Carina Ämmälä

Co-localization of L-type Ca2+ channels and insulin-containing secretory granules and its significance for the initiation of exocytosis in mouse pancreatic B-cells.

We have monitored L‐type Ca2+ channel activity, local cytoplasmic Ca2+ transients, the distribution of insulin‐containing secretory granules and exocytosis in individual mouse pancreatic B‐cells. Subsequent to the opening of the Ca2+ channels, exocytosis is initiated with a latency < 100 ms. The entry of Ca2+ that precedes exocytosis is unevenly distributed over the cell and is concentrated to the region with the highest density of secretory granules. In this region, the cytoplasmic Ca2+ concentration is 5‐ to 10‐fold higher than in the remainder of the cell reaching concentrations of several micromolar. Single‐channel recordings confirm that the L‐type Ca2+ channels are clustered in the part of the cell containing the secretory granules. This arrangement, which is obviously reminiscent of the ‘active zones’ in nerve terminals, can be envisaged as being favourable to the B‐cell as it ensures that the Ca2+ transient is maximal and restricted to the part of the cell where it is required to rapidly initiate exocytosis whilst at the same time minimizing the expenditure of metabolic energy to subsequently restore the resting Ca2+ concentration.

Exocytosis elicited by action potentials and voltage‐clamp calcium currents in individual mouse pancreatic B‐cells.

1. Measurements of membrane capacitance, as an indicator of exocytosis, and intracellular Ca2+ concentration ([Ca2+]i) were used to determine the Ca2+ dependence of secretion in single pancreatic B‐cells. 2. Exocytosis was dependent on a rise in [Ca2+]i and could be evoked by activation of voltage‐dependent Ca2+ currents. The threshold for depolarization‐induced release was 0.5 microM [Ca2+]i. Once the [Ca2+]i threshold was exceeded, exocytosis was rapidly (< 50 ms) initiated. When individual pulses were applied, exocytosis stopped immediately upon repolarization and the Ca2+ channels closed, although [Ca2+]i remained elevated for several seconds. 3. During repetitive stimulation (1 Hz), when [Ca2+]i attained micromolar levels, exocytosis also took place during the interpulse intervals albeit at a slower rate than during the depolarizations. 4. Exocytosis could be initiated by simulated action potentials. Whereas a single action potential only produced a small capacitance increase, and in some cells even failed to stimulate release, larger and more consistent responses were obtained with > or = four action potentials. 5. Comparison of the rates of exocytosis measured in response to depolarization, mobilization of Ca2+ from intracellular stores or infusion of Ca2+ through the patch pipette suggests that [Ca2+]i at the secretory sites attains a concentration of several micromolar. This is much higher than the average [Ca2+]i detected by microfluorimetry suggesting the existence of steep spatial gradients of [Ca2+]i within the B‐cell. 6. Inclusion of inhibitors of Ca2+/calmodulin‐dependent protein kinase II in the intracellular solution reduced the depolarization‐induced exocytotic responses suggesting this enzyme may be involved in the coupling between elevation of [Ca2+]i to stimulation of the secretory machinery. 7. The size of the unitary exocytotic event was 2 fF, corresponding to a secretory granule diameter of 250 nm. 8. Over short periods, exocytosis may be extremely fast (1 pF/s or 500 granules/s), which is much higher than the rate of endocytosis (18 fF/s or 9 granules/s). Since the latter is in better agreement with the maximum rate of insulin secretion from islets (approximately 2 granules/s), we suggest that membrane retrieval may set an upper limit on the rate of exocytosis during extended periods of secretion.

PKC-dependent stimulation of exocytosis by sulfonylureas in pancreatic beta cells.

Hypoglycemic sulfonylureas represent a group of clinically useful antidiabetic compounds that stimulate insulin secretion from pancreatic β cells. The molecular mechanisms involved are not fully understood but are believed to involve inhibition of potassium channels sensitive to adenosine triphosphate (KATP channels) in the β cell membrane, causing membrane depolarization, calcium influx, and activation of the secretory machinery. In addition to these effects, sulfonylureas also promoted exocytosis by direct interaction with the secretory machinery not involving closure of the plasma membrane KATP channels. This effect was dependent on protein kinase C (PKC) and was observed at therapeutic concentrations of sulfonylureas, which suggests that it contributes to their hypoglycemic action in diabetics.

Stimulation of the KATP channel by ADP and diazoxide requires nucleotide hydrolysis in mouse pancreatic beta-cells.

1. The mechanisms by which ADP and the hyperglycaemic compound diazoxide stimulate the activity of the ATP‐regulated K+ channel (KATP channel) were studied using inside‐out patches isolated from mouse pancreatic beta‐cells maintained in tissue culture. 2. The ability of diazoxide and ADP to increase KATP channel activity declined with time following patch excision and no stimulation was observed after 15‐40 min. 3. Activation of KATP channels by ADP required the presence of intracellular Mg2+. The stimulatory effect of ADP was mimicked by AMP but only in the presence of ATP. Replacement of ATP with the non‐hydrolysable analogue beta, gamma‐methylene ATP did not interfere with the ability of ADP to stimulate KATP channel activity. By contrast, enhancement of KATP channel activity was critically dependent on hydrolysable ADP and no stimulation was observed after substitution of alpha,beta‐methylene ADP for standard ADP. 4. The ability of diazoxide to enhance KATP channel activity was dependent on the presence of both internal Mg2+ and ATP. Diazoxide stimulation of KATP channel activity was not observed after substitution of beta,gamma‐methylene ATP for ATP. However, in the presence of ADP, at a concentration which in itself had no stimulatory action (10 microM), diazoxide was stimulatory also in the presence of the stable ATP analogue. 5. The stimulatory action of diazoxide on KATP channel activity in the presence of ATP was markedly enhanced by intracellular ADP. This potentiating effect of ADP was not reproduced by the stable analogue alpha,beta‐methylene ADP and was conditional on the presence of intracellular Mg2+. A similar enhancement of channel activity was also observed with AMP (0.1 mM). In the absence of ATP, diazoxide was still capable of stimulating channel activity provided ADP was present. This effect was not reproduced by AMP. 6. In both nucleotide‐free solution and in the presence of 0.1 mM ATP, the distribution of the KATP channel open times were described by a single exponential with a time constant of approximately 20 ms. Addition of ADP or diazoxide resulted in the appearance of a second component with a time constant of > 100 ms which comprised 40‐70% of the total number of events. Under the latter experimental conditions, the open probability of the channel increased more than fivefold relative to that observed in the presence of ATP alone.(ABSTRACT TRUNCATED AT 400 WORDS)

Separate processes mediate nucleotide-induced inhibition and stimulation of the ATP-regulated K(+)-channels in mouse pancreatic beta-cells.

The mechanisms by which nucleotides stimulate the activity of the ATP regulated K+-channel (KATP-channel) were investigated using inside-out patches from mouse pancreatic β-cells. ATP produces a concentration-dependent inhibition of channel activity with a K1 of 18 μM. The inhibitory action of ATP was counteracted by ADP (0.1 mM) and GDP (0.2 mM) but not GTP (1 mM). Stimulation of channel activity was also observed when ADP, GDP and GTP were applied in the absence of ATP. The ability of ADP and GDP to reactivate KATP-channels blocked by ATP declined with time following patch excision and after 30-60 min these nucleotides were without effect. During the same time period the ability of ADP and GTP to stimulate the channel in the absence of ATP was lost. In fact, ADP now blocked channel activity with 50% inhibition being observed at approximately 0.1 mM. By contrast, GDP remained a stimulator in the absence of ATP even when its ability to evoke channel activity in the presence of ATP was lost. These observations show that nucleotide-induced activation of the KATP-channel does not involve competition with ATP for a common inhibitory site but involves other processes. The data are consistent with the idea that nucleotides modulate KATP-channel activity by a number of different mechanisms that may include both regulation of cytosolic constituents and direct interaction with the channel and associated control proteins.

Cytoplasmic calcium transients due to single action potentials and voltage-clamp depolarizations in mouse pancreatic B-cells.

Changes in the cytoplasmic free calcium concentration ([Ca2+]i) in pancreatic B‐cells play an important role in the regulation of insulin secretion. We have recorded [Ca2+]i transients evoked by single action potentials and voltage‐clamp Ca2+ currents in isolated B‐cells by the combination of dual wavelength emission spectrofluorimetry and the patch‐clamp technique. A 500–1000 ms depolarization of the B‐cell from −70 to −10 mV evoked a transient rise in [Ca2+]i from a resting value of approximately 100 nM to a peak concentration of 550 nM. Similar [Ca2+]i changes were associated with individual action potentials. The depolarization‐induced [Ca2+]i transients were abolished by application of nifedipine, a blocker of L‐type Ca2+ channels, indicating their dependence on influx of extracellular Ca2+. Following the voltage‐clamp step, [Ca2+]i decayed with a time constant of approximately 2.5 s and summation of [Ca2+]i occurred whenever depolarizations were applied with an interval of less than 2 s. The importance of the Na(+)‐Ca2+ exchange for B‐cell [Ca2+]i maintenance was evidenced by the demonstration that basal [Ca2+]i rose to 200 nM and the magnitude of the depolarization‐evoked [Ca2+]i transients was markedly increased after omission of extracellular Na+. However, the rate by which [Ca2+]i returned to basal was not affected, suggesting the existence of additional [Ca2+]i buffering processes.

Hyperinsulinism of infancy: towards an understanding of unregulated insulin release. European Network for Research into Hyperinsulinism in Infancy.

Insulin is synthesised, stored, and secreted from pancreatic β cells. These are located within the islets of Langerhans, which are distributed throughout the pancreas. Less than 2% of the total pancreas is devoted to an endocrine function. When the mechanisms that control insulin release are compromised, potentially lethal diseases such as diabetes and neonatal hypoglycaemia are manifest. This article reviews the physiology of insulin release and illustrates how defects in these processes will result in the pathophysiology of hyperinsulinism of infancy.

Alpha2-adrenoreceptor stimulation does not inhibit L-type calcium channels in mouse pancreatic β-cells

The effects of α2-adrenergic stimulation on the Ca2+-current in mouse pancreatic β-cells were investigated using the patch-clamp technique. When using the conventional whole-cell recording configuration (dialysis of cell interior with pipette solution), addition of adrenaline (1 μM) or the α2-adrenergic agonist clonidine (5 μM) failed to reduce the Ca2+-current, irrespective of whether intracellular GTP (or GTPγ S) was present or not and at both physiological (1.3 mM) and elevated (10.2 mM) Ca2+-concentrations. In fact, in the absence of added guanine nucleotides, the agonists tended toincrease the Ca2+-current amplitude in the presence of the higher Ca2+-concentration. Ca2+-channel activation measured at 1.3 mM Ca2+ was not affected by clonidine. Half-maximal activation was observed at ≈−20 mV. In addition, when Ca2+-currents were recorded from intact β-cells, using the perforated patch whole-cell configuration, clonidine (1 μM) also failed to detectably affect the Ca2+-current. It is therefore suggested that the inhibition of β-cell electrical activity and insulin-secretion produced by α2-adrenoreceptor stimulation does not result from suppression of the L-type Ca2+-current.

Inhibition of L-type calcium channels by internal GTP [gamma S] in mouse pancreatic beta cells.

Pretreatment of pancreatic β cells with pertussis toxin resulted in a 30% increase in peak whole-cell Ca2+ currents recorded in the absence of exogenous intracellular guanine nucleotides. Intracellular application of 90 μM GTP[γS], by liberation from a caged precursor, resulted in 40% reduction of the peak Ca2+ current irrespective of whether the current was carried by Ca2+ or Ba2+. Effects on the delayed outward K+ current were small and restricted to a transient Ca2+-dependent K+ current component. Inhibition by GTP[γS] of the Ca2+ current was not mimicked by standard GTP and could not be prevented either by pretreatment with pertussis toxin or by inclusion of GDP[βS] or cyclic AMP in the intracellular medium. The inhibitory effect of GTP[γS] could be counteracted by a prepulse to a large depolarizing voltage. A similar effect of a depolarizing prepulse was observed in control cells with no exogenous guanine nucleotides. These observations indicate that inhibition of β cell Ca2+ current by G protein activation results from direct interaction with the channel and does not involve second-messenger systems. Our findings also suggest that the β cell Ca2+ current is subject to resting inhibition by G proteins.

Demonstration of a novel apamin-insensitive calcium-activated K+ channel in mouse pancreatic B cells

The whole-cell configuration of the patchclamp technique was used to characterize the biophysical and pharmacological properties of an oscillating K+-current that can be induced by intracellular application of GTP[γS] in mouse pancreatic B cells (Ämmälä et al. 1991). These K+ conductance changes are evoked by periodic increases in the cytoplasmic Ca2+ concentration ([Ca2+]i) and transiently repolarize the B cell, thus inhibiting action-potential firing and giving rise to a bursting pattern. GTP[γS]-evoked oscillations in K+ conductance were reversibly suppressed by a high (300 μM) concentration of carbamylcholine. By contrast, α2-adrenoreceptor stimulation by 20 μM clonidine did not interfere with the oscillatory behaviour but evoked a small sustained outward current. At 0 mV membrane potential, the oscillating K+-current elicited by GTP[γS] was highly sensitive to extracellular tetraethylammonium (TEA; 70% block by 1 mM). The TEA-resistant component, which carried approximately 80% of the current at −40 mV, was affected neither by apamin (1 μM) nor by tolbutamide (500 μM). The current evoked by internal GTP[γS] was highly selective for K+, as demonstrated by a 51-mV change in the reversal potential for a sevenfold change in [K+]o. Stationary fluctuation analysis indicated a unitary conductance of 0.5 pS when measured with symmetric (≈ 140mM) KCl solutions. The estimated singlechannel conductance with physiological ionic gradients is 0.1 pS. The results indicate the existence of a novel Ca2+-gated K+ conductance in pancreatic B cells. Activation of this K+ current may contribute to the generation of the oscillatory electrical activity characterizing the B cell at intermediate glucose concentrations.