Luís M. Rosário

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.

Single-cell fura-2 microfluorometry reveals different purinoceptor subtypes coupled to Ca2+ influx and intracellular Ca2+ release in bovine adrenal chromaffin and endothelial cells.

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 μM) 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 μM) and UTP, but did not respond to either adenosine 5′-[β-thio]diphosphate (ADP[βS]), a P2Y receptor agonist, adenosine 5′-[α,β-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 μM), UTP (EC50=1.6 μM) and 2MeSATP (EC50= 0.38 μM). 2MeSATP behaved as a partial agonist. Ap4A and ADP[βS] 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 Ca2+-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.

A Toxin Fraction (FTX) from the Funnel-Web Spider Poison Inhibits Dihydropyridine-Insensitive Ca2+ Channels Coupled to Catecholamine Release in Bovine Adrenal Chromaffin Cells

Abstract: 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 μM) 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 ∼80 and 70%, respectively, but both substances together abolished the K+‐evoked catecholamine release, as measured by HPLC. The ω‐conotoxin GVIA (0.5 μM) was without effect on K+‐stimulated 45Ca2+ uptake. Our results indicate that FTX blocks dihydropyridine‐ and ω‐conotoxin‐insensitive Ca2+ channels that, together with L‐type voltage‐sensitive Ca2+ channels, are coupled to catecholamine 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.

Electrochemical studies of zinc in zinc–insulin solution

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.

Circumvention of multidrug-resistance in P388 cells is associated with a rise in the cellular content of phosphatidylcholine.

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 resistance 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.

Naloxone inhibits nicotine-induced receptor current and catecholamine secretion in bovine chromaffin cells.

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 (IC(50)=29 microM). In voltage-clamped chromaffin cells, nicotine (10 microM) evoked an average peak inward current of -146 pA that was inhibited by low concentrations of naloxone (42% at 0.1 microM). The antagonist also inhibited total charge influx associated with nicotinic receptor activation (53% at 0.1 microM). 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.

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.