Rosa M. Santos

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.

Neomycin blocks dihydropyridine-insensitive Ca2+ influx in bovine adrenal chromaffin cells

There is evidence that bovine adrenal chromaffin cells are provided with both dihydropyridine-sensitive and -resistant voltage-sensitive Ca2+ influx pathways. Although recent electrophysiological work indicates that the dihydropyridine-resistant pathway is partially mediated by omega-conotoxin-sensitive and -insensitive Ca2+ channels, the pharmacological sensitivity of the latter channels remains elusive. We have now found that combined incubations with nitrendipine (1 microM) and neomycin (0.5 mM) reduced high K+ (50 mM)-evoked intracellular Ca2+ concentration ([Ca2+]i) transients to a larger extent than each drug separately. [Ca2+]i was measured using the fluorescent intracellular Ca2+ indicator fura-2. Neomycin (0.05-2 mM) reduced high K(+)-evoked 45Ca2+ uptake in a dose-dependent manner (IC50 = 0.09 mM). In the presence of nitrendipine (1 microM), the minimal neomycin concentration necessary for total blockade of 45Ca2+ uptake was reduced to 0.3 mM. Moreover, in the absence of nitrendipine the 45Ca2+ 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 [Ca2+]i transient induced by the L-type Ca2+ channel agonist Bay K 8644 (1 microM) 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 (IC50 = 0.8-1.1 mM), the blockade efficiency being enhanced in the presence of 1 microM nitrendipine (IC50 = 0.17-0.19 mM). It is concluded that neomycin (< or = 0.3 mM) blocks preferentially the dihydropyridine-insensitive Ca2+ influx pathway of the chromaffin cell. Moreover, both the dihydropyridine-sensitive and the dihydropyridine-resistant, neomycin-sensitive Ca2+ influx pathways contribute strongly to depolarization-evoked catecholamine secretion.

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.

Protein kinase C activator inhibits voltage-sensitive Ca2+ channels and catecholamine secretion in adrenal chromaffin cells

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, 4α‐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 μM) 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.

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.

Regulation of Ca2+ influx by a protein kinase C activator in chromaffin cells: differential role of P/Q- and L-type Ca2+ 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 microM) inhibited the early and late phases of the ionomycin (2 microM)-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 microM omega-conotoxin GVIA, a blocker of N-type channels, was without effect. A sizeable (< 45%) component of early Ca2+ influx persisted in the combined presence of omega-agatoxin IVA (100 nM) and nitrendipine (1 microM). Simultaneous exposure to omega-agatoxin IVA and PMA inhibited both the early [Ca2+]i transients and Mn2+ quenching to a much greater extent than each drug separately. Inhibition of the [Ca2+]i transients by nitrendipine and PMA did not significantly exceed that produced by PMA alone. It is concluded that phorbol ester-mediated activation of protein kinase C inhibits preferentially L-type VSCCs over P/Q type channels in adrenal chromaffin cells. However, the possibility cannot be ruled out that dihydropyridine-resistant, non-P/Q type channels might also be negatively regulated by protein kinase C. This may represent an important pathway for the specific control of VSCCs by protein kinase C-linked receptors, not only in paraneurones but presumably also in neurones and other excitable cells.

Isoform‐specific inhibition of voltage‐sensitive Ca2+ channels by protein kinase C in adrenal chromaffin cells

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‐α and ‐ϵ isoforms from cytosol to membranes. PKC‐ι and ‐ζ showed no signs of translocation. It is concluded that VSCCs are specifically inhibited by the activation of PKC‐ϵ 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.

Glucose-mediated Ca2+ signalling in single clonal insulin-secreting cells: evidence for a mixed model of cellular activation

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 Ca(2+) concentration ([Ca(2+)](i)) is an established index of beta-cell function. We used fura-2 microfluorescence techniques to assess the [Ca(2+)](i) responsiveness of single BRIN-BD11 cells to glucose and other secretagogues. Glucose (1-16.7 mM) evoked oscillatory [Ca(2+)](i) rises in these cells resembling those found in parental rat pancreatic beta-cells. The percentage of glucose-responsive cells was 11% at 1 mM and increased to 40-70% at 3-16.7 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: 1-5 mM) and a variable dose-dependent amplification of the [Ca(2+)](i) responses (EC(50) range: 2-10 mM), as assessed by a multiple-stimulation protocol. Glyceraldehyde and alpha-ketoisocaproic acid had glucose-like effects on [Ca(2+)](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.

Modulation of glucose-induced insulin secretion by cytosolic redox state in clonal beta-cells.

Nutrient stimulation of pancreatic beta-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 beta-cell line (BRIN-BD11). Long-term exposure to the NAD+ precursor vitamin nicotinic acid (NA, 100 microM) 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.