization into a readily releasible store or fusion at a discharge site.

ATP-sensitive potassium channels in physiology, pathophysiology, and pharmacology.

Potassium-selective ion channels, whose activity is inhibited by micromolar to millimolar concentrations of ATP presented at the cytoplasmic ATP-sensitive K+ (K+[ATP]) surface, have been found in a variety of cell types. These "K+(ATP) channels" have emerged as significant targets for physiologic as well as pharmacologic modulation of cell processes. In insulin-secreting beta cells of the pancreatic islet, closure of these channels on presentation of a metabolite secretogogue, such as glucose, or an oral hypoglycemic sulfonylurea, results in cell depolarization and triggers electrical activity. Ultimately, this results in Ca2+ entry and Ca(2+)-dependent exocytosis of insulin granules. In myocytes, opening of K+(ATP) channels during hypoxia or metabolite deprivation or with exposure to a new class of K+ channel opener drugs results in cell hyperpolarization and myocyte relaxation. This contributes to vasodilation. In renal tubule cells, K+(ATP) channels contribute to cell potassium balance during vectorial bulk solute transfer by the proximal tubule as well as net urinary potassium secretion by the distal nephron. Agents that modulate the activity of these K+(ATP) channels in epithelial cells may prove to be useful as K(+)-sparing diuretics. in epithelial cells may prove to be useful as K(+)-sparing diuretics.

Electrophysiology of stimulus-secretion coupling in human beta-cells.

Herein, we review the applicability to human beta-cells of an electrophysiologically based hypothesis of the coupling of glucose metabolism to insulin secretion. According to this hypothesis, glucose metabolism leads to the generation of intracellular intermediates (including ATP), which leads to closure of ATP-sensitive K+ channels. Channel closure results in membrane depolarization, the onset of electrical activity, and voltage-dependent Ca2+ entry. The resultant rise in cytosolic Ca2+ leads to Ca(2+)-dependent exocytosis of insulin granules. We found that most of the published experimental evidence for human beta-cells supports this hypothesis. In addition, we present three other emerging lines of evidence in support of this hypothesis for human islet beta-cells: 1) the effects of pHi-altering maneuvers on insulin secretion and electrical activity; 2) preliminary identification of LVA and HVA single Ca2+ channel currents; and 3) validation of the feasibility of Cm measurements to track insulin granule exocytosis. On the basis of this last new line of evidence, we suggest that combinations of Cm measurements and electrical activity/membrane current measurements may help define the roles of diverse electrical activity patterns, displayed by human beta-cells, in stimulus-induced insulin secretion.

Parathyroid hormone activation of stretch-activated cation channels in osteosarcoma cells (UMR-106.01).

Cell-attached patches of membrane of osteoblast-like cells UMR-106.01 respond to bath application of parathyroid hormone (PTH) with an increase in the average activity, as well as the single channel conductance, of a stretch-activated non-selective cation channel. Correlations with whole cell membrane potential and conductance changes are considered.

Stimulus-secretion coupling in beta-cells of transplantable human islets of Langerhans. Evidence for a critical role for Ca2+ entry.

With human islets isolated for transplantation, we examined the applicability to humans of a metabolic fuel hypothesis of glucose transduction and a Ca2+ hypothesis of depolarization-secretion coupling, both previously proposed for rodent islet beta-cells. We report that several features of human beta-cell physiology are well accounted for by these hypotheses. With whole-islet perifusion, we demonstrated that insulin secretion induced by glucose, tolbutamide, or elevated K+ is dependent on extracellular Ca2+. Insulin release induced by these secretagogues is enhanced by the dihydropyridine Ca2+ channel agonist BAYk8644 and depressed by the dihydropyridine Ca(2+)-channel antagonist nifedipine. All of the aforementioned secretagogues provoke increases in cytosolic free Ca2+, which are dependent on extracellular Ca2+ and are altered by the dihydropyridine drugs. Individual beta-cells in the islet display diminished resting membrane conductance, graded depolarization, and complex electrical patterns, including bursts of action potentials in response to stimulatory concentrations of glucose or tolbutamide. Individual islet beta-cells display voltage-dependent Ca2+ currents that are activated at membrane potentials traversed during the excursion of the action potential. In most cells, the Ca2+ currents are enhanced by BAYk8644 and depressed by nifedipine at concentrations that have parallel effects on secretagogue-induced increases in cytosolic Ca2+ and insulin secretion. These survey studies should provide the basis for more detailed investigations of the relationship of voltage-dependent ionic currents to electrical activity patterns and of electrical activity patterns to granule exocytosis in single human beta-cells.

Effects of metabolic inhibition by sodium azide on stimulus-secretion coupling in B cells of human islets of Langerhans.

Sodium azide (NaN3), a reversible inhibitor of mitochondrial respiration, blocks glucose-induced electrical activity and insulin secretion in human pancreatic islet B cells. Here we show that brief (10-15 min) application followed by removal of 3 mM NaN3 results in transient overshoot of electrical activity and insulin secretion even at substimulatory levels of glucose (3-5 mM). In addition, application of NaN3, even at very low [Ca2+]o, reversibly increases cytosolic Ca2+ to levels usually associated with substantial insulin release. These results suggest that (i) metabolic inhibition may reset B cell stimulus-secretion coupling and (ii) a rise in free cytosolic Ca2+, by itself, is not sufficient to trigger insulin secretion.

Single cell assay of exocytosis from pancreatic islet B cells.

We have measured changes in membrane capacitance (delta Cm) in pancreatic B cells as a single cell assay of insulin secretion. Evidence that depolarization evoked delta Cm reflects exocytosis includes it's voltage, temperature and Ca2+ o dependence. Decreases in Cm, presumably reflecting endocytosis, occur on a variable time scale. Two features that make B cells unique among excitable cells are the large magnitude of delta Cm, when normalized to Ca2+ current, and the rapid "fatigue" of the response for even minimal stimulation, perhaps due to the depletion of a readily releasable pool of vesicles.