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Glutamatergic vesicles in hippocampal mossy fiber presynaptic boutons release zinc, which plays a modulatory role in synaptic activity and LTP. In this work, a fluorescence microscopy technique and the fluorescent probe for cytosolic zinc, Newport Green (NG), were applied, in a combined study of autofluorescence and zinc changes at the hippocampal mossy fiber-CA3 synaptic system. In particular, the dynamics of flavoprotein (FAD) autofluorescence signals, was compared to that of postsynaptic zinc signals, elicited both by high K+ (20 mM) and by tetraethylammonium (TEA, 25 mM). The real zinc signals were obtained subtracting autofluorescence values, from corresponding total NG-fluorescence data. Both autofluorescence and zinc-related fluorescence were raised by high K+. In contrast, the same signals were reduced during TEA exposure. It is suggested that the initial outburst of TEA-evoked zinc release might activate ATP-sensitive K+ (KATP) channels, as part of a safeguard mechanism against excessive glutamatergic action. This would cause sustained inhibition of zinc signals and a more reduced mitochondrial state. In favor of the “KATP channel hypothesis”, the KATP channel blocker tolbutamide (250 μM) nearly suppressed the TEA-evoked fluorescence changes. It is concluded that recording autofluorescence from brain slices is essential for the accurate assessment of zinc signals and actions.

Current water treatment methods are unable to eliminate artificial sweeteners, which can lead to their accumulation in the environment. Due to this problem, natural sweeteners can be used instead, but it is important to understand their effects in biological systems. Rebaudioside A, one of the main components of stevia, causes an increase in both ROS and in FAD linked autofluorescence in hippocampal CA3 area. These effects may be due to the insulin-mimetic properties of steviol glycosides, with the results suggesting that they cause enhancements in glycolysis and in OXPHOS, with this metabolic pathway being the possible source of the rise in ROS. In excess, these molecules may cause damage to brain cells through oxidative stress. As leftovers from Stevia’s purification process can be used as biomass, with applications ranging from energy production to fertilization, continuous accumulation in the environment should be avoided in order to prevent undesired effects in the ecosystem. Keywords: Autofluorescence, CA3, OXPHOS, Reactive oxygen species, Stevia, Steviol Glycosides

The accumulation of intracellular ionic zinc and pharmaceutical compounds, like the antibiotic sulfamethoxazole, may contribute to various neuropathologies. Sulfamethoxazole and the drug trimethoprim, are inhibitors of enzymes involved in the synthesis of tetrahydrofolate and also of carbonic anhydrases. The inhibition of the latter enzymes, which are localized both intra- and extracellularly and have a key role in pH regulation, causes alkalinization that is associated with higher spontaneous transmitter release. Intense synaptic stimulation causes the entry of released zinc into postsynaptic neurons, through glutamate receptor channels or voltage dependent calcium channels. The aim of this study was to evaluate the effect of sulfamethoxazole (180 μM) on basal postsynaptic zinc and to compare it with that caused by two depolarizing media, containing high potassium or tetraethylammonium, which may induce long term synaptic plasticity. The studies were performed in brain slices from gestating rats, at the mossy fiber synapses from hippocampal CA3 area, using the zinc indicator Newport Green. In the presence of KCl (20 mM) and sulfamethoxazole (180 μM) the zinc signals were enhanced, unlike in tetraethylammonium (25 mM). After sulfamethoxazole the tetraethylammonium evoked zinc signal had reduced amplitude. Thus, the data suggests that sulfamethoxazole enhances transmitter release affecting synaptic zinc physiology.

Zinc, a transition metal existing in very high concentrations in the hippocampal mossy fibers from CA3 area, is assumed to be co-released with glutamate and to have a neuromodulatory role at the corresponding synapses. The synaptic action of zinc is determined both by the spatiotemporal characteristics of the zinc release process and by the kinetics of zinc binding to sites located in the cleft area, as well as by their concentrations. This work addresses total, free and complexed zinc concentration changes, in an individual synaptic cleft, following single, short and long periods of evoked zinc release. The results estimate the magnitude and time course of the concentrations of zinc complexes, assuming that the dynamics of the release processes are similar to those of glutamate. It is also considered that, for the cleft zinc concentrations used in the model (≤ 1 μM), there is no postsynaptic zinc entry. For this reason, all released zinc ends up being reuptaken in a process that is several orders of magnitude slower than that of release and has thus a much smaller amplitude. The time derivative of the total zinc concentration in the cleft is represented by the difference between two alpha functions, corresponding to the released and uptaken components. These include specific parameters that were chosen assuming zinc and glutamate co-release, with similar time courses. The peak amplitudes of free zinc in the cleft were selected based on previously reported experimental cleft zinc concentration changes evoked by single and multiple stimulation protocols. The results suggest that following a low amount of zinc release, similar to that associated with one or a few stimuli, zinc clearance is mainly mediated by zinc binding to the high-affinity sites on the NMDA receptors and to the low-affinity sites on the highly abundant GLAST glutamate transporters. In the case of higher zinc release brought about by a larger group of stimuli, most zinc binding occurs essentially to the GLAST transporters, having the corresponding zinc complex a maximum concentration that is more than one order of magnitude larger than that for the high and low affinity NMDA sites. The other zinc complexes considered in the model, namely those formed with sites on the AMPA receptors, calcium and KATP channels and with ATP molecules, have much smaller contributions to the synaptic zinc clearance.

Glucose-induced insulin secretion from pancreatic beta cells is modulated by several hormones and transmitters, namely adenosine triphosphate (ATP) via purinergic receptors. Although P2Y receptors are well documented in beta cells, the presence of P2X receptors remains elusive. We present the first electrophysiological evidence for the presence of P2X receptors in single beta cells of different species.Ionic currents were recorded from voltage-clamped beta cells near their resting potential using the perforated (nystatin) whole-cell patch-clamp configuration. Receptors were detected by immunocytochemistry.When bathed in substimulatory (2 mM) glucose, mouse beta cells, isolated from islets displaying immunochemical colocalization of P2X1 or P2X3 receptors and insulin, developed large (approximately 250 pA/pF), rapidly activating, and then biexponentially decaying (tau1, approximately 20 milliseconds/tau2, approximately 1 second) inward currents on exposure to micromolar concentrations of ATP and alpha,beta-methylene ATP. The ATP also evoked inward currents (100-300 pA/pF) from porcine and human beta cells, albeit with a slower and more complex inactivation pattern.The ATP-gated ion channels are present in pancreatic beta cells from different species. Specifically, mouse beta cells express rapidly desensitizing P2X1 and P2X3 receptors. Paracrine or neural activation of these receptors may contribute to the initial outburst of glucose- or acetylcholine-evoked insulin release, thus enhancing the islet secretory response.

Nutrient stimulation of pancreatic β-cells increases the cellular reduced pyridine nucleotide content, but the specific role of cytosolic redox state in glucose-induced insulin release (GIIR) remains undetermined. The role of cytosolic redox state has been assessed (as reflected by the lactate/pyruvate ratio) in nutrient- and non-nutrient-induced insulin release using a recently established glucose-sensitive clonal β-cell line (BRIN-BD11). Long-term exposure to the NAD+ precursor vitamin nicotinic acid (NA, 100 μM) was used to promote a more oxidized state in the cytosol. Glucose (2–16 mM) evoked a dose-dependent rise in the cytosolic NADH/NAD+ ratio which was linearly related to the extent of GIIR. NA suppressed the glucose-induced rise in the NADH/NAD+ ratio and concomitantly reduced GIIR by 44%. It also inhibited, by 47%, the average glucose-induced rise in cytosolic free Ca2+ concentration ([Ca2+]i, assessed by fura-2 microfluorometry from single cells). The latter effect was not accounted for by a reduction in the activity of voltage-sensitive Ca2+ channels, inasmuch as both high K+- and tolbutamide-induced [Ca2+]i rises remained insensitive to NA exposure. NA did not affect insulin release evoked by any of the depolarizing agents, indicating that steps in the stimulus-secretion coupling cascade distal to Ca2+ influx are insensitive to changes in the cytosolic redox state. It is concluded that GIIR is partially controlled by the cytosolic redox state. Moreover, the impairment in GIIR, caused by a shift toward a more oxidized state in the cytosol, originates from an attenuated [Ca2+]i response. The latter is likely mediated by the influence of cytosolic redox state on specific metabolic pathways (NADH shuttle systems and/or the malonyl-CoA pathway), leading ultimately to enhancement of the activity of ATP-sensitive K+ channels.

Phorbol esters reduce depolarization-evoked Ca2+ influx in adrenal chromaffin cells, suggesting that voltage-sensitive Ca2+ channels (VSCCs) are inhibited by protein kinase C-mediated phosphorylation. We now address the possibility that L- and P/Q-type Ca2+ channel subtypes might be differentially involved in phorbol ester action. In bovine chromaffin cells, short-term (10 min) incubations with phorbol 12-myristate 13-acetate (PMA) inhibited early high K+-evoked rises in cytosolic free Ca2+ concentration ([Ca2+]i) and the early component of the depolarization-evoked Mn2+ quenching of fura-2 fluorescence in a dose-dependent manner (IC50: 18 and 7 nM; maximal inhibitions: 45 and 48%, respectively). The protein kinase C inhibitor staurosporine (100 nM) reverted the inhibitory action of PMA. PMA (0.1-1 [mu]M) inhibited the early and late phases of the ionomycin (2 [mu]M)-evoked [Ca2+]i transients by 14-23%. [omega]-Agatoxin IVA, a blocker of P/Q-type Ca2+ channels, inhibited high K+-evoked [Ca2+]i rises in a dose-dependent fashion (IC50=50 nM). In contrast, 0.1 [mu]M [omega]-conotoxin GVIA, a blocker of N-type channels, was without effect. A sizeable (

The bioluminescent reaction catalysed by firefly luciferase has become widely established as an outstanding analytical system for assay of ATP. When used in solution, luciferase is unstable and cannot be re-used, a problem that can be partially circumvented by immobilizing the enzyme on solid substrates. Transparent glass is especially advantageous over alternative immobilizing matrices, since it allows most of the emitted photons to be detected. We report a new method for luciferase immobilization on glass which does not require prior silanization and glutaraldehyde activation, thus saving preparation time and minimizing enzyme inactivation. Our method is based on the co-immobilization by adsorption of luciferase (from a firefly lantern extract) and poly-L-lysine (PL) on non-porous glass strips. Luciferase immobilized in this way exhibits minimal variations in intersample activity, high sensitivity for ATP detection (linear luminescence responses down to 50 nmol/L) and good stability (full activity for at least 60 days when stored at −80°C). PL-mediated immobilization of luciferase on glass strips provides an attractive strategy for the design of specific ATP biosensors, with potential in industry, environmental screening, medicine and biological research. © 1998 John Wiley & Sons, Ltd.

Glucose stimulation raises the pHi of pancreatic beta-cells, but the underlying mechanisms are not well understood. We have now investigated the acute effects of metabolizable (glucose and the mitochondrial substrate alpha-ketoisocaproic acid, KIC) and nonmetabolizable (high K+ and the K-ATP channel blocker tolbutamide) insulin secretagogues on the pHi of pancreatic beta-cells isolated from normal mice, as assessed by BCECF fluorescence from single cells or islets in the presence of external bicarbonate. The typical acute effect of glucose (22-30 mM) on the pHi was a fast alkalinization of approximately 0.11 unit, followed by a slower acidification. The relative expression of the alkalinizing and acidifying components was variable, with some cells and islets displaying a predominant alkalinization, others a predominant acidification, and others yet a mixed combination of the two. The initial alkalinization preceded the [Ca2+]i rise associated with the activation of voltage-sensitive Ca2+ channels. There was a significant overlap between the glucose-evoked [Ca2+]i rise and the development of the secondary acidification. Depolarization with 30 mM K+ and tolbutamide evoked pronounced [Ca2+]i rises and concomitant cytosolic acidifications. Blocking glucose-induced Ca2+ influx (with 0 Ca2+, nifedipine, or the K-ATP channel agonist diazoxide) suppressed the secondary acidification while having variable effects (potentiation or slight attenuation) on the initial alkalinization. KIC exerted glucose-like effects on the pHi and [Ca2+]i, but the amplitude of the initial alkalinization was about twice as large for KIC relative to glucose. It is concluded that the acute effect of glucose on the pHi of pancreatic beta-cells is biphasic. While the initial cytosolic alkalinization is an immediate consequence of the activation of H+-consuming metabolic steps in the mitochondria, the secondary acidification appears to originate from enhanced Ca2+ turnover in the cytoplasm. The degree of coupling between glucose metabolism and Ca2+ influx as well as the relative efficacies of these processes determines whether the acute pHi response of a beta-cell (or of a tightly coupled multicellular system such as an islet of Langerhans) is predominantly an alkalinization, an acidification, or a mixed proportion of the two.

We have assessed the relative contribution of Ca2+ entry and Ca2+ release from internal stores to the [Ca2+]i transients evoked by purinergic receptor activation in bovine adrenal chromaffin cells. The [Ca2+]i was recorded from single cells using ratiometric fura-2 microfluorometry. Two discrete groups of ATP-sensitive cells could be distinguished on the basis of their relative capacity to respond to ATP in the virtual absence of extracellular Ca2+. One group of cells (group I) failed to respond to ATP in the absence of Ca2+, was completely insensitive to UTP, and displayed suramin-blockable [Ca2+]i transients when challenged with ATP in the presence of external Ca2+. ATP activated a prominent and rapidly inactivating Mn2+ influx pathway in group I cells, as assessed by monitoring Mn2+ quenching of fura-2 fluorescence. In contrast, a second group of ATP-sensitive cells (group II) exhibited pronounced [Ca2+]i rises when challenged with ATP and UTP in the absence of Ca2+ and was completely insensitive to suramin. ATP and UTP activated a delayed and less prominent Mn2+ influx pathway in group II cells. Contrary to the nicotinic receptor agonist DMPP, which evoked a preferential release of epinephrine, ATP evoked a preferential release of norepinephrine, and UTP had no effect on secretion. Suramin nearly suppressed ATP-evoked norepinephrine release. We conclude that chromaffin cells contain two distinct and cell-specific purinoceptor subtypes. Although some cells express a P2U-type purinoceptor coupled to Ca2+ release from internal stores and to the associated slow Ca2+ refilling mechanism, other cells express a suramin-sensitive and UTP-insensitive purinoceptor exclusively coupled to Ca2+ influx, probably an ATP-gated channel. It is suggested that the ATP-gated channel is preferentially localized to norepinephrine-secreting chromaffin cells and supports specifically hormone output from these cells. Thus, the biochemical pathways involved in the exocytotic release of the two major stress-related hormones appear to be regulated by distinct signaling systems.

ATP and adenosine(5')tetraphospho(5')adenosine (Ap4A), released from adrenal chromaffin cells, are potent stimulators of endothelial cell function. Using single-cell fura-2 fluorescence recording techniques to measure free cytosolic Ca2+ concentration ([Ca2+]i), we have investigated the role of purinoceptor subtypes in the activation of cocultured chromaffin and endothelial cells. ATP evoked concentration-dependent [Ca2+]i rises (EC50 = 3.8 microM) in a subpopulation of chromaffin cells. Both ATP-sensitive and -insensitive cells were potently activated by nicotine, bradykinin and muscarine. Reducing extracellular free Ca2+ concentration to around 100 nM suppressed the [Ca2+]i transient evoked by ATP but not the [Ca2+]i response to bradykinin. ATP-sensitive chromaffin cells were also potently stimulated by 2-methylthioadenosine triphosphate (2MeSATP; EC50 = 12.5 microM) and UTP, but did not respond to either adenosine 5'-[beta-thio]diphosphate (ADP[beta S]), a P2Y receptor agonist, adenosine 5'-[alpha,beta-methylene]triphosphate (pp-[CH2]pA), a P2X agonist or AMP. Adrenal endothelial cells displayed concentration-dependent [Ca2+]i responses when stimulated with ATP (EC50 = 0.86 microM), UTP (EC50 = 1.6 microM) and 2MeSATP (EC50 = 0.38 microM). 2MeSATP behaved as a partial agonist. Ap4A and ADP[beta S] also raised the [Ca2+]i in endothelial cells, whereas AMP and pp[CH2]pA were ineffective. Lowering extracellular free Ca2+ to around 100 nM did not affect the peak ATP-evoked [Ca2+]i rise in these cells. It is concluded that different purinoceptor subtypes are heterogeneously distributed among the major cell types of the adrenal medulla. An intracellular Ca(2+)-releasing P2U-type purinoceptor is specifically localized to adrenal endothelial cells, while a subpopulation of chromaffin cells expresses a non-P2X, non-P2Y subtype exclusively coupled to Ca2+ influx.

Glucose depolarizes the pancreatic beta-cell and induces membrane potential oscillations, but the nature of the underlying oscillatory conductance remains unknown. We have now investigated the effects of the Ca2+ ionophore ionomycin and high external Ca2+ concentration ([Ca2+]o) on glucose-induced electrical activity and whole islet intracellular free Ca2+ concentration ([Ca2+]i), under conditions where the K(ATP) channel was blocked (100 microM tolbutamide or 4 microM glibenclamide). Raising [Ca2+]o to 10.2 or 12.8 mM, but not to 5.1 or 7.7 mM, turned continuous electrical activity into bursting activity. High [Ca2+]o (12.8 mM) regenerated a pattern of fast [Ca2+]i oscillations overshooting the levels recorded in tolbutamide. Ionomycin (10 microM) raised the [Ca2+]i and synergized with 5.1 mM Ca2+ to hyperpolarize the beta-cell membrane. The data indicate that a [Ca2+]i-sensitive and sulphonylurea-insensitive oscillatory conductance underlies the beta-cell bursting activity.

In fura-2 stained drug-sensitive and multidrug-resistant P388 cells, 50 mM KCl failed to provoke an increase in the fluorescent signal, indicating that potential-dependent Ca2+ channels are not present in either cell line. Therefore the circumvention of drug-resistance by verapamil must be related to some other mechanism. In the present study, verapamil and two other circumventors of drug-resistance, tamoxifen and dipyridamole were found to induce an increase in the synthesis of phosphatidylcholine in multidrug-resistant but not in drug-sensitive cells. The relative resistance of multidrug resistant cells to permeabilization by digitonin indicates that the organization of the plasma membrane lipids in these cells must be different from the one occurring in drug-sensitive cells. Extended exposure of multidrug-resistant cells to verapamil negates the resistance to digitonin. This effect of verapamil reflects its ability to modify the lipid organization of the plasma membrane of multidrug-resistant cells. It is suggested that if the lipid composition of the cell membrane is altered by these drugs as was found for whole cells, the change could explain the increase in drug permeability.

The possible physiological role of cytosolic pH changes in adrenal medullary chromaffin cell secretion was examined by investigating the effects of catecholamine secretagogues on cytosolic pH, which was monitored using the intracellular fluorescent indicator 2′,7′-bis-(2-car☐yethyl)-5(6)-car☐yfluorescein (BCECF). Anti-fluorescein antibodies were used to reduce background fluorescence from extracellular 2′,7′-bis-(2-car☐yethyl)-5(6)-car☐yfluorescein (BCECF). Stimulation with both cholinergic agonists (acetylcholine, nicotine) and a depolarizing agent (high K + ) transiently acidified the cytosol of the chromaffin cell. This acidification was antagonized by reducing extracellular Ca 2+ concentration and by Ca 2+ antagonists (Co 2+ , verapamil), indicating that it occurred secondarily to Ca 2+ influx, possibly as a result of exchange of Ca 2+ ions for protons across organelle membranes. Taken together with previously published data [Kuijpers G.A.J. et al . (1989) J. biol. Chem. 264 , 698–705] showing no effect of cytosolic acidification on nicotine-induced catecholamine secretion, these results indicate that secretagogue-induced cytosolic pH changes do not represent a causal step in stimulus-secretion coupling of the chromaffin cell. The cytosolic pH recovery to pre-stimulatory cytosolic pH levels was delayed by amiloride and by5-(N,N-dimethyl)amiloride, at concentrations that otherwise substantially inhibited cytosolic pH recovery from the rebound acidification induced by a 40-fold sudden dilution of NH 4 Cl. This latter type of recovery results from activation of an Na + /H + exchange mechanism. Therefore, the data suggest that Na + /H + exchange is actively involved in the dissipation of the small acid loads generated by secretagogues.

The glucose sensitivity of bursting electrical activity and pulsatile insulin release from pancreatic islets was determined in absence of functional K(ATP) channels. Membrane potential, [Ca(2+)](i) and 5-HT/insulin release were measured by intracellular recording, fura-2 fluorescence and 5-HT amperometry, respectively. Single mouse islets, bathed in tolbutamide or glibenclamide and high extracellular Ca(2+) (Ca(2+)(o)), displayed bursting activity and concomitant fast [Ca(2+)](i) and 5-HT/insulin oscillations. Sulphonylurea block of K(ATP) channel current was unaffected by raising Ca(2+)(o). Raising glucose or alpha-ketoisocaproic acid (KIC) concentration from 3 to 30 mM increased spiking activity and burst plateau duration. Staurosporine did not impair glucose potentiation of electrical activity, ruling out the involvement of serine/threonine kinases. Glucose enhanced both [Ca(2+)](i) and 5-HT/insulin oscillatory activity, causing a approximately 3-fold increase in overall 5-HT release rate. Cells lacking bursting activity in high Ca(2+)(o) and low glucose (or KIC) developed a pattern of intensified spiking in response to 11 mM glucose. It is concluded that beta-cells exhibit graded oscillatory electrical and secretory responses to glucose in absence of functional K(ATP) channels. This suggests that, under physiological conditions, early glucose sensing may involve other channels besides the K(ATP) channel.

Background 2-Methylthioadenosine 5'-triphosphate (2-MeSATP), formerly regarded as a specific P2Y (metabotropic) purinergic receptor agonist, stimulates Ca2+ influx and evokes catecholamine release from adrenal chromaffin cells. These cells express P2Y and P2X (ionotropic) purinoceptors, with the latter providing an important Ca2+ influx pathway. Using single cell calcium imaging techniques, we have determined whether 2-MeSATP might be a specific P2X receptor agonist in bovine chromaffin cells and assessed the relative role of P2X and P2Y receptors on catecholamine secretion from these cells. Results ATP raised the [Ca2+]i in ~50% of the cells. Removing extracellular Ca2+ suppressed the [Ca2+]i-raising ability of 2-MeSATP, observed in ~40% of the ATP-sensitive cells. This indicates that 2-MeSATP behaves as a specific ionotropic purinoceptor agonist in bovine chromaffin cells. The 2-MeSATP-induced [Ca2+]i-rises were suppressed by PPADS. UTP raised the [Ca2+]i in ~40% of the ATP-sensitive cells, indicating that these expressed Ca2+-mobilizing P2Y receptors. UTP-sensitive receptors may not be the only P2Y receptors present, as suggested by the observation that ~20% of the ATP-sensitive pool did not respond to either 2-MeSATP or UTP. The average sizes of the ATP- and 2-MeSATP-evoked [Ca2+]i responses were identical in UTP-insensitive cells. 2-MeSATP stimulated Ca2+ influx and evoked catecholamine release, whereas UTP elicited Ca2+ release from intracellular stores but did not evoke secretion. 2-MeSATP-induced secretion was strongly inhibited by Cd2+ and suppressed by extracellular Ca2+ or Na+ removal. TTX inhibited 2-MeSATP-evoked secretion by ~20%. Conclusion 2-MeSATP is a specific P2X purinoceptor agonist and a potent secretagogue in bovine chromaffin cells. Activation of 2-MeSATP-sensitive receptors stimulates Ca2+ influx mainly via voltage-sensitive Ca2+ channels. For the most part, these are activated by the depolarization brought about by Na+ influx across P2X receptor pores.

Thymeleatoxin (TMX), an activator of Ca2+-sensitive protein kinase C (cPKC) isoforms, was used to assessthe PKC isoform specificity of cholinergic potentiation of glucose (11 mM)-induced pulsatile 5-HT/insulinrelease (PIR) from single mouse pancreatic islets. TMX (100 nM) and carbachol (Cch, 50 μM) enhanced PIR~ 3-fold while reducing the underlying [Ca2+]i oscillations (duration and amplitude) by ~ 40-50%. Botheffects were ablated by the specific PKC inhibitor bisindolylmaleimide and chronic TMX pretreatment. Cchalso evoked an initial transient [Ca2+]i rise and surge of 5-HT release, which remained unaffected by chronicTMX pretreatment. It is concluded that the immediate cholinergic responses are insensitive to cPKC. Incontrast, specific activation of a cPKC isoform mediates sustained cholinergic potentiation of glucose-inducedinsulin secretion. Key terms: Cytosolic free Ca 2+ concentration, 5-HT amperometry, islet of Langerhans, protein kinase C,pulsatile insulin release, thymeleatoxin.

Selective protein kinase C (PKC) activators and inhibitors were used to investigate the involvement of specific PKC isoforms in the modulation of voltage-sensitive Ca2+ channels (VSCCs) in bovine adrenal chromaffin cells. Exposure to the phorbol ester phorbol-12,13-dibutyrate (PDBu) inhibited the Ca2+ currents elicited by depolarizing voltage steps. This inhibition was occluded by the PKC-specific inhibitor Ro 31-8220 but remained unaffected by Gö 6976, a selective inhibitor of conventional PKC isoforms. PDBu treatment caused the translocation of PKC-[alpha] and -[epsilon] isoforms from cytosol to membranes. PKC-[iota] and -[zeta] showed no signs of translocation. It is concluded that VSCCs are specifically inhibited by the activation of PKC-[epsilon] in chromaffin cells. This may be relevant to the action of phospholipase-linked receptors involved in the control of Ca2+ influx, both in catecholaminergic cells and other cell types. http://www.sciencedirect.com/science/article/B6T36-42HFN5W-10/1/3b81ed3b87105497a8f267581ebe6acf

Nicotine-induced catecholamine (CA) secretion and inward ionic currents were inhibited by the opioid antagonist naloxone in cultured bovine chromaffin cells. Naloxone inhibited nicotine-induced CA secretion, as detected by an on-line real-time electrochemical technique, in a dose-dependent manner (IC50=29 [mu]M). In voltage-clamped chromaffin cells, nicotine (10 [mu]M) evoked an average peak inward current of -146 pA that was inhibited by low concentrations of naloxone (42% at 0.1 [mu]M). The antagonist also inhibited total charge influx associated with nicotinic receptor activation (53% at 0.1 [mu]M). This provides strong evidence that naloxone modulation of nicotine-induced CA secretion does not involve opioid receptors but results from the direct interaction with the nicotinic receptor itself, which might also be the case for other related opioid compounds. http://www.sciencedirect.com/science/article/B6SYR-43MC9YT-C/1/1994796a9d2862757ab52186bcacc5f6

Although isolated rat islets are widely used to study in vitro insulin secretion and the underlying metabolic and ionic processes, knowledge on the properties of glucose-induced electrical activity (GIEA), a key step in glucose-response coupling, has been gathered almost exclusively from microdissected mouse islets. Using a modified intracellular recording technique, we have now compared the patterns of GIEA in collagenase-isolated rat and mouse islets. Resting membrane potentials of rat and mouse beta-cells were approximately -50 and -60 mV, respectively. Both rat and mouse beta-cells displayed prompt membrane depolarizations in response to glucose. However, whereas the latter exhibited a bursting pattern consisting of alternating hyperpolarized and depolarized active phases, rat beta-cells fired action potentials from a nonoscillating membrane potential at all glucose concentrations (8.4-22.0 mmol/l). This was mirrored by changes in the intracellular Ca2+ concentration ([Ca2+]i), which was oscillatory in mouse and nonoscillatory in rat islets. Stimulated rat beta-cells were strongly hyperpolarized by diazoxide, an activator of ATP-dependent K+ channels. Glucose evoked dose-dependent depolarizations and [Ca2+]i increases in both rat (EC50 5.9-6.9 mmol/l) and mouse islets (EC50 8.3-9.5 mmol/l), although it did not affect the burst plateau potential in the latter case. We conclude that there are important differences between beta-cells from both species with respect to early steps in the stimulus-secretion coupling cascade based on the following findings: 1) mouse beta-cells have a larger resting K+ conductance in 2 mmol/l glucose, 2) rat beta-cells lack the compensatory mechanism responsible for generating membrane potential oscillations and holding the depolarized plateau potential in mouse beta-cells, and 3) the electrical and [Ca2+]i dose-response curves in rat beta-cells are shifted toward lower glucose concentrations. Exploring the molecular basis of these differences may clarify several a priori assumptions on the electrophysiological properties of rat beta-cells, which could foster the development of new working models of pancreatic beta-cell function.

Using clonal insulin-secreting BRIN-BD11 cells, we have assessed whether the graded response of the whole cell population to glucose can be accounted for by a dose-dependent recruitment of individual cells, an amplification of the response of the recruited cells or both. Cytosolic free Ca2+ concentration ([Ca2+]i) is an established index of [beta]-cell function. We used fura-2 microfluorescence techniques to assess the [Ca2+]i responsiveness of single BRIN-BD11 cells to glucose and other secretagogues. Glucose (1-16.7Â mM) evoked oscillatory [Ca2+]i rises in these cells resembling those found in parental rat pancreatic [beta]-cells. The percentage of glucose-responsive cells was 11% at 1Â mM and increased to 40-70% at 3-16.7Â mM glucose, as assessed by a single-stimulation protocol. This profile was unrelated to possible differences in the cell cycle, as inferred from experiments where the cultured cells were synchronized by a double thymidine block protocol. Individual cells exhibited variable sensitivities to glucose (threshold range: 1-5Â mM) and a variable dose-dependent amplification of the [Ca2+]i responses (EC50 range: 2-10Â mM), as assessed by a multiple-stimulation protocol. Glyceraldehyde and [alpha]-ketoisocaproic acid had glucose-like effects on [Ca2+]i. The data support a mixed model for the activation of insulin-secreting cells. Specifically, the graded secretory response of the whole cell population is likely to reflect both a recruitment of individual cells with different sensitivities to glucose and a dose-dependent amplification of the response of the recruited cells. http://www.sciencedirect.com/science/article/B6TCH-3YW26B7-B/1/53849b978f1051f5cac6464bd7012ee8

http://www.sciencedirect.com/science/article/B6T5Y-49PK0KM-M0/1/4edf2ccfe81c25b6f0ac732004095cd1

1. Glucose-induced insulin release from single islets of Langerhans is pulsatile. We have investigated the correlation between changes in cytosolic free calcium concentration ([Ca2+]i) and oscillatory insulin secretion from single mouse islets, in particular examining the basis for differences in secretory responses to intermediate and high glucose concentrations. Insulin release was monitored in real time through the amperometric detection of the surrogate insulin marker 5-hydroxytryptamine (5-HT) via carbon fibre microelectrodes. The [Ca2+]i was simultaneously recorded by whole-islet fura-2 microfluorometry. 2. In 82 % of the experiments, exposure to 11 mM glucose evoked regular high-frequency (average, 3.4 min-1) synchronous oscillations in amperometric current and [Ca2+]i. In the remaining experiments (18 %), 11 mM glucose induced an oscillatory pattern consisting of high-frequency [Ca2+]i oscillations that were superimposed on low-frequency (average, 0.32 min-1) [Ca2+]i waves. Intermittent high-frequency [Ca2+]i oscillations gave rise to a similar pattern of pulsatile 5-HT release. 3. Raising the glucose concentration from 11 to 20 mM increased the duration of the steady-state [Ca2+]i oscillations without increasing their amplitude. In contrast, both the duration and amplitude of the associated 5-HT transients were increased by glucose stimulation. The amount of 5-HT released per secretion cycle was linearly related to the duration of the underlying [Ca2+]i oscillations in both 11 and 20 mM glucose. The slopes of the straight lines were identical, indicating that there is no significant difference between the ability of calcium oscillations to elicit 5-HT/insulin release in 11 and 20 mM glucose. 4. In situ 5-HT microamperometry has the potential to resolve the high-frequency oscillatory component of the second phase of glucose-induced insulin secretion. This component appears to reflect primarily the duration of the underlying [Ca2+]i oscillations, suggesting that glucose metabolism and/or access to glucose metabolites is not rate limiting to fast pulsatile insulin release.

Previous work has demonstrated that nitric oxide can be an important intracellular messenger in the regulation of neurosecretion in chromaffin cells. Since standard chromaffin cell cultures are mixed populations of noradrenaline and adrenaline producing cells, it would seem important to understand the functional differences between these individual components. The use of fluorescence imaging techniques for the recording of cytosolic calcium from single chromaffin cells together with the immunoidentification of individual cells with specific antibodies against tyrosine hydroxylase, N-phenyl ethanolamine methyl transferase and nitric oxide synthase, has allowed us to measure single-cell calcium responses in identified adrenergic, noradrenergic and nitrergic chromaffin cells, thus helping us to clarify the differential role of nitric oxide in the function of these chromaffin cell types. 53 +/- 2% of chromaffin cells were able to synthesize nitric oxide (nitric oxidesynthase-positive cells), these cells being mainly noradrenergic (82 +/-2%). Results indicate that nitric oxide donors such as sodium nitroprusside, molsidomine and isosorbide dinitrate evoke [Ca2+]i increases in a 62 +/- 4% of chromaffin cells, the response to nitric oxide donors being between 30 and 50% of that of 20 microM nicotine. Cells responding to nitric oxide donors were mainly adrenergic (68 +/- 5%) although 45 +/- 9% of noradrenergic cells also gave [Ca2+]i increasing responses. The distribution of nitric oxide responding cells between nitric oxide synthase-positive and negative was very similar in the whole population (63 +/- 5 and 60 +/- 7%, respectively), but these differences were more prominent when considering the distribution of nitric oxide response between noradrenergic and adrenergic nitric oxide synthase-positive cells; while 73 6% of adrenergic nitric oxide synthase-positive cells evoke [Ca2+]i increases by nitric oxide stimulation, only 35 +/- 11% of noradrenergic nitric oxide synthase-positive cells respond. Taken together these results seem to indicate that (i) nitric oxide could act within adrenal medulla as both an intracellular and intercellular messenger; and (ii) noradrenergic cells seem to be specialized in nitric oxide synthesis while adrenergic cells with an endocrine function could mainly act as a target of neurosecretory action of this second messenger.

The use of the patch-clamp technique has made possible the characterization of the glucose sensitivity of a number of cation channels present in the β-cell membrane. One of these channels, the ATP-dependent K+ (KATP) channel, is specifically blocked by intracellular ATP and appears to play an essential role in the coupling of glucose metabolism to membrane depolarization and, hence, to Ca2+ influx and to the initiation of insulin release[1]. The available data indicate that the activity of the KATP channel is almost fully suppressed at 6 mM glucose, ie. a concentration which brings the membrane potential to a level just below the threshold for the appearance of electrical activity. At glucose concentrations above 7 mM the β-cell displays a pattern of bursting electrical activity which is remarkably sensitive to the hexose concentration, with the silent unexcited period (“silent phase”) being progressively reduced and the depolarized active phase (“active phase”) becoming progressively elongated as the size of the glucose stimulus increases[2]. We conclude that, while the glucose-mediated reduction in the activity of the KATP channels is indeed essential for the glucose-evoked initial depolarization of the β-cell, the modulation of bursting electrical activity by the hexose in the range 7–20 mM is apparently accounted for, at least in part, by a channel distinct from the KATP channel. The present account aims at providing evidence to support this hypothesis.

The electrochemical determination of zinc arising from zinc-insulin complexes was investigated and it was demonstrated that zinc in zinc-insulin solution can be measured in the presence of dissolved oxygen by square-wave anodic stripping voltammetry (SWASV) at mercury thin-film electrodes on glassy carbon disc minielectrode and cylindrical carbon fibre microelectrode substrates. Reoxidation signals arise from complexed zinc at low insulin concentrations (100 nmol l-1) and from labile zinc at higher concentrations; the latter can be quantified through linear calibration curves. Batch injection analysis with SWASV was successfully tested for the determination of zinc in zinc-insulin solutions in small sample volumes. Since intracellularly stored insulin exists in the form of a zinc-insulin complex, these techniques are very promising for the indirect study of insulin release from pancreatic beta-cells.

We report a highly sensitive electrochemical approach suitable for the real time measurement of insulin release from single islets of Langerhans, the functional endocrine units in the pancreas. The method is based on the detection of the insulin surrogate 5-hydroxytryptamine (5-HT) by carbon fibre microelectrodes implanted in the islets. Based on the combination of this novel approach with the simultaneous microfluorometric recording of cytosolic free Ca2+ concentration ([Ca2+]i), we demonstrate that glucose-stimulated islets secrete 5-HT/insulin in a pulsatile fashion under physiological conditions, and that this activity is encoded by synchronous [Ca2+]i oscillations. The sensitivity to detect variations in minute amounts of secreted materials is partially conferred by the fact that the tracer is released into a relatively confined space (the intraislet interstitial space).

In this report we investigate the isoforms of protein kinase C (PKC) present in cultured adrenal chromaffin cells with respect to their modulation by treatment with phorbol ester and their possible differential involvement in the regulation of responses to histamine and bradykinin. The presence of individual isoforms of PKC was investigated by using eight isoform specific antisera, as a result of which PKC-alpha, epsilon, and zeta were identified. To characterize down-regulation of these enzymes, cells were incubated for 6-48 h with 1 microM phorbol myristate acetate (PMA). PKC-epsilon down-regulated more rapidly than PKC-alpha. At 12 h, PMA pretreatment, for example, PKC-epsilon was maximally down-regulated (23 +/- 4% of controls), whereas PKC-alpha was unchanged. PKC-alpha showed partial down-regulation by 24 h of PMA pretreatment. PKC-zeta did not down-regulate at any of the times tested. Translocation from cytosol to membrane in response to PMA was also more rapid for PKC-epsilon than for PKC-alpha. The accumulation of total 3H-inositol (poly) phosphates in response to bradykinin or histamine was essentially abolished by prior treatment with 10-min PMA treatment (1 microM). However, with 12-h exposure to PMA, the bradykinin response was restored to the level seen with no prior PMA exposure. The histamine response showed no recovery by 12 h of PMA, but showed partial recovery by 24 h of PMA pretreatment. These observations showed that the restoration of the response to bradykinin corresponds to the loss of PKC-epsilon, whereas the restoration of the histamine response corresponds to the loss of PKC-alpha. This picture was confirmed with further studies on cytosolic Ca2+. The results show that chromaffin cells exhibit an unusual pattern of down-regulation of PKC isoforms on prolonged exposure to PMA, and that there is a differential effect of exposure to PMA on the histamine and bradykinin responses, suggesting that different PLC-linked receptors in chromafin cells are differentially regulated by PKC isoforms.

A stepwise increase in extracellular Ca2+ concentration ([Ca2+]o) can evoke insulin release from pancreatic islets in the absence of secretagogues. We have investigated the ionic mechanism underlying this secretory response by recording intracellular free Ca2+ concentration ([Ca2+]i) from single mouse islets of Langerhans using ratiometric fura-2 microfluorometry. In the presence of 11 mM glucose, the [Ca2+]i undergoes fast oscillations associated with bursting electrical activity. Nifedipine (10 microM) suppressed these oscillations and markedly lowered the [Ca2+]i. Raising the [Ca2+]o from 2.56 to 12.8 mM in the continued presence of 11 mM glucose and nifedipine evoked pronounced [Ca2+]i rises of variable amplitude and time course. This effect was dose-dependent (EC50 = 3.6 mM) and remained essentially unchanged in the absence of glucose or in the presence of 3 mM glucose and nifedipine, conditions where beta-cells are hyperpolarized by approximately -25 mV. Depleting the acetylcholine-mobilizable internal Ca2+ pools by repetitively challenging the islets with acetylcholine in the absence of Ca2+ actually potentiated the standard high Ca2+ responses. The latter were strongly reduced by millimolar concentrations of Ni2+ (70% reduction at 3 mM) and by diphenylamine-2-carboxylate (DPC; IC50 = 145 microM), a blocker of nonselective cation channels. The standard high Ca2+ responses were relatively insensitive to the glycolytic inhibitor mannoheptulose. It is proposed that the high Ca(2+)-evoked [Ca2+]i responses are primarily accounted for by Ca2+ influx through dihydropyridine- and voltage-insensitive, nonselective cation channels. These channels do not appear to be under the control of glucose metabolism. Although their function is unknown, they may be essential to supplying the beta-cells with Ca2+ in the absence of stimulatory levels of fuel secretagogues.

In glucose-stimulated pancreatic beta-cells, the membrane potential alternates between a hyperpolarized silent phase and a depolarized phase with Ca2+ action potentials. The molecular and ionic mechanisms underlying these bursts of electrical activity remain unknown. We have observed that 10.2-12.8 mM Ca2+, 1 microM Bay K 8644 and 2 mM tetraethylammonium (TEA) trigger bursts of electrical activity and oscillations of intracellular free Ca2+ concentration ([Ca2+]i) in the presence of 100 microM tolbutamide. The [Ca2+]i was monitored from single islets of Langerhans using fura-2 microfluorescence techniques. Both the high-Ca(2+)- and Bay-K-8644-evoked [Ca2+]i oscillations overshot the [Ca2+]i recorded in tolbutamide. Nifedipine (10-20 microM) caused an immediate membrane hyperpolarization, which was followed by a slow depolarization to a level close to the burst active phase potential. The latter depolarization was accompanied by suppression of spiking activity. Exposure to high Ca2+ in the presence of nifedipine caused a steady depolarization of approximately 8 mV. Ionomycin (10 microM) caused membrane hyperpolarization in the presence of 7.7 mM Ca2+, which was not abolished by nifedipine. Charybdotoxin (CTX, 40-80 nM), TEA (2 mM) and quinine (200 microM) did not suppress the high-Ca(2+)-evoked bursts. It is concluded that: (1) the channel underlying the burst is sensitive to [Ca2+]i rises mediated by Ca2+ influx through L-type Ca2+ channels, (2) both the ATP-dependent K+ channel and the CTX- and TEA-sensitive Ca(2+)-dependent K+ channel are highly unlikely to provide the pacemaker current underlying the burst.(ABSTRACT TRUNCATED AT 250 WORDS)

There is evidence that bovine adrenal chromaffin cells are provided with both dihydropyridine-sensitive and -resistant voltage-sensitive Ca 2+ influx pathways. Although recent electrophysiological work indicates that the dihydropyridine-resistant pathway is partially mediated by w -conotoxin-sensitive and -insensitive Ca 2+ channels, the pharmacological sensitivity of the latter channels remains elusive. We have now found that combined incubations with nitrendipine (1 μM) and neomycin (0.5 mM) reduced high K + (50 mM)-evoked intracellular Ca 2+ concentration ([Ca 2+ ] i ) transients to a larger extent than each drug separately. [Ca 2+ ] i was measured using the fluorescent intracellular Ca 2+ indicator fura-2. Neomycin (0.05−2 mM) reduced high K + -evoked 45 Ca 2+ uptake in a dose-dependent manner (IC 50 = 0.09 mM). In the presence of nitrendipine (1 μM), the minimal neomycin concentration necessary for total blockade of 45 Ca 2+ uptake was reduced to 0.3 mM. Moreover, in the absence of nitrendipine the 45 Ca 2+ uptake remaining in 0.3 mM neomycin (26% of maximum) was similar to the fractional inhibition by nitrendipine alone (29%). Neomycin (0.05−2 mM) inhibited the [Ca 2+ ] i transient induced by the L-type Ca 2+ channel agonist Bay K 8644 (1 μM) much more extensively at 2 mM than at 0.3 mM (percent inhibition = 59% and 15%, respectively). Neomycin (0.05−2 mM) blocked high K + -evoked noradrenaline and adrenaline release in a dose-dependent fashion (IC 50 = 0.8−1.1 mM), the blockade efficiency being enhanced in the presence of 1 μM nitrendipine (IC 50 = 0.17−0.19 mM). It is concluded that neomycin (≤ 0.3 mM) blocks preferentially the dihydropyridine-insensitive Ca 2+ influx pathway of the chromaffin cell. Moreover, both the dihydropyridine-sensitive and the dihydropyridine-resistant, neomycin-sensitive Ca 2+ influx pathways contribute strongly to depolarization-evoked catecholamine secretion.

In adrenal chromaffin cells, depolarization-evoked Ca2+ influx and catecholamine release are partially blocked by blockers of L-type voltage-sensitive Ca2+ channels. We have now evaluated the sensitivity of the dihydropyridine-resistant components of Ca2+ influx and catecholamine release to a toxin fraction (FTX) from the funnel-web spider poison, which is known to block P-type channels in mammalian neurons. FTX (1:4,000 dilution, with respect to the original fraction) inhibited K(+)-depolarization-induced Ca2+ influx by 50%, as monitored with fura-2, whereas nitrendipine (0.1-1 microM) and FTX (3:3), a synthetic FTX analogue (1 mM), blocked the [Ca2+]i transients by 35 and 30%, respectively. When tested together, FTX and nitrendipine reduced the [Ca2+]i transients by 70%. FTX or nitrendipine reduced adrenaline and noradrenaline release by approximately 80 and 70%, respectively, but both substances together abolished the K(+)-evoked catecholamine release, as measured by HPLC. The omega-conotoxin GVIA (0.5 microM) was without effect on K(+)-stimulated 45Ca2+ uptake. Our results indicate that FTX blocks dihydropyridine- and omega-conotoxin-insensitive Ca2+ channels that, together with L-type voltage-sensitive Ca2+ channels, are coupled to catecholamine release.

Pancreatic beta cells, tightly organized in the islet of Langerhans, secrete insulin in response to glucose in a calcium-dependent manner. The calcium input required for this secretory activity is thought to be provided by an oscillatory electrical activity occurring in the form of "bursts" of calcium action potentials. The previous observation that islet intracellular free Ca2+ levels undergo spontaneous oscillations in the presence of glucose, together with the fact that islet cells are coupled through gap junctions, hinted at a highly effective co-ordination between individual islet cells. Through the use of simultaneous recordings of intracellular calcium and membrane potential it is now reported that the islet calcium waves are synchronized with the beta cell bursting electrical activity. This observation suggests that each calcium wave is due to Ca2+ entering the cells during a depolarized phase of electrical activity. Moreover, fura-2 fluorescence image analysis indicates that calcium oscillations occur synchronously across the whole islet tissue. The maximal phase shift between oscillations occurring in different islet cells is estimated as 2 s. This highly co-ordinated oscillatory calcium signalling system may underlie pulsatile insulin secretion and the islet behaviour as a secretory "syncytium". Since increasing glucose concentration lengthens calcium wave and burst duration without significantly affecting wave amplitude, we further propose that it is the fractional time at an enhanced Ca2+ level, rather than its amplitude, that encodes for the primary response of insulin-secreting cells to fuel secretagogues.

The role of intracellular pH in stimulus-secretion coupling was investigated in cultured bovine adrenal medullary chromaffin cells. NH4Cl (1-25 mM) did not affect basal catecholamine or ATP release but markedly inhibited nicotine- or high K+-induced release by up to 60%. The inhibition had a rapid onset (less than 1 min) and was maximal at about 5 mM NH4Cl. The effect of NH4Cl was largely sustained over 20 min and was reversed upon NH4Cl removal. Sodium propionate did not affect secretion but partially reversed the inhibition by NH4Cl in a concentration-dependent manner. Methylamine (10 mM) produced a similar, but slower, inhibition than NH4Cl. Monensin (1-10 microM) inhibited catecholamine secretion by 30-60%, and its effect was reduced in the presence of NH4Cl. Using the fluorescent Ca2+ probe Fura-2, we found that the increase of [Ca2+]i following stimulation was not altered by concentrations of NH4Cl which inhibited secretion maximally. Measurement of cytosolic pH (pHi) with the fluorescent probe 2',7'-bis-carboxyethyl-5(6)-carboxyfluorescein (BCECF) revealed an alkalinization by NH4Cl (2.5-25 mM) of 0.1-0.23 pH units and acidification by sodium propionate (10-20 mM) of 0.2-0.25 pH units, with intermediate combined effects. Monensin (1 microM) caused a cytosolic acidification of 0.26 pH units. All pHi changes were partly recovered in 15 min. Fluorescence quenching measurements using the weakly basic fluorescent probe acridine orange indicated the accumulation of the probe into acidic compartments, presumably the chromaffin granules, which was strongly reduced by both NH4Cl and monensin. From these findings we conclude that the pH of the chromaffin granule modulates secretion by affecting some step in the secretory process unrelated to the rise in [Ca2+]i.

The existence of [Ca2+]i-activated K+-channels in the pancreatic beta-cell membrane is based in two observations: quinine inhibits K+-permeability and, increasing intracellular Ca2+ stimulates it. The changes in K+-permeability of the beta-cell have been monitored electrically by combining measurements of the dependence of the membrane potential on external K+ concentration and input resistance. The changes in the passive 42K and 86Rb efflux from the whole islet have been measured directly. Intracellular Ca2+ has been increased by various means, including increasing extracellular Ca2+, addition of the Ca2+-ionophore A23187 or noradrenaline and application of mitochondrial uncouplers and blockers. In addition to quinine, many other substances have been found to inhibit or modulate the [Ca2+]i-activated K+-channel. The most important of these is the natural stimulus for insulin secretion, glucose. Glucose may inhibit K+-permeability by lowering intracellular Ca2+. Glibenclamide, a hypoglycaemic sulphonylurea, is about 25 times more active than quinine in blocking the K+-channel in beta-cells. The methylxanthines, c-AMP, various calmodulin inhibitors and Ba2+ also inhibit K+-permeability. Genetically diabetic mice have been studied and show an alteration in the [Ca2+]i-activated K+-channel. It is concluded that the [Ca2+]i-activated K+-channel plays a major role in the normal function of the pancreatic beta-cell. The study of its properties should prove valuable for the understanding and treatment of diabetes.

High-resistance microelectrodes were used to measure membrane potential changes in response to increased extracellular K+ concentration ([K+]o; or a test cation X+ such as Li+, Rb+, Cs+, NH+4) in B cells from mouse islets of Langerhans. In the absence of glucose, a sudden increase in [K+]o (or [X+]o), keeping the sum [Na+]o + [K+]o constant (or [Na+]o + [K+]o + [X+]o), induced a rapid depolarization of the membrane. The membrane potential changes were essentially unchanged in the presence of 20 mM tetraethylammonium (TEA). The Goldman-Hodgkin-Katz equation was fitted to the experimental relationship between membrane potential and [K+]o (or [X+]o), and permeability (P) ratios were estimated. In the absence of TEA, P Na/PK was estimated to be approximately 0.046. In the presence of TEA the following ratios were estimated: P Rb/PK = 0.74, P Cs/PK = 0.62, and P NH4/PK = 0.36. From these ratios the following sequence of permeabilities was obtained, PK greater than P Rb greater than P Cs greater than P NH4 greater than P Na. It is proposed that this sequence reflects the selectivity of the intracellular [Ca2+]-activated K+ channel of the pancreatic B cell.

Intracellular Ca2+ levels were monitored in single, acutely isolated mouse islets of Langerhans by dual emission Indo-1 fluorometry. High-frequency (3.1 min−1) [Ca2+]i, oscillations with a brief rising time (1–2 s) and 10 s half-width (‘fast’ oscillations) were detected in 11 mM glucose. Raising the glucose concentration to 16.7 mM increased the duration of these oscillations, which were otherwise absent in 5.5 mM glucose. [Ca2+]i waves of lower frequency (0.5 min−1) and longer rising time (‘slow’ oscillations) were also recorded. The data indicate that “fast” oscillations are directly related to β-cell bursting electrical activity, and suggest the existence of extensive networks of electrically coupled cells in the islet.

The study of the influence of intracellular pH (pHi) changes on the mechanism underlying pancreatic beta-cell bursting has been hampered by concomitant effects on the activity of background ATP-dependent K+ (K-ATP) channels. beta-cells were made to burst in the absence of active K-ATP channels by raising external Ca2+ in the presence of 11 mM glucose and tolbutamide. An alkalinizing pHi shift (exposure to 20 mM NH4Cl) increased the burst active phase duration. Conversely, an acidifying shift (NH4Cl withdrawal) suppressed the electrical activity. This is the mirror image of the effects recorded in the absence of tolbutamide. Glibenclamide and quinine suppressed the alkalinization-evoked hyperpolarization. This study emphasizes the differential sensitivity of different beta-cell ion channels to pHi and the prevalent role of K-ATP channels as electrical transducers of cytoplasmic pH changes under regular physiological conditions.

We have investigated the effects of the phorbol ester 12-myristate 13-acetate (PMA) on depolarization-evoked Ca2+ influx and catecholamine secretion in bovine adrenal chromaffin cells. PMA (100 nM) strongly inhibited K(+)-evoked [Ca2+]i transients and Mn2+ quenching of fura-2 fluorescence. In contrast, 4 alpha-phorbol 12,13-didecanoate, a phorbol ester inactive on protein kinase C (PKC), had no effect. Maximal PMA-mediated inhibition occurred at 5-10 min incubations and were variable from cell to cell, ranging from 25 to 65% of controls. The [Ca2+]i transients evoked by the L-type Ca2+ channel activator Bay K 8644 were strongly inhibited by 100 nM PMA. PMA (0.1-10 microM) inhibited K(+)-evoked adrenaline and noradrenaline release by 23-44%. The data indicate that phorbol ester-mediated activation of PKC inhibits voltage-sensitive Ca2+ channels in chromaffin cells, leading to a prominent depression of depolarization-evoked catecholamine secretion.

The fluorescent Ca2+ indicator FURA-2 was used to characterize the depolarization-related intracellular Ca2+ signalling process in bovine adrenal chromaffin cells. Depolarization with high K+ (10-65 mM) gave rise to a very rapid increase in intracellular free Ca2+ concentration, which subsequently decayed slowly towards a "plateau". The size of this initial increase varied sigmoidally with the calculated membrane potential, the relationship being described well by a Boltzmann distribution function for a transition between two states (transition potential, -23 mV). A dihydropyridine calcium channel agonist [(+)202-791, 1 microM] raised intracellular free Ca2+ concentration further in the presence of 30 mM K+, and it enhanced the initial intracellular Ca2+ response to depolarization. Voltage-sensitive calcium channels in chromaffin cells are believed to include the L-type. Several dihydropyridine calcium channel antagonists [(-)202-791, nifedipine, nitrendipine; 1-5 microM], known to be active on L-type channels, caused only modest inhibition of K+ -induced increase in intracellular free Ca2+ concentration: c. 50% (at 30 mM K+) and 25% (at 40-70 mM K+). In addition, omega-conotoxin GVIA (1-10 microM), a blocker of neuronal N- and L-type calcium channels, reduced the initial increase in intracellular free Ca2+ concentration only slightly at 55 mM K+. Further, the dihydropyridine-insensitive component of the intracellular Ca2+ signal was also insensitive to omega-conotoxin, which was otherwise quite active in a central nervous rat in vivo preparation Gd3+ (40 microM), a potent calcium antagonist in the chromaffin cell, blocked the intracellular Ca2+ response to depolarization. When added at different times after K+ stimulation, however, Gd3+ reduced intracellular free Ca2+ concentration to control levels along a slow time course of several minutes. Similar results were obtained when EGTA was added to reduce extracellular Ca2+ concentration to sub-nanomolar levels, in the presence of high K+. We conclude that bovine chromaffin cells are equipped with at least two different classes of voltage-dependent calcium channels, only one of which is likely to be the L-type channel. We also propose that depolarization, in addition to stimulating Ca2+ influx, may also lead to enhancement of Ca2+ release from an intracellular store.

Multi-drug resistance (MDR) in cancer cells is associated with reduced drug accumulation. Although intensively studied, the mechanism of this process remains ill-defined. We have now developed a new, rapid and quantitative method of measuring uptake of doxorubicin by these cells, in which the fluorescence of accumulated drug is rapidly quenched by DNA in the cell nucleus. Pre-treatment of cells with deoxyribonuclease eliminates DNA from non-viable, permeable cells, and this obviates the spurious fluorescence quenching that made previous application of this technique useless. Our data strongly suggest that the drug passively diffuses into cells. The rate of this diffusion into drug-resistant cells is considerably lower than that found in drug-sensitive cells. The ratio of the rates of drug entry in these cell types could fully account for the differences between the cell lines in doxorubicin growth-inhibitory activity. In these experiments no evidence for the previously proposed active efflux mechanism was found in either cell line.

Since Ca-action potentials were first measured in β-cells, it has been assumed that the resultant Ca2+ influx provides a critical Ca2+-dependent step in the events leading to insulin secretion.4,6. The correlation between electrical activity and insulin release remained indirect, however, since membrane potentials were recorded from single β-cells within an isolated islet and insulin release was measured from batches of collagenase isolated islets (usually more than 100) or from the perfused pancreas (containing over 1000 islets).

Several mouse mutants exhibiting obesity or diabetes-like syndromes (e.g. yellow obese, obese, diabetes, KK, New Zealand obese) have been used as animal models of diabetes mellitus11,12,28. They all have hyperglycemia, hyperinsulinemia, and obesity as common metabolic characteristics28. Among these mouse mutants, particular attention has been paid to the homozygous diabetes mouse carrying the diabetes gene (commonly known as the db/db mouse), and the homozygous obese mouse carrying the obese gene (the ob/ob mouse). Besides the metabolic disorders and the insulin receptor defects characteristic of the diabetes-like syndromes, little is known about the possible malfunction of the pancreatic β-cell. In this context, the earlier membrane potential measurements carried out by Meissner and Schmidt in pancreatic β-cells from C57BL/KsJ db/db-mouse islets33 assume a special significance. These authors have shown that the db/db β-cells exhibit a pattern of bursting or continuous electrical activity which is virtually insensitive to the concentration of the natural secretagogue D-glucose, thus suggesting an impairment of the db/db β-cell K+-permeability9. In pancreatic β-cells from normal mouse islets, K+-permeability is now known to play a key role in both the generation and regulation of the glucose-induced bursts of electrical activity3–5,7.

The effect of methylamine on electrical activity and simultaneously measured insulin release was investigated in single perifused islets of normal mice. Methylamine, (2 mmol/L or 6 mmol/L) failed to affect beta-cell input resistance and only caused a modest and transient inhibition of electrical activity of islets exposed to 11.1 mmol/L glucose. Methylamine (2 mmol/L) inhibited insulin release evoked by a five-minute rise in glucose concentration from 5.6 to 22.2 mmol/L, even when the glucose-induced electrical activity remained unaltered. Methylamine, at 2 or 5 mmol/L, partially inhibited insulin release but failed to affect the continuous electrical activity in islets exposed throughout to 22.2 mmol/L glucose. At 10 mmol/L, methylamine reduced both insulin release and electrical activity. These data reinforce the idea that the glucose-induced changes in beta-cell membrane potential represent an early event in the process of stimulus-secretion coupling and can be dissociated from the subsequent process of insulin release.

The effects of apamin and quinine on glucose-induced electrical activity in pancreatic islets from ob/ob mice (Norwich colony) were compared. Apamin (40–400 nM) increased the duration of the bursts of electrical activity, whereas quinine (50–100 μM) affected only slightly the steady-state electrical response to glucose. This sensitivity to apamin and poor response to quinine contrast with the resistance to apamin and sensitivity to quinine previously reported for pancreatic islets from albino mice. The results give further support to the idea that pancreatic β-cells from ob/ob mice have a modified Ca2+-activated K+ permeability.

Measurements of the effects of NH 3 /NH 4 + on glucose-induced electrical activity in β-cells from microdis-sected mouse islets of Langerhans and on intracellular pH in single collagenase-isolated islets pre-loaded with a fluorescent pH probe were performed and are reported here. Application of NH 3 /NH + 4 (15 mM) in the presence of glucose (11 mM) promptly hyperpolarized the β-cell membrane, reduced input resistance by 60% and blocked electrical activity. These changes were paralleled by an increase in islet fluorescence indicative of a cytosolic pH increase. Removal of NH 4 Cl initially stumulated electical activity, which returned to resting level with a time constant of 51 s. Concomitant with the removal of NH 4 C1 there was a drop in pH i followed by a slow return to resting level with a time constant of 83 s. The results suggest that the [Ca 2+ ]-dependent K + channel in the β-cell membrane is activated by a rise in cytosolic pH.

High-resistance micro-electrodes were used to measure membrane potentials in beta-cells from islets of Langerhans of ob/ob obese mice (Norwich colony). In the presence of glucose the burst pattern of electrical activity recorded in ob/ob beta-cells, although similar to the burst pattern recorded from normal beta-cells, presents important differences. The membrane potential of the ob/ob beta-cells in the presence of 11 mM glucose in the modified Krebs solution oscillates between a silent-phase level at -48 mV and an active-phase level at -36 mV, similarly to normal mouse islet beta-cells. However, the average active-phase duration is 20 s in ob/ob beta-cells compared with 5 s in normal beta-cells. The average burst frequency is 1.8 bursts/min in ob/ob beta-cells compared with 3 bursts/min in normal beta-cells. While normal beta-cells show continuous spike activity above 16 mM glucose, ob/ob beta-cells often exhibit a burst pattern of electrical activity at glucose concentrations as high as 33 mM. Compared with normal beta-cells, the relationship between spike frequency and glucose concentration is shifted towards lower concentrations in ob/ob beta-cells. Thus, the concentration for half-maximal spike frequency is 6.9 mM for the ob/ob beta-cells and 10.2 mM for the normal beta-cells. In ob/ob beta-cells, the mitochondrial inhibitor carbonyl-cyanide m-chlorophenylhydrazone induces hyperpolarization of the membrane, consistent with its effect of stimulating K+ permeability in normal islets. However, quinine and the sulphonylurea glibenclamide did not block the silent phase between the bursts of electrical activity. Both drugs block the [Ca2+]i-activated K+ permeability thought to control the membrane potential at the silent phase in normal beta-cells. The modified pattern of response to glucose and decreased sensitivity to quinine and glibenclamide suggest that the beta-cell membrane of the ob/ob islet of Langerhans has a modified [Ca2+]i-activated K+ permeability.