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P Rorsman

Publications and source records attributed to P Rorsman.

At least 37 records · Page 2Linked to original sources
PubMed1999

Significance of Na/Ca exchange for Ca2+ buffering and electrical activity in mouse pancreatic beta-cells.

We have combined the patch-clamp technique with microfluorimetry of the cytoplasmic Ca2+ concentration ([Ca2+]i) to characterize Na/Ca exchange in mouse beta-cells and to determine its importance for [Ca2+]i buffering and shaping of glucose-induced electrical activity. The exchanger contributes to Ca2+ removal at [Ca2+]i above 1 microM, where it accounts for >35% of the total removal rate. At lower [Ca2+]i, thapsigargin-sensitive Ca2+-ATPases constitute a major (70% at 0.8 microM [Ca2+]i) mechanism for Ca2+ removal. The beta-cell Na/Ca exchanger is electrogenic and has a stoichiometry of three Na+ for one Ca2+. The current arising from its operation reverses at approximately -20 mV (current inward at more negative voltages), has a conductance of 53 pS/pF (14 microM [Ca2+]i), and is abolished by removal of external Na+ or by intracellularly applied XIP (exchange inhibitory peptide). Inhibition of the exchanger results in shortening (50%) of the bursts of action potentials of glucose-stimulated beta-cells in intact islets and a slight (5 mV) hyperpolarization. Mathematical simulations suggest that the stimulatory action of glucose on beta-cell electrical activity may be accounted for in part by glucose-induced reduction of the cytoplasmic Na+ concentration with resultant activation of the exchanger.

Action Potentials↗
PubMed1999

Activation of Ca(2+)-dependent K(+) channels contributes to rhythmic firing of action potentials in mouse pancreatic beta cells.

We have applied the perforated patch whole-cell technique to beta cells within intact pancreatic islets to identify the current underlying the glucose-induced rhythmic firing of action potentials. Trains of depolarizations (to simulate glucose-induced electrical activity) resulted in the gradual (time constant: 2.3 s) development of a small (<0.8 nS) K(+) conductance. The current was dependent on Ca(2+) influx but unaffected by apamin and charybdotoxin, two blockers of Ca(2+)-activated K(+) channels, and was insensitive to tolbutamide (a blocker of ATP-regulated K(+) channels) but partially (>60%) blocked by high (10-20 mM) concentrations of tetraethylammonium. Upon cessation of electrical stimulation, the current deactivated exponentially with a time constant of 6.5 s. This is similar to the interval between two successive bursts of action potentials. We propose that this Ca(2+)-activated K(+) current plays an important role in the generation of oscillatory electrical activity in the beta cell.

ATP-Binding Cassette Transporters↗
PubMed1999

Hormone-sensitive lipase, the rate-limiting enzyme in triglyceride hydrolysis, is expressed and active in beta-cells.

Triglycerides in the beta-cell may be important for stimulus-secretion coupling, through provision of a lipid-derived signal, and for pathogenetic events in NIDDM, where lipids may adversely affect beta-cell function. In adipose tissues, hormone-sensitive lipase (HSL) is rate-limiting in triglyceride hydrolysis. Here, we investigated whether this enzyme is also expressed and active in beta-cells. Northern blot analysis and reverse transcription-polymerase chain reaction demonstrated that HSL is expressed in rat islets and in the clonal beta-cell lines INS-1, RINm5F, and HIT-T15. Western blot analysis identified HSL in mouse and rat islets and the clonal beta-cells. In mouse and rat, immunocytochemistry showed a predominant occurrence of HSL in beta-cells, with a presumed cytoplasmic localization. Lipase activity in homogenates of the rodent islets and clonal beta-cells constituted 2.1 +/- 0.6% of that in adipocytes; this activity was immunoinhibited by use of antibodies to HSL. The established HSL expression and activity in beta-cells offer a mechanism whereby lipids are mobilized from intracellular stores. Because HSL in adipocytes is activated by cAMP-dependent protein kinase (PKA), PKA-regulated triglyceride hydrolysis in beta-cells may participate in the regulation of insulin secretion, possibly by providing a lipid-derived signal, e.g., long-chain acyl-CoA and diacylglycerol.

Adipocytes↗
PubMed1998

Cellular regulation of islet hormone secretion by the incretin hormone glucagon-like peptide 1.

Glucagon-like peptide 1 is a gastrointestinally derived hormone with profound effects on nutrient-induced pancreatic hormone release. GLP-1 modulates insulin, glucagon and somatostatin secretion by binding to guanine nucleotide binding protein-coupled receptors resulting in the activation of adenylate cyclase and generation of cyclic adenosine monophosphate (cAMP). In the B-cell, cAMP, via activation of protein kinase A, interacts with a plethora of signal transduction processes including ion channel activity, intracellular Ca2+ handling and exocytosis of the insulin-containing granules. The stimulatory action of GLP-1 on insulin secretion, contrary to that of the currently used hypoglycaemic sulphonylureas, is glucose dependent and requires the presence of normal or elevated concentrations of the sugar. For this reason, GLP-1 attracts much interest as a possible novel principle for the treatment of human type-2 diabetes. Here we review the actions of GLP-1 on islet cell function and attempt to integrate current knowledge into a working model for the control of pancreatic hormone secretion.

Animals↗
PubMed1998

Glucagon-like peptide 1 (7-36) amide stimulates exocytosis in human pancreatic beta-cells by both proximal and distal regulatory steps in stimulus-secretion coupling.

The effect of glucagon-like peptide 1(7-36) amide [GLP-1(7-36) amide] on membrane potential, whole-cell ATP-sensitive potassium channel (K[ATP]) and Ca2+ currents, cytoplasmic Ca2+ concentration, and exocytosis was explored in single human beta-cells. GLP-1(7-36) amide induced membrane depolarization that was associated with inhibition of whole-cell K(ATP) current. In addition, GLP-1(7-36) amide (and forskolin) produced greater than fourfold potentiation of Ca2+-dependent exocytosis. The latter effect resulted in part (40%) from acceleration of Ca2+ influx through voltage-dependent (L-type) Ca2+ channels. More importantly, GLP-1(7-36) amide (via generation of cyclic AMP and activation of protein kinase A) potentiated exocytosis at a site distal to a rise in the cytoplasmic Ca2+ concentration. Photorelease of caged cAMP produced a two- to threefold potentiation of exocytosis when the cytoplasmic Ca2+ concentrations were clamped at > or =170 nmol/l. The effect of GLP-1(7-36) amide was antagonized by the islet hormone somatostatin. Similar effects on membrane potential, ion conductances, and exocytosis were observed with glucose-dependent insulinotropic polypeptide (GIP), the second major incretin. The present data suggest that the strong insulinotropic action of GLP-1(7-36) amide and GIP in humans results from its interaction with several proximal as well as distal important regulatory steps in the stimulus-secretion coupling.

Adult↗
PubMed1998

Stimulation of insulin release by repaglinide and glibenclamide involves both common and distinct processes.

The action of repaglinide, a novel insulin secretagogue, was compared with the sulfonylurea glibenclamide with regard to the hypoglycemic action in vivo, binding to betaTC-3 cells, insulin secretion from perifused mouse islets, and capacity to stimulate exocytosis by direct interaction with the secretory machinery in single voltage-clamped mouse beta-cells. Two binding sites were identified: a high-affinity repaglinide (KD = 3.6 nmol/l) site having lower affinity for glibenclamide (14.4 nmol/l) and one high-affinity glibenclamide (25 nmol/l) site having lower affinity for repaglinide (550 nmol/l). In contrast to glibenclamide, repaglinide (in concentrations as high as 5 micromol/l) lacked the ability to enhance exocytosis in voltage-clamped beta-cells. Repaglinide was more potent than glibenclamide in stimulating insulin release from perifused mouse islets (EC50 29 vs. 80 nmol/l). The greater potency of repaglinide in vitro was paralleled by similar actions in vivo. The ED50 values for the hypoglycemic action were determined to be 10.4 and 15.6 microg/kg after intravenous and oral administration, respectively. The corresponding values for glibenclamide were 70.3 microg/kg (intravenous) and 203.2 microg/kg (oral). Further, repaglinide (1 mg/kg p.o.) was effective (P < 0.001) as an insulin-releasing agent in a rat model (low-dose streptozotocin) of type 2 diabetes. These observations suggest that the insulinotropic actions of repaglinide and glibenclamide in vitro and in vivo are secondary to their binding to the high-affinity repaglinide site and that the insulinotropic action of repaglinide involves both distinct and common cellular mechanisms.

Animals↗
PubMed1997

Rapid ATP-dependent priming of secretory granules precedes Ca(2+)-induced exocytosis in mouse pancreatic B-cells.

1. The glucose and ATP dependence of exocytosis were investigated in single mouse pancreatic B-cells by monitoring changes in cell capacitance evoked by voltage-clamp depolarizations, infusion of high [Ca2+]i buffers or photorelease of caged Ca2+ or ATP. 2. In intact B-cells, using the perforated patch whole-cell technique, glucose (5 mM) increased exocytotic responses evoked by membrane depolarization 5-fold over that observed in the absence of the sugar. Increasing the glucose concentration to 20 mM produced a further doubling of exocytosis. The stimulatory action of glucose was attributable to glucose metabolism and abolished by mannoheptulose, an inhibitor of glucose phosphorylation. 3. Exocytosis triggered by infusion of high [Ca2+]i and ATP was reduced by 80% when ATP was replaced by its non-hydrolysable analogue adenosine 5'-[beta, gamma-methylene]triphosphate (AMP-PCP) in standard whole-cell experiments. Exocytosis elicited by GTP gamma S was similarly affected by replacement of ATP with the stable analogue. 4. Photoreleasing ATP in the presence of 170 nM [Ca2+]i, following the complete wash-out of endogenous ATP produced a prompt (latency, < 400 ms) and biphasic stimulation of exocytosis. 5. Elevation of [Ca2+]i to exocytotic levels by photorelease from Ca(2+)-nitrophenyl EGTA preloaded into the cell evoked a biphasic stimulation in the presence of Mg-ATP. The response consisted of an initial rapid (completed in < 200 ms) phase followed by a slower (lasting > or = 10 s) sustained component. Replacement of ATP with AMP-PCP abolished the late component but did not affect the initial phase. The latency between elevation of [Ca2+]i and exocytosis was determined as < 45 ms. Inclusion of N-ethylmaleimide (NEM; 0.5 mM for 3 min) in the intracellular solution exerted effects similar to those obtained by substituting AMP-PCP for ATP. 6. We conclude that the B-cell contains a small pool (40 granules) of primed granules which are immediately available for release and which are capable of undergoing exocytosis in an ATP-independent fashion. We propose that this pool of granules is preferentially released during first phase glucose-stimulated insulin secretion. The short latency between the application of ATP and the onset of exocytosis finally suggests that priming takes place with sufficient speed to participate in the rapid adjustment of the secretory capacity of the B-cell.

Adenosine Triphosphate↗
PubMed1997

Protein kinase A-dependent and -independent stimulation of exocytosis by cAMP in mouse pancreatic B-cells.

1. The mechanisms by which cAMP stimulates Ca(2+)-dependent insulin secretion were investigated by combining measurements of whole-cell Ca2+ currents, the cytoplasmic free Ca2+ concentration ([Ca2+]i) and membrane capacitance in single mouse B-cells maintained in tissue culture. 2. Cyclic AMP stimulated exocytosis > 4-fold in whole-cell experiments in which secretion was evoked by intracellular dialysis with a Ca(2+)-EGTA buffer with a [Ca2+]i of 1.5 microM. This effect was antagonized by inhibitors of protein kinase A (PKA). 3. Photorelease of cAMP from a caged precursor potentiated exocytosis at Ca2+ concentrations which were themselves stimulatory (> or = 60 nM), but was without effect in the complete absence of Ca2+. 4. Elevation of intracellular cAMP (by exposure to forskolin) evoked a 6-fold PKA-dependent enhancement of the maximal exocytotic response (determined as the maximum increase in cell capacitance that could be elicited by a train of depolarizations) in perforated-patch whole-cell recordings. 5. Exocytosis triggered by single depolarizations in standard whole-cell recordings was strongly potentiated by cAMP, but in this case the effect was unaffected by PKA inhibition. 6. When exocytosis was triggered by Ca2+ released from Ca(2+)-NP-EGTA ('caged Ca2+'), cAMP exerted a dual stimulatory effect on secretion: a rapid (initiated within 80 ms) PKA-independent phase and a late PKA-dependent component. 7. We conclude that cAMP stimulates insulin secretion both by increasing the release probability of secretory granules already in the readily releasable pool and by accelerating the refilling of this pool.

Animals↗
PubMed1997

G protein-dependent inhibition of L-type Ca2+ currents by acetylcholine in mouse pancreatic B-cells.

1. The effect of acetylcholine (ACh) on voltage-dependent Ca2+ currents in mouse pancreatic B-cells was studied using the whole-cell configuration of the patch-clamp technique. 2. ACh (0.25-250 microM) reversibly and dose-dependently inhibited the Ca2+ current elicited by depolarizations from -80 mV to +10 mV. Maximal inhibition was observed at concentrations > 25 microM where it amounted to approximately 35%. The effect was voltage independent and prevented by atropine (10 microM) suggesting that it was mediated by muscarinic receptors. 3. The inhibitory action of ACh on the Ca2+ current was abolished when the cytoplasmic solution contained GDP beta S (2 mM) and became irreversible when the non-hydrolysable GTP analogue GTP gamma S (10 microM) was included in the pipette. This indicates the participation of G proteins in the inhibitory effect of ACh but pretreatment of the cells with either pertussis or cholera toxin failed to prevent the effect of ACh on the Ca2+ current. 4. ACh remained equally effective as an inhibitor of the whole-cell Ca2+ current in the presence of the L-type Ca2+ channel agonist (-)-Bay K 8644 and after partial inhibition of the current by nifedipine. Addition of omega-agatoxin IVA, omega-conotoxin GVIA or omega-conotoxin MVIIC neither affected the peak Ca2+ current amplitude nor the extent of inhibition produced by ACh. These pharmacological properties indicate that ACh acts by inhibiting L-type Ca2+ channels. 5. The inhibitory action of ACh on the B-cell Ca2+ current was not secondary to elevation of [Ca2+]i and ACh remained equally effective as an inhibitor when Ba2+ was used as the charge carrier, when [Ca2+]i was buffered to low concentrations using EGTA and under experimental conditions preventing the mobilization of Ca2+ from intracellular stores. 6. These results suggest that ACh reduces the whole-cell Ca2+ current in the B-cell through a G protein-regulated, voltage- and Ca(2+)-independent inhibition of L-type Ca2+ channels.

Acetylcholine↗
PubMed1997

The pancreatic beta-cell as a fuel sensor: an electrophysiologist's viewpoint.

The pancreatic beta cell serves as the fuel sensor of the entire body and controls, via secretion of the hypoglycaemic hormone insulin, the blood glucose concentrations within narrow limits by regulation of glucose uptake and release. During the last 30 years, a combination of biochemical and ultrastructural approaches has resulted in dramatic progress in the understanding of the processes by which glucose and other nutrients modulate the release of insulin. The beta cells have also been investigated using electrophysiological techniques and were thus found to be electrically excitable and to undergo complex changes in their membrane potential when exposed to glucose and other stimulators of secretion. The application of the patch-clamp technique to the pancreatic islet preparations has revolutionized the understanding of how bioelectrical processes participate in the fuel-sensing of the beta cell. An important achievement was the identification of an ATP-sensitive K(+)-channel as the resting and glucose-sensitive membrane conductance of the beta cell. This channel also constitutes the target of the hypoglycaemic sulphonylureas: a group of compounds which have been used successfully in the treatment of insulin-dependent diabetes mellitus for several decades.

Animals↗
PubMed1997

Multisite regulation of insulin secretion by cAMP-increasing agonists: evidence that glucagon-like peptide 1 and glucagon act via distinct receptors.

The mechanisms by which glucagon-like peptide 1(7-36)amide (GLP-1[7-36]amide) potentiates insulin secretion were investigated by measurements of whole-cell K+ and Ca2+ currents, membrane potential, the cytoplasmic Ca2+ concentration ([Ca2+]i) and exocytosis in mouse pancreatic B-cells. GLP-1(7-36)amide (10 nM) stimulated glucose-induced (10 mM) electrical activity in intact pancreatic islets. The effect was manifested as a 34% increase in the duration of the bursts of action potentials and a corresponding 28% shortening of the silent intervals. GLP-1(7-36)amide had no effect on the electrical activity at subthreshold glucose concentrations (< or = 6.5 mM). In cultured B-cells, GLP-1(7-36)amide produced a decrease of the whole-cell ATP-sensitive K+ (KATP) conductance remaining at 5 mM glucose by approximately 30%. This effect was associated with membrane depolarization and the initiation of electrical activity. GLP-1(7-36)amide produced a protein-kinase-A-(PKA-) and glucose-dependent fourfold potentiation of Ca(2+)-induced exocytosis whilst only increasing the Ca2+ current marginally. The stimulatory action of GLP-1(7-36)amide on exocytosis was mimicked by the pancreatic hormone glucagon and exendin-4, a GLP-1 receptor agonist. Whereas the stimulatory action of GLP-1(7-36)amide could be antagonized by exendin-(9-39), this peptide did not interfere with the ability of glucagon to stimulate exocytosis. We suggest that GLP-1(7-36)amide and glucagon stimulate insulin secretion by binding to distinct receptors. The GLP-1(7-36)amide-induced stimulation of electrical activity and Ca2+ influx can account for (maximally) a doubling of insulin secretion. The remainder of its stimulatory action results from a cAMP/PKA-dependent potentiation of Ca(2+)-dependent exocytosis exerted at a stage distal to the elevation of [Ca2+]i.

Animals↗
PubMed1997

Adrenaline stimulates glucagon secretion in pancreatic A-cells by increasing the Ca2+ current and the number of granules close to the L-type Ca2+ channels.

We have monitored electrical activity, voltage-gated Ca2+ currents, and exocytosis in single rat glucagon-secreting pancreatic A-cells. The A-cells were electrically excitable and generated spontaneous Na+- and Ca2+-dependent action potentials. Under basal conditions, exocytosis was tightly linked to Ca2+ influx through omega-conotoxin-GVIA-sensitive (N-type) Ca2+ channels. Stimulation of the A-cells with adrenaline (via beta-adrenergic receptors) or forskolin produced a greater than fourfold PKA-dependent potentiation of depolarization-evoked exocytosis. This enhancement of exocytosis was due to a 50% enhancement of Ca2+ influx through L-type Ca2+ channels, an effect that accounted for <30% of the total stimulatory action. The remaining 70% of the stimulation was attributable to an acceleration of granule mobilization resulting in a fivefold increase in the number of readily releasable granules near the L-type Ca2+ channels.

Animals↗
PubMed1997

Glucagon-like peptide I and glucose-dependent insulinotropic polypeptide stimulate Ca2+-induced secretion in rat alpha-cells by a protein kinase A-mediated mechanism.

High-resolution capacitance measurements were used to explore the effects of the gut hormones GLP-I(7-36) amide [glucagon-like peptide I(7-36) amide] and GIP (glucose-dependent insulinotropic polypeptide) on Ca2+-dependent exocytosis in glucagon-secreting rat pancreatic alpha-cells. Both peptides produced a greater than threefold potentiation of secretion evoked by voltage-clamp depolarizations, an effect that was associated with an approximately 35% increase of the Ca2+ current. The stimulatory actions of GLP-I(7-36) amide and GIP were mimicked by forskolin and antagonized by the protein kinase A (PKA)-inhibitor Rp-8-Br-cAMPS. The islet hormone somatostatin inhibited the stimulatory action of GLP-I(7-36) amide and GIP via a cyclic AMP-independent mechanism, whereas insulin had no effect on exocytosis. These data suggest that the alpha-cells are equipped with receptors for GLP-I and GIP and that these peptides, in addition to their well-established insulinotropic capacity, also stimulate glucagon secretion. We propose that the reported inhibitory action of GLP-I on glucagon secretion is accounted for by a paracrine mechanism (e.g., mediated by stimulated release of somatostatin that in turn suppresses exocytosis in the alpha-cell).

Animals↗
PubMed1997

[Electrophysiological studies of insulin secretion, physiology and pathophysiology].

Recent progress in experimental diabetes research is reviewed in this article. The application of the patch-clamp technique to the insulin-secreting pancreatic beta-cell has revolutionised our understanding of how an increase in the blood glucose concentration is linked to stimulation of insulin secretion. A major advance was the identification of an ATP-regulated K-channel (KATP-channel) as the glucose-sensitive membrane conductance of the beta-cell. Closure of these channels underlies the glucose-induced depolarisation of the beta-cell, which then triggers the opening of voltage-gated Ca(2+)-channels and the onset of Ca(2+)-dependent exocytosis. With the patch-clamp technique, it was also possible to demonstrate that the KATP-channel represents the molecular target of the hypoglycaemic sulphonylureas, compounds which are used clinically in the treatment of type 2 diabetes.

Adenosine Triphosphate↗
PubMed1996

Ca(2+)- and GTP-dependent exocytosis in mouse pancreatic beta-cells involves both common and distinct steps.

1. The effects of GTP and Ca2+ on secretion from single pancreatic beta-cells were studied using capacitance measurements as an indicator of exocytosis. 2. GTP or GTP gamma S produced a concentration-dependent increase in cell capacitance in the absence of intracellular calcium. There was no effect of cyclic AMP or BAPTA an GTP-induced secretion. 3. In the absence of GTP, the relationship between intracellular calcium concentration and the maximum rate of secretion was fitted by the Hill equation with a slope factor of 2.5 and half-maximal activation at 1.6 microM intracellular Ca2+. Similar values were obtained in the presence of GTP gamma S, suggesting GTP does not alter the sensitivity of the secretory machinery to Ca2+. 4. GDP beta S alone had no effect on cell capacitance but caused a dose-dependent inhibition of exocytosis induced by infusion of either GTP gamma S or Ca2+, suggesting both stimuli involve G-protein activation. GDP beta S was without effect on exocytosis evoked by depolarization-mediated Ca2+ entry. 5. The time course of exocytosis following rapid elevation of GTP gamma S by photolysis of a caged precursor was dependent on the intracellular Ca2+ and cyclic AMP concentrations. 6. Our results are interpreted in terms of a model in which the secretory pathways stimulated by Ca2+ and GTP contain both common and separate parts.

Animals↗
PubMed1996

Cooling inhibits exocytosis in single mouse pancreatic B-cells by suppression of granule mobilization.

1. The mechanisms by which cooling inhibits insulin secretion were investigated by capacitance measurements of exocytosis in single mouse pancreatic B-cells maintained in short-term tissue culture. 2. A reduction of the bath temperature from 34 to 24 degrees C produced a gradual inhibition of exocytosis. Inhibition of exocytosis was use dependent rather than time dependent. The steady-state inhibition amounted to 90%, which was paralleled by a 30% reduction of the peak Ca2+ current. 3. The Q10 values (between 27 and 37 degrees C) for inhibition of exocytosis and the peak Ca2+ current amplitude were determined as > 5 and 1.6, respectively. From the temperature dependence of exocytosis, an energy of activation was estimated as 145 kJ mol-1. 4. Suppression of exocytosis was not the result of a reduction of Ca2+ influx. When the Ca2+ currents were blocked by 30% (comparable to that produced by cooling) by using a low concentration of Co2+, exocytosis was reduced by < 25%. 5. Elevation of cytoplasmic free Ca2+, by photorelease of 'caged' Ca2+ from Ca(2+)-nitrophenyl-EGTA preloaded into the B-cell, was equally effective at eliciting exocytosis at 24 and 34 degrees C. 6. Cooling produced 70% inhibition of exocytosis evoked by infusion of Ca2+ through the recording electrode. Omission of either MgATP or cAMP from the electrode solution resulted in a comparable reduction of exocytosis. Cooling had no additional inhibitory effect when exocytosis was already suppressed by removal of cytoplasmic MgATP. 7. Our data indicate that exocytosis of granules already docked beneath the membrane is little affected by cooling in the B-cell. Instead, the high overall temperature sensitivity of insulin secretion arises because the replenishment of the readily releasable pool is temperature dependent.

Animals↗
PubMed1996

Endocytosis of secretory granules in mouse pancreatic beta-cells evoked by transient elevation of cytosolic calcium.

1. To investigate the mechanisms regulating the reuptake of secretory granule membranes following regulated exocytosis, we have monitored changes in cell capacitance in single pancreatic beta-cells. 2. Membrane retrieval (endocytosis) occurred both in a continuous manner and in abrupt steps, corresponding to the simultaneous retrieval of 50-100 granules. The large endocytotic steps were associated with a conductance change of about 1 nS which we attribute to the formation of a fission pore with a pore radius of approximately 1 nm. 3. In some cells, we observed large amplitude capacitance fluctuations, suggesting that aggregates of granules are connected to the plasma membrane by a single pore and are subsequently retrieved as a single unit. 4. Endocytosis was evoked by elevation of cytosolic [Ca2+]i, but once initiated, a sustained increase in [Ca2+]i was not required for endocytosis to continue. 5. The [Ca2+]i dependence of exo- and endocytosis was studied by photorelease of Ca2+ from the 'caged' precursor Ca(2+)-nitrophenyl-EGTA (Ca(2+)-NP-EGTA). Both exo- and endocytosis were initiated at between 0.5 and 2 microM Cai(2+). The rate of endocytosis saturated above 2 microM Cai(2+), whereas exocytosis continued to increase up to 4 microM Cai(2+). The maximum rate of endocytosis was < 25% of that of exocytosis. 6. Unlike exocytosis, endocytosis proceeded equally well in the presence or absence of Mg-ATP. 7. Our data indicate that in the pancreatic beta-cell, exocytosis and endocytosis are regulated by different mechanisms.

Adenosine Triphosphate↗