Amélia M. Silva

Control of pulsatile 5-HT/insulin secretion from single mouse pancreatic islets by intracellular calcium dynamics.

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.

Bursting electrical activity in pancreatic β-cells: evidence that the channel underlying the burst is sensitive to Ca2+ influx through L-type Ca2+ channels

In glucose-stimulated pancreatic β-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 μM 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 μM tolbutamide. The [Ca2+]i was monitored from single islets of Langerhans using fura-2 microfluorescence techniques. Both the high-Ca2+ and Bay-K-8644 evoked [Ca2+]i oscillations overshot the [Ca2+]i recorded in tolbutamide. Nifedipine (10–20 μM) 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 μM) 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 μM) did not suppress the high-Ca2+-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 Ca2+-dependent K+ channel are highly unlikely to provide the pacemaker current underlying the burst. We propose that the burst is mediated by a distinct Ca2+-dependent K+ channel and/or by [Ca2+]idependent slow processes of inactivation of Ca2+ currents.

Electrophysiological and Immunocytochemical Evidence for P2X Purinergic Receptors in Pancreatic β Cells

Objectives: Glucose-induced insulin secretion from pancreatic &bgr; cells is modulated by several hormones and transmitters, namely adenosine triphosphate (ATP) via purinergic receptors. Although P2Y receptors are well documented in &bgr; cells, the presence of P2X receptors remains elusive. We present the first electrophysiological evidence for the presence of P2X receptors in single &bgr; cells of different species. Methods: Ionic currents were recorded from voltage-clamped &bgr; cells near their resting potential using the perforated (nystatin) whole-cell patch-clamp configuration. Receptors were detected by immunocytochemistry. Results: When bathed in substimulatory (2 mM) glucose, mouse &bgr; cells, isolated from islets displaying immunochemical colocalization of P2X1 or P2X3 receptors and insulin, developed large (~250 pA/pF), rapidly activating, and then biexponentially decaying (&tgr;1, ~20 milliseconds/&tgr;2, ~1 second) inward currents on exposure to micromolar concentrations of ATP and &agr;,&bgr;-methylene ATP. The ATP also evoked inward currents (100-300 pA/pF) from porcine and human &bgr; cells, albeit with a slower and more complex inactivation pattern. Conclusions: The ATP-gated ion channels are present in pancreatic &bgr; cells from different species. Specifically, mouse &bgr; 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.

Multiphasic action of glucose and alpha-ketoisocaproic acid on the cytosolic pH of pancreatic beta-cells. Evidence for an acidification pathway linked to the stimulation of Ca2+ influx.

Glucose stimulation raises the pH of pancreatic β-cells, but the underlying mechanisms are not well understood. We have now investigated the acute effects of metabolizable (glucose and the mitochondrial substrate α-ketoisocaproic acid, KIC) and nonmetabolizable (high K and the K-ATP channel blocker tolbutamide) insulin secretagogues on the pH of pancreatic β-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 pH 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 [Ca] rise associated with the activation of voltage-sensitive Ca channels. There was a significant overlap between the glucose-evoked [Ca] rise and the development of the secondary acidification. Depolarization with 30 mM K and tolbutamide evoked pronounced [Ca] rises and concomitant cytosolic acidifications. Blocking glucose-induced Ca influx (with 0 Ca, 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 pH and [Ca], 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 pH of pancreatic β-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 Ca turnover in the cytoplasm. The degree of coupling between glucose metabolism and Ca influx as well as the relative efficacies of these processes determines whether the acute pH response of a β-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.

High external Ca2+ levels trigger membrane potential oscillations in mouse pancreatic β-cells during blockade of K(ATP) channels

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.

Electrical activity and exocytotic correlates of biphasic insulin secretion from β-cells of canine islets of Langerhans

Biphasic insulin secretion in response to glucose, consisting of a transient first phase followed by a progressive second phase, is a well described in pancreatic islets. Using single canine β-cells we have compared the time courses of electrical activity and insulin granule exocytosis to biphasic insulin secretion. Short trains of action potentials, similar those found during first phase insulin secretion, trigger phasic exocytosis from a small pool of insulin granules, likely an immediately releasable pool docked near voltage activated Ca2+ channels. In contrast, plateau depolarizations to between −35 and −20 mV resembling those during second phase insulin secretion, trigger tonic exocytosis from a larger pool of insulin granules, likely a highly Ca2+-sensitive pool farther from Ca2+ channels. Both phasic and tonic modes of exocytosis are enhanced by glucose, via its metabolism. Hence, in canine β-cells two distinct components of exocytosis, tuned to two components of electrical activity, may contribute significantly to biphasic insulin secretion.

Ion channels underlying stimulus-exocytosis coupling and its cell-to-cell heterogeneity in β-cells of transplantable porcine islets of Langerhans

Given the growing interest in porcine islets as model tissue for studying the pathogenesis of human diabetes mellitus and its treatment by transplantation, we investigated stimulus-exocytosis coupling in single porcine β-cells using patch clamp electrophysiology, Ca2+ imaging, capacitance tracking and amperometry. We establish that porcine β-cells display several features prominently seen in β-cells from human islets of Langerhans. These include: (i) wide heterogeneity of electrical responsiveness to glucose; (ii) dependence of action potential activity on voltage-dependent Na+ as well as high voltage activated Ca2+ current; (iii) heterogeneity of time course of depolarization-evoked insulin granule exocytosis; and (iv) the dependence of vigorous single cell electrical activity and insulin granule exocytosis on the presence of agents that enhance cytosolic cAMP concentration. These findings promote the usefulness of porcine β-cells as a model for studying β-cell function in large mammals, including humans, as well as an appropriate source of tissue for xenotransplantation.

Phasic and tonic modes of depolarization-exocytosis coupling in β-cells of porcine islets of Langerhans

In response to depolarizations that open voltage dependent Ca2+ channels single porcine β-cells display heterogeneous time courses of exocytosis. Some cells display phasic exocytosis that is triggered by individual or short burst of action potentials typically characteristic of glucose-induced electrical activity or brief voltage clamp depolarization. Other cells, singularly or additionally, display tonic exocytosis that (i) is triggered during prolonged (up to seconds-long) depolarizations to voltages (-30 to -20 mV), and (ii) coincides with rises in global cytosolic [Ca2+] > 500 nM. We suggest that tonic exocytosis (i) likely results from a recently described pool of granules that is more Ca2+ sensitive and less co-localized with voltage-sensitive Ca2+ entry channels than that contributing to phasic exocytosis and (ii) helps tune exocytosis to glucose-induced electrical activity when the latter consists of spike activity followed by intervals of plateau depolarization to nearly -20 mV.

Bursting Electrical Activity Generated in the Presence of KATP Channel Blockers

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.

New strategies for the treatment of autoimmune diseases using nanotechnologies

Abstract The immune system is composed of organs, cells, and soluble factors, which together act to defend the organism against foreign invaders, and is divided into innate and adaptive immune system (or acquired). Antigens are substances (usually foreign macromolecules) recognized by adaptive immune system receptors as dangerous non-self and that immediately initiate a response. While, response against normal body components (itself reaction) may occur as a result of failure in self-tolerance mechanisms, producing different autoimmune diseases which are, in general, progressive, chronic, and self-perpetuating. The available treatments aim to ameliorate or treat symptoms but have serious side-effects due to the high doses needed, as used drugs have in general low water solubility and long treatment periods. Recent advances with resource of nanotechnology aiming to achieve better results in terms of drug bioavailability and targeting to the treatment site of action as well as reduction of side effects will be discussed. In this chapter, five autoimmune diseases were selected, due to their incidence to the general population: the organ-specific diseases diabetes mellitus type I, psoriasis, multiple sclerosis, lupus erythematosus, and rheumatoid arthritis that affect several organs.